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British Journal Of Ophthalmology
December 2005
(Volume 89, Number 12).
BJO at a glance
BJO at a glance
Creig Hoyt
Br J Ophthalmol 2005; 89: 1547. doi:10.1136/bjo.2005.atag
Editorials
ROP and neurodevelopmental disabilities
G E Quinn
Br J Ophthalmol 2005; 89: 1547. doi:10.1136/bjo.2005.077388
Overcoming the technical challenges of deep lamellar keratoplasty
M Yamada
Br J Ophthalmol 2005; 89: 1548-1549. doi:10.1136/bjo.2005.077396
Mouse models may provide new insight into the relation between cholesterol and
age related macular degeneration
J L Duncan
Br J Ophthalmol 2005; 89: 1549-1551. doi:10.1136/bjo.2005.077370
World view
Characteristic clinical features as an aid to the diagnosis of suppurative keratitis
caused by filamentous fungi
P A Thomas, A K Leck, and M Myatt
Br J Ophthalmol 2005; 89: 1554-1558. doi:10.1136/bjo.2005.076315
A population based survey of the prevalence and types of glaucoma in rural
West Bengal: the West Bengal Glaucoma Study
A Raychaudhuri, S K Lahiri, M Bandyopadhyay, P J Foster, B C Reeves, and G J
Johnson
Br J Ophthalmol 2005; 89: 1559-1564. doi:10.1136/bjo.2005.074948
Cover
Are you calling me primitive?
I R Schwab and S P Collin
Br J Ophthalmol 2005; 89: 1553. doi:10.1136/bjo.2005.078006
Clinical science - Scientific reports
Intermittent exotropia increasing with near fixation: a "soft" sign of
neurological disease
P H Phillips, K J Fray, and M C Brodsky
Br J Ophthalmol 2005; 89: 1120-1122. doi:10.1136/bjo.2004.063123
"Near misses" in a cataract theatre: how do we improve understanding and
documentation?
K Mandal, W Adams, and S Fraser
Br J Ophthalmol 2005; 89: 1565-1568. doi:10.1136/bjo.2005.072850
Effect of cataract extraction on frequency doubling technology perimetry in
patients with glaucoma
M A R Siddiqui, A Azuara-Blanco, and S Neville
Br J Ophthalmol 2005; 89: 1569-1571. doi:10.1136/bjo.2005.080655
The relative effects of corneal thickness and age on Goldmann applanation
tonometry and dynamic contour tonometry
A Kotecha, E T White, J M Shewry, and D F Garway-Heath
Br J Ophthalmol 2005; 89: 1572-1575. doi:10.1136/bjo.2005.075580
Aniseikonia associated with epiretinal membranes
M Ugarte and T H Williamson
Br J Ophthalmol 2005; 89: 1576-1580. doi:10.1136/bjo.2005.077164
Comparison of optical coherence tomography models OCT1 and Stratus OCT
for macular retinal thickness measurement
V Pierre-Kahn, R Tadayoni, B Haouchine, P Massin, and A Gaudric
Br J Ophthalmol 2005; 89: 1581-1585. doi:10.1136/bjo.2005.069815
Development of Microelectromechanical Systems (MEMS) forceps for
intraocular surgery
R B Bhisitkul and C G Keller
Br J Ophthalmol 2005; 89: 1586-1588. doi:10.1136/bjo.2005.075853
Is routine biopsy of the lacrimal sac wall indicated at dacryocystorhinostomy? A
prospective study and literature review
C Merkonidis, C Brewis, M Yung, and M Nussbaumer
Br J Ophthalmol 2005; 89: 1589-1591. doi:10.1136/bjo.2005.072199
Clinical science - Extended reports
Variation in rates of severe retinopathy of prematurity among neonatal intensive
care units in the Australian and New Zealand Neonatal Network
B A Darlow, J L Hutchinson, J M Simpson, D J Henderson-Smart, D A Donoghue, N
J Evans on behalf of the Australian and New Zealand Neonatal Network
Br J Ophthalmol 2005; 89: 1592-1596. doi:10.1136/bjo.2005.073650
Deep lamellar keratoplasty by deep parenchyma detachment from the corneal
limbs
T Senoo, K Chiba, O Terada, J Mori, M Kusama, K Hasegawa, and Y Obara
Br J Ophthalmol 2005; 89: 1597-1600. doi:10.1136/bjo.2005.072215
Contact lenses and special back surface design after penetrating keratoplasty to
improve contact lens fit and visual outcome
C Gruenauer-Kloevekorn, U Kloevekorn-Fischer, and G I W Duncker
Br J Ophthalmol 2005; 89: 1601-1608. doi:10.1136/bjo.2005.069500
Lower limits of fluorescein and indocyanine green dye for digital cSLO
fluorescence angiography
A Bindewald, O Stuhrmann, F Roth, S Schmitz-Valckenberg, H-M Helb, A Wegener,
N Eter, and F G Holz
Br J Ophthalmol 2005; 89: 1609-1615. doi:10.1136/bjo.2005.070409
Systemic carboplatin for retinoblastoma: change in tumour size over time
D H Abramson, S D Lawrence, K L Beaverson, T C Lee, I S Rollins, and I J Dunkel
Br J Ophthalmol 2005; 89: 1616-1619. doi:10.1136/bjo.2005.072033
Unilateral electronegative ERG of non-vascular aetiology
A G Robson, E C Richardson, A H C Koh, C E Pavesio, P G Hykin, A Calcagni, E M
Graham, and G E Holder
Br J Ophthalmol 2005; 89: 1620-1626. doi:10.1136/bjo.2005.071357
Laboratory science - Scientific reports
Increased expression of vascular endothelial growth factor associated with
accumulation of lipids in Bruch’s membrane of LDL receptor knockout mice
M Rudolf, B Winkler, Z Aherrahou, L C Doehring, P Kaczmarek, and U SchmidtErfurth
Br J Ophthalmol 2005; 89: 1627-1630. doi:10.1136/bjo.2005.071183
Advanced glycation end products can induce glial reaction and neuronal
degeneration in retinal explants
A Lecleire-Collet, L H Tessier, P Massin, V Forster, G Brasseur, J A Sahel, and S
Picaud
Br J Ophthalmol 2005; 89: 1631-1633. doi:10.1136/bjo.2005.079491
5,10-Methylenetetrahydrofolate reductase C677T gene polymorphism in
Behçet’s patients with or without ocular involvement
Y Özkul, C Evereklioglu, M Borlu, S Taheri, M Calis, M Dündar, and Ö Ilhan
Br J Ophthalmol 2005; 89: 1634-1637. doi:10.1136/bjo.2005.076836
Laboratory science - Extended reports
Gremlin gene expression in bovine retinal pericytes exposed to elevated glucose
R Kane, L Stevenson, C Godson, A W Stitt, and C O’Brien
Br J Ophthalmol 2005; 89: 1638-1642. doi:10.1136/bjo.2005.069591
Expression of FGFR-2 and FGFR-3 in the normal human fetal orbit
S H Khan, J A Britto, R D Evans, and K K Nischal
Br J Ophthalmol 2005; 89: 1643-1645. doi:10.1136/bjo.2005.075978
Perspective
Hypertensive retinopathy revisited: some answers, more questions
A Grosso, F Veglio, M Porta, F M Grignolo, and T Y Wong
Br J Ophthalmol 2005; 89: 1646-1654. doi:10.1136/bjo.2005.072546
Impression cytology of the ocular surface
R Singh, A Joseph, T Umapathy, N L Tint, and H S Dua
Br J Ophthalmol 2005; 89: 1655-1659. doi:10.1136/bjo.2005.073916
Letters
Ultrahigh resolution optical coherence tomography of birdshot
retinochoroidopathy
A J Witkin, J S Duker, T H Ko, J G Fujimoto, and J S Schuman
Br J Ophthalmol 2005; 89: 1660-1661. doi:10.1136/bjo.2005.076570
Unilateral necrotising toxoplasmic retinochoroiditis as the main clinical
manifestation of a peptide transporter (TAP) deficiency
A Parissiadis, A Dormoy, D Fricker, D Hanau, H de la Salle, J-P Cazenave, P
Lenoble, and L Donato
Br J Ophthalmol 2005; 89: 1661-1662. doi:10.1136/bjo.2005.078097
Scleral contact lenses are not optically inferior to corneal lenses
A Salam, B Melia, and A J Singh
Br J Ophthalmol 2005; 89: 1662-1663. doi:10.1136/bjo.2005.074377
Optometric referrals: towards a two way flow of information?
B J W Evans, D E Harle, and B Cocco
Br J Ophthalmol 2005; 89: 1663. doi:10.1136/bjo.2005.075531
Retinal changes in juvenile X linked retinoschisis using three dimensional optical
coherence tomography
Y Minami, S Ishiko, Y Takai, Y Kato, H Kagokawa, A Takamiya, T Nagaoka, R
Kinouchi, and A Yoshida
Br J Ophthalmol 2005; 89: 1663-1664. doi:10.1136/bjo.2005.075648
Linezolid induced toxic optic neuropathy
K Kulkarni and L V Del Priore
Br J Ophthalmol 2005; 89: 1664-1665. doi:10.1136/bjo.2005.074237
Delayed progressive visual loss following wrapping of bilateral clinoidal
aneurysms: recovery of vision and improvement in neuroimaging during
corticosteroid treatment
P S Subramanian, N R Miller, V Renard, and R J Tamargo
Br J Ophthalmol 2005; 89: 1666-1668. doi:10.1136/bjo.2005.078626
Mailbox
Visual loss may be due to silicone oil tamponade effect rather than silicone oil
removal
P K Rani, R Raman, P Bhende, and T Sharma
Br J Ophthalmol 2005; 89: 1667. doi:10.1136/bjo.2005.082602
Visual loss after silicone oil removal
E N Herbert, S H M Liew, and T H Williamson
Br J Ophthalmol 2005; 89: 1667-1668. doi:10.1136/bjo.2005.082610
Visual loss following silicone oil removal
R S B Newsom, R Johnston, P Sullivan, B Aylward, G Holder, and Z Gregor
Br J Ophthalmol 2005; 89: 1668. doi:10.1136/bjo.2005.082644
Obituary
Marshall Miller Parks, MD, 1918–2005
D Taylor
Br J Ophthalmol 2005; 89: 1552. doi:10.1136/bjo.2005.081695
Corrections
CORRECTION
Br J Ophthalmol 2005; 89: 1668. doi:10.1136/bjo.2005.63123corr1
From the library
From the Library
Br J Ophthalmol 2005; 89: 1672. doi:10.1136/bjo.2005.bjdec05ftl
EDITORIAL
1547
Retinopathy of prematurity
.......................................................................................
ROP and neurodevelopmental
disabilities
G E Quinn
...................................................................................
The dilemma in premature babies
T
here are essentially three ways in
which blindness from retinopathy
of prematurity (ROP) can be prevented, including elimination of premature birth, changes in neonatal care, and
improved detection and treatment of
established sight threatening retinopathy. It is on the latter that the attention
and efforts of the ophthalmic community have been focused, leaving the first
two to the paediatricians, perinatologists, neonatologists, nurses, and others
who care for these tiny babies. What
Darlow et al have done in the paper in
this issue of the BJO (p 1592) is direct
our attention to the second possibility—
that is, neonatal care may be able to be
altered to decrease the incidence of
potentially blinding disease.
Darlow et al document a remarkable
variability in the prevalence of severe
ROP among the nurseries in the neonatal intensive care units in the Australian
and New Zealand Neonatal Network.
Such variability is generally expected
when morbidity or mortality results are
compared across centres, even in an
essentially population based study that
this report represents (the four tertiary
paediatric care referral centres are
excluded from the analysis reported by
Darlow et al). The variability in such
reports can usually be explained by a
combination of several factors, including case mix, assessment of the primary
outcome measure, viability considerations by neonatologists at the various
centres, and variations in clinical practice. For example, the percentage of high
risk deliveries may differ dramatically
among nurseries, leading to more at-risk
infants in one nursery than another.
Smaller nurseries may also have greater
variation in the incidence of severe ROP
because of sampling variability. In addition, standardisation of outcome measures in multicentre studies represents a
huge challenge and can seriously impact
the perceived prevalence of severe disease.
Thanks to a large enough sample and
novel analytical techniques, Darlow et al
have been able to take into account
many of the potential confounding
variables using logistic regression models. They find that variability across
nurseries in the rates of severe ROP
requiring treatment is not explained by
case mix, sampling, or outcome assessment, but rather is probably the result of
clinical practice in the individual nurseries. In their analysis, they wisely
chose to highlight the improvement in
rates of serious ROP that could be
reached if clinical practice was altered
to achieve the ‘‘best practice’’ rates
achieved in the top 20th percentile of
the nurseries, rather than focusing on
the ‘‘problem’’ nurseries. Using this
systemwide approach to changes in
clinical care would lower the rate of
severe ROP to 5.9% in the top 20% of
nurseries from the overall rate of 9.6%.
They estimate that 79 cases of severe
ROP would have been prevented.
A major problem that arises when
practice patterns are changed is that
interventions aimed at decreasing the
rate of one serious problem may
increase the rate of another, equally
serious problem. A very clearcut example of this was provided in the 1950s
when oxygen was found to be the root
cause of blinding ROP and its delivery
markedly restricted.1 This led to an
increase in mortality2 and spastic diplegia among premature babies,3 and it was
estimated that 16 infants died from
curtailed oxygenation for each case of
blindness prevented.4 This observation
has particular clinical relevance when
evaluating possible changes in clinical
care of premature babies, a population
that has had an increased survival rate
as a result of advances in perinatal and
neonatal medicine over the past
20 years in countries with high levels
of human development5; it is also a
population in which we have only an
evolving understanding of the developmental disabilities that are detected
later in life.6–8 Since clinical care of the
premature baby is directed broadly at
prevention of illness, undernutrition,
and infection during a time of rapid
brain growth and development, it is not
hard to postulate that systemwide
changes in practice may differentially
impact on the occurrence of cerebral
palsy, visual and hearing impairments,
behavioural and social problems, and
learning difficulties.9 When considering
changes in practice patterns, clearly the
possible effect of any change must
concentrate not only on survival, but
also on lifelong disabilities that may
occur in this vulnerable population.
Identifying and implementing practice pattern changes may decrease the
prevalence of severe ROP and, with
appropriate treatment, of blindness.
However, most children being blinded
from ROP do not live in countries with
high levels of human development, but
rather they are born in countries with
middle levels of human development,
where neonatal services are rapidly
expanding and where limited resources
may severely impact delivery of the
highest levels of neonatal care.9 Thus,
the alterations in clinical practice that
may arise from examining the ‘‘best
practice’’ nurseries in Australia and New
Zealand may have limited generalisability to the nurseries that care for the
largest proportion of babies at risk of
blindness because of severe ROP.
Br J Ophthalmol 2005;89:1547.
doi: 10.1136/bjo.2005.077388
Correspondence to: G E Quinn, Pediatric
Ophthalmology, 1st Floor, Wood Building, The
Children’s Hospital of Philadelphia,
Philadelphia, PA 19104, USA; quinn@email.
chop.edu
REFERENCES
1 Patz A, Hoeck LE, de la Cruz E. Studies on the
effect of high oxygen administration in retrolental
fibroplasia. Am J Ophthalmol 1952;35:1248–52.
2 Avery ME, Oppenheimer EH. Recent increase in
mortality from hyaline membrane disease.
J Pediatr 1960;57:553–9.
3 McDonald A. Cerebral palsy in children of very
low birth weight. Arch Dis Child
1963;38:579–88.
4 Cross KW. Cost of preventing retrolental
fibroplasias. Lancet 1973;2:954–6.
5 Tucker J, McGuire W. Epidemiology of preterm
birth. BMJ 2004;329:675–8.
6 MacDonald H. Committee on Fetus and
Newborn. Perinatal care at the threshold of
viability. Pediatrics 2002;110:1024–7.
7 Colvin M, McGuire W, Fowlie PW.
Neurodevelopmental outcomes after preterm
birth. BMJ 2004;329:1390–3.
8 Hintz SR, Kendrick DE, Vohr BR, et al. Changes in
neurodevelopmental outcomes at 18 to 22
months’ corrected age among infants of less than
25 weeks’ gestational age born in 1993–1999.
Pediatrics 2005;115:1645–51.
9 Gilbert C, Fielder A, Gordillo L, on behalf of the
International NO-ROP Group, et al.
Characteristics of infants with severe retinopathy
of prematurity in countries with low, moderate
and high levels of development: Implications for
screening programs. Pediatrics
2005;115:e518–25.
www.bjophthalmol.com
EDITORIAL
1548
Deep lamellar keratoplasty
.......................................................................................
Overcoming the technical challenges of
deep lamellar keratoplasty
M Yamada
...................................................................................
With continued improvements in surgical technique it may
become the procedure of choice
L
amellar keratoplasty was the first
form of corneal transplantation
attempted, with a history over a
century, and has been regarded mainly
as a therapeutic technique.1 2 Lamellar
grafting offers several advantages over
penetrating keratoplasty, including the
elimination of allograft rejection and the
avoidance of intraocular complications.
In addition, more donor cornea can be
used in lamellar keratoplasty since the
procedure does not require donor
endothelium. This is particularly important in countries where donor corneas
are scarce. However, the use of lamellar
keratoplasty has been limited by difficulties such as irregularity and scarring
of tissue interfaces, leading to poor
visual outcomes compared with penetrating keratoplasty,1–3 as well as technical
difficulties
and
prolonged
operating time. Penetrating keratoplasty
has thus been the most common corneal
transplantation procedure for visual
restoration for many years. Although
penetrating keratoplasty has been
shown to be effective and safe for most
anterior segment pathologies, there are
persistent long term risks such as
endothelial failure and immunological
graft rejection.4
Deep lamellar keratoplasty (DLK) is a
logical step in the surgical management
of corneal stromal opacification in the
setting of functional endothelium.1 In
DLK, pathological stroma is excised
down to Descemet’s membrane, and
offers the promise of better visual outcomes compared with conventional
lamellar grafting. Since this procedure
was first reported by Arichila in 1985,5
several large case series have described
favourable visual results after DLK.3 6 A
report of 120 cases by Sugita and Kondo
demonstrated that corrected visual
acuity improved by 0.09 to 0.6 on
average after DLK.6 Anwar and
Teichmann reported that 89% of 181
eyes treated by DLK achieved visual
acuity of 20/40 or better.3 More recently,
Shimazaki and associates performed a
randomised prospective trial of DLK
versus penetrating keratoplasty,7 showing that visual function after DLK, as
www.bjophthalmol.com
measured by corrected visual acuity,
contrast visual acuity, the glare test,
and corneal topography, was comparable to that achieved in penetrating
keratoplasty. It should be noted that
DLK was superior to penetrating keratoplasty in measures of operative morbidity such as continuous endothelial
cell loss and intraocular complications.7 8 These results suggest that DLK
is a safe alternative to penetrating
keratoplasty in eyes without endothelial
abnormalities. These promising findings
regarding DLK underscore the importance of overcoming technical challenges such as achieving thorough
stromal tissue excision without perforation of Descemet’s membrane.
Deep lamellar keratoplasty is a
logical step in the surgical management of corneal stromal opacification in the setting of functional
endothelium
Most corneal surgeons have confronted the technical challenge of deep
lamellar dissection and the attendant
risk of puncturing Descemet’s membrane during DLK. To facilitate the
dissection of stromal tissue while reducing the risk of perforation, Arichila5
and Price9 employed an air injection
technique to separate tissue planes.
Sugita and Kondo6 and Amayem and
Anwar10 used hydrodelamination to
separate the deep stromal fibres from
Descemet’s membrane. Manche and
associates11 used a hyaluronic acid for
viscodelamination. These dissection
based procedures appear to be useful
both in the identification of stromal
layers, and in the separation of stromal
layers from Descemet’s membrane. An
alternative approach involves improving
visualisation of the deep stromal layers
during surgery. Balestrzzi et al12 used
trypan blue to stain and visualise the
posterior stromal fibres. Moore and
associates13 attempted to use an ocular
endoscope to visualise the posterior
corneal surface. However, despite all
these efforts, perforation of Descemet’s
membrane remains common in DLK.
Even experienced DLK surgeons have
been reported to demonstrate a perforation rate of as high as 25%.2 6 Sugita and
Kondo6 also reported that stromal tissue
was incompletely removed in one third
of cases. Thus, thoroughly baring
Descemet’s membrane without perforation has proved to be a difficult, time
consuming, and elusive operative goal
for most corneal surgeons.
When employing air, fluid, or viscoelastic injection to dissect Descemet’s
membrane from the stromal tissue
planes, it is ideal to achieve complete
separation of these tissue planes with a
single injection. When this is achieved, as
described by Anwar and Teichmann,3 a
large bubble is formed, indicating perfect
separation of tissue planes. However, this
result is not always achieved, in large part
because the proper depth of dissection is
difficult to estimate. Melles and associates14 described a technique for the
visualisation of the posterior corneal surface by filling the anterior chamber with
air. Through a scleral incision, a deep
stromal pocket was created across the
cornea, using the mirror image of a
30 gauge needle as a reference for dissection depth. Senoo and associates, in this
issue of the BJO (p 1597), describe
another approach to determining the
proper depth of dissection. A sclerocorneal flap, as is employed during trabeculectomy, is made, and direct microscopic
visualisation is used to guide dissection of
stromal tissues to the region directly
overlying Descemet’s membrane. The
continued development of such techniques promises to make DLK easier, safer,
and less time consuming.
Trends in keratoplasty have been
changing over the past decade. Ocular
surface reconstruction, consisting of
limbal transplantation combined with
amniotic membrane transplantation,
has enabled us to improve the management
of
cicatrising
diseases.15 16
Posterior lamellar keratoplasty, also
referred to as deep lamellar endothelial
keratoplasty, was developed for patients
with endothelial dysfunction.2 17 These
procedures are based on the concept
that only the pathological part of the
cornea, such as the epithelium or
endothelium, should be replaced by
donor tissue, leaving the healthy portion
of the host cornea intact. DLK is
consistent with this paradigm, and can
be viewed as a procedure designed to
remove pathological stroma from
healthy corneas. With continued
improvements in surgical technique,
including the advance described by
Senoo and associates in this issue, DLK
may become the procedure of choice for
keratoplasty in most eyes without
endothelial abnormalities.
EDITORIAL
Br J Ophthalmol 2005;89:1548–1549.
doi: 10.1136/bjo.2005.077396
Correspondence to: Masakazu Yamada, MD,
Division for Vision Research, National Institute
of Sensory Organs, National Tokyo Medical
Center, 2-5-1 Higashigaoka, Meguro-ku,
Tokyo 152-8902, Japan; yamadamasakazu@
kankakuki.go.jp
The author has no proprietary interest in any
materials in this manuscript.
1549
4
5
6
7
REFERENCES
1 Aggarwal RK. Deep lamellar keratoplasty—an
alternative to penetrating keratoplasty.
Br J Ophthalmol 1997;81:178–9.
2 Terry MA. The evolution of lamellar grafting
techniques over twenty-five years. Cornea
2000;19:611–16.
3 Anwar M, Teichmann KD. Deep lamellar
keratoplasty; surgical techniques for anterior
lamellar keratoplasty with and without baring of
8
9
10
Descemet’s membrane. Cornea
2002;21:374–83.
Thompson RW, Price MO, Bowers PJ, et al. Longterm graft survival after penetrating keratoplasty.
Ophthalmology 2003;110:1396–402.
Arichila E. Deep lamellar keratoplasty dissection
of host tissue with intrastromal air injection.
Cornea 1985;3:217–18.
Sugita J, Kondo J. Deep lamellar keratoplasty
with complete removal of pathological stroma for
vision improvement. Br J Ophthalmol
1997;81:184–8.
Shimazaki J, Shimmmura S, Ishioka M, et al.
Randomized clinical trial of deep lamellar
keratoplasty vs penetrating keratoplasty.
Am J Ophthalmol 2002;134:159–65.
Panda A, Bageshwar LMS, Ray M, et al. Deep
lamellar keratoplasty versus penetrating
keratoplasty for corneal lesions. Cornea
1999;18:172–5.
Price F. Air lamellar keratoplasty. Refract Corneal
Surg 1989;5:240–3.
Amayem AF, Anwar M. Fluid lamellar
keratoplasty in keratoconus. Ophthalmology
2000;107:76–80.
Age related macular degeneration
.......................................................................................
Mouse models may provide new insight
into the relation between cholesterol
and age related macular degeneration
J L Duncan
...................................................................................
With the goal of preventing vision loss from this disease, it is
important to identify modifiable risk factors that may be targets for
intervention
A
ge related macular degeneration
(AMD) is the leading cause of
severe vision loss among the
elderly in the United States, Europe,
and Australia. However, the cause of
this blinding disease remains a topic of
active investigation. Most agree the
pathogenesis of AMD is multifactorial
and that it results from the interaction
of genetic, environmental and ageing
effects. Evidence from population based
studies has supported a role for heredity
in the pathogenesis of AMD. Recent
studies have identified a polymorphism
in the gene for complement factor H
which may be present in up to half of all
white AMD patients.1–4 However, polymorphisms in this gene are also frequently found in Japanese5 and
Chinese6 populations, where AMD infrequently causes vision loss. Clearly,
development of AMD depends on the
interaction of genetic background with
other, presumably environmental, factors.
With the goal of preventing vision loss
from this disease, it is important to
identify modifiable risk factors that may
be targets for intervention. Some, but
not all, epidemiological studies have
identified an association between cardiovascular disease risk and AMD.7
Cigarette smoking, a well recognised
risk factor for cardiovascular disease, is
the most consistently demonstrated
modifiable risk factor contributing to
AMD,8 and its role in complement
activation has been considered supportive evidence of the part played by
complement factor H mutations.3
Recent case-control9 and prospective10
studies have identified elevated C reactive protein, an inflammatory biomarker
associated with cardiovascular disease,
as a risk factor for AMD and AMD
progression. Systemic hypertension has
been associated with neovascular AMD11
and a poorer response to laser therapy
for choroidal neovascularisation (CNV)
in patients with AMD.12 Some studies
have found an association between
markers of systemic atherosclerosis
and AMD,13 14 but other large population
based studies have found no consistent
11 Manche EE, Holland GN, Maloney RK. Deep
lamellar keratoplasty using viscoelastic dissection.
Arch Ophthalmol 1999;117:1561–5.
12 Balestrazzi E, Balestrazzi A, Mosca L, et al. Deep
lamellar keratoplasty with trypan blue
intrastromal staining. J Cataract Refract Surg
2002;28:929–31.
13 Moore JE, Herath G, Sharma A. Endoscopic
visualization to aid deep anterior lamellar
keratoplasty. Eye 2004;18:188–91.
14 Melles GRJ, Lander F, Rietveld FJ, et al. A new
surgical technique for deep stromal, anterior
lamellar keratoplasty. Br J Ophthalmol
1999;83:327–33.
15 Tseng SC, Prabhasawat P, Barton K, et al.
Amniotic membrane transplantation with or
without limbal allografts for corneal surface
reconstruction in patients with limbal stem cell
deficiency. Arch Ophthalmol 1998;116:431–41.
16 Tsubota K, Satake Y, Ohyama M, et al. Surgical
reconstruction of the ocular surface in advanced
ocular cicatricial pemphigoid and Stevens-Johnson
syndrome. Am J Ophthalmol 1996;122:38–52.
17 Melles GR, Eggink FA, Lander F, et al. A surgical
technique for posterior lamellar keratoplasty.
Cornea 1998;17:618–26.
association.11 15 Increased dietary consumption of saturated fat,16 17 monounsaturated and polyunsaturated fat and
vegetable fat17 and cholesterol16 has been
associated with early and late AMD in
various studies. Some recent studies
have identified an association between
use of cholesterol lowering medications,
such as statins, and reduced risk of early
or late AMD,18–22 while others have
found
no
such
association.23–25
Although total serum cholesterol has
been associated with neovascular AMD
in a large case-control study,26 many
large population based studies have
found no association.8 27 Some studies
have suggested an association between
different lipoprotein polymorphisms
and risk of AMD, including apoE,28 29
apo B, and apo A1.30 Certainly the
relation between cardiovascular risk
factors, lipid metabolism, and AMD
remains confusing.
The findings described in LDL receptor
deficient mice may provide insight
into the mechanism of early AMD
Insight into the role lipid metabolism
has in the development of early AMD
has come from the study of preclinical
models. Although no murine model
exists that exactly replicates the phenotype seen in human AMD, studies have
shown that C57Bl/6 mice fed a high fat
diet and briefly exposed to blue-green
light develop basal laminar deposits,31 a
histological feature of human eyes with
AMD.32 Mice with null mutations in
apoE have shown basal linear deposits
and thickened Bruch’s membranes,
similar to findings in human eyes with
AMD.33 34 However, neither of these
models develops choroidal neovascularisation or geographic atrophy, the stages
www.bjophthalmol.com
EDITORIAL
1550
of AMD associated with vision loss in
patients, limiting our understanding of
the mechanisms responsible for these
sight threatening complications.
In this issue of the BJO (p 1627),
Rudolf and colleagues present novel
information about mice with a null
mutation for the low density lipoprotein
(LDL) receptor, which have been studied as a murine model of atherosclerosis. After receiving a high fat diet, LDL
receptor deficient mice develop membrane bound translucent particles
within
a
significantly
thickened
Bruch’s membrane, while control mice
with normal LDL receptors show no
Bruch’s membrane abnormalities. The
membrane bound translucent particles
observed in the LDL receptor deficient
mice resemble vesicles observed in
histological sections of basal linear
deposits and large drusen, findings
specific for early AMD.35 Although
plasma cholesterol is significantly elevated in LDL receptor deficient mice fed
both normal and high fat diets, it is not
clear from the present work that the
changes in Bruch’s membrane in LDL
receptor deficient mice derive from
plasma cholesterol rather than from an
intraocular source.36 Further ultrastructural analysis of the lipid composition of
Bruch’s membrane in LDL receptor
deficient
mice,
using
previously
described methods to preserve neutral
lipids,36 may provide insight into
whether these deposits result from
elevated plasma lipid levels or an
intraocular source. Such information
may clarify the discrepancies noted
between plasma lipid abnormalities
and risk of AMD in epidemiological
studies.
Of interest, the authors demonstrate
immunohistochemical reactivity for vascular endothelial growth factor (VEGF)
in the basal retinal pigment epithelial
(RPE) cells, the outer plexiform layer,
and the photoreceptor inner segments of
LDL receptor deficient mice, which
increased after the mice received a high
fat diet. The authors state that no
spontaneous CNV was observed in the
mice studied despite high levels of VEGF
expression. However, the mice in this
study were investigated at 4 months of
age. It will be interesting to observe LDL
receptor deficient mice at senescent ages
to determine if the changes described in
Bruch’s membrane progress with
advanced age or are accompanied by
the development of CNV or RPE atrophy. Other mutant mice with phenotypes similar to human AMD develop
fundus and histological changes only
after the age of 9 months, with geographic atrophy and CNV developing
only after 16 months and 18 months of
age, respectively.37
www.bjophthalmol.com
Even in the absence of correlates of
late AMD, the findings described in LDL
receptor deficient mice may provide
insight into the mechanism of early
AMD. The fact that the mice develop
abnormally thickened Bruch’s membranes, similar to early AMD, and
demonstrate VEGF upregulation suggests that ischaemia or oxidative stress
occurs even in early stages of AMD,
perhaps as a result of compromised
diffusion from the choriocapillaris to
the outer retina. LDL receptor deficient
mice will serve as a useful model of early
AMD and may allow investigators to
determine the part abnormalities of
cholesterol metabolism may play in its
pathogenesis. Whether or not deficiencies in the LDL receptor are associated
with AMD in humans, the ocular
phenotype of LDL receptor deficient
mice described in the present work
should encourage investigators to study
murine models of atherosclerosis with
careful attention to the eyes.
ACKNOWLEDGEMENTS
This work was supported by a Career
Development Award from Research to
Prevent Blindness, New York, New York;
grants EY00415 and EY02162 from the
National Eye Institute, Bethesda, Maryland;
and grants from the Bernard A Newcomb
Macular Degeneration Fund and That Man
May See, Inc, San Francisco, California, and
the Foundation Fighting Blindness, Owings
Mills, Maryland, USA.
Br J Ophthalmol 2005;89:1549–1551.
doi: 10.1136/bjo.2005.077370
Correspondence to: Jacque L Duncan, MD,
University of California, San Francisco, 10
Koret Way, K129, San Francisco, CA 941430730, USA; [email protected]
Competing interests: none declared
REFERENCES
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factor H polymorphism in age-related macular
degeneration. Science 2005;308:385–9.
2 Haines JL, Hauser MA, Schmidt S, et al.
Complement factor H variant increases the risk of
age-related macular degeneration. Science
2005;308:419–21.
3 Hageman GS, Anderson DH, Johnson LV, et al.
A common haplotype in the complement
regulatory gene factor H (HF1/CFH)
predisposes individuals to age-related macular
degeneration. Proc Natl Acad Sci USA
2005;102:7227–32.
4 Edwards AO, Ritter R 3rd, Abel KJ, et al.
Complement factor H polymorphism and agerelated macular degeneration. Science
2005;308:421–4.
5 Nakamura S, Ohue O, Sawaguchi A. Genetic
polymorphism of human factor H (beta 1H
globulin). Hum Hered 1990;40:121–6.
6 Tsunenari S, Higashi T, Kibayashi K, et al.
Genetic polymorphism of human factor H (HF,
beta 1H globulin) in Chinese Han population in
northeast China. Jpn J Hum Genet
1992;37:145–8.
7 Snow KK, Seddon JM. Do age-related macular
degeneration and cardiovascular disease share
common antecedents? Ophthalmic Epidemiol
1999;6:125–43.
8 Smith W, Assink J, Klein R, et al. Risk factors for
age-related macular degeneration: pooled
findings from three continents. Ophthalmology
2001;108:697–704.
9 Seddon JM, Gensler G, Milton RC, et al.
Association between C-reactive protein and agerelated macular degeneration. JAMA
2004;291:704–10.
10 Seddon JM, George S, Rosner B, et al.
Progression of age-related macular
degeneration: prospective assessment of Creactive protein, interleukin 6, and other
cardiovascular biomarkers. Arch Ophthalmol
2005;123:774–82.
11 Hyman L, Schachat AP, He Q, et al.
Hypertension, cardiovascular disease, and agerelated macular degeneration. Age-Related
Macular Degeneration Risk Factors Study Group.
Arch Ophthalmol 2000;118:351–8.
12 Macular Photocoagulation Study Group. Laser
photocoagulation for juxtafoveal choroidal
neovascularization: five-year results from
randomized clinical trials. Arch Ophthalmol
1994;112:500–9.
13 van Leeuwen R, Ikram MK, Vingerling JR, et al.
Blood pressure, atherosclerosis, and the incidence
of age-related maculopathy: the Rotterdam Study.
Invest Ophthalmol Vis Sci 2003;44:3771–7.
14 Klein R, Klein BE, Tomany SC, et al. The
association of cardiovascular disease with the
long-term incidence of age-related maculopathy:
the Beaver Dam Eye Study. Ophthalmology
2003;110:636–43.
15 Tomany SC, Wang JJ, Van Leeuwen R, et al. Risk
factors for incident age-related macular
degeneration: pooled findings from 3 continents.
Ophthalmology 2004;111:1280–7.
16 Mares-Perlman JA, Brady WE, Klein R, et al.
Dietary fat and age-related maculopathy. Arch
Ophthalmol 1995;113:743–8.
17 Seddon JM, Cote J, Rosner B. Progression of agerelated macular degeneration: association with
dietary fat, transunsaturated fat, nuts, and fish
intake. Arch Ophthalmol 2003;121:1728–37.
18 Hall NF, Gale CR, Syddall H, et al. Risk of
macular degeneration in users of statins: cross
sectional study. BMJ 2001;323:375–6.
19 McCarty CA, Mukesh BN, Guymer RH, et al.
Cholesterol-lowering medications reduce the risk
of age-related maculopathy progression.
Med J Aust 2001;175:340.
20 McGwin G Jr, Owsley C, Curcio CA, et al. The
association between statin use and age related
maculopathy. Br J Ophthalmol 2003;87:1121–5.
21 McGwin G Jr, Xie A, Owsley C. The use of
cholesterol-lowering medications and age-related
macular degeneration. Ophthalmology
2005;112:488–94.
22 Wilson HL, Schwartz DM, Bhatt HR, et al. Statin
and aspirin therapy are associated with
decreased rates of choroidal neovascularization
among patients with age-related macular
degeneration. Am J Ophthalmol
2004;137:615–24.
23 Klein R, Klein BE, Tomany SC, et al. Relation of
statin use to the 5-year incidence and progression
of age-related maculopathy. Arch Ophthalmol
2003;121:1151–5.
24 Van Leeuwen R, Vingerling JR, de Jong PT. Risk
of macular degeneration with statin use should be
interpreted with caution. BMJ 2001;323:1308.
25 Van Leeuwen R, Vingerling JR, Hofman A, et al.
Cholesterol lowering drugs and risk of age related
maculopathy: prospective cohort study with
cumulative exposure measurement. BMJ
2003;326:255–6.
26 Seddon JM, Ajani UA, Sperduto RD, et al. Dietary
carotenoids, vitamins A, C, and E, and advanced
age-related macular degeneration. Eye Disease
Case-Control Study Group. JAMA
1994;272:1413–20.
27 Cruickshanks KJ, Hamman RF, Klein R, et al. The
prevalence of age-related maculopathy by
geographic region and ethnicity. The ColoradoWisconsin Study of Age-Related Maculopathy.
Arch Ophthalmol 1997;115:242–50.
28 Klaver CC, Kliffen M, van Duijn CM, et al. Genetic
association of apolipoprotein E with age-related
macular degeneration. Am J Hum Genet
1998;63:200–6.
EDITORIAL
1551
29 Simonelli F, Margaglione M, Testa F, et al.
Apolipoprotein E polymorphisms in
age-related macular degeneration in an
Italian population. Ophthalmic Res
2001;33:325–8.
30 Nowak M, Swietochowska E, Marek B, et al.
Changes in lipid metabolism in women with agerelated macular degeneration. Clin Exp Med
2005;4:183–7.
31 Cousins SW, Espinosa-Heidmann DG,
Alexandridou A, et al. The role of aging, high fat
diet and blue light exposure in an experimental
mouse model for basal laminar deposit formation.
Exp Eye Res 2002;75:543–53.
32 Green WR. Histopathology of age-related
macular degeneration. Mol Vis 1999;5:27.
33 Dithmar S, Curcio CA, Le NA, et al.
Ultrastructural changes in Bruch’s membrane of
apolipoprotein E-deficient mice. Invest
Ophthalmol Vis Sci 2000;41:2035–42.
34 Ong JM, Zorapapel NC, Rich KA, et al. Effects of
cholesterol and apolipoprotein E on retinal
abnormalities in ApoE-deficient mice. Invest
Ophthalmol Vis Sci 2001;42:1891–900.
35 Curcio CA, Millican CL. Basal linear deposit and
large drusen are specific for early age-related
maculopathy. Arch Ophthalmol
1999;117:329–39.
36 Curcio CA, Presley JB, Millican CL, et al. Basal
deposits and drusen in eyes with age-related
maculopathy: evidence for solid lipid particles.
Exp Eye Res 2005;80:761–75.
37 Ambati J, Anand A, Fernandez S, et al. An
animal model of age-related macular
degeneration in senescent Ccl-2- or Ccr-2deficient mice. Nat Med 2003;9:1390–7.
Clinical Evidence—Call for contributors
Clinical Evidence is a regularly updated evidence-based journal available worldwide both as
a paper version and on the internet. Clinical Evidence needs to recruit a number of new
contributors. Contributors are healthcare professionals or epidemiologists with experience in
evidence-based medicine and the ability to write in a concise and structured way.
Areas for which we are currently seeking contributors:
Pregnancy and childbirth
Endocrine disorders
Palliative care
Tropical diseases
N
N
N
N
We are also looking for contributors for existing topics. For full details on what these topics
are please visit www.clinicalevidence.com/ceweb/contribute/index.jsp
However, we are always looking for others, so do not let this list discourage you.
Being a contributor involves:
Selecting from a validated, screened search (performed by in-house Information
Specialists) epidemiologically sound studies for inclusion.
Documenting your decisions about which studies to include on an inclusion and exclusion
form, which we keep on file.
Writing the text to a highly structured template (about 1500-3000 words), using evidence
from the final studies chosen, within 8-10 weeks of receiving the literature search.
Working with Clinical Evidence editors to ensure that the final text meets epidemiological
and style standards.
Updating the text every 12 months using any new, sound evidence that becomes available.
The Clinical Evidence in-house team will conduct the searches for contributors; your task is
simply to filter out high quality studies and incorporate them in the existing text.
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N
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N
If you would like to become a contributor for Clinical Evidence or require more information
about what this involves please send your contact details and a copy of your CV, clearly
stating the clinical area you are interested in, to [email protected].
Call for peer reviewers
Clinical Evidence also needs to recruit a number of new peer reviewers specifically with an
interest in the clinical areas stated above, and also others related to general practice. Peer
reviewers are healthcare professionals or epidemiologists with experience in evidence-based
medicine. As a peer reviewer you would be asked for your views on the clinical relevance,
validity, and accessibility of specific topics within the journal, and their usefulness to the
intended audience (international generalists and healthcare professionals, possibly with
limited statistical knowledge). Topics are usually 1500-3000 words in length and we would
ask you to review between 2-5 topics per year. The peer review process takes place
throughout the year, and out turnaround time for each review is ideally 10-14 days.
If you are interested in becoming a peer reviewer for Clinical Evidence, please complete the
peer review questionnaire at www.clinicalevidence.com/ceweb/contribute/peerreviewer.jsp
www.bjophthalmol.com
1552
Obituary
Br J Ophthalmol 2005;89:1552
.........................................................................................................
Marshall Miller Parks, MD, 1918–2005
M
arshall Miller Parks died on 27
July 2005. Marshall’s death
marks the passing of the archetypal American gentleman paediatric
ophthalmologist. With changing life
styles, ambitions, ethics, and training
we are unlikely to see anyone remotely
similar. But many of his gifts in life have
been passed on to the paediatric
ophthalmologists that he influenced as
surely as if the message were sent
unerringly in a double helix.
Born in Old Mission, MI, USA, son of
Ruth and Reuben Parks, he was one of
four siblings. He received his BS from
Illinois College in 1939, and graduated
from St Louis University School of
Medicine in 1943 before entering the
US Navy during the second world war,
serving as a medical officer on destroyers in the South Pacific. He studied
paediatric ophthalmology under Frank
D Costenbader, the first American paediatric ophthalmologist, and they
started the first fellowship programme
at what became the Children’s National
Medical Center in Washington, DC. He
held many other posts in Washington,
DC, and later in Dallas, Texas, during
his career as a full time private practitioner.
Among many elected positions, Dr
Parks was a founding member and first
president of the American Association
for Pediatric Ophthalmology and
Strabismus and was president of the
American Academy of Ophthalmology.
Dr Parks’s academic contributions
included the diagnosis and treatment
of strabismus and amblyopia, description of the monofixation syndrome, the
benefits of early strabismus surgery, the
management of infantile cataracts, and
innovative surgical techniques. He wrote
several meticulously researched and
illustrated books, contributed chapters
to 30 others, and had over 70 papers
published. He presented 45 named
lectures, one of which was the 1977
Doyne lecture on ‘‘The superior oblique
tendon.’’ Like other lectures that he
gave, and the associated publications,
www.bjophthalmol.com
Marshall Miller Parks. Picture taken in 1977.
it was clear, beautifully illustrated,
contained observations based on his
vast clinical experience, and one felt
that he made things look so easy that if
you just followed the rules he laid down
you could not go wrong!
He was an energetic person and a
prodigious worker. Following him in his
practice was not for the faint hearted:
for about three days a week he saw the
first patient at 07.30 then several
patients an hour (all beautifully organised and drilled by his secretary with
letters dictated at the end of the
consultation and given to the patient
before departure). He had half an hour
for lunch and then saw patients through
to 19.30. Academic work came after
dinner and, if fortunate enough to be
staying at his house, a visitor got to take
part in that too! On top of his vast
outpatient practice he had a very brisk
referral surgical practice as a result of
his meticulous and technically excellent
surgery, and he served on numerous
committees.
Just to eulogise the work side of Dr
Parks’s life is to miss the point. He and
his late wife Angeline raised 11 children
in a suitably sized house on
Massachusetts Avenue, next to the
British embassy. One son, Peter, predeceased him. He had 25 grandchildren
and 10 great grandchildren. He had a
strong religious faith and was a great
family man, most relaxed when surrounded by family and ready to move
mountains for any of them. Dr Parks
had a summer retreat in Maine, which
he referred to as his ‘‘sacred place.’’
There, work and teaching were never far
away, though, and many paediatric
ophthalmologists went there in the
summer months to take part in teaching
and learning sessions.
Marshall could listen and, although it
was rather difficult to get him to change
his views on a subject, once he did he
gave credit to who had changed them.
He had that great knack of making one
feel that one was the only person at that
moment that he was engaging with. He
was sociable and liked to share with
guests his passion for the occasional
strong martini. His legendary sense of
humour even survived when, over a
mistake in the Anglo-American meaning of the word quite, he was booked
into the most expensive suite in the
most expensive hotel in London!
Angeline and Marshall made a great
team with common goals and a shared
philosophy of life that their children
have benefited from and thus Angeline’s
death left a big gap in his life. Later, he
had the great good fortune to meet and
later marry Martha, also distinguished
in her own right, who formed another
enduring anchor point till the end of his
87 year long and fruitful life in July
2005.
‘‘Happy are those who have died in
the Lord; let them rest from their
labours for their good deeds go with
them.’’
D Taylor,
Visual Sciences Unit, Institute of Child Health,
30 Guilford Street, London WC1N 1EH, UK;
[email protected]
Br J Ophthalmol 2005;89:1553
1553
COVER ILLUSTRATION
.........................................................
Are you calling me primitive?
L
ampreys are animals without bones
or jaws, and yet they are prototype
vertebrates. Related to hagfishes, the
lampreys are cartilaginous fish with
sucker-like mouths and are 550–450
million years old. These two groups
comprise the agnathans and are reminiscent of Pikaia gracilens, the Cambrian
fossil believed to be close to the first
vertebrate. Despite lampreys and hagfish lacking bone and tooth enamel or
other hard parts, they are still surprisingly well preserved in the fossil record.
However, as a result of a paucity of
fossils, the phylogeny of these groups is
controversial and incomplete. Current
evolutionary evidence suggests that
hagfishes preceded the lampreys, with
all three extant lamprey families coming
from a common ancestor. The southern
lamprey, Geotria australis, spends the first
4–5 years of its life in freshwater
streams of southern hemisphere lands
including Australia, New Zealand,
South Africa, and South America.
During this time, its eyes are only poorly
developed. Although usually thought of
as parasites, lampreys actually begin life
as filter feeders, feasting mainly on
detritus and unicellular algae. During
metamorphosis into the pelagic stage,
which takes 6 months, the lamprey’s
eyes enlarge and develop the visual
pigments necessary for managing its
impending pelagic lifestyle. In the second stage of its life, the lamprey
descends into the Southern Ocean,
where it attaches onto the flanks of
other fish. It uses its sucker mouth and
rasp-like teeth, illustrated on the right
of this month’s cover, to attach and tear
into the body tissues to feed on muscle.
After approximately 2 years of this
predatory life, it leaves the ocean and
swims back into freshwater streams to
spawn. This journey can take up to
18 months and, during this time, the
lamprey does not feed. Like salmon,
lampreys are anadromous and return to
fresh water from the sea to spawn, lay
eggs, and die. Once spawning takes
place by the female, the eggs are
fertilised by the male, and both die.
Although depauperate (lacking in
species variety), lampreys occur in three
families with one family found only in
the northern hemisphere and the other
two families found only in the southern
hemisphere. All three extant lamprey
families have probably changed little
during the past 280 million years, and
illustrate just how ancient the basic
mechanisms of much of our visual
system are likely to be.
Evolutionarily,
recent
molecular
research suggests that the most recent
common ancestor of the agnathans
possessed four of the five major classes
of the visual pigments found in the
radiation of gnathostome vertebrates.
Geotria australis, pictured on the left of
the cover and this page, has a pure cone
retina with five visual pigments, three of
which are orthologous to opsins found
in jawed vertebrates. These three common cone opsins have peak sensitivities
in the long wavelengths (red), short
wavelengths (blue), and very short
wavelengths (ultraviolet). A fourth cone
opsin with a peak sensitivity in the
medium wavelengths (green) underwent an independent gene duplication
within the jawless (lampreys and hagfishes) and jawed (all others) fish,
leading to two different cone opsins)
in G australis, and a different cone opsin
in addition to a rod opsin in the jawed
fishes (Collin SP et al, Curr Biol
2003;13:R864–5).
This molecular research has several
profound implications. Importantly,
rods and rhodopsin are relative latecomers to the visual party as they probably did not appear until at least the
mid or late Cambrian (approximately
500 million years ago). This actually
makes sense, since an opsin with a
peak in the short wavelengths (blue)
was probably the first visual pigment
(bacteriorhodopsin)
and
probably
appeared with very early prokaryotes.
Surprisingly, this is not the only
unusual feature of the eye of G australis.
The lens is asymmetrical with an
increased anterior to posterior diameter
as a result of a small posterior protrusion of lens material, much as if it were
posterior lenticonus. This pyriform lens
is multifocal although its function is not
understood. The central visual axis and
more dorsal retina are moderately to
highly myopic and the more peripheral
and ventral retina would be hyperopic
(Collin SP et al, Brain Behav Evol
1999;54:96–118). Since G australis is
preyed upon by albatrosses during the
epipelagic phase, a predation that comes
from above, the ventral retina may be
useful for avoiding predation.
The only other family of southern
lamprey is Mordaciidae. These animals
are nocturnal and have lost the variation
in visual pigment and photoreceptor
type. They have a single large photoreceptor which is much more rod-like,
while still retaining some cone characteristics, suggesting that this family has
traded diurnal vision for nocturnal
vision. As might be expected, the
Mordaciidae species has a tapetum to
maximise photon capture, helping
further with their nocturnal lifestyle
(Collin SP et al, Brain Behav Evol
2000;55:120–38).
G australis are survivors indeed, with
five visual pigments in its retina, a
bizarre lifestyle, and an unusual eye.
Perhaps they aren’t so primitive, after
all.
I R Schwab,
University of California Davis, 4860 Y Street,
Suite 2400, Sacramento, CA 95817, USA;
[email protected]
S P Collin,
The Vision, Touch, and Hearing Research
Centre, School of Biomedical Scinces,
University of Queensland, Brisbane 4072,
Australia
Cover photographs by Stephen Moore,
Taranaki Regional Council, New Zealand.
Advice and comments on the essay by
Stephen Moore, Ian Potter, and Robert
McDowell. Pictures on cover published
with permission from Cambridge University
Press, from Collin et al, (Visual Neuroscience
2003;20:119–30).
www.bjophthalmol.com
1554
WORLD VIEW
Characteristic clinical features as an aid to the diagnosis of
suppurative keratitis caused by filamentous fungi
P A Thomas, A K Leck, M Myatt
...............................................................................................................................
Br J Ophthalmol 2005;89:1554–1558. doi: 10.1136/bjo.2005.076315
See end of article for
authors’ affiliations
.......................
Correspondence to:
Astrid Leck, PhD,
Department of Infectious
and Tropical Diseases,
London School of Hygiene
and Tropical Medicine,
Keppel Street, London
WC1E 7HT, UK; astrid.
[email protected]
Accepted for publication
28 July 2005
.......................
S
Aim: To assess whether the presence of characteristic clinical features can be used as a diagnostic aid for
suppurative keratitis caused by filamentous fungi.
Methods: Patients presenting with suppurative keratitis in India underwent detailed clinical examination
followed by microbiological investigation of corneal scrapes. A partial diagnostic score based upon the
strength of the association, as estimated by the odds ratio, between reported clinical features and
laboratory confirmed diagnoses was devised and subsequently tested using a case series from Ghana.
Results: Serrated margins, raised slough, dry texture, satellite lesions and coloration other than yellow
occurred more frequently in cases of filamentous fungal keratitis than bacterial keratitis (p,0.05).
Hypopyon and fibrinous exudate were observed more frequently in bacterial keratitis (p,0.05). When
incorporated into a backwards stepwise logisitic regression model only serrated margins, raised slough,
and colour were independently associated with fungal keratitis; these features were used in the scoring
system. The probability of fungal infection if one clinical feature was present was 63%, increasing to 83% if
all three features were present.
Conclusions: Microbiological investigations should be performed whenever possible; however, where
facilities are not available, a rapid presumptive diagnosis of suppurative keratitis may be possible by
scoring clinical features.
uppurative keratitis is an important cause of monocular
blindness worldwide, in particular in developing countries in tropical latitudes. Most ophthalmologists in the
developing world do not have recourse to specialised ocular
microbiological laboratory facilities and may have to depend
on their own clinical acumen for diagnosis, with empirical
treatment of patients with these infections. Kaufman and
Wood described certain clinical manifestations that they
believed to be characteristic of mycotic keratitis, including a
severe ocular reaction; folds in Descemet’s membrane;
‘‘hyphate’’ margin; firm and elevated lesions; hypopyon;
endothelial plaque and immune ring.1 However, these
descriptions were based on a study of only 15 patients with
culture proved fungal keratitis. According to Jones, fungal
infection is likely in a suppurative corneal lesion with one or
more of the following features: a dry, raised ulcer with
crenate, spiculate or pseudohyphate border; satellite lesions;
recurrent hypopyon or posterior chamber endophthalmitis
with progressive shallowing of the anterior chamber; failure
to respond to antibacterial treatment.2 These guidelines were
based on observations in just 29 patients with laboratory
diagnosed fungal keratitis.
Since then these clinical features have generally been
accepted as being diagnostic of filamentous fungal keratitis.
Keratitis caused by yeast-like fungi such as Candida spp has
been described as resembling bacterial keratitis.3–5 Recent
studies on large numbers of patients with culture proved
mycotic keratitis have tended to document only the
frequency of occurrence of these features and there has
hitherto been no attempt to compare the occurrence of these
features in a large series of patients with bacterial and fungal
keratitis with a view to assessing their suitability as
diagnostic aids for suppurative keratitis.6 7 In this study we
have attempted to determine to what extent the presence of
certain clinical features may be used to provide a rapid,
presumptive diagnosis of suppurative keratitis caused by
filamentous fungi.
www.bjophthalmol.com
METHODS
A prospective study of suppurative keratitis (defined as loss
of corneal epithelium with underlying stromal infiltrate and
suppuration associated with signs of inflammation, with or
without hypopyon) was conducted at three centres in
southern India and in three centres in Ghana between June
1999 and May 2001. The aetiology of the infection in 1090
consecutive cases (800 from India and 290 from Ghana) has
already been reported.8
Clinical findings
Ophthalmologists examined patients at the different centres
using a standardised protocol and proforma. A detailed
examination was performed on each patient at the slit lamp;
clinical features were documented, drawings made for
patient records, and a photograph was taken of the affected
eye. The length of the slit beam was used to assess the vertical
diameter of the corneal ulceration; this was then turned 90˚
to assess the horizontal diameter. Using the slit beam
parallelopiped the depth of ulceration and infiltrate were
assessed. The presence or absence and form of the following
clinical features were documented: elevation of slough
(raised, flat), texture of slough (wet, dry), ulcer margins
(serrated, well defined), satellite lesions, immune ring,
hypopyon, keratic precipitates, or perineural infiltrate, fibrin,
flare or cells in the anterior chamber (AC), and deep lesions
(posterior corneal abscess, endothelial plaque).
Microbiological investigations
Laboratory diagnosis was determined using microscopy and
culture.9 Microbial cultures were considered to be significant
if (i) growth of the same organism was demonstrated on two
or more solid culture media; (ii) there was semi-confluent
growth at the site of inoculation or growth on one solid
medium consistent with microscopy (that is, appropriate
staining and morphology with Gram stain); or (iii) semiconfluent growth at the site of inoculation on one solid
Suppurative keratitis caused by filamentous fungi
1555
Table 1 Univariate analysis of clinical features occurring in fungal and bacterial keratitis
Clinical feature
Frequency
(% fungal)
Frequency
(% bacterial)
x2
OR (CI)
Serrated margins
Raised slough
Dry texture of slough
Satellite lesions
Hypopyon
Fibrin
Colour (not yellow)
180/228 (79%)
135/228 (59%)
101/228 (44%)
51/222 (23%)
105/219 (48%)
21/210 (10%)
213/228 (93%)
63/132 (48%)
52/132 (39%)
37/132 (28%)
17/132 (13%)
83/128 (65%)
28/125 (22%)
106/132 (80%)
37.14
13.50
9.36
4.91
9.29
9.65
14.26
4.09
2.23
2.04
1.95
0.50
0.38
3.47
(2.57
(1.44
(1.29
(1.08
(0.32
(0.20
(1.77
to
to
to
to
to
to
to
6.56)
3.55)
3.26)
3.61)
0.78)
0.70)
6.98)
p Value
Sens
Spec
PPV
0.00
0.00
0.00
0.04
0.00
0.00
0.00
0.79
0.59
0.44
0.22
0.48
0.10
0.93
0.52
0.61
0.72
0.87
0.35
0.78
0.20
0.74
0.72
0.73
0.75
0.56
0.43
0.67
PPV, positive predictive value.
medium (if bacteria); or (iv) growth of the same organism on
repeated scraping. Culture positivity was the ‘‘gold standard’’
used to establish the diagnosis of the bacterial ulcers. If
fungal hyphae were observed in corneal tissue, but failed to
grow in culture, the causative organism was reported as
fungal.
Verbal patient consent was obtained but written consent
was not considered appropriate as the study did not involve
any deviation from routine diagnostic or treatment protocol.
Patients were excluded from the study if they did not agree to
investigation and treatment.
Probability of fungal infection
1.0
Cases included in this study
In this study only patients with confirmed bacterial or fungal
infection were included in the analysis. The following
patients were excluded from the 1090 consecutive cases:
those with suspected or confirmed viral keratitis, corneas at
risk of perforation (where corneal scrapes were not possible)
and small ulcers; defined as an epithelial defect ,2 mm
(greatest diameter) and/or an infiltrate of ,4 mm (greatest
diameter) (128) as these comprised early stage corneal ulcers
which may present with non-specific signs, and characteristic
clinical features may not be present. Those with a mixed
infection (44), Acanthamoeba keratitis (seven), unconfirmed
laboratory diagnosis (249), or where clinical features had not
been adequately recorded (12) were excluded. The clinical
features of 360/800 patients in India with confirmed bacterial
or fungal infection were analysed further to devise a
diagnostic score. The diagnostic score was subsequently
applied to 115/290 cases of bacterial or fungal infection from
the Ghanaian patient dataset (after excluding patients
according to the criteria applied to the Indian dataset).
0.6
0.4
0.2
0.0
0
1
2
3
Score
Figure 1 Operating characteristic (OC) curve for diagnostic score
(India data).
simulation with the prevalence of fungal infection at each
score estimated in 1000 bootstrap (that is, with replacement)
samples of size 1000 taken from the clinical dataset. The
probability of fungal infection was estimated using the
median prevalence found at each score in the bootstrap
samples. A 95% confidence interval around the probability of
fungal infection was estimated using the 2.5% and 97.5%
quantiles of the distribution of the prevalences found at each
score in the bootstrap samples. Simulations were performed
using the R language for data analysis and graphics.11–13 Data
were plotted using R. The Ghanaian patient dataset was
tested using the diagnostic score devised from the Indian
data, and an OC curve was created by computer based
simulation, as for the Indian data, and compared.
Analysis of data
From the Indian dataset pairwise associations between
clinical features and diagnosis were investigated using SPSS
(SPSS for Windows, Release 10.0.5, SPSS Inc, 1989–2001)
and EpiCalc.10 Significant associations were entered into a
logistic regression model and non-significant associations
removed using backwards elimination. A score was created
from a count of signs positively and independently associated
with a fungal aetiology. An operating characteristic (OC)
curve for the diagnostic score was created by computer based
Table 2
keratitis
0.8
RESULTS
Clinical data for 360 patients from India with confirmed
fungal (228) and bacterial (132) keratitis were analysed.
Features which occurred significantly (p,0.05) more frequently in fungal than in bacterial corneal ulcers by
univariate analysis were as follows: serrated margins, raised
slough, dry textured slough, satellite lesions, and colour
Multivariate analysis of clinical features occurring in fungal and bacterial
Clinical feature
OR (CI)
Serrated margins
Raised slough
Fibrin
Colour (not yellow)
3.45
2.32
0.39
2.85
(2.12
(1.43
(0.20
(1.34
p Value
to
to
to
to
5.64)
3.74)
0.77)
6.03)
0.00
0.00
0.01
0.01
www.bjophthalmol.com
1556
Thomas, Leck, Myatt
Table 3 Screening test indices at each level OC curve
Score
Sensitivity
Specificity
PPV
NPV
.0
.1
.2
0.99 (0.97, 1.00)
0.88 (0.83, 0.92)
0.45 (0.38, 0.51)
0.08 (0.04, 0.14)
0.41 (0.33, 0.50)
0.84 (0.76, 0.90)
0.65 (0.60, 0.70)
0.72 (0.66, 0.77)
0.83 (0.75, 0.89)
0.83 (0.51, 0.97)
0.66 (0.54, 0.76)
0.47 (0.40, 0.53)
PPV, positive predictive value; NPV, negative predictive value.
Table 4 Screening test indices at each level OC curve (Ghana test data)
Score
Sensitivity
Specificity
PPV
NPV
.0
.1
.2
0.94 (0.87, 0.98)
0.64 (0.53, 0.74)
0.25 (0.16, 0.35)
0.15 (0.05, 0.36)
0.46 (0.27, 0.66)
0.92 (0.73, 0.99)
0.79 (0.70, 0.86)
0.80 (0.69, 0.88)
0.92 (0.72, 0.99)
0.44 (0.15, 0.77)
0.27 (0.15, 0.43)
0.26 (0.18, 0.37)
PPV, positive predictive value; NPV, negative predictive value.
(other than yellow). Features found more frequently in
bacterial than in fungal corneal ulcers were hypopyon and
fibrin in the anterior chamber (table 1).
No significant differences were observed between the
frequency of occurrence of an immune ring, keratic precipitates, perineural infiltrates, endothelial plaque, and flare
or cells in the AC. Clinical features found to be characteristic
of fungal or bacterial infection were entered into a logistic
regression model.
Serrated margins, raised slough, coloration other than
yellow, and fibrin were statistically independent features as
determined by the logistic regression model (table 2).
Using the three clinical features associated with fungal
infection a score was devised, with the presence of a
significant feature scoring +1. The higher the score, the
greater the probability of fungal infection (fig 1/table 3).
The probability of fungal infection if one of the clinical
features was present; either a serrated margin, raised slough
or coloration other than yellow; was 63%; the presence of all
three signs indicated an 83% chance of fungal infection.
The results obtained from testing the cases from Ghana
closely correlated with those from the Indian dataset and the
presence of all three signs resulted in a probability of .90%
that the infection was fungal (fig 2/table 4).
Approximately 17% of the corneal ulcers in the Indian
dataset and .50% of corneal ulcers in Ghana scored zero.
These comprised bacterial corneal ulcers and fungal corneal
Probability of fungal infection
1.0
0.8
0.6
0.4
0.2
Test – Ghana data
Train – India data
0.0
0
1
2
3
Score
Figure 2 Operating characteristic (OC) curve for diagnostic score
(Ghana data).
www.bjophthalmol.com
ulcers for which all of the scored clinical features were
absent.
DISCUSSION
Filamentous fungi are the commonest cause of mycotic
keratitis in many countries in tropical latitudes and it is vital
that a specific diagnosis is made as quickly as possible to
ensure prompt institution of antifungal therapy.6–8 14–18
Although a detailed clinical examination may help to reach
a rapid presumptive diagnosis, fungal keratitis continues to
be confused with other causes of inflammatory keratitis.19
Certain clinical characteristics of corneal ulcers may suggest a
specific pathogen, but it is now generally accepted that a
reliable diagnosis cannot be made by clinical appearance
alone and that microbiological investigations should be
performed.20 21 Unfortunately, many ophthalmologists working in developing countries do not have access to basic ocular
microbiological investigations such as microscopy or culture
of corneal scrapes. Even in the United States, patients with
corneal ulcers are frequently not referred for microbiological
investigations and ophthalmologists tend to depend on their
clinical acumen when prescribing.22 23 Thus, it is imperative to
assess the reliability of what are considered to be ‘‘characteristic’’ clinical features in the diagnosis of suppurative
keratitis.
The observations of Kaufman and Wood and Jones are
now firmly established in the ophthalmic literature, however,
both of these and other similar observational studies have
limitations. There was no comparison of the frequency of
occurrence of these clinical features in fungal, bacterial, or
other types of microbial ulcers and there was no assessment
of the relative importance of these different characteristics in
establishing a diagnosis. It is essential to determine the
validity of these concepts, an issue which most published
case series of microbial keratitis have not attempted to
address.6 7 14 15 18 24–26
Only in recent studies have authors compared clinical
features in fungal and bacterial keratitis. In a study of 142
patients with suppurative keratitis in Bangladesh, patients
with culture proved fungal keratitis (almost all caused by
filamentous fungi) reported a longer history of symptoms
than bacterial ulcers (p,0.01) and a dry, raised, necrotic or
fluffy surface were more frequent (p,0.01). Endothelial
rings were also more frequent in fungal than in bacterial
ulcers, whereas dacryocystitis was significantly more common in bacterial ulcers.16 Wong et al compared fungal and
bacterial keratitis in a hospital based retrospective study in
Singapore and reported anterior chamber involvement to be
more common in fungal ulcers (45% v 35%).17 In many of the
Suppurative keratitis caused by filamentous fungi
studies serrated margins are commonly reported in association with fungal infection.1–5 In our study 79% of fungal
ulcers had serrated margins, but this sign was not pathognomonic, as 48% of bacterial ulcers also had serrated
margins.
Another important feature of fungal corneal ulcers is
believed to be raised, dry, necrotic slough1 2 4 In our study dry
textured slough was seen more frequently in fungal corneal
ulcers. Satellite lesions, which are reported to occur frequently in fungal corneal ulcers, were also seen in cases of
bacterial keratitis although less frequently occurring.1–3 An
immune ring is believed to be a frequent occurrence in a
filamentous fungal corneal ulcer; in the present study, an
immune ring was only observed in very few cases. Anterior
chamber pathology (hypopyon, fibrin) was more frequently
observed in bacterial than in fungal ulcers. This is in contrast
with the observations of some authors but consistent with
the findings of others.1–3 14 17 25 26
An ophthalmologist confronted with a patient with
suppurative keratitis may be uncertain as to which clinical
features should be given more importance in differentiating
between fungal and bacterial infection. Although certain
clinical features appear to be more strongly associated with
fungal infection we do not believe it is wise to depend on a
single clinical feature to reach a presumptive diagnosis. The
rationale for devising a scoring scheme was based on the
premise that the simultaneous presence of several ‘‘characteristic’’ clinical features in a corneal ulcer should permit a
more precise diagnosis of fungal infection than individual
characteristics considered in isolation. If a high score is
obtained then the observer can be 83% certain of fungal
infection. Conversely, a low score (including zero) is
associated with increased probability of bacterial aetiology.
The greatest benefit of a clinical score such as this may be
to clinicians who are working in eye centres where there are
no facilities for laboratory investigation. The score may be
used to provide a rapid indication of the type of infection,
essential in guiding treatment choice. The management of
corneal ulcers using this scheme will vary depending on the
setting in which it is used. For example, a clinician working
in a region where fungal keratitis is thought to be common
may decide to use antifungals to treat ulcers with a score of 1
or 2, whereas a clinician working in an area in which fungal
keratitis is less common may reserve antifungals for ulcers
with a maximum score of 3. Choosing a low score as a guide
for management as a fungal ulcer may result in unnecessary
treatment of bacterial ulcers with antifungals; however,
patients with very early disease would not be missed. In
contrast, the selection of a highest score as a cut-off point
would permit more specific diagnosis, and a more selective
institution of antifungal therapy; however, this may mean
that for patients with very early disease, where the clinical
features may be vague and non-specific, appropriate treatment may be delayed. For ophthalmologists living in areas
where fungal keratitis is a common or frequent cause of
keratitis (southern India, sub-tropical Africa, and south
Florida), low scoring ulcers may be given antifungals
whereas for those working in more temperate climates (the
United Kingdom, northern United States) the decision to
treat with antifungals might be confined to those cases which
score highly. The decision as to when to treat an ulcer with
antifungals may also be influenced by other factors such as
financial constraints and availability of antifungal agents. It
is recommended that fungal infections are treated with a
combination of an antibiotic and an antifungal agent in case
of mixed infection.
We did not stratify clinical presentation based on the
duration of the symptoms as this information was thought to
be unreliable as the majority of patients reported short
1557
duration of symptoms that did not concur with the clinical
picture. It is possible that if duration of symptoms were
included the results might have been different. We have also
used only two broad categories—namely, culture proved
bacterial keratitis, and microscopy or culture proved fungal
keratitis, and not introduced subsets based on the infecting
fungal or bacterial genus in each category, as a larger number
of cases would have been needed. However, from our results
it is clear that bacterial and fungal ulcers can exhibit the
same features and therefore analysing the data based on
genera may reduce the usefulness of such a test.
Further investigations to test and refine this scoring
scheme are currently under way and there are plans to
validate the proposed scheme in different settings.
CONCLUSIONS
The clinical features of microbial keratitis may vary considerably and no one clinical feature can be considered as
absolutely pathognomonic of a particular type of aetiological
agent.
Ophthalmologists are urged to send corneal scrapes for
microbiology examination where facilities for ocular microbiology are available. However, where such facilities are not
available, a rapid presumptive clinical diagnosis of filamentous fungal keratitis may be possible using a tool such as the
scoring scheme presented here. When fungal infection is
suspected a combination of antibiotic and antifungal therapy
is recommended.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the work of the ophthalmologists and microbiologists in India and Ghana who were involved in
the collection of the original data.8 This research was funded by the
Community Fund, UK.
.....................
Authors’ affiliations
P A Thomas, Institute of Ophthalmology, Joseph Eye Hospital,
Tiruchirapalli, India
A K Leck, International Centre for Eye Health, Clinical Research Unit,
London School of Hygiene and Tropical Medicine, London, UK
M Myatt, Division of Epidemiology, Institute of Ophthalmology, London,
UK
Competing interests: none declared
Ethical approval was obtained from the ethics committees at Moorfields
Eye Hospital, Joseph Eye Hospital (India) and Korle Bu Teaching Hospital
(Ghana).
REFERENCES
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1965;59:993–1000.
2 Jones BR. Principles in the management of oculomycosis. Am J Ophthalmol
1975;79:719–51.
3 Foster CS. Fungal keratitis. Infect Dis Clin N America 1992;6:851–7.
4 Thomas PA. Mycotic keratitis:an underestimated mycosis. J Med Vet Mycol
1994;32:235–54.
5 Klotz SA, Penn CC, Negvesky GJ, et al. Fungal and parasitic infections of the
eye. Clin Microbiol Rev 2000;13:662–85.
6 Rosa RH Jr, Miller D, Alfonso E. The changing spectrum of fungal keratitis in
South Florida. Ophthalmology 1994;101:1005–13.
7 Garg P, Gopinathan U, Choudhary K, et al. Keratomycosis—clinical and
microbiologic experience with dematiaceous fungi. Ophthalmology
2000;107:574–80.
8 Leck AK, Thomas PA, Hagan M, et al. Aetiology of suppurative corneal ulcers
in Ghana and south India, and epidemiology of fungal keratitis.
Br J Ophthalmol 2002;86:1211–15.
9 Jones DB. Strategy for the initial management of suspected microbial keratitis.
In: Barraquer J, Binder PS, Buxton JN, et al, eds. Transactions of the New
Orleans Academy of Ophthalmology. Symposium on Medical and Surgical
Diseases of the Cornea. St Louis: CV Mosby, 1980:86–119.
10 Gilman J, Myatt M. Epicalc 2000 Ver 1.02, Brixton Books, 1998.
11 Cleveland WS. Robust locally weighted regression and smoothing scatterplots.
J Am Statist Assoc 1979;74:829–36.
12 Cleveland WS. LOWESS: a program for smoothing scatterplots by robust
locally weighted regression. Am Stat 1981;35:54.
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13 Ihaka R, Gentleman R. R: a language for data analysis and graphics.
J Comput Graph Statist 1996;5:299–314.
14 Gugnani HC, Talwar RS, Njoku-Obi ANU, et al. Mycotic keratitis in Nigeria.
A study of 21 cases. Br J Ophthalmol 1976;60:607–13.
15 Liesegang TJ, Forster RK. Spectrum of microbial keratitis in South Florida.
Am J Ophthalmol 1980;90:38–47.
16 Dunlop AA, Wright ED, Howlader SA, et al. Suppurative corneal
ulceration in Bangladesh: a study of 142 cases, examining the
microbiological diagnosis, clinical and epidemiological features
of bacterial and fungal keratitis. Aust NZ J Ophthalmol
1994;22:105–10.
17 Wong T-Y, Ng T-P, Fong K-S, et al. Risk factors and clinical outcomes
between fungal and bacterial keratitis: a comparative study. CLAO J
1997;23:275–81.
18 Gopinathan U, Garg P, Fernandes M, et al. The epidemiological features and
laboratory results of fungal keratitis. A 10 year review at a referral eye care
center in South India. Cornea 2002;21:555–9.
19 Sridhar MS, Gopinathan U, Garg P, et al. Aspergillus fumigatus keratitis with
wreath pattern infiltrates. Cornea 2001;20:534–5.
20 Florakis GJ, Moasami G, Schubert H, et al. Scanning slit confocal microscopy
of fungal keratitis. Arch Ophthalmol 1997;115:1461–3.
21 Winchester K, Mathers WD, Sutphin JE. Diagnosis of Aspergillus keratitis in
vivo with confocal microscopy. Cornea 1997;16:27–31.
22 McDonnell PJ, Nobe J, Gauderman WJ, et al. Community care of corneal
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23 McLeod SD, DeBacker CM, Viana MA. Differential care of corneal ulcers in
the community based on apparent severity. Ophthalmology
1996;103:479–84.
24 Wilhelmus KR, Jones DB. Curvularia keratitis. Trans Am Ophth Soc
2001;99:111–32.
25 Cruz OA, Sabir SM, Capo H, et al. Microbial keratitis in childhood.
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1559
WORLD VIEW
A population based survey of the prevalence and types of
glaucoma in rural West Bengal: the West Bengal Glaucoma
Study
A Raychaudhuri, S K Lahiri, M Bandyopadhyay, P J Foster, B C Reeves, G J Johnson
...............................................................................................................................
Br J Ophthalmol 2005;89:1559–1564. doi: 10.1136/bjo.2005.074948
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr B C Reeves, Department
of Public Health and Policy,
London School of Hygiene
and Tropical Medicine,
London WC1E 7HT, UK;
[email protected].
uk
Accepted for publication
15 July 2005
.......................
G
Aim: To determine (i) the prevalence of glaucoma in people aged >50 years, (ii) the proportions of
different types of glaucoma, (iii) the distributions of intraocular pressure and vertical cup disc ratio.
Method: Population based prevalence survey in rural West Bengal. People aged >50 years in randomly
selected villages in 24 Parganas South district. The main outcome measures were diagnosis of glaucoma,
based on criteria described by the International Society for Geographic and Epidemiological
Ophthalmology.
Results: 1594 people aged >50 years were enumerated in nine villages; 1324 (83.1%) were surveyed
and 1269 people adequately examined. 42 definite cases of glaucoma were identified, with prevalence
increasing from 2.7% (95% CI 1.7 to 3.7) in people aged 50–59 years to 6.5% (95% CI 0.0 to 14.1) in
those aged >80 years. The age standardised estimate for the prevalence of all glaucoma in people aged
>50 years was 3.4%. Only three cases of primary angle closure glaucoma (PACG) were identified, giving
a crude ratio of primary open angle glaucoma (POAG) to PACG of more than 10:1. Three people with
glaucoma were blind in one eye but none was blind in both eyes.
Conclusion: Compared to other surveys of glaucoma in India, the age standardised prevalence observed
was less than in Hyderabad, but similar to Tamil Nadu and Dhaka. The ratio of POAG to PACG was much
higher than found previously, suggesting that PACG may be less prevalent in Bengalis than in Indian
populations living in south India. The authors conclude that ophthalmic services in West Bengal should
focus on detecting POAG. Since there is still no satisfactory method of screening for POAG, there is no
alternative to case detection (opportunistic screening) in eye clinics.
laucoma has been established, in most regions of the
world as well as globally, as the second most frequent
cause of blindness after cataract.1 According to this
World Health Organization model, based on the most recent
available data, glaucoma accounted for 12.3% of blindness in
2002. The authors concluded that countries should be
encouraged to carry out periodic population based surveys
of the magnitude and causes of visual impairment, particularly in densely populated countries, and countries in regions
where data are scarce.
Until the last few years, no robust population based data
for glaucoma have been available from India. It has generally
been assumed from clinic studies that the proportion of
primary open angle glaucoma (POAG) to primary angle
closure glaucoma (PACG) is approximately equal.2 However,
the complex patterns of migration across India,3 contributing
to marked ethnic differences between different regions, may
mean that both the overall prevalence and the proportion of
PACG may vary from one part of the country to another.
A population based study of 972 people aged 30–60 years
suggested that PACG is about five times as common as POAG
in Vellore, Tamil Nadu.4 A larger study of an urban
population in Hyderabad, Andra Pradesh, found that the
prevalence of POAG was more than twice that for PACG.5 6 A
recent comprehensive survey in Madurai, also in Tamil Nadu,
gave an estimate of the prevalence of POAG three times that
for PACG.7 These three reports come from southern India. No
epidemiological data have been available for glaucoma in
eastern India. Recently, however, data have been published
from the Bengali population of Dhaka in Bangladesh.8
This paper reports a survey of a rural population in West
Bengal. The objectives were to determine: (i) the prevalence
of glaucoma in people aged 50 years or more, (ii) the proportion
of different types of glaucoma, (iii) the distribution of
intraocular pressure (IOP) and vertical cup disc ratio (VCDR).
METHODS
Study population
The district of 24 Parganas South in West Bengal was chosen
for the survey because there were existing, well established
community links because of a child health programme
(ICDS). These links were considered likely to improve local
collaboration and participation. Three of 30 ICDS blocks in
the district within a distance of 50 kilometres from the
Regional Institute of Ophthalmology, Calcutta, were chosen
by simple random selection. Three of about 100 villages in
each block were randomly selected. All people aged 50 years
and over in these villages were enumerated and considered
eligible for inclusion in the study. For each household, the
name, age, sex, and number of family members were
recorded. A history was taken for each family member, with
particular attention to the duration of dimness of vision, if
any; symptoms of painful dimness of vision with red eyes, or
of seeing halos around lights; any previous surgical procedures undertaken on the eyes; trauma; past glaucoma
diagnosis, and if any family member suffered from glaucoma.
Clinical examination
Visual acuity was recorded using a Snellen distance vision
chart at 6 metres. An ophthalmologist carried out refraction
Abbreviations: IOP, intraocular pressure; PACG, primary angle closure
glaucoma; PMOA, paramedical ophthalmic assistant; POAG, primary
open angle glaucoma; VCDR, vertical cup disc ratio
www.bjophthalmol.com
1560
Raychaudhuri, Lahiri, Bandyopadhyay, et al
Table 1 Age and sex distribution of subjects attending for clinical examination (n = 1324)
Age group
Males in sample (not able
to examine adequately)
% of sample
% in population
(n = 777)
Females in sample (not able
to examine adequately)
% of sample
% in population
(n = 817)
50–59 years
60–69 years
70–79 years
80+ years
Total
318 (4)
196 (4)
93 (9)
22 (1)
629 (18)
50.6
31.2
14.8
3.5
100.0
51.0
30.8
14.5
3.7
100.0
319 (5)
233 (16)
111 (9)
32 (7)
695 (37)
45.9
33.5
16.0
4.6
100.0
45.0
32.8
15.8
6.4
100.0
and recorded the corrected visual acuity. Visual field
examination was carried out by a paramedical ophthalmic
assistant (PMOA) using a static, semi-automated (computerised) visual field analyser (Henson CFA 3200, Tinsley,
Newbury, UK). An automated threshold related single
stimulus suprathreshold program was used to check 68
points in the central 25 degrees in each eye. If a test location
was not seen, the stimulus intensity was automatically
increased in stages from 0.5 to 0.8 and ultimately to 1.2 log
units. The visual field was classified as normal if no defect
was observed. The central field analyser itself classified visual
field results as normal/suspect/defect; these results were
recorded for correlation with the other clinical findings.
Oblique flash light test, examinations under slit lamp,
tonometry, fundus photography, and interpretation of the
findings for the purpose of diagnosis were done by one of the
authors (ARC). Any atrophic patch on the iris, signs of
exfoliation, and the condition of the lens were noted.
Tonometry was performed using a Goldmann applanation
tonometer on the slit lamp; three readings were taken and
the mean (the nearest whole number) was recorded as the
IOP. Gonioscopy was carried out using a Goldmann two
mirror gonio lens (Haag-Streit). The angle of the anterior
chamber was graded according to Shaffer’s angle grading
system.9 Any peripheral anterior synechiae were noted. The
iris profile and the insertion of the iris were noted according
to Spaeth’s system.9 The optic disc was examined after
dilating each pupil with one drop of a mixture of tropicamide
0.8% and phenylephrine 5%. A +90D lens (Volk) was used at
the slit lamp for biomicroscopy of the disc. A ratio of the
longest vertical diameter of the cup to the longest vertical
diameter of the disc was estimated as the VCDR for each
eye.10 Any asymmetry of the VCDRs between the two eyes,
the narrowest neuroretinal rim, any notching at the cup
margin, disc pallor, and haemorrhage were noted. A red free
light was used to check for any defect in the nerve fibre layer.
The macula of each eye was examined with the same lens.
Fundus photography (Topcon Retinal camera TRC-50XT)
was done routinely except in subjects where dense cataractous change in the lens prevented visualisation of the disc.
One non-stereoscopic photograph and two paired stereoscopic photographs were taken for each eye using
Kodachrome 100 ASA film.
Criteria for classification of glaucoma
We applied criteria for diagnosing glaucoma previously
described by the International Society for Geographic and
Epidemiological Ophthalmology (ISGEO), using ‘‘three levels
of evidence’’11:
(a)
a VCDR of 0.7 or greater or asymmetry between the
right and left VCDRs of 0.2 or more, and a visual field
defect consistent with glaucoma (an abnormal 68 point
field test);
(b) a VCDR of 0.9 or greater in either eye or asymmetry
between the right and left VCDRs of 0.3 or more, and a
reliable field test result could not be obtained;
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(c)
an IOP greater than 26 mm Hg and visual acuity worse
than 3/60, or evidence of previous glaucoma filtering
surgery, when the optic disc could not be examined
because of media opacity.
The VCDR and IOP criteria described above were based on
the 97.5th and 99.5th percentiles for ‘‘hypernormals’’ in
surveys described by Foster et al,11 rather than on the basis of
the population sample studied in West Bengal. There were
three reasons for this: (a) extreme percentiles are intrinsically
unstable; (b) the study sample for the present survey was
relatively small compared to the studies reviewed by Foster et
al11; (c) the criteria for population based samples of
hypernormals in different countries are similar,11 despite
some concern about variations in disc size affecting VCDR.12
Open angle glaucoma
In the presence of open anterior chamber angles, a patient
was given a diagnosis of POAG if one or both eyes met any of
the criteria outlined above, unless there was any other sign of
retinal or optic nerve disease—for example, diabetes mellitus,
branch or central retinal vein occlusion, or signs of
pseudoexfoliation, trauma or pigment dispersion. If any of
the latter signs were present, a diagnosis of secondary open
angle glaucoma was made.
A diagnosis of suspected POAG was made in the presence
of an open angle of the anterior chamber, a VCDR of 0.7 or
more, or asymmetry between the right and left VCDRs of 0.2
or more without an associated definite visual field abnormality.
Angle closure
The presence of an occludable angle was the essential feature
for diagnosing angle closure glaucoma, if one or both eyes
met any of the criteria outlined above. An angle in which the
pigmented trabecular meshwork was not visible throughout
three quarters or more of the angle circumference in the
primary position without manipulation or indentation was
classified as occludable. In the absence of any other cause for
angle closure, patients with an occludable angle meeting any
of the criteria for glaucoma described above were diagnosed
as having chronic PACG. Patients were diagnosed as having
acute PACG if they had signs of past attack of acute angle
closure on iris and lens surfaces, or if they reported a clear
history of seeing a rainbow halo around light, sudden or
intermittent attacks of painful red eye, and dimness of vision.
If there were characteristic disc changes but no field changes
in the presence of an occludable angle, a diagnosis of
suspected PACG was made. Angle closure glaucoma associated with signs of other primary causes was classified as
secondary angle closure glaucoma.
In addition to applying the ISGEO criteria described above
to clinical findings, optic disc photographs and visual field
assessments were reviewed by three ophthalmologists. As a
result of this review, some patients with suspected glaucoma
without definite field abnormalities were classified as
probable cases of glaucoma—for example, when there was
Prevalence of glaucoma in West Bengal
1561
Table 2 Profiles of the distributions of intraocular pressure (IOP) and vertical cup disc
ratio (VCDR) among ‘‘hypernormals’’—that is, people confirmed not to have glaucoma
(n = 1170)11 (and in the whole sample of people attending for examination, n = 1324)
Observations
Missing data
Mean
Median
SD
97.5th percentile
99.5th percentile
IOP right
IOP left
VCDR right
VCDR left
VCDR asymmetry*
1153
(1282)
17
(42)
13.8
(14.0)
13
(14)
3.1
(3.7)
20
(20)
24
(26)
1148
(1280)
22
(44)
13.7
(13.8)
13
(14)
2.9
(3.1)
20
(20)
24
(26)
1149
(1250)
21
(74)
0.40
0.42
0.4
(0.4)
0.13
(0.15)
0.6
(0.7)
0.6
(0.9)
1143
(1246)
27
(78)
0.40
(0.42)
0.4
(0.4)
0.12
0.15
0.6
(0.7)
0.6
(0.8)
1124
(1222)
46
(102)
0.02
(0.03)
0
(0)
0.04
(0.05)
0.1
(0.2)
0.2
(0.3)
*Modulus of VCDR left – VCDR right.
consensus that the optic disc appeared to be clearly
pathological. Therefore, prevalences are reported both for
cases of definite glaucoma, and cases of definite or probable
glaucoma.
subjects. All of these had non-glaucomatous fellow eyes and
no signs of glaucoma in the eye that could not be examined
adequately. They were classified as not having glaucoma and
were included in the denominator for calculation of
prevalence.
It was possible to classify all but two of the people who
attended for examination as blind or not in either eye. The
number blind, and prevalences of blindness, in one or
both eyes (males and females combined) were 50–59 years,
14/636, 2.2% (95% confidence interval 1.2 to 3.2);
60–69 years, 21/428, 4.9% (2.3 to 7.5); 70–79 years, 26/204,
12.7% (9.1 to 16.4); 80+ years, 13/54, 24.1% (15.6 to 32.5).
The prevalences of blindness in both eyes (males and females
combined) were: 50–59 years, 1/636, 0.2% (0.0 to 0.4);
60–69 years, 4/428, 0.9% (0.0 to 2.0); 70–79 years, 7/204,
3.4% (1.5 to 5.3); 80+ years, 1/54, 1.9% (0.0 to 5.2).
The distributions of VCDR and intraocular pressure for
those who were confirmed not to have glaucoma (that is,
excluding glaucoma cases and suspects, and people who
could not be adequately examined), and for all people who
attended for examination, are shown in table 2. It was not
possible to examine or photograph the optic discs, or to
measure the intraocular pressure in a small number of
patients (3–6%), either because of media opacities or because
of refusal to be examined.
Table 3 summarises the numbers and prevalences of
definite, and definite and probable, glaucoma cases detected.
Data analysis
We used simple random sampling at block and village level.
Therefore, point prevalence estimates are unbiased.
Confidence intervals were calculated with Stata (version
8.2; Stata Corporation, TX, USA) ‘‘svy’’ commands to take
into account the clustering of individuals in villages.
RESULTS
Study population
Out of a total population of 13 215 enumerated in the nine
villages, 1594 people aged 50 years or more were identified
and were eligible to undergo clinical examination for
glaucoma. Of these, 1324 people (83.1%) responded to the
invitation to attend for examination at clinics established in
the villages between September 1998 and December 1999;
distributions of their age and sex are given in table 1. One or
both eyes of 1269 people could be adequately examined for
glaucoma. Of 55 people in whom neither eye could be
adequately examined, 25 had dense cataract in both eyes, 24
refused examination, and six had one eye that was phthisical
or had a corneal scar and had dense cataract in the other eye.
One eye could not be adequately examined in a further 40
Table 3 Number and prevalences of definite (and definite and probable) glaucoma by sex and glaucoma type in people who
could be examined adequately (n = 1269)
Age
group
(years)
50–59
60–69
70–79
80+
Total
Males
Females
Diagnostic category
Diagnostic category
POAG
PACG
Secondary
glaucoma
10
(10)
7
(10)
5
(6)
0
(0)
22
(26)
1
(1)
0
(0)
1
(1)
1
(1)
3
(3)
0
(0)
1
(1)
0
(0)
0
(0)
1
(1)
Number
314
192
84
21
611
Prevalence,
95% CI
3.5, 1.3 to 5.7
(3.5, 1.3 to 5.7)
4.2, 1.6 to 6.7
(5.7, 2.8 to 8.7)
7.1, 1.5 to 12.8
(8.3, 2.4 to 14.2)
4.8, 0.0 to 14.4
(4.8, 0.0 to 14.4)
POAG
PACG
Secondary
glaucoma
6
(7)
4
(4)
4
(7)
2
(2)
16
(20)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
0
(0)
Total
prevalence
Number
Prevalence
314
1.9, 0.3 to 3.5
(2.2, 0.1 to 4.3)
1.8, 0.0 to 4.7
(1.8, 0.0 to 4.7)
3.9, 0.8 to 7.0
(6.9, 2.5 to 11.2)
8.0, 0.0 to 18.8
(8.0, 0.0 to 18.8)
217
102
25
658
2.7, 1.7 to 3.7
(2.9, 1.8 to 3.9)
2.9, 1.5 to 4.3
(3.7, 1.7 to 5.6)
5.4, 2.8 to 8.0
(7.5, 4.1 to 10.9)
6.5, 0.0 to 14.1
(6.5, 0.0 to 14.1)
3.4 *
POAG, primary open angle glaucoma; PACG, primary angle closure glaucoma; CI confidence interval.
*Overall prevalence for people aged 50 years or more, age standardised against the total Indian population as described in the 2001 census (see also table 5)
(www.censusindia.net/agedata/index.html).
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1562
Raychaudhuri, Lahiri, Bandyopadhyay, et al
Table 4 Cases of definite (and definite and probable) glaucoma by sex and glaucoma type in people who could be examined
adequately (n = 1269)
Definite (and definite and probable) glaucoma cases
Characteristic
POAG
PACG
Sex (M:F)
22:16
(26:20)
61.7
(62.5)
50 to 85
(50 to 85)
3:0
(3:0)
68.3
(68.3)
55 to 80
(55 to 80)
Mean age
Age range
Secondary
glaucoma
1:0
(1:0)
65.0
(65.0)
–
Total
POAG
PACG
Secondary
glaucoma
Total
26:16
(30:20)
62.3
(62.9)
50 to 85
(50 to 85)
24:28
(20:24)
60.3
(57.5)
50 to 75
(50 to 75)
1:1
(1:1)
57.5
(57.5)
50 to 65
(50 to 65)
1:2
(1:2)
58.3
(58.3)
50 to 65
(50 to 65)
26:31
(22:27)
60.1
(59.1)
50 to 75
(50 to 75)
There were 42 definite cases of glaucoma and a further eight
probable cases. All but one of the definite cases met the
criteria for ‘‘level 1 evidence’’11; the remaining case was
diagnosed as having secondary glaucoma on the basis of
clinical signs, no perception of light and extreme cupping in
one eye, and intraocular pressure of 62 in the blind eye and
15 in the fellow eye (level 3 evidence11). Five of the probable
cases met the VCDR or asymmetry criteria for level 1 evidence
of glaucoma (see Methods) but had only suspicious visual
field results based on the Henson algorithm, consistent with
glaucomatous optic nerve damage (for example, classic,
arcuate pattern but few points missed), rather than definite
field defects. The remaining three probable cases satisfied the
VCDR or VCDR asymmetry criteria for level 2 evidence of
glaucoma (see Methods) but had visual field results classified
as normal by the Henson algorithm; suspicion about the
reliability of the visual field result—for example, evidence of
poor fixation, combined with the extreme VCDR findings, led
to these cases being described as probable. Except in people
aged 80 years or more, the prevalence of definite glaucoma
was higher in males than in females. The prevalence of
definite glaucoma among males and females combined
increased from 2.7% (95% CI 1.7 to 3.7) in people aged 50–
59 years to 6.5% (95% CI 0.0 to 14.1) in those aged 80 years
or more. These patterns, of increasing prevalence with age
and higher prevalence in males, were unchanged if probable
glaucoma cases were included.
Table 4 describes the age and sex characteristics of people
with different types of glaucoma and glaucoma suspects.
Nine of every 10 glaucoma cases were classified as POAG.
More definite cases were male than female for each glaucoma
type. PACG and secondary glaucoma cases tended to be
slightly older than POAG cases, but this finding is based on
very few PACG and secondary cases. Based on the WHO
definition for blindness (worse than Snellen 3/6013), two
POAG and one secondary glaucoma cases (all definite) were
blind in one eye only; no one with glaucoma was found to be
blind in both eyes. No eye was found to be blind from PACG.
As was found for definite glaucoma cases, approximately
nine of every 10 glaucoma suspects were classified as POAG
suspects. There were slightly more female than male
suspects. PACG and secondary glaucoma suspects had similar
ages compared POAG suspects, but again this finding is
based on very few PACG and secondary cases. Based on the
WHO definition, none of the suspects were blind in either
eye.
DISCUSSION
Of the 1594 people aged 50 years or over in this sample, 83%
responded to the invitation to attend for examination for
glaucoma. This is a reasonable response for a survey of this
kind, similar to the study in urban Hyderabad (85%),5 6 less
than the response in Tamil Nadu (93%),7 but higher than was
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Suspect and probable (and suspect) glaucoma cases
achieved in the rural and urban areas of Dhaka (66%).8 It
should also be pointed out that response rates depend on the
accuracy of determination of the population being surveyed—that is, there is likely to be some uncertainty about
the true population denominators.
Of those who were not adequately examined for glaucoma,
more than half had dense cataracts, or cataract in one eye
and the second eye phthisical. It is possible that some of these
subjects also had glaucoma so that the overall estimate for
the prevalence of glaucoma (3.4%) in this age group may be
conservative.
Those who did not attend for examination made up a
larger proportion than those who could not be examined
adequately. Non-attendance may have introduced bias, either
because non-attendees had a higher prevalence of blindness
than those who responded (on the grounds of poorer
mobility) or because they had a lower prevalence of blindness
than those who responded (on the grounds of being more
socioeconomically active and unwilling to give up the time to
attend). The age and sex distributions for the sample and the
whole population were very similar, except for females aged
80 years or over who were under-represented in the sample
(see table 1), suggesting that any such bias was small.
The district used for the survey was not chosen randomly
but for logistical reasons. This may have introduced bias but
we are not aware of any reason why the prevalence of
glaucoma or blindness should be different in the chosen
district compared to neighbouring ones.
The prevalence of glaucoma in surveys such as the one
described here depends on the exact criteria for diagnosis
used in each study. For example, using a VCDR of 0.7 as the
criterion for diagnosing glaucoma resulted in slightly lower
estimates of glaucoma prevalence than if we had used the
observed 97.5th percentile for the study sample—that is, 0.6.
Therefore, direct comparisons between population based
surveys in India can give only approximations of the
prevalences and proportions of POAG and PACG. Table 5
compares data from recently published papers that have used
the ISGEO criteria, or broadly similar criteria, for diagnosis.
The age standardised prevalence of primary glaucoma among
people aged 50 years or more in this sample from West
Bengal was less than in Hyderabad,5 6 but similar to rural
Tamil Nadu7 and Dhaka (expected to be an ethnically similar
population).8
What is striking about this present survey is the very small
number of cases of PACG that were identified; only three
people with PACG out of 1324 were found, a crude prevalence
of 0.23% in people aged 50 years or over and a crude ratio of
POAG to PACG of more than 10:1. The prevalence of PACG
was also relatively low in Dhaka, 0.5% in those aged 40 and
over, with the ratio of POAG to PACG of about 4:1. In Tamil
Nadu, the ratio of POAG to PACG was 3.4:1, and in
Hyderabad 2.4:1 in the same age range (1.95:1 among people
Madurai, Tamil Nadu
Dhaka, Bangladesh
Calcutta, West Bengal
Ramakrishnan et al, 20037`
Rahman et al, 20048$
Raychauduri et al
Rural
Rural and urban
Rural
Urban
Setting
83.1% (1324/1594)
65.9% (2347/3562)
93.0% (5150/5539)
85.4% (2522/2954)
% of population
examined
>50 years (1269)
>35 years (1102)
>40 years (3084)
All ages (539)
Age range of study population
(number >50 years)
50–59 years: 2.5
60–69 years: 2.7
70–79 years: 4.8
>80 years: 4.3
40–49 years: 1.1
50–59 years: 1.9
60–69 years: 2.0
70–79 years: 1.9
>80 years: 1.1
50–59 years: 0.2
60–69 years: 0.0
70–79 years: 0.5
>80 years: 2.2
40–49 years: 0.4
50–59 years: 0.6
60–69 years: 0.6
70–79 years: 0.6
>80 years: 0.4
40–49 years: 0.5
50–59 years: 0.5
60–69 years: 0.5
>70 years: 0.5
50–59 years: 1.5
60–69 years: 2.2
>70 years: 3.2
50–59 years: 2.3
60–69 years: 4.9
>70 years: 6.3
40–49 years: 0.3
50–59 years: 1.6
60–69 years: 1.8
>70 years: 2.9
40–49 years: 0.0
Prevalence of PACG
40–49 years: 1.3
Prevalence of
POAG %
50–59 years: 2.7
60–69 years: 2.9
70–79 years: 5.4
>80 years: 6.5
40–49 years: 1.5
50–59 years: 2.5
60–69 years: 2.6
70–79 years: 2.5
>80 years: 1.5
40–49 years: 1.6
50–59 years: 2.8
60–69 years: 3.1
>70 years: 4.1
50–59 years: 3.8
60–69 years: 7.1
>70 years: 9.5
40–49 years: 1.3
Prevalence of all
glaucoma
3.4%
2.4%
3.2%
6.1%
Age standardised
prevalence >50 years *
*Age standardised prevalences among people aged >50 years were estimated on the basis of the total Indian population as described in the 2001 census (www.censusindia.net/agedata/index.html).
Dandona and colleagues5 6 only reported cases of POAG and PACG; the estimated age specific prevalences of all glaucoma, and the overall age standardised prevalence of glaucoma among people aged >50 years, are therefore based only
on cases of POAG and PACG.
`Ramakrishnan and colleagues7 only reported overall prevalences of PACG and other glaucomas; the estimated age specific prevalences of PACG and all glaucoma, and the overall age standardised prevalence of glaucoma among people
aged >50 years, assume PACG and other glaucomas had similar prevalences across age groups.
8
$Rahman and colleagues only reported the overall numbers of POAG and PACG; the estimated age specific prevalences of POAG, PACG and all glaucoma, and the overall age standardised prevalence of glaucoma among people aged
>50 years, assume the ratio of POAG and PACG was constant across age groups and there were no cases diagnosed with glaucoma other than POAG and PACG.
Hyderabad, Andra
Pradesh
Dandona et al, 20005 6
Location
Comparison of prevalences of total glaucoma and types of glaucoma from population based surveys in India
Author and year of
publication
Table 5
Prevalence of glaucoma in West Bengal
1563
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1564
aged 50 years or more in Hyderabad). These proportions
suggest that PACG may be less prevalent in Bengalis than in
Indian populations living further south in the subcontinent.
Calculation of prevalence estimates and confidence intervals
needs to be tailored to the sampling methods used to prevent
bias and to avoid underestimating the precision of estimates.
These issues are particularly important if sampling has been
stratified by a factor likely to be associated with the health
state of interest or, when multistage sampling has been used,
the prevalence of the health state of interest varies markedly
between clusters. The surveys described in table 5 used
various sampling methods and appear to have reported
appropriate analyses.
There was one case of secondary glaucoma and two
suspects. The definite case was caused by a hypermature
cataract. The two suspects had pseudoexfoliation.
The Vellore Eye Study4 (excluded from table 5) concluded
that PACG was about five times as common as POAG in that
part of Tamil Nadu. However, in that study people were
classified as PACG cases on the basis of peripheral anterior
synechiae and raised intraocular pressure in the presence of
closed angles on gonioscopy, without necessarily having
evidence of optic nerve damage. Only nine subjects were
found to have glaucomatous field defects, four of whom were
classified as POAG and five as PACG giving a ratio of about
1:1.
It is also notable from the present study that none of the
1269 people who could be adequately examined had a blind
eye as a result of PACG. Indeed, no one was found to be
bilaterally blind because of glaucoma, although two people
were blind unilaterally as a result of POAG and one as a
result of secondary glaucoma. PACG, therefore, does not
seem to be a major public health problem among rural
Bengalis.
An unexpected observation in the Dhaka survey was that
the prevalence of glaucoma was relatively high in younger
people (age 35–49 years) and did not increase with age.
Although the present study only included people aged
50 years and over, glaucoma prevalence increased with
increasing age, which does not support the finding in the
Dhaka survey.
From the point of view of eye care programmes and
prevention of blindness, the available survey data imply that
the emphasis in both West Bengal and Dhaka should be on
the detection of POAG. There is still no satisfactory method of
screening for POAG which can be applied to populations,
especially in low income countries. As Thomas and colleagues
concluded in their letter on glaucoma in southern India,14
‘‘On balance, we believe there is no current alternative to case
detection (opportunistic screening), developing our infrastructure, and making routine gonioscopy the norm.’’
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Raychaudhuri, Lahiri, Bandyopadhyay, et al
ACKNOWLEDGEMENTS
We are grateful to all the people who participated in the survey, to
villagers and the members of Gran Panchayat for their help in
establishing the clinics in their villages, and to ICDS, Block and
District administrators and directors of the Regional Institute of
Ophthalmology and Department of Community Medicine, Kolkata,
who facilitated the survey.
.....................
Authors’ affiliations
A Raychaudhuri, Department of Ophthalmology, Institute of Post
Graduate Medical Education and Research, Kolkata, India
S K Lahiri, Department of Community Medicine, Medical College,
Kolkata, India
M Bandyopadhyay, Regional Institute of Ophthalmology, Kolkata, India
P J Foster, Department of Epidemiology, Institute of Ophthalmology,
London, UK
B C Reeves, Department of Public Health and Policy, London School of
Hygiene and Tropical Medicine, London WC1E 7HT, UK
G J Johnson, International Centre for Eye Health, London School of
Hygiene and Tropical Medicine, London, UK
Funding: The survey was funded by the UK Department for International
Development, as part of the Indo-UK Community Ophthalmology
Project. The design of the survey, its execution, analysis, interpretation,
and publication were carried out independently of the funder.
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13 World Health Organization. Adaptation of the international classification of
diseases. 1975 revision. Geneva: WHO, 1977.
14 Thomas R, Muliyil J, George R. Glaucoma in south India. Ophthalmology
2001;108:1173–4.
1565
SCIENTIFIC REPORT
‘‘Near misses’’ in a cataract theatre: how do we improve
understanding and documentation?
K Mandal, W Adams, S Fraser
...............................................................................................................................
Br J Ophthalmol 2005;89:1565–1568. doi: 10.1136/bjo.2005.072850
Aim: Near miss event reporting is widely used in industry to
highlight potentially unsafe areas or practice. The aim of this
study was to see if a descriptive method of recording near
misses was an appropriate method for use in an ophthalmic
operating theatre and to quantify how many untoward events
were recorded using this system.
Methods: The study was wholly conducted in a cataract
theatre in the United Kingdom. The theatre nurse assigned to
the patient in their journey through the operating theatre was
asked to note any untoward events. As, at present, there is no
consensus definition of near misses in ophthalmology the
nurses recorded, in free text, any events that they considered
to be a deviation from the normal routine in that theatre.
Results: Of the 500 cases randomly chosen, 96 ‘‘deviations
from normal routine’’ were described in 93 patients—that is,
19% of cases. All forms distributed to the nurses were
returned (100% response rate). The commonest abnormal
events were intraoperative (69), with a lesser number being
recorded preoperatively (27). When these events were
further classified, it was thought that 25 could be classified
as near misses. One true adverse event was recorded during
the study.
Conclusions: The results suggest that experienced nursing
staff in an ophthalmic theatre are a reliable source for
collecting data regarding near misses. A consensus is now
required to define near misses in ophthalmology and to
devise a user friendly input system that can use these
definitions to consistently record these potentially vital events.
they can offer are ignored because the patient has not come
to harm.
In most hospitals, adverse events are reported through an
established incident reporting system tailored to individual
hospitals. However, near misses are not usually systematically recorded, unlike in industry.4–7 Near misses outnumber adverse events in a relationship that was quantified
over 60 years ago by Heinrich, who estimated that for every
300 near misses there would be 29 minor injuries and one
major injury.1
If we are to utilise lessons from near misses, we need to
know how often they occur and in what circumstances.
Before this can be done near miss events need to be defined
on the basis of their likelihood and consequences. In
ophthalmology there is, as yet, no clear definition of near
misses. It is therefore difficult to subject them to quantitative
methods of analysis. Other specialties have indicated that the
operating theatre is one of the commonest sites of errors and
near misses—and the area with the greatest potential for
serious harm from these.8 We thought that cataract surgery
represented a good model for analysing operating theatre
errors—it is a common operation, complications can be sight
threatening, and it has the highest indemnity claims in
ophthalmology.9–11
The purpose of this study was thus to devise an acceptable
method of recording near misses in a theatre dedicated to
cataract surgery. Since near misses in ophthalmology have
not been fully defined, we decided to use a descriptive
method to record experienced theatre nurses’ perception of
what they considered to be ‘‘deviations from the routine.’’
METHOD
I
t is increasingly recognised that within health systems
patients can sometimes come to harm.1 The prevalence of
iatrogenic harm has been shown to be much higher than
previously thought.2 3 This has led, in recent years, to an
increased interest in the study of patient safety within
healthcare systems. Central to this study has been the
development of definitions of harm or near harm that
patients can and do suffer. Thus a ‘‘adverse healthcare
event’’ is an event or omission during clinical care causing
physical or psychological injury to a patient. A healthcare
‘‘near miss’’ is a situation in which an event or omission, or a
sequence of errors or omissions, arising during clinical care
fails to develop further (as a result of compensating action),
thus preventing injury to a patient.1
In most hospitals, adverse incidents are reported using a
central adverse incident reporting system. Few systems
however encompass near misses. Near misses occur more
frequently than actual adverse incidents and they provide a
valuable opportunity for learning by quantitative analysis
about the nature, frequency, and types of safety issues.2 The
importance of near misses is that they can indicate where
flaws in systems lie. The weakness of them is that the lessons
The study was conducted at a single specialty ophthalmic
hospital with a dedicated high volume cataract theatre. The
theatre has an annual turnover of over 5000 patients.
The system employed in this theatre is that on the day of
the operation the patient is assigned a named nurse called
the ‘‘primary nurse.’’ The duties of the primary nurse are to
accompany the patient throughout their time in theatre—
they are also responsible for filling in the operative notes.
Because they chaperone the patient throughout their visit to
the cataract theatre but are not involved in the mechanics of
the surgery, we thought them to be in the best position to
record the events surrounding the operation. We thus asked
the primary nurses to report any event during their patient’s
visit to the theatre that they considered to be a ‘‘deviation
from routine.’’
All the nurses who participated in the study were trained
ophthalmic nurses experienced in cataract surgery. They were
not asked to categorise their comments into near misses or
adverse events but simply to describe events that struck them
as ‘‘deviations from routine’’ during surgery. The method of
reporting was anonymous in that the nurses did not need to
identify themselves or any other member of the team.
www.bjophthalmol.com
1566
Mandal, Adams, Fraser
Table 1 Reasons given for delay in starting the
operation
Delay
Number
Late surgeon
Late anaesthetist
Autoclave delay
Trial phaco machine
Complicated procedure in previous patient
Preoperative refraction required
Delay in acquiring patient’s notes
No cause stated
2
1
2
2
2
1
1 (NM)
1
Table 3 Other preoperative deviations recorded
Miscellaneous
Number
Biometry error
Incorrect patients’ notes
Consent form not signed by patient
Checklist not signed by surgeon
5% povidone iodine not available
5 (NM)
3 (NM)
1
1
1 (NM)
NM, near miss.
Table 4 Defective instrument events recorded
NM, near miss.
Table 2 Anaesthetic problems noted
Anaesthetic problems
Number
Painful peribulbar injection
Venous cannulation tissued
Difficult cannulation
1
1
2
Defects
Number
Phaco machine failure
Phaco probe blocked
Irrigation aspiration canula blocked
Defective forceps
Problem with microscope settings
Mark on intraocular lens
2 (NM)
10 (NM)
1 (NM)
5
1
1 (NM)
NM, near miss.
(3) Postoperative (from completion of surgery to
discharge) ‘‘departures from routine.’’
A total of 500 cases in the year 2002 were randomly
chosen. The selected patients’ names, hospital identification
numbers, and dates of operation were printed on A4 sheets
and distributed to the primary nurses at the start of every list.
The sheets were otherwise blank to allow free text.
RESULTS
All 500 distributed sheets were returned (response rate
100%). Most response sheets had ‘‘uncomplicated,’’ ‘‘nothing
to report,’’ or ‘‘nothing untoward’’ written on them. Ninety
six sheets had responses that the nurses thought described
deviations from the routine. Three patients had more than
one deviation during their operation. No patients had more
than two recorded. Therefore, in this study 93 (19%) patients
had, what the primary nurse considered to be, a deviation
from the normal routine during their visit.
Although these deviations were reported by the primary
nurses in a descriptive manner, for ease of presentation and
analysis we have categorised their responses into preoperative, intraoperative, and postoperative events. These are
summarised below:
(1) Preoperative ‘‘deviations from routine’’
N
N
N
Delay in starting operation, 12 (2.5%) cases (table 1)
Anaesthetic problems, four (1%) cases (table 2)
Miscellaneous, 11 (2%) cases (table 3).
(2) Intraoperative ‘‘departures from routine’’
N
Extended surgery, four (1%) cases.
(Analysis of the theatre logbook indicated they were all over
20 minutes. None involved any surgical complication.)
N
N
N
N
Defective instruments, 20 (4%) cases (table 4)
Difficult operation, 14 (3%) cases (appendix, table A1)
Complications, 26 (5%) (appendix, table A2)
Miscellaneous, five (1%)
(One case incorrect intraocular lens brought by the floor nurse. Three
cases of ‘‘contamination’’ of surgical field by the patient. One case of a
patient with known allergy to cefuroxime was given the drug
subconjunctivally.)
www.bjophthalmol.com
No incident was documented in this study.
After categorising these deviations, we assessed which
deviations we thought could be classified as near misses and
which as true adverse events. This was inevitably a subjective
interpretation. Although we have given standard definitions in
our introduction there remains considerable debate surrounding these definitions (see www.safetyandquality.org/definition/
smhome.htm for further discussion of this). The results of our
deliberations are documented in table 5. Near misses (which we
defined as having the potential to cause harm if correcting
action was not taken) are denoted by ‘‘NM’’ in the remaining
tables. As far as true adverse events (that is, where the patient
did come to temporary or permanent harm) are concerned, we
thought there was only one of these—the patient who was
known to have a cefuroxime allergy and was given the drug.
The patient developed some itchiness and was observed until
this abated—resulting in some delay to the patients discharge.
This adverse event was reported via the standard hospital
system.
DISCUSSION
General findings
This descriptive study found 96 occurrences in 93 patients
(out of a total of 500), which the primary nurse thought
deviated enough from the normal routine to record.
Although it was one of the commonest recorded events,
‘‘complications’’ or ‘‘difficult surgery’’ has been excluded
from this analysis as we thought that these represented well
recognised and unavoidable variations in surgical difficulty.
For full details of these see the tables in the appendix. It is
important to note that none of the near misses described
resulted in a complication in this study.
Excluding the above, the commonest deviation was
defective instruments—described in 20 cases. The most
common problem was blocking of the phaco tip, followed
by defective forceps and then failure of the phaco machine
itself. The majority of deviations in this category we thought
could be categorised as potential near misses.
A number of preoperative events could also be classified as
near misses. These included biometry errors, incorrect patient
notes (corrected before commencement), and iodine not
available. ‘‘Delay in starting operation’’ cannot be called a near
miss in itself but could contribute to creating an environment in
which near misses/adverse events are more likely to occur.
‘‘Near misses’’ in a cataract theatre
1567
Table 5 Recorded deviations from routine, assessment of number of near misses within
this and number of adverse events
Number of deviations
from routine
Preoperative
Delay in starting operation
Anaesthetic problems
Miscellaneous
Total
Intraoperative
Extended surgery
Defective instruments
Difficult operation
Complications
Miscellaneous
Total
Postoperative
Totals
Number of
near misses
Number of
adverse events
12
4
11
27
0
0
9
9
0
0
0
0
4
20
14
26
5
69
0
14
0
0
2
16
0
0
0
0
1
1
0
96
0
25
0
1
No postoperative complications were described in this
study—this is the time the primary nurse helps the patient
leave the theatre before preparing them for discharge and it is
likely that they had too limited a time to record any untoward
events. In our study this was therefore not a useful method of
recording deviations.
Overall, we assessed that 25 of the ‘‘deviations’’ could be
classified as near misses. Thus, 5% (25/500) of cases had a
near miss. With one case classified as a true adverse event, we
have calculated the Heinrich ratio from this study as 25:1.
Usefulness of study method
The aim of this study was to test if a simple, open ended
method of recording untoward events in theatre would be
feasible. Our response rate (100%) suggests that the method
itself was efficient and that the personnel chosen (the
primary nurse) was best placed to record these events.
The nurses thought that in 19% of operations, there were
deviations from the routine that were worth reporting. These
positive responses are interesting in that they provide us with
information which probably would not have been documented elsewhere but could have a significant impact on the
outcome of cases in any operating list. The figure does need
to be treated with some caution as we did not assess any
deviations that the nurses either missed or did not record.
Indeed, it may be speculated that they are likely to underrecord these events in the ‘‘heat of the moment.’’
As described previously, there are, at present, no universally agreed definitions of near misses in ophthalmology.
This study suggests that our method may be a useful first step
in creating these definitions.
The advantages of this ‘‘blank canvas’’ recording method
include the fact that the observer is not forced to choose from
a predetermined range of choices making it more likely that a
wider range of deviations are noted. For example ‘‘phaco
probe blocked’’ is not something that would be reported in
any adverse event system but it represents a situation where
if the blockage is suddenly relieved the posterior capsule may
be ruptured. It was shown in this study to be relatively
common and is preventable.
The descriptive method is only really useful though as a
starting point as it produces a large amount of data, not all of
which may be relevant (for example, variations in surgical
difficulty). For a near miss reporting system to be generalisable there needs to be a consensus from ophthalmologists,
theatre nurses, and other theatre personnel. Our definitions
of near misses in this study were subjective and future work
needs to use this consensus as a basis for devising a practical
near miss recording system.
CONCLUSION
The aim of the medical profession is to provide safe, humane,
and up to date care individualised to every patient. In order to
provide our patients with a safer healthcare system, errors
need to be documented, types of errors and trends and factors
contributing to errors need to be identified. Near misses often
appear insignificant but when analysed systematically can
provide valuable information about ‘‘weak links’’ in a system.
Our study suggests that experienced nursing staff in an
ophthalmic theatre appear to be a reliable observers and the
descriptive methods they used appeared acceptable.
The results of the study now need to be refined to produce
a definition of important near misses in cataract theatres.
Once these definitions and guidelines have been devised a
user friendly but flexible input system needs to be developed.
This will then allow us to analyse the frequency and patterns
of near misses and in the long term increase patient safety in
this commonest of operations.
.....................
Authors’ affiliations
K Mandal, W Adams, S Fraser, Sunderland Eye Infirmary, Queen
Alexandra Road, Sunderland SR2 9HP, UK
S Fraser, School of Health, Natural and Social Sciences, University of
Sunderland, UK
Competing interests: none declared
Correspondence to: S Fraser, Sunderland Eye Infirmary, Queen
Alexandra Road, Sunderland SR2 9HP, UK; [email protected]
Accepted for publication 7 July 2005
APPENDIX
Table A1 Reasons given for ‘‘difficult’’ surgery
Difficulties recorded
Number
Bulging eye
Small eye
Small pupil
Iris prolapse
Hyphaema
Dense cataract
Subluxated lens preoperatively
Patient positioned upright because of orthopnoea
Patient moving at operation
2
1
1
1
2
2
1
1
3
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1568
Table A2
Mandal, Adams, Fraser
Complications noted during surgery
Complication recorded
Number
Posterior capsule rupture
Zonule rupture
Anterior vitrectomy
Conversion to extracapsular technique
IOL damaged at folding
IOL damaged at insertion
Painful operation
5
1
2
2
1
11
4
REFERENCES
1 Department of Health. An organisation with a memory: report of an expert
group on learning from adverse events in the NHS. London: Stationery Office,
2000.
2 Killen AR, Beyea SC. Learning from near misses in an effort to promote patient
safety. AORN J 2003;77:423–5.
www.bjophthalmol.com
3 Williams J. Risk management in surgery. In: Vincent C, ed. Clinical risk
management. Enhancing patient safety. London: BMJ Publishers,
2001:137–51.
4 Billings CE. Some hopes and concerns, regarding medical event reporting
systems: lessons from NASA Aviation Safety Reporting System (ASRS). Arch
Pathol Med 1998;122:214–15.
5 Davies JB, Wright LB, Courtney E. CIRAS. A confidential reporting system for
the railway industry. Proceedings: confidential human factors and incident and
near miss reporting programmes. London: The Royal Society of Medicine:
Sheffield: Health and Safety Executive Press, 1998.
6 Van der Schaaf TW, Lucas DA, Hale AR. Near miss reporting as a safety tool.
Oxford: Butterworth-Heinemann, 1991.
7 Berman JVF, Collier GD. The collection and use of near miss data: is ‘no
blame’ just wishful thinking? In: Cox RF, ed. Managing risks in a changing
organizational climate. Proceedings of a symposium held in Swindon, UK, 9
October 1996. The Safety and Reliability Society, Manchester 1–12 June, 1996.
8 Reed MWR, Phillips WS. Operating theatre lists-accidents waiting to happen?
Ann R Coll Surg Eng (Suppl) 1994;76:279–80.
9 Brick DC. Risk management lessons from a review of 168 cataract surgery
claims. Surv Ophthalmol 1994;43:356–60.
10 Krausher F, Robb JH. Ophthalmic malpractice lawsuits with large monetary
awards. Arch Ophthalmol 1996;114:333–40.
11 Bettman JW. Seven hundred medicolegal cases in ophthalmology.
Ophthalmology 1997;10:1379–84.
1569
SCIENTIFIC REPORT
Effect of cataract extraction on frequency doubling
technology perimetry in patients with glaucoma
M A R Siddiqui, A Azuara-Blanco, S Neville
...............................................................................................................................
Br J Ophthalmol 2005;89:1569–1571. doi: 10.1136/bjo.2005.080655
Aim: To evaluate the effect of cataract surgery on frequency
doubling technology (FDT) perimetry in patients with coexisting cataract and glaucoma.
Methods: In this consecutive prospective cohort study 27
patients with open angle glaucoma scheduled for cataract
extraction alone or combined with trabeculectomy were
enrolled. All patients underwent FDT threshold C-20 visual
fields within 3 months before and 3 months after surgery.
Changes in mean deviation (MD) and pattern standard
deviation (PSD) were evaluated. Additionally, changes in
best corrected logMAR visual acuity (VA), intraocular
pressure (IOP), and number of glaucoma medications were
also studied.
Results: 22 patients completed the study. VA improved after
surgery, from 0.47 (SD 0.19) to 0.12 (0.17) (p,0.001). The
visual indexes changed after cataract extraction: MD
improved (from 210.9 (SD 4.6) dB to 27.0 (4.6) dB;
p,0.001) while PSD worsened (from 7.1 (SD 3.5) dB to 8.5
(3.8) dB; p = 0.001).
Conclusion: In patients with co-existing cataract and glaucoma, examined with FDT, MD improved and PSD worsened
after cataract surgery. Global indexes of FDT should be
interpreted with caution in patients with glaucoma and
cataracts.
V
isual field (VF) assessment provides essential information for the diagnosis and management of glaucoma. In
the past decade novel VF tests such as frequency
doubling technology (FDT), have emerged. FDT utilises the
frequency doubling illusion, in which a sine wave grating of
low spatial frequency (,1 cycle/degree) undergoing counterphase flicker at high temporal frequency (.15 Hz) appears to
the observer to have double the number of bars than are
actually present.1 This psychophysical illusion is thought to
be mediated by My ganglion cells which are a subset of the
magnocellular system.2
FDT has been released commercially and provides suprathreshold and threshold strategies. Suprathreshold modes
(C-20-5 and C-20-1), requiring less than 1 minute per eye,
have shown good diagnostic performance and have been
used for glaucoma screening.3 4 Another positive aspect of
FDT is that it may be able to detect glaucomatous field loss
earlier than the standard white on white perimetry,5 6
perhaps because large diameter magnocellular ganglion cells
may be preferentially damaged early in glaucoma.7
Glaucoma and cataract have an increased prevalence in
elderly populations and they often co-exist. Evaluating the
results of VF tests in patients with cataract and glaucoma is a
common challenge. Previous studies have shown the effect of
cataract on standard white on white perimetry in patients
with glaucomatous VF loss.8–13 Similarly, cataract has been
shown to affect short wavelength automated perimetry
(SWAP).14 Recent work has demonstrated the effect of
cataract on FDT perimetry in normal subjects.15 16 The aim
of this study was to evaluate the effect of cataract extraction
on FDT perimetry results in patients with glaucoma.
PATIENTS AND METHODS
Glaucoma patients were prospectively recruited from the
glaucoma unit in an academic hospital. Patients included in
this study were scheduled for cataract extraction alone or in
combination with trabeculectomy. The indication for cataract
surgery was visually significant cataract that was thought to
contribute to limitations in daily activities. The indication for
phaco-trabeculectomy included uncontrolled intraocular
pressure or medically controlled intraocular pressure with
more than two medications and advanced glaucoma related
damage to the optic nerve associated with a visually
significant cataract. The study adhered to the tenets of the
Declaration of Helsinki and informed consent was obtained
from all participants. Ethics committee approval was available for this study.
Patients with chronic open angle glaucoma (primary open
angle glaucoma, normal tension glaucoma, pseudoexfoliation
glaucoma, or pigmentary glaucoma) with a Snellen visual
acuity (VA) of 6/36 or better were included in the study. The
diagnosis of glaucoma was based on the presence of
glaucomatous optic disc cupping (for example, cup to disc
ratio of more than 0.8, disc asymmetry, thinning of the
neuroretinal rim). Exclusion criteria included the use of
miotics drops, ocular morbidity other than glaucoma or
cataract, and inability to perform reliable white on white VF
(more than 33% of false positive, false negatives, or fixation
losses). Patients with surgical complications, consistent
postoperative IOP of more than 35 mm Hg, or unreliable
FDT tests (more than 33% abnormal responses on any of the
reliability indexes) were also excluded.
Threshold FDT C-20 perimetry (Carl-Zeiss Meditec, Dublin,
CA, USA; and Welch Allyn, Skaneateles Falls, NY, USA) was
performed within 3 months before and after surgery. A
screening FDT test was used in each patient as a training tool.
All eyes were refracted 1 month after surgery. Best corrected
logMAR acuity, using EDTRS vision charts under appropriate
illumination, and IOP, using Goldmann applanation tonometry, were evaluated at the time of preoperative and
postoperative VF testing. The number of antiglaucoma
medications was also recorded.
All patients undergoing phacoemulsification had a
clear cornea procedure with a foldable intraocular lens
implant (MA60BM, Alcon Surgical, Fort Worth, TX, USA)
under topical anaesthesia. Patients undergoing combined
Abbreviations: FDT, frequency doubling technology; IOP, intraocular
pressure; MD, mean deviation; PSD, pattern standard deviation; SWAP,
short wavelength automated perimetry; VA, visual acuity; VF, visual
fields
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1570
Siddiqui, Azuara-Blanco, Neville
Table 1 Participant demographics
Age, mean (SD) (range) years
Sex (male: female ratio)
Race (white: non-white ratio)
Type of glaucoma
POAG, n (%)
PXF, n (%)
NTG, n (%)
Type of surgery
Phaco-trabeculectomy, n (%)
Phacoemulsification, n (%)
Preoperative
Postoperative
76.6 (8.4) (54–90)
13:9
22:0
19 (86.4%)
2 (9.1%)
1 (4.5%)
30°
Total
deviation
30°
30°
Pattern
deviation
30°
16 (72.7%)
6 (27.3%)
POAG, primary open angle glaucoma; PXF, pseudoexfoliation
glaucoma; NTG, normal tension glaucoma.
phaco-trabeculectomy had a one site procedure under subTenon’s anaesthesia. A fornix based conjunctival dissection
was made and 0.4 mg/ml of mitomycin C (MMC) was
applied with soaked sponges over the bare sclera and covered
with the conjunctival flap for at least 1 minute. The area
exposed to MMC was vigorously irrigated with balanced salt
solution. A 4 mm63 mm partial thickness rectangular scleral
flap was constructed in the superior limbus. A block of
corneal tissue was removed with a punch and a small
peripheral iridectomy was performed. Phacoemulsification
was carried out before removing the block of tissue. The
scleral flap was sutured with at least two interrupted and one
releasable 10/0 nylon sutures. The conjunctival flap was
sutured watertight with 10/0 nylon sutures. Ocular massage,
laser suturelysis, and 5-fluorouracil subconjunctival injection
was carried out according to the surgeon’s criteria.
Data were entered in a statistical software package SPSS
(version 10-0, Chicago, IL, USA). Normality of the data was
assessed by Kolmogorov-Smirnov test. Changes between
preoperative and postoperative VF indexes were evaluated
by paired t test. Associations between improvement of VA
and changes in MD and PSD values of FDT after cataract
extraction were analysed with the Pearson correlation test.
Probability values of less than 0.05 were considered to be
statistically significant.
RESULTS
Twenty seven patients were enrolled in the study. Two
patients failed to attend for postoperative VF test within
3 months. One patient complained of a red and watery eye
after surgery and was unable to perform the test postoperatively. Another patient died of unrelated ruptured
abdominal aortic aneurysm 2 months after surgery. One
patient developed visual distortion after surgery and was
found to have a choroidal neovascular membrane. A total of
22 patients were included in the final analysis.
Demographics of the participants are summarised in
table 1. Changes in VA, IOP, and number of glaucoma
medications are shown in table 2. The median number of
antiglaucoma medications was two before surgery and none
Table 2
>=5%
< 5%
< 2%
< 1%
< 0.5%
Postoperative value
MD = +0.19 dB
PSD = +22.28 dB
Figure 1 FDT perimetry printout (total and pattern deviation plots, and
MD and PSD values) before and after surgery of a patient with glaucoma
and cataract who underwent phaco-trabeculectomy.
after surgery. In patients undergoing phaco-trabeculectomy
(n = 16) the mean IOP (SD) before and after surgery was
20.2 (5.0) and 15.9 (3.7), respectively (p = 0.014).
The MD value improved after surgery (p,0.001), while
PSD deteriorated (p = 0.001) (fig 1). These changes were
statistically significant (table 2). The extent of VA improvement correlated with the deterioration of PSD score. The
Pearson correlation test showed a statistically significant
correlation between the postoperative VA improvement and
the PSD change (p = 0.024, R2 = 0.478). However, the
changes of MD and VA were not correlated (p = 0.252,
R2 = 0.252).
DISCUSSION
In white on white conventional perimetry cataract usually
results in a diffuse reduction of sensitivity with a worsening
of the MD index. However, indexes designed to quantify
localised defects would not expected to be affected by
cataract. Several studies have evaluated the effects of cataract
on white on white perimetry in normal subjects and also in
patients with glaucoma. In the majority of evaluations on
normal volunteers an improvement in MD was noted after
cataract surgery, while the PSD remained unchanged.16–18
However, in glaucoma patients examined with standard
perimetry a worsening of PSD was observed after cataract
extraction (table 3).9–13
Changes in VA, IOP, number of glaucoma medications, and FDT global indexes
Best corrected logMAR acuity
IOP (all patients, n = 22) (mm Hg)
IOP (phaco-trabeculectomy, n = 16) (mm Hg)
No of medications (all patients, n = 22)
No of medications (phaco-trabeculectomy, n = 16)
Mean deviation (dB)
Pattern standard deviation (dB)
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Preoperative value
MD = –7.36 dB
PSD = +18.74 dB
p
p
p
p
p
Preoperative
Postoperative
Mean (SD)
Mean (SD)
p Value(paired
t test)
0.47 (0.19)
18.8 (5.5)
20.2 (5.0)
1.8 (0.8)
2.2 (0.5)
210.9 (4.6)
7.1 (3.5)
0.12 (0.17)
16.6 (4.0)
15.9 (3.7)
0.5 (0.7)
0.2 (0.5)
27.0 (4.6)
8.5 (3.8)
,0.001
0.063
0.014
,0.001
,0.001
,0.001
0.001
Effect of cataract extraction on FDT
1571
Table 3 Studies assessing effect of cataract on visual field tests
Study
17
Lam et al
Yao et al18
8
Stewart et al
9
Smith et al
Chen et al10
11
Gillies et al
Kim et al14
Kim et al14
12
Hayashi et al
Kook et al16
16
Kook et al
15
Tanna et al
Koucheki et al13
Our study
Year
Test
Glaucoma
MD*
CPSD, PSD, or CLV*
1991
1993
1995
1997
1998
1998
2001
2001
2001
2004
2004
2004
2004
2005
Humphrey threshold 30-2
Octopus (program G1)
Humphrey 30-2
Humphrey threshold 24-2 or 30-2
Humphrey threshold 24-2 or 30-2
Humphrey threshold 24-2
White-on-white 24-2 (FASTPAC)
Blue on yellow 24-2 (FASTPAC)
Humphrey threshold 30-2
FDT C-20
SITA-fast 30-2
FDT threshold C-20
Humphrey threshold 24-2
FDT threshold C-20
No
No
Yes
Yes
Yes
Yes
No
No
Yes
No
No
No
Yes
Yes
q
q
«
q
q
q
q
q
q
q
q
q
q
q
«
« (CLV)
«
Q
Q
Q
«
«
Q
«
«
«
Q
Q
*Post-cataract extraction: («) no change; (q) improved; (Q) worsened.
MD, mean deviation; PSD, pattern standard deviation; CPSD, corrected pattern standard deviation; CLV, corrected loss variance.
The impact of cataract on the results of FDT perimetry has
been evaluated recently in normal subjects. Tanna et al15
found that cataract has an adverse effect on MD but not on
PSD. Similarly, Kook et al16 evaluated the effect of cataract in
healthy individuals undergoing both FDT threshold C-20
perimetry and SITA-fast. Their results showed a generalised
reduction of sensitivity in FDT and SITA-fast associated with
cataract, whereas PSD remained unchanged in both tests.
However, to our knowledge, there are no reports on the effect
of cataract on FDT in patients with glaucoma. As it has been
shown that FDT perimetry is resistant to refractive blur,19
researchers have thought that it may also be resistant to the
effects of cataract to some extent. It has been suggested that,
in screening mode of FDT, cataracts are not a cause of
abnormal results.20
In the current study, among patients with cataract and
glaucoma examined with FDT perimetry both MD and PSD
indexes changed after cataract surgery. The improvement of
MD was not surprising. However, the PSD worsening after
cataract extraction highlights the potential of underestimating
the true extent of VF defects on sequential observations in
glaucoma patients. This finding has also been observed in
standard white on white full threshold perimetry (see above).
It may be possible that a cataract renders margins of a localised
field defect less distinct, hence masking ‘‘true’’ PSD. It is
difficult to attribute these changes to a learning effect. Previous
studies evaluating the learning effect of patients with glaucoma
using FDT did not show any differences in PSD values.21
FDT has been advocated as a useful test for screening in
glaucoma. Because PSD may be masked by cataract, some
patients with increased MD but ‘‘normal’’ PSD may have
glaucoma. This could diminish the sensitivity of FDT to detect
glaucoma in patients with co-existing cataract. As cataract
also worsens MD, it could affect the specificity of the test in
patients with only cataract.
A limitation of our study was that a standard system to
quantify lenticular opacities was not used. The effect of lens
opacities on VF tests may vary with the type of cataract.
Similarly, the sample size was small and it was not possible to
analyse the effects of different severities and types of
glaucoma on FDT parameters.
In conclusion, FDT is affected by lens opacities in glaucoma
patients. Global indexes of FDT should be used with caution
in patients with coexisting cataract and glaucoma.
.....................
Authors’ affiliations
M A R Siddiqui, A Azuara-Blanco, S Neville, Department of
Ophthalmology, NHS Grampian, University of Aberdeen AB25 2ZN,
UK
Competing interests: none declared
Correspondence to: Augusto Azuara-Blanco, PhD, FRCS(Ed), The Eye
Clinic, Aberdeen Royal Infirmary, Aberdeen AB25 2ZN, UK;
[email protected]
Accepted for publication 21 August 2005
REFERENCES
1 Kelly DH. Frequency doubling in visual responses. J Opt Soc Am A
1966;56:1628–33.
2 Johnson CA, Samuels SJ. Screening for glaucomatous visual field loss with
frequency-doubling perimetry. Invest Ophthalmol Vis Sci 1997;38:413–25.
3 Quigley HA. Identification of glaucoma-related visual field abnormality with
the screening protocol of frequency doubling technology. Am J Ophthalmol
1998;125:819–29.
4 Wadood AC, Azuara-Blanco A, Aspinall P, et al. Sensitivity and specificity of
frequency-doubling technology, tendency-oriented perimetry, and Humphrey
Swedish interactive threshold algorithm-fast perimetry in a glaucoma practice.
Am J Ophthalmol 2002;133:327–32.
5 Sample PA, Bosworth CF, Blumenthal EZ, et al. Visual function-specific
perimetry for indirect comparison of different ganglion cell populations in
glaucoma. Invest Ophthalmol Vis Sci 2000;41:1783–90.
6 Medeiros FA, Sample PA, Weinreb RN. Frequency doubling technology
perimetry abnormalities as predictors of glaucomatous visual field loss.
Am J Ophthalmol 2004;137:863–71.
7 Quigley HA, Dunkelberger GR, Green WR. Chronic human glaucoma causing
selectively greater loss of large optic nerve fibers. Ophthalmology
1988;95:357–63.
8 Stewart WC, Rogers GM, Crinkley CM, et al. Effect of cataract extraction on
automated fields in chronic open-angle glaucoma. Arch Ophthalmol
1995;113:875–9.
9 Smith SD, Katz J, Quigley HA. Effect of cataract extraction on the results of
automated perimetry in glaucoma. Arch Ophthalmol 1997;115:1515–19.
10 Chen PP, Budenz DL. The effects of cataract extraction on the visual field of
eyes with chronic open-angle glaucoma. Am J Ophthalmol
1998;125:325–33.
11 Gillies WE, Brooks AM. Effect of lens opacity on the glaucomatous field of
vision. Aust N Z J Ophthalmol 1998;26(suppl):19–21.
12 Hayashi K, Hayashi H, Nakao F, et al. Influence of cataract surgery on
automated perimetry in patients with glaucoma. Am J Ophthalmol
2001;132:41–6.
13 Koucheki B, Nouri-Mahdavi K, Patel G, et al. Visual field changes after cataract
extraction: the AGIS experience. Am J Ophthalmol 2004;138:1022–8.
14 Kim YY, Kim JS, Shin DH, et al. Effect of cataract extraction on blue-on-yellow
visual field. Am J Ophthalmol 2001;132:217–20.
15 Tanna AP, Abraham C, Lai J, et al. Impact of cataract on the results of
frequency-doubling technology perimetry. Ophthalmology
2004;111:1504–7.
16 Kook MS, Yang SJ, Kim S, et al. Effect of cataract extraction on frequency
doubling technology perimetry. Am J Ophthalmol 2004;138:85–90.
17 Lam BL, Alward WL, Kolder HE. Effect of cataract on automated perimetry.
Ophthalmology 1991;98:1066–70.
18 Yao K, Flammer J. Relationship cataract density and visual field damage.
Eur J Ophthalmol 1993;3:1–5.
19 Anderson AJ, Johnson CA. Frequency-doubling technology perimetry and
optical defocus. Invest Ophthalmol Vis Sci 2003;44:4147–52.
20 Sponsel WE, Trigo Y, Hendricks J, et al. Frequency doubling perimetry.
Am J Ophthalmol 1998;126:155–6.
21 Matsuo H, Tomita G, Suzuki Y, et al. Learning effect and measurement
variability in frequency-doubling technology perimetry in chronic open-angle
glaucoma. J Glaucoma 2002;11:467–73.
www.bjophthalmol.com
1572
SCIENTIFIC REPORT
The relative effects of corneal thickness and age on
Goldmann applanation tonometry and dynamic contour
tonometry
A Kotecha, E T White, J M Shewry, D F Garway-Heath
...............................................................................................................................
Br J Ophthalmol 2005;89:1572–1575. doi: 10.1136/bjo.2005.075580
Aims: To establish the effects of central corneal thickness
(CCT) on intraocular pressure (IOP) measured with a
prototype Pascal dynamic contour tonometer (DCT), to
evaluate the effect of CCT and age on the agreement
between IOP measured with the Pascal DCT and Goldmann
applanation tonometer (GAT), and to compare the interobserver and intraobserver variation of the DCT with the
GAT.
Methods: GAT and DCT IOP measurements were made on
130 eyes of 130 patients and agreement was assessed by
means of Bland-Altman plots. The effect of CCT and age on
GAT/DCT IOP differences was assessed by linear regression
analysis. Interobserver and intraobserver variations for GAT
and DCT were assessed in 100 eyes of 100 patients.
Results: The mean difference (95% limits of agreement)
between GAT and DCT was 20.7 (26.3 to 4.9) mm Hg.
GAT/DCT IOP differences increased with thicker CCT (slope
0.017 mm Hg/mm, 95% CI 0.004 to 0.03, r2 = 0.05,
p = 0.01), and with greater age, slope 0.05 mm Hg/year
(95% CI 0.012 to 0.084, r2 = 0.05, p = 0.01). The intraobserver variability of GAT and DCT was 1.7 mm Hg and
3.2 mm Hg, respectively. The interobserver variability was
(mean difference (95% limits of agreement)) 0.4 (23.5 to
4.2) mm Hg for GAT and 0.2 (24.9 to 5.3) mm Hg for DCT.
Conclusions: GAT is significantly more affected than DCT by
both CCT and subject age. The effect of age suggests an age
related corneal biomechanical change that may induce
measurement error additional to that of CCT. The prototype
DCT has greater measurement variability than the GAT.
T
he current ‘‘gold standard’’ for intraocular pressure (IOP)
measurement is the Goldmann applanation tonometer
(GAT), which makes a static measurement of the force
required to flatten a fixed area of the cornea. In designing the
tonometer, Goldmann recognised that corneal effects, such as
central corneal thickness (CCT) and the precorneal tear film,
would influence the pressure measurements, and this has
been shown in various studies.1–6
A new slit lamp mounted tonometer, the Pascal dynamic
contour tonometer (DCT; Swiss Microtechnology AG, Port,
Switzerland) has been developed to remove the corneal
biomechanical effects from IOP measurement by using a
direct transcorneal method. When the Pascal DCT is placed
on the eye, the cornea takes the contour of the tip so that its
biomechanical effects on IOP measurement are reduced. The
DCT gathers 100 IOP readings per second. Recent studies
have indicated that its IOP measurements are independent of
CCT7 and are unchanged following thinning of the CCT with
laser in situ keratomileusis (LASIK).8 9
www.bjophthalmol.com
The primary purpose of this study was to assess the
agreement between the GAT and Pascal DCT, and to establish
the effects of CCT on IOP measurements made with these
two devices. A secondary aim was to evaluate the intraobserver and interobserver variability of the Pascal DCT.
METHODS
Patients attending their routine appointment in the ocular
hypertension or pigment dispersion clinics at the Glaucoma
Research Unit (Moorfields Eye Hospital, London, UK)
between February and May 2004 were invited to take part
in the study (see table 1 for demographic data). Informed
consent, according to the tenets of the Declaration of
Helsinki, was obtained before examination. The study had
local ethics committee approval.
For the tonometer comparison study, 130 eyes of 130
patients were examined; 71 patients (55%) were on topical
ocular hypotensive therapy (table 2). Two GAT and three DCT
(prototype version 1.2) readings were obtained in a randomised order. Measurements were performed by either one of
two technicians (ETW or JMS) and by a clinician (AK) also in
a randomised order. With GAT measurements, the tonometer
was set at 10 mm Hg before each reading. For DCT, three
readings of ‘‘good’’ quality were saved (score (3 on a scale
up to 5, as recommended by the manufacturer). The
observers were masked to each other’s results. Keratometry
was performed with the IOLMaster (Carl Zeiss Meditec, AG,
Germany) before tonometry, and CCT was measured with the
Altair ultrasonic pachymeter (20 MHz solid tip probe;
Optikron 2000, Rome, Italy) after tonometry. The sample
size chosen allows a study power of 90% (p,0.05) to detect a
correlation of r = 0.3 between CCT and IOP.
For the intraobserver and interobserver variability study,
100 eyes (49 left eyes) of 100 (45 female) patients were
examined. The mean patient age was 60 years (range 26–83;
SD 13.2 years). This group was a subset of that used in the
previous study. The order of GAT/DCT and technician/
clinician was randomised with a 5 minute break between
GAT and DCT measurements. Measurements were obtained
as already described, and the agreement between technicians
and clinician was assessed.
Data analysis
Bland-Altman plots10 were used to assess the agreement in
IOP measurements between techniques (DCT measurements by the clinician versus GAT measurements by the
technicians), and between observers for each of the two
techniques. Mean difference and 95% limits of agreement
were calculated. Linear regression analysis was used to
Abbreviations: CCT, central corneal thickness; DCT, dynamic contour
tonometer; GAT, Goldmann applanation tonometer; IOP, intraocular
pressure; LASIK, laser in situ keratomileusis
Effects of corneal thickness and age on GAT and DCT
1573
Mean
Eye (left/number)
Sex (female/number)
Age (years)
CCT (mm)
Keratometry (mm)
Corneal astigmatism (mm)
GAT (mm Hg)
DCT (mm Hg)
60
52
61
556
7.74
0.17
19
19
SD
13.3
36.9
0.21
0.13
4.6
4.0
Difference [GAT – DCT] (mm Hg)
Table 1 Demographic data of comparison group
(n = 130)
Range
22–83
468–642
7.10–8.44
0–0.88
9–33
11–29
10
8
+1.96 SD
4.9
6
4
2
Mean
–0.7
0
–2
–4
–1.96 SD
–6.3
–6
–8
–10
10
15
20
25
30
35
Average [GAT and DCT] (mm Hg)
where SD is the standard deviation, and observations 1 and 2
are the recorded IOP measurements. The wsSD was only
calculated if the magnitude of SD was unrelated to that of
mean IOP readings.
To establish the effect of the choice of reading, repeatability
was calculated for DCT readings 1 and 2, and 2 and 3. The
repeatability of GAT and DCT measurements was calculated
for the clinician and technicians.
To assess the effect of DCT reading quality on repeatability,
the association between the SD of DCT IOP measurements
and average reading quality was determined.
All statistical analyses were performed using Medcalc
Version 7.4.2.0 (Medcalc Software, Mariakerke, Belgium).
Figure 1 Bland-Altman plot of GAT/DCT IOP differences against GAT/
DCT mean.
Difference [GAT – DCT] (mm Hg)
determine the associations between GAT/DCT differences,
and CCT and age.
Repeatability (intraobserver variability) of IOP measurements with GAT and DCT was calculated as 2.77 times the
within subject standard deviation (wsSD)11:
10
8
6
4
2
0
–2
–4
–6
–8
–10
450
500
550
600
650
CCT (µm)
Figure 2 Relation between CCT and GAT/DCT IOP differences.
RESULTS
Difference [GAT – DCT] (mm Hg)
IOP measurements satisfying the quality criteria were
obtained for all patients. Tables 1 and 2 summarise the
demographic data of the study group.
Agreement between GAT and DCT measurements
The average of two GAT readings was compared with that of
DCT readings 2 and 3 (see ‘‘Intraobserver and interobserver
variability,’’ below). The mean difference (95% limits of
agreement) between GAT and DCT was 20.7 (26.3 to 4.9)
mm Hg, and no relation between GAT/DCT differences and
average was found (fig 1).
Effect of CCT on GAT and DCT IOP measurements
There was a relation between GAT/DCT IOP differences and
CCT (slope 0.017, 95% CI 0.004 to 0.03, r2 = 0.05, p = 0.01)
(fig 2). Analysing treated and untreated eyes separately
showed no relation between GAT/DCT IOP differences and
Table 2
10
8
6
4
2
0
–2
–4
–6
–8
–10
20
30
40
50
60
70
80
90
Age (years)
Figure 3 Relation between age and GAT/DCT IOP differences.
Status and treatment of ‘‘comparison’’ group (total n = 130, treated eyes n = 71)
Diagnosis
No of eyes
(number
treated)
Prostaglandin
analogue
(number)
b blocker
(number)
a agonist
(number)
CAI
(number)
OHT
POAG
PDS
Glaucoma suspect
29/58
21/21
14/30
7/21
15
17
7
3
12
2
6
3
0
1
1
1
2
1
0
0
OHT, ocular hypertension; POAG, primary open angle glaucoma; PDS, pigment dispersion syndrome.
www.bjophthalmol.com
1574
Kotecha, White, Shewry, et al
CCT in treated eyes (n = 71, CCT slope 0.004, 95% CI 0.012 to
0.020, r2 = 0.003, p = 0.65), but an association in untreated
eyes (n = 59, CCT slope 0.025; 95% CI 0.003 to 0.046,
r2 = 0.083, p = 0.03). Analysis of variance (ANOVA) performed to test whether treatment status had an effect on
either CCT or GAT/DCT differences for the whole dataset
showed no significant effect (F ratio 0.23, p = 0.6 for CCT and
F ratio 1.7, p = 0.2 for GAT/DCT difference).
Effect of age on GAT and DCT IOP measurements
GAT/DCT differences were associated with age (GAT/DCT
difference = 0.05 6 age 2 3.6, slope 95% CI 0.012 to 0.084,
r2 = 0.05, p = 0.01) (fig 3). There was no relation between
GAT/DCT IOP differences and age in treated eyes (n = 71, age
slope 20.031, 95% CI 20.086 to 0.024, r2 = 0.018; p = 0.27),
whereas an association was found in untreated eyes (n = 59,
age slope 0.079, 95% CI 0.029 to 0.130, r2 = 0.14, p = 0.003).
Linear regression analysis showed no significant association
between CCT and age (slope 0.174, 95% CI 0.65 to 20.31,
r2 = 0.004, p = 0.48). Multiple regression analysis demonstrated an association between GAT/DCT differences, and
both CCT and age (GAT/DCT difference = 0.045 6age + 0.016
6 CCT 2 12.5, r2 = 0.09, p = 0.01 for both age and CCT).
Intraobserver and interobserver variability
With both techniques, IOP reading SDs were independent of
the mean IOP. The repeatability of IOP measurements for
each observer are summarised in table 3. The difference
between two measurements on the same subject would be
less than the value for repeatability in 95% of pairs of
observations.
The repeatability of DCT measurements improved when
the first reading was discarded. For this reason, the average
of readings 2 and 3 was used for the interobserver variability
study. The mean difference (95% limits of agreement) in
average GAT readings between clinician and technician was
0.4 (23.5 to 4.2) mm Hg. The mean difference (95% limits of
agreement) in average DCT measurements between clinician
and technician was 0.2 (24.9 to 5.3) mm Hg. The association
between DCT recording quality and DCT measured IOP
variability approached, but did not reach, significance
(Pearson r = 0.18; 95% CI 20.02 to 0.37; p = 0.08).
DISCUSSION
In this study, a significant positive association between GAT/
DCT IOP differences and CCT was found. The association of
CCT with GAT/DCT IOP differences was studied as 55% of our
subject population were on topical hypotensive therapy,
which may confound the effect of CCT on IOP measurements.
The association was shown not to be affected by treatment
status, although a separate analysis indicated that GAT/DCT
differences in untreated eyes were more affected by CCT
compared with treated eyes. This finding, however, was not
significant, perhaps because of the small subject numbers.
DCT IOP measurements have been reported to be
independent of CCT,8 9 while studies have shown an increase
in GAT IOP measurements with CCT, with slopes of
0.23 mm Hg12 and 0.27 mm Hg13 increase per 10 mm increase
Table 3 Repeatability of measurement technique
(mm Hg)
in CCT reported in UK populations. Our findings indicate a
slope of 0.17 mm Hg per 10 mm increase in CCT. A steeper
slope would be expected if the DCT was not affected by CCT,
and a study with greater power is required to test the
hypothesis that CCT has a weak effect on DCT measured IOP.
It is also possible that the association between IOP measurement and CCT is affected by a change in corneal biomechanics through the use of topical hypotensive therapy,
especially prostaglandin analogues. Prostaglandin analogues
are known to alter the extracellular matrix of the ciliary
muscle,14 and they may also affect the extracellular matrix of
the cornea, altering its rigidity. Approximately 32% of
patients in our study were on prostaglandin therapy, and it
is possible that this may have affected IOP measurement.
Overall, GAT/DCT differences were positively associated
with age (slope 0.05, r2 = 0.05, p = 0.01) and this relation
increased when untreated eyes alone were assessed (slope
0.08, r2 = 0.14, p = 0.003). In younger eyes, DCT readings
were greater than GAT readings, and this difference reversed
in older eyes. It has been suggested that age related increase
in corneal ‘‘stiffness’’ may induce a further measurement
error with GAT,15 and studies have shown age related changes
that may contribute to this increase.16–18 DCT measurements
may be less affected as the effect of corneal biomechanics on
IOP measurement is reduced with this technique. However,
the association found here requires to be validated in further
studies powered to detect this effect.
In this study, the intraobserver variability of IOP readings
was greater with the DCT compared with the GAT, although
opposite findings were reported in a recent paper by
Kauffmann et al.7 Possible explanations for the difference
include differences in study subjects (patients versus hospital
staff volunteers), order of testing (randomised order versus
GAT followed by DCT), instrument model, and DCT reading
quality. The association of reading quality and measurement
variability approached significance (p = 0.08) in this study,
therefore tighter quality control may result in improved
repeatability. The better repeatability of GAT in this study
may have been because the observers were not masked to
their own results. Although the GAT drum was rotated to
10 mm Hg between readings, an element of digit preference
may have remained. The DCT, on the other hand, provides
objective IOP measurements and the operator cannot directly
manipulate the readings.
The interobserver variability was relatively low for GAT and
DCT measurements, with mean difference (95% confidence
intervals) being 0.4 (23.5 to 4.2) mm Hg for GAT and 0.2
(24.9 to 5.3) mm Hg for DCT. This value is in concordance
with most reports of GAT reproducibility.19
This study shows that the DCT is less affected by CCT than
the GAT. Age accounted for as much intersubject variation in
GAT/DCT differences as did CCT, suggesting a significant
effect of age related corneal stiffening on IOP measurement
with GAT. However, measurement variability was higher
with the prototype DCT compared with the GAT. A nonsignificant trend relating reading quality and measurement
variation suggests that tonometry technique may be a source
of variation.
ACKNOWLEDGEMENTS
Method
Technician
Clinician
The authors thank Carleton Optical for the loan of the Pascal DCT
and Tuan Ho for editing the manuscript. Aachal Kotecha is funded by
the Special Trustees of Moorfields Eye Hospital.
GAT 1 and 2
DCT 1 and 2
DCT 2 and 3
1.6
4.2
3.2
1.7
3.3
2.6
.....................
Authors’ affiliations
A Kotecha, E T White, J M Shewry, D F Garway-Heath, Glaucoma
Research Unit, Moorfields Eye Hospital, London, UK
www.bjophthalmol.com
Effects of corneal thickness and age on GAT and DCT
Correspondence to: D F Garway-Heath, MD FRCOphth, Glaucoma
Research Unit, Moorfields Eye Hospital, 162 City Road, London EC1V
2PD, UK; [email protected]
Accepted for publication 3 August 2005
REFERENCES
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1957;134:221–42.
2 Ehlers N, Bramsen T, Sperling S. Applanation tonometry and central corneal
thickness. Acta Ophthalmol (Copenh) 1975;53:34–43.
3 Whitacre MM, Stein RA, Hassanein K. The effect of corneal thickness on
applanation tonometry. Am J Ophthalmol 1993;115:592–6.
4 Whitacre MM, Stein R. Sources of error with use of Goldmann-type
tonometers. [Review]. Surv Ophthalmol 1993;38:1–30.
5 Gordon MO, Beiser JA, Brandt JD, et al. The Ocular Hypertension Treatment
Study: baseline factors that predict the onset of primary open-angle glaucoma.
Arch Ophthalmol 2002;120:714–20.
6 Feltgen N, Leifert D, Funk J. Correlation between central corneal thickness,
applanation tonometry, and direct intracameral IOP readings. Br J Ophthalmol
2001;85:85–7.
7 Kaufmann C, Bachmann LM, Thiel MA. Comparison of dynamic contour
tonometry with goldmann applanation tonometry. Invest Ophthalmol Vis Sci
2004;45:3118–21.
8 Kaufmann C, Bachmann LM, Thiel MA. Intraocular pressure measurements
using dynamic contour tonometry after laser in situ keratomileusis. Invest
Ophthalmol Vis Sci 2003;44:3790–4.
1575
9 Siganos DS, Papastergiou GI, Moedas C. Assessment of the Pascal dynamic
contour tonometer in monitoring intraocular pressure in unoperated eyes and
eyes after LASIK. J Cataract Refract Surg 2004;30:746–51.
10 Bland JM, Altman DG. Statistical methods for assessing agreement between
two methods of clinical measurement. Lancet 1986;1:307–10.
11 Bland JM, Altman DG. Statistics notes: measurement error. BMJ
1996;313:744.
12 Bhan A, Browning AC, Shah S, et al. Effect of corneal thickness on
intraocular pressure measurements with the pneumotonometer, Goldmann
applanation tonometer, and Tono-Pen. Invest Ophthalmol Vis Sci
2002;43:1389–92.
13 Gunvant P, Baskaran M, Vijaya L, et al. Effect of corneal parameters on
measurements using the pulsatile ocular blood flow tonograph and Goldmann
applanation tonometer. Br J Ophthalmol 2004;88:518–22.
14 Weinreb RN, Toris CB, Gabelt BT, et al. Effects of prostaglandins on the
aqueous humor outflow pathways. Surv Ophthalmol 2002;47(Suppl
1):S53–64.
15 Friedenwald JS. Contribution to the theory and practice of tonometry.
Am J Ophthalmol 1937;20:985–1024.
16 Daxer A, Misof K, Grabner B, et al. Collagen fibrils in the human corneal
stroma: structure and aging. Invest Ophthalmol Vis Sci 1998;39:644–8.
17 Malik NS, Moss SJ, Ahmed N, et al. Ageing of the human corneal stroma:
structural and biochemical changes. Biochim Biophys Acta
1992;1138:222–8.
18 Sherrard ES, Novakovic P, Speedwell L. Age-related changes of the corneal
endothelium and stroma as seen in vivo by specular microscopy. Eye
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19 Tonnu PA, Ho T, Sharma K, et al. A comparison of four methods of tonometry:
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11th European Forum on Quality Improvement in Health Care
26–28 April 2006, Prague, Czech Republic
For further information please go to: www.quality.bmjpg.com
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www.bjophthalmol.com
1576
SCIENTIFIC REPORT
Aniseikonia associated with epiretinal membranes
M Ugarte, T H Williamson
...............................................................................................................................
Br J Ophthalmol 2005;89:1576–1580. doi: 10.1136/bjo.2005.077164
Aims: To determine whether the computerised version of the
new aniseikonia test (NAT) is a valid, reliable method to
measure aniseikonia and establish whether aniseikonia
occurs in patients with epiretinal membranes (ERM) with
preserved good visual acuity.
Methods: With a computerised version of the NAT, horizontal and vertical aniseikonia was measured in 16
individuals (mean 47 (SD 16.46) years) with no ocular
history and 14 patients (mean 67.7 (14.36) years) with ERM.
Test validity was evaluated by inducing aniseikonia with size
lenses. Test reliability was assessed by the test-retest method.
Results: In normal individuals, the mean percentage (SD)
aniseikonia was 20.24% (0.71) horizontal and 0% (0.59)
vertical. Validity studies revealed mean (SD) 0.990 (0.005)
horizontal and 0.991 (0.004) vertical correlation coefficients, 0.985 (0.111) horizontal and 0.989 (0.102) vertical
slope. Repeatability coefficients were 1.04 horizontal and
0.88 vertical. Aniseikonia in patients with ERM ranged from
4% to 14%. Eight patients showed 2% or more size difference
between horizontal and vertical meridians.
Conclusions: The aniseikonia test used in this study can be
considered a simple, fast, valid and reliable method to
measure the difference in image size perceived by each eye.
Aniseikonia does occur in symptomatic patients with ERM.
The effect of ERM on image size is heterogeneous across the
retinal area affected.
E
piretinal membranes (ERM) are non-vascular fibrocellular proliferations on the retinal inner surface. They
develop either spontaneously, in association with ocular
diseases (for example, retinal detachment, chorioretinitis,
retinal vein occlusions), or following surgery (for example,
scleral buckling, cataract extraction, retinal cryopexy). The
5 year Blue Mountain Study found a 5.3% cumulative
incidence of spontaneous ERM above the age of 49.1 ERM
are generally located in the macula and their ability to
contract2 can distort the photoreceptor distribution in the
fovea. This would affect image perception causing an object
to appear larger (macropsia) or smaller (micropsia). Some of
the symptoms in patients with ERM may result from
aniseikonia (Greek: anisos, unequal; eikon, image) or the
perception of the same image as being of different size with
each eye. A few studies have reported aniseikonia in macular
disease such as ERM,3 4 vitreoretinal traction,4 and central
serous retinopathy.5 However, the prevalence of aniseikonia
in ERM is unknown since it is not tested routinely in the
clinic.
Aniseikonia can be measured by two dimensional and
three dimensional methods. The space eikonometer6 depends
on the observed effect of size lenses on a three dimensional
array of cords and rods. It is very accurate but the
information provided is difficult to interpret. The use of the
instrument was discontinued in the 1970s. A simplified test
www.bjophthalmol.com
consisting of graded stereoscopic cards reproducing the space
eikonometer target was developed later on.7 The performance
of this test requires good stereopsis. This is known to be
reduced in patients with aniseikonia, making its results
unreliable. The NAT measures aniseikonia directly, by
presenting a red and a green semicircle to each eye by means
of dissociation with red/green goggles. It is easy and rapid to
perform2 8 and we consider it ideal for clinical use. We used a
computerised version of the NAT after confirming its validity
and reliability to measure aniseikonia in symptomatic
patients with unilateral macular ERM.
MATERIALS AND METHODS
Sixteen volunteers, mean age 47 (SD 16.46), 10 women and
six men, without ocular history and less than 1 dioptre (D)
anisometropia were included in the control group. Fourteen
patients, mean age 67.7 (SD 14.36), five women and nine
men, with ERM were recruited between October 2003 and
December 2004. Inclusion criteria were visual complaints,
less than 1 D anisometropia, logarithmic minimum angle of
resolution (logMAR) visual acuity (VA) 0.5 or better in each
eye, and unilateral macular ERM. The research carried out
followed the tenets of the World Medical Association
Declaration of Helsinki. Subjects underwent ocular examination, refraction, best corrected VA, orthoptic assessment,
metamorphopsia analysis with Amsler chart, threshold
horizontal and vertical aniseikonia measurement, slit lamp
examination, and funduscopy.
The computerised NAT consisted of matched pairs of red/
green semicircles with a white, round fixation target on a
black background. The fixation target was 3 cm in diameter
and the red semicircle 15 cm. The diameter of the green
semicircle varied in 1% steps (from 214% to +14%). Subjects
viewed the monitor from 66 cm with appropriate correction
and red/green goggles. The white target projected an image
1.5˚ around fixation and the red semicircle 7.5˚; 1% variation
in the green semicircle diameter corresponded to 0.15˚
(9 minutes of arc) increases/decreases in retinal image size.
Two series of matched semicircles (horizontal and vertical)
were presented at random. The individual had to identify the
pair in which both semicircles appeared equal in size. The size
difference represented the percentage of aniseikonia.
Threshold aniseikonia was measured by bracketing.
Different pairs were shown reversing about threshold. The
average of three reversals was taken as the threshold.
The precision of our measurements was assessed by
evaluating the test validity and reliability. Validity was
analysed by calculating the agreement between our measurements and the true value obtained by inducing micropsia/
macropsia in one eye of the 16 controls using four size lenses
(magnification (m): +3%, +5%, +7% and +9%, spectacle
magnification (M), M = 1 + (m/100): 1.03, 1.05, 1.07, and
1.09, respectively). By placing the concave or convex lens
surface facing the eye the image size was increased or
Abbreviations: ERM, epiretinal membranes; NAT, new aniseikonia test;
VA, visual acuity
Aniseikonia in epiretinal membranes
1577
Table 1 Profile and clinical characteristics of subjects in the control group
Control
No
Sex
Age
(years)
1
2
3
4
5
6
7
8
9
F
M
F
M
F
F
M
M
F
20
26
27
31
37
40
43
47
50
10
F
51
11
F
53
12
M
55
13
14
F
M
59
66
15
F
69
16
F
78
Mean (SD)
47 (16.46)
Right eye
Left eye
Refraction (D)
VA
Refraction (D)
VA
Plano
Plano
Plano
Plano
20.50 sphere
22.75/20.56056
Plano
+0.75/20.756176
Plano
Add +2.00
Plano
Add +2.00
+1.50/20.506094
Add +2.00
Plano
Add +2.00
20.75/21.256067
Plano
Add +3.00
Plano
Add +2.75
+0.50/21.006049
Add +3.00
20.1
20.2
20.1
20.2
20.2
20.1
20.1
20.1
20.1
Plano
Plano
Plano
Plano
20.50/20.256025
23.25/20.756014
20.50 sphere
+1.00/20.256121
Plano
Add +1.75
Plano
Add +2.00
+1.50 sphere
Add +2.00
Plano
Add +2.00
20.75
Plano
Add +3.00
Plano
Add +2.50
20.50/22.006074
Add +3.00
20.1
20.2
20.1
20.2
20.2
0
0.1
20.1
20.1
20.1
20.1
20.1
20.1
+0.1
20.1
20.1
20.1 (0.07)
20.1
20.2
20.1
20.1
+0.1
0
20.1
20.1 (0.10)
VA, logMAR visual acuity.
reduced, respectively. Correlation coefficient, the slope of the
best fit linear regression, and y axis intercept were calculated.
Reliability was examined by measuring agreement between
repeated measurements9 recorded 2 weeks apart on 10
control individuals (20–69 years old). The magnification
used in reliability studies was 29%, 25%, 0%, +5%, and
+9%. The coefficient of repeatability10 was calculated. The
unpaired t test was used to compare aniseikonia results in
controls and patients.
RESULTS
A summary of the ophthalmic examination of the 16 controls
and 14 patients with unilateral ERM is shown in tables 1 and
2. The logMAR VA (mean (SD)) was 20.1 (0.1) in the
controls, 0.18 (0.25) in the eye with ERM in our patients, and
0 (0.13) in the unaffected eye. All patients with ERMs had
visual symptoms.
The NAT validity was assessed by comparing our measurements with the true value obtained by inducing increments/
10
A
8
6
4
2
0
–2
–4
–6
–8
–10
–9
–7
–5
–3
0
3
5
7
Induced change in image size
using afocal size lenses (%)
Figure 1 Horizontal (A) and vertical
(B) aniseikonia measured in 16 normal
subjects after inducing changes in the
green semicircle size (magnification
(m): 29%, 27%, 25%, 23%, 3%, 5%,
7%, and 9%) with size lenses. The
straight line represents the ideal test, 1
correlation coefficient, 1 slope and Yaxis intercept at 0.
(Mean (SD))
Correlation coefficient 0.991 (0.004)
Slope: 0.989 (0.102)
Y axis intercept: –0.06 (0.339)
Threshold vertical aniseikonia (%)
Threshold horizontal aniseikonia (%)
(Mean (SD))
Correlation coefficient 0.990 (0.005)
Slope: 0.985 (0.111)
Y axis intercept: –0.08 (0.468)
reductions (29%, 27%, 25%, 23%, 0%, +3%, +5%, +7%, and
+9%) in the green semicircle using size lenses (fig 1). This
revealed (mean (SD)): (1) correlation coefficient of 0.990
(0.005) for horizontal and 0.991 (0.004) vertical aniseikonia,
confirming the agreement between our measurements and
its true value; (2) slope of 0.985 (0.111) horizontal and 0.989
(0.102) vertical aniseikonia, suggesting there is a small
underestimation, (3) y axis intercept of 20.08 (0.468)
horizontal and 20.06 (0.339) vertical. The test-retest method
revealed coefficient of repeatability, 1.04 horizontal and 0.88
vertical (table 3). In both meridians the differences between
readings 1 and 2 were within 2 SD of the mean. The results
from this test can, therefore, be considered reproducible and
reliable.
The range of horizontal and vertical threshold aniseikonia
in patients with unilateral ERM was 4–14% (fig 2). Eleven
patients perceived the image of the affected eye as larger and
three as smaller than in the fellow eye. In eight patients,
there was 2% or more difference between the amount of
9
10
B
8
6
4
2
0
–2
–4
–6
–8
–10
–9
–7
–5
–3
0
3
5
7
9
Induced change in image size
using afocal size lenses (%)
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www.bjophthalmol.com
M
M
F
F
M
F
M
F
M
F
M
M
M
M
3
4
5
6
7
8
9
10
11
12
13
14
Mean
(SD )
VA, logMAR visual acuity.
Sex
1
2
54
67.7 (14.36)
36
72
45
71
84
65
61
79
83
78
73
79
67
Age (years)
Poor stereopsis
Rivalry
Asthenopia, poor stereopsis, reading
difficulties
Binocular diplopia, difficulty reading
Rivalry, occipital headache
Blurred vision
Difficulty judging distances
Blurred vision
Photophobia
Blurred vision
Macropsia
Blurred vision
Rivalry
Objects looked further away, diplopia
watching TV
Blurred vision, difficulty reading
Symptoms
Buckle and cryopexy in
affected eye
Nil of note
Cystoid macular oedema postphaco, affected eye
Nil of note
Bilateral pseudophakia
Bilateral pseudophakia
Nil of note
Nil of note
Nil of note
Bilateral pseudophakia
Nil of note
Pseudophakia in unaffected eye
Bilateral pseudophakia
Nil of note
Ocular history
Profile and clinical characteristics of patients with unilateral ERM
Case
No
Table 2
23.00/20.756089
Plano/21.006083
Add +2.00
Plano
+1.75/22.256076
+2.00 sphere
Add +1.75
21.75/21.256076
Add +2.5
22.50 D sphere
Add +1.75
21.75/21.506105
Add +2.50
+5.00 D/20.506100
Add +1.25
Plano/23.256023
Add +2.50
+0.50/21.006155
20.50/20.506069
Add +1.50
+0.25/20.506026
Add +2.00 D
Plano/21.256096
Refraction (D)
Affected eye
VA
20.1
0.18 (0.25)
20.1
+0.1
20.2
+0.1
+0.5
+0.3
+0.3
+0.5
0
+0.1
+0.5
0
+0.5
23.25/20.506106
+0.75/22.006077
Add +2.00
Plano
Plano/216026
+2.25 sphere
Add +1.75
21.50 D sphere
Add +2.75 D
21.50 D sphere
add +2.00
Plano/20.506026
Add +2.50
+5.50 D/21.006100
Add +1.50
+0.50/23.006167
Add +2.50
Plano/21.506111
Plano/21.256180
Add +1.50
Plano/21.006175
Add +2.00 D
Plano/20.506070
Refraction (D)
Unaffected eye
VA
20.1
0 (0.13)
20.1
20.1
20.1
0
+0.3
0
-0.1
+0.2
0
0
+0.1
20.1
+0.1
1578
Ugarte, Williamson
Aniseikonia in epiretinal membranes
1579
Table 3 Assessment of agreement between repeated measurements of horizontal (A)
and vertical (B) threshold aniseikonia
1st reading
2nd reading
Difference
between
readings
Mean (SD)
Mean (SD)
Mean (SD)
Coefficient of
repeatability
1 (0.53)
0.3 (0.42)
0.5 (0.53)
0.5 (0.41)
0.6 (0.52)
0.58 (0.519)
1.05
0.84
1.05
0.82
1.03
1.03 (0.119)
0.7 (0.53)
0.3 (0.42)
0.45 (0.37)
0.5 (0.41)
0.8 (0.42)
0.57 (0.440)
1.06
0.88
0.74
0.82
0.84
0.84 (0.119)
(A) Induced horizontal aniseikonia with size lenses
29%
28.4 (1.10)
27.8 (0.54)
25%
24.5 (0.47)
24.7 (0.42)
0%
20.2 (0.59)
20.05 (0.55)
5%
4.0 (0.71)
4.3 (0.63)
9%
8.0 (1.07)
8.1 (0.96)
Mean (SD)
(B) Induced vertical aniseikonia with size lenses
29%
27.8 (1.14)
27.9 (0.85)
25%
24.6 (0.83)
24.7 (0.42)
0%
0.1 (0.62)
0.3 (0.43)
5%
4.7 (1.13)
4.5 (1.10)
9%
8.1 (0.70)
7.8 (0.48)
Mean (SD)
N, 10.
Two readings were taken on 10 subjects in the control group (Nos 1, 3, 5, 7, 9, 10, 11, 12, 13, and 15) with a
2 week interval. Induced aniseikonia with size lenses, magnification (m): 29%, 25%, 0%, 5%, 9%.
aniseikonia detected in the vertical and horizontal meridians.
The unpaired t test comparing horizontal and vertical
aniseikonia in controls and patients with ERMs revealed
p = 0.0419 and p = 0.0024, respectively.
DISCUSSION
ERM can alter retinal morphology11 and function.11–14 The
compression, separation, and/or tilt of photoreceptors can
result in the perception of an image as being larger
(macropsia)/smaller (micropsia) and simultaneous stimulation of corresponding retinal regions by uncorrelated images.
If one of them is not suppressed the inability to integrate
information from each eye can cause an extremely confusing
experience. We propose that some of the symptoms in ERM
may be due to aniseikonia, a condition in which each eye
perceives the same image as being of different size.
It is essential to have clinical methods to measure
aniseikonia accurately. In this study, we used a computerised
14
Horizontal
Vertical
12
10
Threshold aniseikonia (%)
8
6
4
2
0
–2
–4
–6
–8
–10
–12
–14
1
2
3
4
5
6
7
8
9
10 11 12 13 14
Patients with unilateral macular epiretinal membranes
Figure 2 Horizontal and vertical aniseikonia (%) measured in 14
patients with unilateral macular ERMs. In 11 patients the image
perceived by the affected eye was larger than in the fellow eye
(macropsia) and in the remaining three, the eye with the ERM detected
images as smaller (micropsia). Eight patients demonstrated 2% or more
difference in image size between horizontal and vertical meridia.
version of the NAT after confirming its validity and
repeatibility. Validity, the extent to which the test measures
what it purports to measure, was evaluated by inducing
aniseikonia with size lenses in 16 controls and comparing our
measurements with the true value. Correlation coefficients
(mean (SD)) were 0.990 (0.005) horizontal and 0.991 (0.004)
vertical, in good accordance with studies by others.8 15
Horizontal and vertical slope (mean (SD)) was 0.985
(0.111) and 0.989 (0.102), respectively, suggesting a small
underestimation. In this context, NAT comparisons with the
space eikonometer16 and phase difference haploscope17 have
also shown an underestimation. This may be due to a greater
sensory fusion range when dissociating with red/green
anaglyphs. The Y axis intercepts (representing ‘‘inherent’’
aniseikonia) (mean (SD)) were 20.08 (0.468) horizontal and
20.06 (0.339) vertical. Repeatability coefficients were 1.04
horizontal and 0.88 vertical; 1% difference in target size
corresponds to 0.15˚ or 9 minutes of arc variation in the
retinal image size projected 7.5˚ around fixation. The
logarithmic VA (mean (SD)) in the centre of the fovea of
our controls was 20.1 (0.01) (equivalent to 0.75 minutes of
arc minimal angle of resolution). Given that the VA reduces
as a function of eccentricity18; 7.5˚ away from the fovea the
VA would be about 10–20% of the maximum (5–10 minutes
of arc); therefore, repeatability coefficients of 1.04 and 0.88
can be considered acceptable.
Threshold aniseikonia (mean (SD)) in controls was
20.24% (0.71%) horizontal and 0% (0.59%) vertical. Several
studies have advocated different values of ‘‘normal’’ aniseikonia based on binocular fusion tolerance thresholds (1.5%,19
5–8%,20 and 18%21). This great variation could depend on the
type of aniseikonia investigated (for example, axial or
refractive anisometropia) or the method used (target size,
distance from target). It would, therefore, be advisable to
standardise the methodology in order to be able to compare
results. In symptomatic patients with unilateral ERMs
(n = 14) aniseikonia ranged from 4% to 14%, similar to other
studies.3 Because of the heterogeneous shape of epiretinal
proliferation the effect on retinal morphology would be
expected to vary across the area affected. This was confirmed
by a more than 2% difference in horizontal and vertical
aniseikonia in eight patients. This heterogeneity implies that
the compensatory mechanisms of the visual system or
optical correction with iseikonic lenses would be ineffective.
In our own experience, surgical removal of ERM improves
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1580
Ugarte, Williamson
micropsia/macropsia. Further studies are currently being carried
out by us to determine the effect of surgical intervention on
image size, aniseikonia, and patients’ symptoms.
The results presented here support the idea that the
computerised version of the NAT is a simple, fast, reliable
method to measure aniseikonia clinically. Aniseikonia occurs
in symptomatic patients with macular ERMs with good VA.
The change in image size caused by the ERM is heterogeneous across the retinal area being distorted. This can
result in intolerable symptoms when working with both eyes
simultaneously.
10
ACKNOWLEDGEMENT
11
The authors thank Mr Ken Clarke for his invaluable help with the
computer program of the MAT.
4
5
6
7
8
9
12
.....................
Authors’ affiliations
M Ugarte, T H Williamson, Department of Ophthalmology, Queen
Mary’s Hospital, Sidcup, Kent DA14 6LT and St Thomas’s Hospital,
London SE1 7EH, UK
Correspondence to: Marta Ugarte, Department of Ophthalmology, St
Thomas’s Hospital, London SE1 7EH, UK; [email protected]
Accepted for publication 28 July 2005
REFERENCES
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ultrastructural characteristics and clinicopathologic correlation.
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3 Enoch JM, Schwartz A, Chang D, et al. Aniseikonia, metamorphopsia and
perceived entoptic pattern: some effects of a macular epiretinal membrane,
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macular electroretinogram in eyes with epiretinal membrane and macular
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Mosby Year Book, 1992:531–47.
Crone RA, Leuridan OM. Tolerance for aniseikonia. I. Diplopia thresholds in
the vertical and horizontal meridians of the visual field. Albrecht Von Graefes
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1581
SCIENTIFIC REPORT
Comparison of optical coherence tomography models
OCT1 and Stratus OCT for macular retinal thickness
measurement
V Pierre-Kahn, R Tadayoni, B Haouchine, P Massin, A Gaudric
...............................................................................................................................
Br J Ophthalmol 2005;89:1581–1585. doi: 10.1136/bjo.2005.069815
Aims: To compare the values measured for retinal macular
thickness with the first and last generations of the optical
coherence tomograph (OCT1 and Stratus OCT, Zeiss,
Humphrey Division).
Methods: This was a cohort study. 59 eyes were examined:
17 had a normal macula and 42 had a diabetic macular
oedema. In each eye, mean retinal thickness (RT) was
measured automatically in the nine macular Early Treatment
Diabetic Retinopathy Study areas and at the foveal centre,
using OCT1 and Stratus OCT. The paired mean RT values for
each area and the type and proportion of artefacts were
compared.
Results: Of the 590 automatic measurements, 505 had no
artefact, either with OCT1 or Stratus OCT. The mean
difference between the OCT1 and Stratus OCT measurements was 25 (SD 26.2) mm (p,0.0001). With Stratus OCT,
RT values were significantly higher, by 8.1% (7.8%), than
with OCT1. Artefacts were only observed in cases of diabetic
macular oedema and were significantly more frequent with
OCT1 than Stratus OCT (10.5% versus 4.4, p,0.0001).
Conclusion: The macular retinal thickness values measured
with Stratus OCT were significantly higher than those
measured with OCT1. Stratus OCT has the advantage of
producing fewer artefacts than OCT1 in pathological cases.
T
he introduction of optical coherence tomography (OCT)
in clinical practice in 1996 made it possible to measure
retinal thickness (RT) routinely. The first version, OCT1
(Carl Zeiss Meditec, Humphrey Division, Dublin CA. USA),
had an axial resolution of about 15 mm. In 2002, the new
Stratus OCT came into use and reduced axial resolution by up
to 7 mm. The repeatability and reliability of RT measurements
with OCT1 have been demonstrated in several studies.1 2 It
was therefore of interest to test the new Stratus OCT
instrument, to see if it gave the same retinal thickness values
as the OCT1, and whether artefacts were less frequent with
Stratus OCT than with OCT1. We then compared the macular
retinal thickness values obtained with OCT1 and Stratus OCT
for normal and diabetic patients.
PATIENTS AND METHODS
Patients
Retinal macular thickness was measured in 59 eyes of 37
patients using OCT1 and Stratus OCT. Seventeen eyes of 13
healthy emmetropic volunteers (six men and seven women,
mean age 37.5 (SD 8.6) years) as well as 42 eyes of 24
diabetic patients with untreated focal or diffuse macular
oedema (15 men and nine women, mean age 56.5 (11.2)
years) were included in the study. The volunteers gave
informed consent to participate. For diabetic patients, OCT
examination was part of the usual assessment of the fundus.
Diabetic patients with the following characteristics were
excluded: opaque media, treated macular oedema, or loss of
central fixation.
Optical coherence tomography
OCT1 and Stratus OCT were performed on each eye on the
same day and by the same examiners (BH or VPK). Radial
line scan protocol was used for both OCT instruments to map
the macular areas. With OCT1, RT was measured automatically using the latest software version (A6.2, Carl Zeiss
Meditec, Dublin CA, USA), which enabled us to obtain, in
1 second, cross sectional tomographic images 6 mm long
(radial B-scan), comprising 100 axial measurements (Ascans). Axial resolution was about 13 mm. The retinal map
was calculated by integrating the results of the six radial
scans (fig 1A). Mean macular RT was displayed for the nine
Early Treatment Diabetic Retinopathy Study (ETDRS)-type
areas,3 including a central 1000 mm disc and inner and outer
rings of 3000 mm and 6000 mm, respectively. Each ring was
divided into four quadrants (fig 1B). Average RT was
calculated automatically for each of the nine quadrants
(A1–A9). Central foveal RT was also calculated at the point of
intersection of the six radial lines. A paired comparison was
performed of the two sets of 590 values.
With Stratus OCT, the latest software version (2.0, Carl
Zeiss Meditec, Dublin, CA, USA) was used for RT measurements. Tomographic images 6 mm long were obtained in
1.2 seconds and integrated 512 A-scans with consequent
higher longitudinal resolutions than those of OCT1. Axial
resolution was also better, at about 7 mm. Mean macular RT
was displayed on the same radial spoke pattern grid as with
OCT1 (fig 2).
Artefacts
The proportion of artefacts on radial B-scans was also
compared for OCT 1 and Stratus OCT. Artefacts were defined
as the discordance between the automatically detected
anterior and posterior retinal boundaries and the boundaries
detected by the examiner. Such artefacts can either increase
or reduce the RT values measured manually in the quadrants
concerned (fig 3).
Data analysis
Comparison of OCT1 and Stratus OCT macular RT estimations was based on quadrant by quadrant paired comparison.
The coefficient of correlation and the mean difference
between RT measurements with OCT1 and Stratus OCT were
calculated. Results are expressed as means (SD). Fisher’s
Abbreviations: IS, inner segment; OCT, optical coherence tomography;
OS, outer segment; RPE, retinal pigment epithelium; RT, retinal thickness
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A
Pierre-Kahn, Tadayoni, Haouchine, et al
B
A6
A2
A9
A5 A1
A3
A7
Figure 1 Retinal mapping protocol.
(A) Radial spoke pattern of six 6 mm
long scans centred on the subject’s
fixation point in a healthy left eye, (B)
ETDRS-type areas of the macula in a left
eye. A1, central zone 1000 mm in
diameter; A2–A5, superior, temporal,
inferior and nasal areas of a disc
3000 mm in diameter; and A6–A9 of a
disc 6000 mm in diameter.
A4
A8
0
1
2 mm
Figure 2 (Top) 6 mm long vertical
scan line on (left) OCT1 and (right)
Stratus OCT, with the automatically
positioned anterior and posterior retinal
boundaries (white lines). (Bottom) Right
eye of a diabetic patient with macular
oedema, as seen with (left) OCT1 and
(right) Stratus OCT macular mapping
with colour coded map and numerical
RT.
Figure 3 Stratus OCT scan line 6 mm
long centred on the fovea, at an angle
of 30˚ from the horizontal line, in a
diabetic right eye with focal temporal
macular oedema. Two intraretinal
hyper-reflective exudates (white arrows)
generate (left) a lower automatically
positioned inner retinal boundary with
(right) two subsequent artefacts (black
arrows) in the retinal thickness mapping
(quadrants A7 and A9).
exact test was used for the correlation studies, and paired
Student t test, for the comparative studies. Statistical analysis
was performed with Apple iMac StatView software (SAS
Institute Inc, Version 5.0). All analyses were performed on
the two complete sets of 590 values and also on the sets of
values that remained after the exclusion of quadrants with
artefactual measurements. The type and proportion of the
artefacts obtained with OCT1 and Stratus OCT were also
compared (x2 test).
Retinal outer boundary reference line
Careful examination of the outer boundary reference line,
automatically aligned by the mapping software of OCT,
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showed that, in Stratus OCT, it was constantly located on the
inner segment/outer segment (IS/OS) photoreceptor line
rather than on the retinal pigment epithelium (RPE) line.
This misalignment was not noted in OCT1, which did not
distinguish between these two lines because of its lower axial
resolution. To evaluate and quantify this error more
accurately, we measured the mean distance between the IS/
OS and the RPE lines, at the foveal centre of healthy maculas
mapped with Stratus OCT, using the ‘‘Scan Profile’’ protocol
of the software. The outer boundary of the retina was
characterised by two peaks, the outermost being the signal of
the RPE. Callipers easily allowed measurement of the
distance between the two peaks. This measurement was
Comparison of OCT1 and Stratus OCT for macular RT measurement
1583
Figure 4 Examples of artefacts
resulting in lower RT values due to (A)
the presence of hard exudates with
OCT1, (B) erroneous analyses of a
cystoid macular oedema with both
OCT1 and (C) Stratus OCT. Examples
of artefacts resulting in increased RT
values due to misinterpretation of a
detached posterior hyaloid as the
internal limiting membrane by both (D)
OCT1, and (E) Stratus OCT.
repeated at the five contiguous pixels on each side. The
average of the 11 measurements performed was considered
as the distance between the two lines at the foveal centre.
RESULTS
Artefacts
Of the two sets of 590 paired values, 85 exhibited artefacts
with either OCT1 or Stratus OCT or both. With OCT1,
artefacts were present in 19 eyes, and with Stratus OCT, in
nine. They were only observed in eyes with diabetic macular
oedema (DMO). Of the two sets of 420 values obtained with
OCT1 and Stratus OCT in the 42 eyes with DMO, 62 (10.5%)
and 26 (4.4%), respectively, exhibited artefacts (p,0.0001).
Artefacts resulting in lower RT values than with manual
measurement were observed in 44/62 quadrants (71%) in the
OCT1 scans, and in 16/26 quadrants (61.5%) in the Stratus
OCT scans. These underestimations were mainly the result of
the presence of intraretinal hard exudates, or by erroneous
analysis of cystoid macular oedema (fig 4A, B, and C). There
were a few overestimated artefacts, the result of erroneous
positioning of the inner boundary of the retina. The detached
posterior hyaloid was, for example, misinterpreted as the
internal limiting membrane (figu 4D and E).
Retinal thickness values
On the basis of the two complete series of 590 values—that is,
including the artefacts, the mean RT in the 6 mm diameter
area was 322.3 (126.3) mm with OCT1 and 358.9 (156) mm
with Stratus OCT. There was good agreement between the
two instruments (r = 0.899, p,0.0001). However, the RT
values obtained with Stratus OCT were always higher than
those obtained with OCT1, by 11.6% (23.1%) (mean
difference: 36.6 (70.1) mm p,0.0001); the greater the retinal
thickness, the greater the difference.
On the basis of the two series of 505 values—that is,
excluding the artefacts, the mean RT in the 6 mm diameter
area, was 308.8 (116.8) mm with OCT1 and 333.8 (126.7) mm
with Stratus OCT. The agreement between the OCT1 and
Stratus OCT measurements was better than when the
artefactual quadrants were included (r = 0.98, p,0.0001).
However, these measurements still remained unequal
(fig 5A). The mean difference between Stratus OCT and
Table 1 Distribution of (1) the retinal thickness (RT) measured with OCT1 and with Stratus OCT, (2) the difference between the
RT values measured with Stratus OCT and OCT1, and (3) the RT ratio (Stratus OCT – OCT1)/OCT1 based on the 505 nonartefactual RT measurements for 42 eyes with diabetic macular oedema and 17 eyes with healthy maculae
Number of values
Mean
SD
Error of mean
Minima
Maxima
OCT 1
(mm)
Stratus OCT
(mm)
Stratus OCT 2 OCT 1 (mm)
(Stratus OCT 2 OCT 1)/OCT 1
505
308.8
116.8
5.2
124
761
505
333.8
126.7
5.6
126
824
505
25.0
26.2
1.2
268
+216
505
0.08
0.078
0.003
20.192
+0.557
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900
Pierre-Kahn, Tadayoni, Haouchine, et al
A
800
r = 0.98
OCT 3 (µm)
700
600
500
r=1
400
300
200
100
100
200
300
400
500
600
700
800
600
700
800
OCT 1 (µm)
0.6
B
(OCT 3 – OCT 1)/OCT 1
0.5
0.4
0.3
0.2
0.1
0.0
–0.1
–0.2
–0.3
100
200
300
400
500
OCT 1 (µm)
Figure 5 Non-artefactual measurements of macular retinal thickness
obtained with OCT1 compared to those obtained with Stratus OCT. (A)
Graph showing the correlation between RT measurements with OCT1
and Stratus OCT (solid line: observed correlation r = 0.98; broken line:
equal correlation r = 1). (B) The values for macular retinal thickness
measured with Stratus OCT were higher than those measured with
OCT1. The excess ranged from 7.5% to 8.7% (mean 8.1) in 95% of the
measures.
OCT1 RT values was 25 (26.2) mm (p,0.0001). The RT
measurements obtained with Stratus OCT always exceeded
those obtained with OCT1, by 8.1% (7.8%) (fig 5B and
table 1).
In the 17 healthy eyes, the mean RT in the central area
1 mm in diameter, according to the mapping software, was
178.4 (11.5) mm with OCT1, and 191.4 (17.6) mm with Stratus
OCT. These measurements were also correlated (r = 0.76,
p = 0.0002). In this area, mean difference between Stratus
OCT and OCT1 RT measurements was 13 (11.8) mm,
(p = 0.0004).
Distance between the two outer hyper-reflective lines
on stratus OCT
The distance between these two outer lines, measured at the
foveal centre on Stratus OCT A-Scan as the mean value of 11
contiguous measurements, was 46.6 (9) mm.
DISCUSSION
Axial resolution is twice as good with Stratus OCT as with
OCT1 (about 7 mm with 1000 pixels for each A-scan versus
13 mm with 500 pixels for OCT1). Longitudinal resolution is
also better with Stratus OCT (512 axial profiles per
tomographic line, instead of 100 with OCT1). In the present
study, RT was therefore measured in each 6 mm diameter
area at a total of 600 points using OCT1 and 3072 points
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using Stratus OCT. In both OCT instruments, computer
image processors measure RT from retinal tomograms as the
distance between the highly reflective inner and outer
boundaries of the retina, which are located by a thresholding
algorithm.5
The mapping software of OCT1 has been shown to have
good reproducibility for RT measurements in healthy
subjects, and in patients with DMO.1 2 It also appeared to
be a sensitive tool for detecting early retinal thickening in
diabetic patients.6 7 We therefore compared the RT measurements obtained with OCT1 (software A6.2) and Stratus OCT
(software 2.0) and showed that both give measurements that
are highly correlated but nevertheless slightly different. We
do not know which OCT gives RT values closest to the in vivo
reality, and can only compare the macular RT measured with
both instruments. Stratus OCT tended to overestimate the RT
measured with OCT1, by 25 (26.2) mm (p,0.0001). As stated
in Results, the greater the RT, the greater the difference
between OCT1 and Stratus OCT values. The equation:
RT(Stratus
OCT) = 1.064
RT(OCT1)
+
5.43
was
extrapolated from the regression graph ‘‘ RT(Stratus
OCT) = f(RT(OCT1))’’ (fig 5A) obtained with StatView software. The mean percentage by which Stratus OCT exceeded
OCT1 was 8.1% (7.8%) (fig 5B). Despite the wide range
observed for this ratio (–19% to +56%), the standard error of
the mean was only 0.3%—that is, in 95% of the RT measures,
Stratus OCT exceeded OCT1 by 7.5% to 8.7%. This means that
by adding 8% to our OCT1 values, we can estimate the Stratus
OCT values with an error of less than 0.6% in 95% of cases.
Conversely, by reducing the Stratus OCT values by 7%, the
OCT1 values can be estimated with an error of less than 0.6%
in 95% of cases.
Because of its better axial resolution, Stratus OCT displays
two different outer hyper-reflective lines. The innermost is
generated by the IS/OS junction, as shown by Drexler et al4
with an ultra high resolution OCT prototype, and the
outermost, by the RPE. Stratus OCT takes the IS/OS line as
the outer boundary of the retina, thus underestimating RT.
The difference between the RT measurements obtained with
the two OCT instruments would have been greater if the
outer boundary line of the Stratus OCT had been correctly
located at the RPE level.
The significantly lower artefact rate with Stratus OCT
than with OCT1 for DMO (4.4% v 10.5%) may be the result
of the better definition of A-scans with the former
instrument. Artefact locations were also different. Most of
the artefacts encountered in OCT1 were not seen in Stratus
OCT and vice versa. Maculopathies with hard exudates
are the most likely to generate artefacts. However, even in
these eyes, artefacts were fewer with Stratus OCT than with
OCT 1.
In conclusion, retinal thickness measured with Stratus
OCT (version 2.0) was significantly greater than with OCT1
(version A6.2). Therefore, extrapolation of retinal thickness
measurements from OCT1 to Stratus OCT should take into
account a correcting value. This value would be even higher,
by up to 46 mm, if the outer reference line for macular
thickness measurement were correctly located on the RPE
and not on the IS/OS line. Although Stratus OCT has the
advantage of being more accurate and producing fewer
artefacts than OCT1 in pathological cases, the retinal
thickness values provided by its mapping software should
be carefully reappraised.
.....................
Authors’ affiliations
V Pierre-Kahn, R Tadayoni, B Haouchine, P Massin, A Gaudric,
Department of Ophthalmology, Hospital Lariboisiere, Assistance
Publique-Hôpitaux de Paris, Université Paris 7, Paris, France
Comparison of OCT1 and Stratus OCT for macular RT measurement
Correspondence to: Ramin Tadayoni, MD, Service d’Ophtalmologie,
Hôpital Lariboisière 2, rue Ambroise Paré, 75475 Paris Cedex 10,
France; [email protected]
Accepted for publication 1 August 2005
REFERENCES
1 Massin P, Vicaut E, Haouchine B, et al. Reproducibility of retinal thickness
measurements in healthy and diabetic subjects using optical coherence
tomography. Arch Ophthalmol 2001;119:1135–42.
2 Baumann M, Gentile R, Liebmann J, et al. Reproducibility of retinal thickness
measurements in normal eyes using optical coherence tomography.
Ophthalmic Surg Lasers 1998;29:280–85.
1585
3 Early Treatment Diabetic Retinopathy Study Research Group.
Photocoagulation for diabetic macular edema. ETDRS report number 1. Arch
Ophthalmol 1985;103:1796–806.
4 Drexler W, Sattmann H, Hermann B, et al. Enhanced visualization of macular
pathology with the use of ultrahigh-resolution optical coherence tomography.
Arch Ophthalmol 2003;121:695–706.
5 Hee MR, Puliafito CA, Duker JS, et al. Topography of diabetic
macular edema with optical coherence tomography. Ophthalmology
1998;105:360–70.
6 Massin, Erginay A, Haouchine B, et al. Retinal thickness in healthy and
diabetic subjects measured using optical coherence tomography mapping
software. Eur J Ophthalmol 2002;12:102–8.
7 Otani T, Kishi Sh, Maruyama Y. Patterns of diabetic macular
edema with optical coherence tomography. Am J Ophthalmol
1999;127:688–93.
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1586
SCIENTIFIC REPORT
Development of Microelectromechanical Systems (MEMS)
forceps for intraocular surgery
R B Bhisitkul, C G Keller
...............................................................................................................................
Br J Ophthalmol 2005;89:1586–1588. doi: 10.1136/bjo.2005.075853
Aim: To develop silicon microforceps for intraocular surgery
using Microelectromechanical Systems (MEMS) technology,
the application of microchip fabrication techniques for the
production of controllable three dimensional devices on the
micrometre scale.
Methods: Prototype MEMS forceps were designed and manufactured for intraocular surgery. Scanning electron microscopy
was used to evaluate device tip construction. Designs using both
thermal expansion actuators and conventional mechanical
activation were tested in human cadaver eyes and in vivo rabbit
eyes to assess functionality in standard vitreoretinal surgery.
Results: MEMS forceps were constructed with various tip
designs ranging from 100 mm to 2 mm in length. Scanning
electron microscopy confirmed accurate construction of micro
features such as forceps teeth as small as tens of micrometres. In
surgical testing, the silicon forceps tips were effective in surgical
manoeuvres, including grasping retinal membranes and
excising tissue. The mechanical actuator design on a 20 gauge
handle was more operational in the intraocular environment
than the thermal expansion actuator design. While handheld
operation was possible, the precision of the forceps was best
exploited when mounted on a three axis micromanipulator.
Conclusion: MEMS microforceps are feasible for conventional vitreoretinal surgery, and offer advances in terms of
small scale, operating precision, and construction tolerance.
W
hile vitreoretinal surgical instrumentation has
undergone refinements over the past several decades, basic aspects of retinal instruments—materials, scale, and construction—have not departed in a
fundamental way from established designs. The ability to
achieve surgical objectives in ophthalmology is determined at
least in part by the limits of instrumentation in terms of size
and precision. Microelectromechanical Systems (MEMS) is a
broad technology that utilises the materials and techniques of
silicon microchip fabrication to create movable, controllable
devices on the scale of micrometres to millimetres.1 2 From
commercial and industrial applications, increasing interest is
directed towards biomedical applications of MEMS.3–6
Using MEMS technology, we have designed and manufactured micro-forceps prototypes for intraocular surgery,
with instrument tips of single crystal silicon construction on
the scale of 100 mm and design features on the scale of
10 mm. Testing under standard vitreoretinal surgery conditions demonstrated their feasibility and functionality. To our
knowledge this represents the first report of MEMS instruments for intraocular surgery.
two dimensional designs using boron doped silicon wafers
(orientation (100), resistivity 0.02 ohm-cm). Photoresist was
spun onto wafers and patterned using a photomask, then
hard baked at 125˚C. Deep etching was performed using the
Bosch process in an STS plasma etcher. After cleaning wafers
with acetone, piranha (H2SO4/H2O2), and deionised (DI)
water rinse, a 1 mm thermal oxide coat was grown (1100˚C,
O2, steam). Oxide was removed from the backside of the
wafer using 5:1 buffered oxide etch (aqueous hydrofluoric
acid (HF) with ammonium fluoride). A ring of oxide was
preserved at the outer diameter of the wafer, to preserve the
full wafer thickness around the edge to prevent it from
becoming too fragile to handle. Wafers were placed in 25%
tetramethyl ammonium hydroxide at 60˚C until the etch
front reached the bottom of the plasma etched pattern
(where only oxide windows remain). Wafers were rinsed in
DI water and all oxide was removed with 49% HF. A 1 mm
thick layer of wet thermal oxide was grown (1100˚C, O2,
steam) to remove sharp corners and stress concentrations,
then all oxide was removed with 49% HF. Finally, silicon
parts were obtained, and for some designs assembled with
epoxy to a forceps actuator shaft.
Scanning electron microscopy
Scanning electron microscopy (EM) was done with a Jeol
6400. Silicon specimens were mounted on standard aluminium scanning EM stubs using colloidal carbon paint. Gold
coating was not necessary for visualisation as the silicon
alone is sufficiently conductive. Micrographs were obtained
and stored digitally.
MATERIALS AND METHODS
Forceps design and construction
Two different actuator designs were developed to control
forceps operation. The initial design was an electrically
heated thermal expansion actuator, which incorporated
tweezer tips and heat sink fins as a single piece (fig 1A). A
manual potentiometer was used to apply current to rapidly
heat and cool the semiconductive silicon of the thermal
actuator, causing expansion and contraction in length, to
open and close the tips via a lever linkage.
The second generation of forceps used a more conventional
mechanical actuation (figs 1B and 2). MEMS silicon forceps
tips were joined to a 20 gauge stainless steel instrument shaft
enclosing a spring loaded opening mechanism, which
included a microcalibration system for fine adjustments of
the forceps tip excursion. To maximise stability, the
mechanical actuator itself is electrically activated, via wire
connections to a control switch, much like the automated
MPC scissor familiar to retinal surgeons.7
Several tip configurations were designed to be suitable for
intraocular surgery. The stiffness of tips made for these forceps
ranged from 1 nanonewton/mm to 100 micronewtons/mm.
MEMS fabrication process
Original designs for a variety of MEMS forceps were
manufactured at the University of California Berkeley
Microfabrication Laboratory. Forceps were constructed from
Abbreviations: DI, deionised; EM, electron microscopy; HF,
hydrofluoric acid; MEMS, Microelectromechanical Systems; MVR,
microvitreoretinal
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Development of MEMS forceps
1587
Figure 1 Prototype MEMS intraocular forceps. (A) Scanning electron
micrograph of an early version of micro-tweezers with thermal
expansion actuator. The device incorporates heat sink fins into the body
of the tweezers; electric current actuates the tips by thermal expansion
(calibration bar = 100 mm). (B) Later forceps designs employed a
mechanical actuator system within a 20 gauge instrument shaft. (The
forceps are shown with a 25 gauge needle in the background.)
Intraocular surgery
For surgical testing in human cadaver eyes, an eye cup was
formed by excising the cornea, iris, and lens, then filling the
vitreous cavity with balanced salt solution after vitrectomy.
Surgery was done in an ‘‘open sky’’ fashion under the
operating microscope (Zeiss Op-Mi6, Carl Zeiss, Germany).
For in vivo surgical testing, standard three port 20 gauge
vitrectomy (Storz Millenium, Rochester, NY, USA) was
performed on adult New Zealand White rabbits (Charles
River Laboratories Inc, Wilmington, MA, USA), anaesthetised
with 3–5% isoflourane mask inhalation (Baxter, Deerfield, IL,
USA). Both lensectomy and lens sparing vitrectomies were
done. The MEMS instruments were introduced into the eye
through standard sclerotomies made with a 20 gauge
microvitreoretinal (MVR) blade. At the completion of surgery
animals were euthenised with intramuscular ketamine (30–
50 mg/kg, Fort Dodge Animal Health, Ft Dodge, IA, USA)
and xylazine (5–10 mg/kg, Phoenix Pharmaceutical Inc, St
Joseph, MO, USA), followed by intramuscular sodium
pentobarbital (.150 mg/kg, Schering-Plough, Kenilworth,
NJ, USA) and bilateral thoracotomy. All rabbit experiments
were done in accordance with UCSF committee on animal
research guidelines.
RESULTS
The instruments were manufactured at tip lengths from
100 mm to 2 mm; scanning EM confirmed the accurate
construction of serrated teeth as small as 10 mm. Figure 2
shows a MEMS forceps with a serrated jaw design alongside
a commercial stainless steel subretinal forceps, demonstrating the relative scale of the instrument tips as well as the high
design tolerance made possible with MEMS technology.
Two different actuator systems were designed for the
MEMS forceps. In the first prototypes, a thermal expansion
Figure 2 Scanning electron micrographs at different magnifications (A,
B) of mechanically actuated MEMS forceps with serrated jaws. In the
background is a commercial stainless steel subretinal forceps. The shaft
of the mechanical actuator to which the MEMS tips are glued is shown in
(C). Calibration bars = 100 mm.
actuator adapted from engineering applications was redesigned for intraocular surgery. With this design, electrically
heated beams (fig 1A), allow tip excursions as small as
several micrometres. This actuator was usable in the eye only
with an ‘‘open sky’’ approach, and frequently had thermal
coagulation of materials on its surface after repeated current
application which interfered with its mechanism.
Therefore, a redesign was made, based on a mechanical
actuation system. A variety of MEMS designs were incorporated
in a more conventional 20 gauge stainless steel spring loaded
system. The circuit for its automated activation was enclosed
within the shaft of the instrument and no thermal coagulation
of surface materials was noted with repeated activation. For in
vivo surgery in rabbit eyes with standard three port, 20 gauge
vitrectomy, operation of the forceps was done in a conventional
handheld fashion, and also by mounting the forceps on a Sutter
Instruments three axis micromanipulator. Handheld operation
was found to be feasible, but the micromanipulator provided
greater stability and movement precision commensurate with
the small scale of the instrument tips.
Surgical tests confirmed the viability of silicon as a
material for intraocular instruments. The tensile properties
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1588
Bhisitkul, Keller
thermal expansion actuator was adapted from engineering
and microscopy applications, but had disadvantages during
eye surgery, exemplifying the disparities in transferring this
technology from the laboratory setting to the intraocular
environment. Improved systems for operative imaging,
stabilisation, and micromanipulation may be required to
realise the potential of MEMS microsurgery. In this project,
we have not departed substantially from standard designs,
but instead have advanced the scale and material of
conventional forceps archetypes. However, MEMS technology may offer the capability to evolve instrument designs for
surgical applications not foreseen presently.
ACKNOWLEDGEMENTS
This study was supported by That Man May See Foundation and by
Research to Prevent Blindness.
See video on www.bjophthalmol.com/supplemental
.....................
Authors’ affiliations
Figure 3 MEMS forceps in use during three port vitrectomy in the rabbit
eye in vivo. (A) MEMS forceps grasping posterior hyaloid to manipulate
detached retina. (B) MEMS forceps used to peel a fine preretinal
membrane (arrows).
of silicon proved to be non-distensible and not plastically
deformable under conditions of standard vitreoretinal surgery. Also, silicon was found to be antireflective with
standard endoillumination, aiding visibility of the small tips.
In surgical manoeuvres with serrated forceps (fig 3, and see
video on BJO website), we were able to firmly engage tissues
and displace membranes without slippage. No breakage or
fracture of the silicon tips was observed in multiple trials.
DISCUSSION
This project indicates the viability of MEMS forceps for
intraocular surgery. The miniaturisation and construction
tolerance of the MEMS forceps surpass that of commercially
available stainless steel instruments. The tensile properties of
silicon confer durability and function at small scales at which
stainless steel would be plastically deformable. The material
biocompatibility and sterilisability of MEMS devices and
materials appear from initial studies to be satisfactory.5 8
MEMS fabricated instruments have potential applications
in eye surgery. For example, the innovation of the 25 gauge
vitrectomy system9 10 has necessitated a rescaling of the full
array of handheld vitreoretinal instruments, which MEMS
instruments could complement and expand. Besides forceps,
MEMS technology could be used to design membrane picks,
blades, scissors, etc, on a scale much smaller than current
products. The miniaturisation allowed by MEMS instruments
could be used to advantage in advances such as nonvitrectomy retinal surgery.11 12 Furthermore, since any two
dimensional design template can be rapidly fabricated at a
large scale, MEMS processes may facilitate advances in
instrument design and even allow customisability for
individual surgeons. Emerging research in biomedical
MEMS includes drug delivery devices, micro-pumps, sensors,
and retinal prostheses.13–16
Challenges remain with the development of MEMS
intraocular instruments. In these tests, the initial prototype
www.bjophthalmol.com
R B Bhisitkul, Beckman Vision Center, Department of Ophthalmology,
University of California San Francisco, San Francisco, CA, USA
C G Keller, MEMS Precision Instruments, Richmond, CA, USA
Financial disclosure: CGK is founder of MEMS Precision Instruments; RBB
has no financial interest.
Correspondence to: Robert B Bhisitkul, MD, PhD, Beckman Vision
Center, Department of Ophthalmology, University of California San
Francisco, 10 Koret Way, K301, San Francisco, CA 94143, USA;
[email protected]
Accepted for publication 1 August 2005
REFERENCES
1 Petersen KE. Silicon as a mechanical material. Proc IEEE 1982;70:420–57.
2 Wise KD, Najafi K. Microfabrication techniques for integrated sensors and
microsystems. Science 1991;254:1335–42.
3 Roy S, Ferrara LS, Fleischman AJ, et al. Microelectromechanical Systems and
neurosurgery: a new era in a new millennium. Neurosurgery
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4 Bashir R. BioMEMS: state-of-the-art in detection, opportunities and prospects.
Adv Drug Deliv Rev 2004;56:1565–86.
5 Kotzar G, Freas M, Abel P, et al. Evaluation of MEMS materials of construction
for implantable medical devices. Biomaterials 2002;23:2737–50.
6 Polla DL, Erdman AG, Robbins WP, et al. Microdevices in medicine. Ann Rev
Biomed Eng 2000;2:551–76.
7 Machemer R, Parel JM, Hickingbotham D, et al. Membrane peeler cutter:
automated vitreous scissors and hooked needle. Arch Ophthalmol
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8 Weisenberg BA, Mooradian DL. Hemocompatibility of materials used in
microelectromechanical systems: Platelet adhesion and morphology in vitro.
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9 Fujii GY, De Juan E Jr, Humayun MS, et al. A new 25-gauge instrument system
for transconjunctival sutureless vitrectomy surgery. Ophthalmology
2002;109:1807–12.
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transconjunctival sutureless vitrectomy system for vitreoretinal surgery.
Ophthalmology 2002;109:1814–20.
11 Saito Y, Lewis JM, Park I, et al. Non-vitrectomizing vitreous surgery: a strategy
to prevent postoperative nuclear sclerosis. Ophthalmology
1999;106:1541–5.
12 Sawa M, Saito Y, Hayashi A, et al. Assessment of nuclear sclerosis after
nonvitrectomizing vitreous surgery. Am J Ophthalmol 2001;132:356–62.
13 Liepmann D, Pisano AP, Sage B. Microelectromechanical systems technology
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14 Polla DI, Erdmann AG, Robbins WP, et al. Microdevices in medicine. Annu
Rev Biomed Eng 2000;2:551–76.
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16 Zhou DD, Greenberg RJ. Microsensors and microbiosensors for retinal
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1589
SCIENTIFIC REPORT
Is routine biopsy of the lacrimal sac wall indicated at
dacryocystorhinostomy? A prospective study and literature
review
C Merkonidis, C Brewis, M Yung, M Nussbaumer
...............................................................................................................................
Br J Ophthalmol 2005;89:1589–1591. doi: 10.1136/bjo.2005.072199
Objective: To determine whether routine biopsy of the
lacrimal sac wall at dacryocystorhinostomy (DCR) is indicated.
Methods: A prospective study and literature review. In 193
consecutive endoscopic DCRs performed on 164 patients
(108 females and 56 males) part of the medial wall of the
lacrimal sac was sent for histological examination. The mean
age of the patients was 64 years with a range of 2.5–
89 years. Previous reported series were reviewed.
Results: Of the 193 specimens, 44 (23%) showed normal
histology, 146 (76%) showed varying degrees of non-specific
chronic inflammation, and three (1.2%) showed specific
pathology. Of the three specimens that showed specific
pathology two showed sarcoidosis and one showed transitional cell papilloma. The two specimens with sarcoidosis
were obtained from one patient who underwent bilateral
surgery. In this and the six previous reported series only
seven of 1294 specimens (0.5%) showed specific pathology,
which was definitely not suspected preoperatively or
intraoperatively, and only one of these (0.08%) was found
to be malignant (a lymphoma).
Conclusions: Biopsy of the lacrimal sac wall at DCR is not
indicated routinely and is only indicated if there is a reason
to suspect pathology other than chronic inflammation
preoperatively or intraoperatively.
L
acrimal obstruction may be proximal (single or common
canalicular obstruction), distal (sac or duct obstruction),
functional, or a combination of these.1 A diagnosis of
functional obstruction is made when syringing and probing
demonstrate no obstruction of the lacrimal system and yet
the more physiological investigation of scintigraphy demonstrates reduced passage of radiolabelled tracer through the
lacrimal system.
The surgical treatment for lacrimal obstruction is dacryocystorhinostomy (DCR) which involves marsupialisation of
the lacrimal sac into the nasal cavity. DCR can be performed
either externally or endoscopically and the results of both
techniques are similar.2 3
A Medline search performed by the authors has identified
six publications reporting the results of histological examination of specimens taken from the outflow system at DCR.
These have shown that lacrimal obstruction is associated
with non-specific chronic inflammation of the outflow
system in most cases and with specific pathologies in
between zero and 14.3% of cases (table 1). These specific
pathologies may be inflammatory or neoplastic (table 2). In
view of this some authors have advocated routine histological
examination of the lacrimal sac at DCR to avoid missing
specific pathologies.4–6
The senior author (MWY) has been performing endoscopic
DCR for patients with proximal, distal, and functional
lacrimal obstruction since 1994. We report the results of
and discuss the value of routine histological examination of
the lacrimal sac at DCR.
MATERIALS AND METHODS
In 193 consecutive endoscopic DCRs performed on 164
patients between January 1999 and December 2001, a part
of the medial wall of the lacrimal sac was routinely sent for
histological examination. The DCRs were bilateral in 23
patients. The mean age of the patients was 64 years with a
range of 2.5–89 years; 108 were female and 56 were male.
The indications for surgery and level of obstruction for these
193 DCRs are shown in table 3.
Preoperative assessment included syringing and probing,
dye testing, and (in selected cases) dacryocystography or
scintigraphy.2 At operation, bone of the frontal process of the
maxilla was removed to expose the lacrimal duct and sac and
the medial wall of the sac was removed with a keratome and
through-cutting forceps.3 The operation was performed under
local anaesthetic using a lacrimal fossa block7 and sedation in
139 cases and under general anaesthetic in 31 cases.
RESULTS
Of the 193 specimens, 44 (23%) showed normal histology,
146 (76%) showed varying degrees of non-specific chronic
inflammation, and three (1.2%) showed specific pathology
(table 4). Of the 146 specimens that showed non-specific
chronic inflammation a number also showed other changes
of the epithelial lining of the sac or duct, including erosion
(three), ulceration (two), hyperplasia (five), oncocytic
metaplasia (one), flattening of the epithelium (two),
thickening of the basement membrane (one), polyp formation (one), and cyst formation (four). Of the three specimens
that showed specific pathology, two showed sarcoidosis and
one showed transitional cell papilloma (mixed exophytic and
inverted type). The two specimens with sarcoidosis were
obtained from one patient who underwent bilateral surgery.
The cases of the two patients with specific pathology are
described below.
Case 1
A 53 year old woman was referred with a 2 year history of
bilateral epiphora. She had a history consistent with
rhinosinusitis and examination showed an oedematous nasal
mucosa and a superiorly thickened nasal septum. Skin prick
testing showed no allergies and a computed tomograph (CT)
scan showed no sinus disease. A dacrocystogram showed
obstruction of the lacrimal sac on the right. A nasal steroid
spray was commenced and a submucosal resection and
Abbreviations: DCR, dacrocystorhinostomy
www.bjophthalmol.com
1590
Merkonidis, Brewis, Yung, et al
Table 1 Number and percentage of lacrimal sac specimens with specific pathology and with specific pathology that was
unsuspected preoperatively and intraoperatively in seven series of dacryocystorhinostomies
Patients
Specimens
Specimens with specific
pathology
Specimens with unsuspected specific
pathology
Reference
No
No
No
%
No
%
Case selection
Linberg5
11
Mauriello
Tucker6
Lee-Wing10
4
Anderson
Bernardini9
Present study
Total
13
44
150
166
316
258
164
1111
14
44
162
202
377
302
193
1294
2
0
4
0
31
10
3
50
14.3
0
2.5
0
8.2
3.3
1.6
3.9
1
0
1
0
>3*
0
2
7
7.1
0
0.6
0
>0.8*
0
1.0
0.5
Unselected
Unselected
Unselected
Unselected
? Selected*
Unselected
Unselected
*See Discussion.
bilateral endoscopic DCR were performed. At operation both
lacrimal sacs were found to be oedematous but otherwise
normal and histology of both lacrimal sacs showed sarcoidosis. The patient was subsequently investigated with a chest
x ray that showed bilateral hilar lymphadenopathy and
angiotensin converting enzyme levels, which were raised. A
course of oral corticosteroids was prescribed for the pulmonary sarcoidosis. Three years postoperatively she has no
epiphora and has only mild lower respiratory tract symptoms.
Case 2
A 31 year old woman was referred with a 2 year history of
right epiphora. A dye test and scintigram showed delayed
emptying of the lacrimal sac and syringing showed a patent
lacrimal system. A diagnosis of functional blockage was
made and endoscopic DCR was performed. At operation the
lumen of the sac and duct were found to be filled with
granulation tissue and histology showed a transitional cell
papilloma (mixed exophytic and inverted type) of the sac.
The remainder of the sac and duct were subsequently
removed via a combined endoscopic and external approach.
Three years postoperatively there has been no recurrence and
it is planned to reconstruct the lacrimal system with a
pedicled nasal septal tube.8
Of the 193 specimens the obstruction was proximal in 31,
distal in 138, mixed in 10, and functional in 15 (table 4). Of
the 31 cases with proximal obstruction, histology was normal
in 12 and indicated non-specific chronic inflammation in 19.
Of the 138 cases with distal obstruction, histology was
normal in 27, there was non-specific chronic inflammation in
109, and sarcoidosis in two. Of the 10 cases with mixed
obstruction, histology was normal in five and there was nonspecific chronic inflammation in five. Of the 15 cases with
functional obstruction, histology showed non-specific
Table 2 Type and number of specific pathologies
identified in 1294 lacrimal sac specimens in seven series
of dacryocystorhinostomies
Pathology
Number
Reference
Sarcoidosis
Lymphoma
Papilloma
Lymphoplasmacytic infiltrate
Leukaemia
Oncocytoma
Transitional cell carcinoma
Adenocarcinoma
Granular cell tumour
Plasmacytoma
Poorly differentiated carcinoma
Transitional cell papilloma
16
11
7
4
3
2
2
1
1
1
1
1
4, 5, 6, 8, present study
4, 6, 8
4, 8
4
4, 5, 8
4, 6
4
4
4
4
4
Present study
www.bjophthalmol.com
chronic inflammation in 14 and transitional cell papilloma
in one.
DISCUSSION
In our series lacrimal obstruction was associated with nonspecific chronic inflammation of the lacrimal sac in 146 out
of 193 specimens (76%). This is in keeping with previous
series and is consistent with a pathophysiology of chronic
inflammation leading to epithelial and subepithelial changes
and lacrimal obstruction.4–6 9–11
Lacrimal obstruction may also be associated with specific
pathology. Previous series have shown specific pathology in
between zero and 14.3% of specimens (table 1). The most
common specific pathologies were sarcoidosis, lymphoma,
and papilloma (table 2). Specific pathology was found in 31
out of 377 specimens in Anderson’s series (eight sarcoidosis,
seven lymphoma, four papilloma, four lymphoplasmacytic
infiltrate, two transitional cell carcinoma, one oncocytoma,
one granular cell tumour, one adenocarcinoma, one poorly
differentiated carcinoma, one plasmacytoma, and one leukaemia)4; in 10 out of 302 specimens in Bernardini’s series
(four sarcoidosis, three squamous papilloma, two lymphoma,
one leukaemia)9; in four out of 162 specimens in Tucker’s
series (two lymphoma, one sarcoidosis, one oncocytoma)6;
and in two out of 14 specimens in Linberg’s series (one
sarcoidosis and one leukaemia).5 No specific pathology was
found in 44 specimens in Mauriello’s series11 and in 202
specimens in Lee-Wing’s series.10 In our series specific
pathology was found in three out of 193 specimens (two
sarcoidosis and one transitional cell papilloma). All these
series were described as being of unselected, consecutive
surgical specimens. However, the specimens in Anderson’s
series may have been selected as they were identified from
laboratory rather than surgical records, and as 10 were from
another laboratory and appeared to have a high proportion of
specific pathology. Overall, 50 out of 1294 specimens (3.9%)
in these seven series showed specific pathology.
The aim of this study was to determine whether biopsy of
the lacrimal sac wall at DCR is indicated in all cases or only in
those selected cases in which specific pathology is suspected
either preoperatively (from the history or examination) or
intraoperatively (from the appearance of the lacrimal sac). If
biopsy was performed only in selected cases it is possible that
specific pathology that was unsuspected preoperatively or
intraoperatively might be overlooked. It is therefore important to know in how many specimens with specific pathology
this was unsuspected. As described above, specific pathology
was identified in four previously published series (table 1). In
Lindberg’s series,5 of the two specimens with specific
pathology this was unsuspected in one specimen with
sarcoidosis. In Tucker’s series,6 of the four specimens with
specific pathology this was unsuspected in one specimen with
Is routine biopsy of the lacrimal sac wall indicated at DCR?
Table 3
1591
Indication for surgery and level of obstruction in 193 dacryocystorhinostomies
Indication for
surgery
No
%
Silent epiphora
163
85
Recurrent dacryocystitis
13
7
Pyocele
Mucocele
7
10
3
5
Table 4
Level of
obstruction
No
Proximal
Distal
Multiple levels
Functional
Distal
Multiple levels
Distal
Distal
31
110
7
15
10
3
7
10
Level of obstruction and histology in 193 dacryocystorhinostomies
Proximal
Distal
Multiple levels
Functional
Total
Normal
Chronic
inflammation
Specific
12
27
5
0
44
19
108
5
14
146
0
2 (sarcoidosis*)
0
1 (transitional cell papilloma)
3
*Two specimens from one patient.
oncocytoma. In Bernardini’s series,9 of the 10 specimens with
specific pathology this was suspected in all specimens. In
Anderson’s series,4 of the 31 specimens with specific
pathology it was stated this was unsuspected preoperatively
in at least eight. However, it was not stated what the nature
of the pathology was (except one lymphoma) or whether it
was suspected intraoperatively (except two unspecified
epithelial tumours and one lymphoma which were not
suspected preoperatively or intraoperatively).12 In our series,
of the three specimens with specific pathology this was
unsuspected in two specimens with sarcoidosis (both from
the same patient). Overall, only seven out of 1294 specimens
(0.5%) in these seven series showed specific pathology, which
was definitely unsuspected, and in only one of these was this
malignant (a lymphoma).
In view of this we believe that biopsy of the lacrimal sac
wall at DCR is not indicated routinely and is only indicated if
there is a reason to suspect specific pathology preoperatively
or intraoperatively. To minimise the risk of overlooking
specific pathology it is important to inquire about symptoms
or history of systemic disease preoperatively, to assess the
appearance of the lacrimal sac intraoperatively, and to biopsy
the lacrimal sac in those cases where specific pathology is
suspected. The only specific pathology that might be overlooked in practice with such an approach is sarcoidosis.
Although most patients with sarcoidosis of the lacrimal sac
have a history of sarcoidosis or an abnormal appearance of
the nasal mucosa or lacrimal sac,13 some cases, including
ours, do not.14
In conclusion, we believe that this prospective study and
literature review demonstrates that routine biopsy of the
lacrimal sac wall at dacryocystorhinostomy is not indicated.
.....................
Authors’ affiliations
C Merkonidis, C Brewis, M Yung, M Nussbaumer, Department of
Otolaryngology, Ipswich Hospital NHS Trust, Heath Road, Ipswich IP4
5PD, UK
Correspondence to: Matthew Yung, PhD, FRCS, DLO, Department of
Otolaryngology, Ipswich Hospital NHS Trust, Heath Road, Ipswich IP4
5PD, UK; [email protected]
Accepted for publication 4 July 2005
REFERENCES
1 Paulsen F. The human nasolacrimal ducts. Adv Anat Embryol Cell Biol
2003;170:1–106.
2 Yung MW, Hardman-Lea S. Analysis of the results of surgical endoscopic
dacryocystorhinostomy: effect of the level of obstruction. Br J Ophthalmol
2002;86:792–4.
3 Yung MW, Hardman-Lea S. Endoscopic inferior dacryocystorhinostomy. Clin
Otolaryngol 1998;23:152–7.
4 Anderson NG, Wojno TH, Grossniklaus HE. Clinicopathologic findings from
lacrimal sac biopsy specimens obtained during dacryocystorhinostomy.
Ophthalmol Plast Reconstr Surg 2003;19:173–6.
5 Linberg JV, McCormick SA. Primary acquired nasolacrimal duct obstruction.
A clinicopathologic report and biopsy technique. Ophthalmology
1986;93:1055–63.
6 Tucker N, Chow D, Stockl F, et al. Clinically suspected primary acquired
nasolacrimal duct obstruction: clinicopathologic review of 150 patients.
Ophthalmology 1997;104:1882–6.
7 Smith MG, Kong AS, Yung MW, et al. Lacrimal fossa block: an audit of a
minimally invasive regional anaesthetic technique for endoscopic
dacryocystorhinostomy (DCR). Clin Otolaryngol 2001;26:407–10.
8 Yung MW, Hardman-Lea S. Ipswich lacrimal tube: pedicle nasal septal tube
for the reconstruction of lacrimal drainage passage. J Laryngol Otol
2003;117:130–1.
9 Bernardini FP, Moin M, Kersten RC, et al. Routine histopathologic evaluation
of the lacrimal sac during dacryocystorhinostomy: how useful is it?
Ophthalmology 2002;109:1214–17.
10 Lee-Wing MW, Ashenhurst ME. Clinicopathologic analysis of 166 patients
with primary acquired nasolacrimal duct obstruction. Ophthalmology
2001;108:2038–40.
11 Mauriello JA Jr, Palydowycz S, DeLuca J. Clinicopathologic study of lacrimal
sac and nasal mucosa in 44 patients with complete acquired nasolacrimal
duct obstruction. Ophthal Plast Reconstr Surg 1992;8:13–21.
12 Hurwitz JJ. Re: ‘‘Clinicopathologic findings from lacrimal sac biopsies
obtained during dacryocystorhinostomy.’’ Ophthal Plast Reconstr Surg
2003;19:412; author reply 412–13.
13 Chapman KL, Bartley GB, Garrity JA, et al. Lacrimal bypass surgery in
patients with sarcoidosis. Am J Ophthalmol 1999;27:443–6.
14 Kay DJ, Saffra N, Har-el G. Isolated sarcoidosis of the lacrimal sac without
systemic manifestations. Am J Ophthalmol 2002;23:53–5.
www.bjophthalmol.com
1592
EXTENDED REPORT
Variation in rates of severe retinopathy of prematurity
among neonatal intensive care units in the Australian and
New Zealand Neonatal Network
B A Darlow, J L Hutchinson, J M Simpson, D J Henderson-Smart, D A Donoghue, N J Evans, on
behalf of the Australian and New Zealand Neonatal Network*
...............................................................................................................................
Br J Ophthalmol 2005;89:1592–1596. doi: 10.1136/bjo.2005.073650
*See appendix
See end of article for
authors’ affiliations
.......................
Correspondence to:
Professor Brian Darlow,
Department of Paediatrics,
Christchurch School of
Medicine and Health
Sciences, PO Box 4345,
Christchurch, New
Zealand; brian.darlow@
chmeds.ac.nz
Accepted for publication
1 July 2005
.......................
S
Aim: To analyse variations in rates of severe retinopathy of prematurity (ROP) among neonatal intensive
care units (NICUs) in the Australian and New Zealand Neonatal Network (ANZNN), adjusting for
sampling variability and for case mix.
Methods: 25 NICUs were included in the study of 2105 infants born at less than 29 weeks in 1998 and
1999, who survived to 36 weeks post-menstrual age and were examined for ROP. The observed NICU
rates of severe ROP were adjusted for case mix using logistic regression on gestation, weight for
gestational age and sex, and for sampling variability using shrinkage estimates. The corrected rate in the
best 20% of NICUs was identified and NICU variations in rates were compared with those in 2000–1.
Results: The overall (unadjusted) rate of severe ROP in the NICUs was 9.6% (interquartile range
5.4212.8%). After adjusting for both case mix and sampling variability there remained significant
variation among the NICUs. 20% of NICUs had a rate of severe ROP (5.9%. Variation in rates among
NICUs showed a similar pattern in both time periods. If the overall network rate was reduced to 5.9%, the
20th centile of the adjusted rates, there would be 79 fewer cases in a 2 year period, in contrast with 26
fewer if rates in the two units with excess rates improved to the average.
Conclusions: Considerable variation in rates of severe ROP among NICUs remained after adjustment for
case mix and sampling variability. These data will facilitate investigation of potentially better practices
associated with a reduced risk of severe ROP.
ince the first description of retrolental fibroplasia by
Terry in 1942, a notable feature of the condition (now
known as retinopathy of prematurity, ROP) has been the
variation in incidence among hospitals.1 Recent hospital
cohorts2 3 and population based reports4–8 continue to
demonstrate widespread variability in reported incidence
and severity of ROP.
Such variation in outcome, across different specialties and
disease entities, has been the subject of considerable debate.
Differences in outcome ascertainment, case mix, and
sampling variability, as well as different clinical practices
may all contribute to the observed differences.9 In order to be
able to identify NICUs with significantly better outcomes,
and thus investigate potentially better practices associated
with those outcomes, it is necessary to control for these
sources of variation. No previous study has taken this
approach to reduction in the incidence of severe ROP.
The Australia and New Zealand Neonatal Network
(ANZNN) consists of all 29 level III neonatal intensive care
units (NICUs) in Australia and New Zealand. Since 1995 a
dataset of 60 perinatal variables, using agreed definitions, has
been collected prospectively on all infants of less than
32 weeks gestation or birth weight less than 1500 g. Both
countries have a regionalised system of neonatal intensive
care, meaning that the ANZNN dataset for infants with
gestation less than 29 weeks represents essentially population based data. Previously, we analysed data on 21 antenatal
and intrapartum variables for all infants ,29 weeks gestation born in the 2 year period 1998–9 and who survived to
36 weeks post-menstrual age (PMA) to identify risk factors
for severe (stage 3 or more10) ROP.11 That study showed that
decreasing gestation, lower birth weight for gestation, and
male sex were significantly associated with increased risk.11
www.bjophthalmol.com
We have now analysed the differences in severe ROP
among NICUs of the ANZNN, adjusting for differences in
case mix and sampling variability, using our previously
reported methodology.9 Our aim was to identify true
variations in severe ROP in order to provide a framework
for reducing the overall network incidence of severe ROP.
METHODS
The four children’s hospitals out of the 29 NICUs in the
ANZNN were excluded from the study since they represent a
different population of infants (all outborn and many
requiring surgery). The remaining 25 NICUs included 2830
infants born at ,29 weeks in 1998–9, of whom 2286 (80.8%)
survived to 36 weeks PMA. The 181 (7.9%) infants who did
not have an eye examination reported were excluded, leaving
2105 eligible infants.
To analyse variation in outcome rates among NICUs we
compared the observed frequency (O) of severe ROP in each
unit with the expected frequency (E).9 The unadjusted value
of E for each NICU is simply the overall rate of severe ROP for
all NICUs multiplied by the number of eligible admissions to
that NICU. To adjust for bias caused by case mix, we used the
logistic regression model to predict the probability of severe
ROP for each infant from its gestation, weight for gestational
age and sex.11 The adjusted value of E for each NICU is the
sum of the predicted probabilities for all infants in that NICU.
Abbreviations: ANZNN, Australian and New Zealand Neonatal
Network; CRIB, Clinical Risk Index for Babies; GA, gestational age; IQR,
interquartile range; NICU, neonatal intensive care unit; PMA, postmenstrual age; ROP, retinopathy of prematurity; SNAP-PE, Score for
Neonatal Acute Physiology
Variation in rates of severe ROP
We also allowed for the effect of sampling variability,
which is smaller when the outcome rate is closer to 0% or to
100%, assuming the rates have a binomial distribution. It is
also smaller for larger NICUs, because the standard error of
the estimated rate decreases as sample size increases. Because
of sampling variability the observed rate varies about the true
rate for each NICU. We allowed for this by calculating
shrinkage estimators using a Bayesian approach. Details are
given elsewhere9 but, briefly, each NICU is assumed to have a
true underlying rate of severe ROP. Shrinkage estimators use
data from all NICUs to improve the estimate of the true rate
of severe ROP for each individual NICU, by shrinking the
observed rate towards the overall rate for all units. The
shrinkage is greater for smaller NICUs, so shrinkage
estimates are more conservative and less variable than the
observed rates. The shrinkage estimates were obtained using
maximum likelihood methods assuming a gamma Poisson
model, which also gave an estimate, v, of the standard
deviation of the true incidence ratios (O/E) for all NICUs.12
The corrected excess (O 2 E) number of infants with
severe ROP was calculated for each NICU using the shrunken
estimators. The 95% control limits were obtained similarly, to
show the limits within which this excess should lie when the
expected number of cases is E.9
To inform quality improvement, we explored the benefit of
a network-wide approach rather than targeting NICUs
performing poorly. The 20th centile was estimated as the
(n+1)/5th value of the adjusted rates of severe ROP for all
NICUs sorted in ascending order. This is used as an
achievable estimate of ‘‘best practice,’’ because it is already
achieved by 20% of NICUs. The ‘‘centile gain’’ is the number
of potential cases of severe ROP that could be prevented in a
Percentage severe ROP
30
A
Observed
Adjusted
25
20
15
10
5
0
X E Q P GM I L F R B N V C Y U KW J D S T A O H
NICU ordered by decreasing number of eligible infants
Percentage severe ROP
30
25
B
Observed
Shrunken, adjusted
20
15
10
5
0
X E Q P GM I L F R B N V C Y U KW J D S T A O H
NICU ordered by decreasing number of eligible infants
Figure 1 Comparison of observed percentage of cases with severe ROP
in each NICU (the NICUs are identified by letter on the X-axis, as in
table 1) by decreasing number of eligible infants (1998–9) and (A)
percentage adjusted for case mix only. (B) percentage adjusted for case
mix and sampling variability.
1593
2 year period if the overall NICU rate was reduced to the 20th
centile rate. This was compared with the ‘‘outlier gain,’’ the
reduction in the number of cases that would result if NICUs
that were exceeding their 95% limits improved to their
expected number of cases.
The same methods were applied separately to data for a
similar cohort born in 2000–1 (that is, infants of ,29 weeks
gestation, registered with ANZNN in 2000–1, who survived to
36 weeks PMA and were examined for ROP) for comparison
(n = 2277) (full data for 2000–1 not shown).
We also explored the potential measurement bias resulting
from the failure to examine eligible infants. Kendall’s rank
correlation coefficient was used to assess the relations
between the rate of severe ROP and the observed mortality
rate (that is, the proportion of infants of ,29 weeks gestation
who died before 36 weeks PMA), the number of infants of
,29 weeks gestation alive at 36 weeks PMA, and the
percentage of these eligible infants not examined for ROP
per NICU. In addition, sensitivity analysis was performed
using best and worst case scenarios (worst case treating
babies not examined for ROP as cases of severe ROP, best
case as no ROP11).
Lastly, the NICUs were surveyed on their clinical practice
with respect to indirect ophthalmoscopy. All NICUs in the
ANZNN confirmed that examination guidelines13 14 with
respect to timing and technique, including adequate pupil
dilatation, were followed and that infants are screened until
the retina is fully or nearly fully (extreme zone 3)
vascularised. Examination findings are reported using the
International Classification of ROP.10
Approval for the project was given by Royal Prince Alfred
Hospital ethics review committee and University of Sydney
human ethics committee.
RESULTS
In 1998–9 ROP of any stage was reported in 42% of examined
infants, stage 3 or more in 9.6%, and 57% of the latter
received treatment by laser or cryotherapy. The unadjusted
rates of severe ROP for individual NICUs ranged from 0% to
25% (column 4, table 1), interquartile range (IQR) 5.4–12.8%.
The effects of both the shrinkage and case mix adjustment
are shown in table 1. Adjusting for sampling variability alone
(column 6) has a relatively minor impact on the order of the
NICUs (except for ‘‘H’’). Adjusting for case mix has a more
marked effect on the order, most notably for ‘‘P’’ and ‘‘T.’’
The relative impact of the adjustments is best demonstrated
graphically. Figure 1 compares the observed rate of severe
ROP with the rates adjusted for (a) case mix only and (b)
both case mix and sampling variability (corrected rates).
Figure 2 shows the number of infants involved in this
variation by depicting the corrected excess (O 2 E) number
of severe ROP cases for each NICU in 1998–9 (black
diamonds), with the NICUs ordered by decreasing number
of examined infants and the upper and lower 95% control
limits shown. NICU ‘‘X’’ had almost 20 more cases of severe
ROP than expected over the 2 year period. NICU ‘‘Y’’ had six
excess cases and NICU ‘‘B’’ had five fewer cases of severe
ROP than expected.
For infants in the 2000–1 cohort, the overall rate of severe
ROP was 10.2% (60% of these infants receiving treatment)
and the variability among NICUs increased slightly (fig 2,
open squares). There were an additional two NICUs (‘‘O’’ and
‘‘G’’) with fewer than expected cases and another two NICUs
(‘‘R’’ and ‘‘N’’) with more than expected. Individual unit
performance for this outcome can be easily compared over
the two time periods using figure 2 because the NICUs are in
the same order. The NICU with almost 20 more cases than
expected in 1998–9 had 25 excess cases in 2000–1.
www.bjophthalmol.com
1594
Darlow, Hutchinson, Simpson, et al
Table 1 Rates of severe ROP in infants born at ,29 weeks gestational age who survived to 36 weeks PMA and had eyes
examined, for 25 ANZNN NICUs (1998–9)
Observed (unadjusted)
Adjusted for sampling
variability only
Adjusted for sampling
variability and case mix
NICU
Total
infants
Severe ROP
Rate (%)
Order
Rate (%)
Order
Rate (%)
Order
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
Total
37
91
76
47
173
100
119
17
111
47
59
100
116
88
25
138
141
98
39
39
60
84
48
188
64
2105
0
2
3
2
8
5
7
1
7
3
4
7
9
7
2
12
14
11
5
5
9
13
9
42
16
203
0.0
2.2
3.9
4.3
4.6
5.0
5.9
5.9
6.3
6.4
6.8
7.0
7.8
7.9
8.0
8.7
9.9
11.2
12.8
12.8
15.0
15.5
18.7
22.3
25.0
9.6
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19.5
19.5
21
22
23
24
25
5.3
4.7
6.1
6.9
5.7
6.5
6.9
8.6
7.3
8.0
8.0
7.8
8.3
8.5
9.1
8.9
9.9
10.7
11.1
11.1
12.7
13.4
14.3
19.8
18.6
2
1
4
6
3
5
7
14
8
10
11
9
12
13
16
15
17
18
19.5
19.5
21
22
23
25
24
4.2
4.5
5.9
6.5
5.4
5.5
6.8
7.9
7.4
8.0
8.3
8.2
8.3
8.3
10.3
8.2
10.5
10.2
12.1
9.2
10.7
14.1
15.2
20.3
18.6
1
2
5
6
3
4
7
9
8
10
14
12
13
15
18
11
19
17
21
16
20
22
23
25
24
Shrunken: adjusted for sampling variability using Bayesian shrinkage estimators
Case mix: adjusted for differences in gestational age (GA), weight for GA and sex, using logistic regression
Rates presented are (a) observed (unadjusted), (b) adjusted for sampling variability (shrunken), and (c) adjusted for sampling variability and case mix.
Excess number of severe ROP cases
Estimates of the variation (v) among the NICUs’ true
incidence ratios are given in table 2 for three different
scenarios: with adjustment for none, two, and three predictor
variables. The variability among NICUs actually increased as
we adjusted for more variables. Adjusting for the three
predictors (gestational age, birth weight for gestational age,
and sex), the best 20% of NICUs were estimated to have a
rate of 5.9% or less while the overall rate in this period was
9.6%. If the network rate was reduced to 5.9%, there would be
79 fewer cases of severe ROP over a 2 year period (‘‘centile
gain’’), in contrast with only 26 fewer cases by taking the
‘‘outlier gain’’ approach—that is, by improving the outcome
in the two NICUs that were in excess of their 95% limits.
30
Excess 1998–9
Excess 2000–1
95% limits 1998–9
95% limits 2000–1
25
20
15
10
5
0
–5
–10
–15
X E Q P GM I L F R B N V C Y U KW J D S T A O H
NICU ordered by decreasing number of eligible infants
in 1998–9 cohort
Figure 2 Excess number of cases of severe ROP (O 2 E) by NICU,
calculated using estimates adjusted for both case mix and sampling
variability, for 1998–9 and 2000–1 cohorts. NICUs are ordered by
decreasing number of eligible cases in 1998–9. Upper and lower 95%
control limits are shown for each cohort. (The NICUs are identified by
letter on the X-axis, as in table 1).
www.bjophthalmol.com
There was no relation between the observed rate of severe
ROP and a NICU’s mortality rate before 36 weeks PMA
(Kendall’s rank correlation, T = 0.10, p = 0.47), or size of
NICU, as judged by the number of eligible infants (T = 0.04,
p = 0.78). Measurement bias could also result from excluding
those infants not examined for ROP, with the potential for
NICUs with high rates of ‘‘not examined’’ to have low rates of
severe ROP. Figure 3 displays these data with the size of each
point proportional to the number of infants in the cohort
from each NICU. There was a significant negative correlation
between the rate of severe ROP and the rate of ‘‘not
examined’’ (Kendall’s rank correlation, T = 20.30,
p = 0.04). The sensitivity analysis using the worst case
scenario—that is, assuming all non-examined infants had
severe ROP, resulted in an additional two NICUs having
corrected excess above their 95% limits. However, these
NICUs had a greater than average proportion of larger, more
mature infants, who have a relatively low risk of severe ROP.
DISCUSSION
This study has demonstrated that even when data are
corrected for case mix and sampling variability there remains
considerable variation in rates of severe ROP among 25
NICUs in the ANZNN.
Our analysis did demonstrate a significant relation
between the percentage of infants not examined for ROP
and the rate of severe ROP. NICUs with lower rates of ROP
tended to have a greater proportion of eligible infants not
examined. Overall, only a small proportion of eligible infants
(7.9%) were not examined and our previous analysis showed
the majority of these were larger and more mature, with 82%
being in the upper quartile for birth weight and 75% being
born at 27 weeks gestation or beyond.11 One possibility is that
NICUs with lower rates of ROP in infants most at risk were
more likely not to examine larger and more mature infants.
However, these data do emphasise the need for all infants to
Variation in rates of severe ROP
1595
Table 2 Estimates of variation (v) in severe ROP rates among 25 ANZNN NICUs (19989), based on adjustment for 0, 2, and 3 predictor variables (gestational age (GA), weight
for gestational age, sex)
Predictor variables
adjusted for
v
20th centile
rate (%)
Centile
gain
Outlier
gain
None
GA, sex
GA, sex, weight for GA
0.4747
0.4986
0.5247
6.46
6.09
5.88
67
75
79
25
26
26
Variation is expressed as: (a) v, the standard deviation of the true incidence ratios (O/E) for all study NICUs; (b)
20th centile of the rates of severe ROP adjusted for sampling variability and predictors; (c) ‘‘centile gain’’’ the
number of cases that might be prevented in 2 years if the overall rate of severe ROP was reduced to the 20th centile
rate; (d) ‘‘outlier gain,’’ the number of cases that might be prevented in 2 years if NICUs with excess rates
improved to the average.
Percentage not examined for ROP
have appropriate screening for ROP according to the national
guidelines.
To examine variation in practice among NICUs, we focused
on antenatal and intrapartum variables that affect the
infant’s condition before arrival in the NICU when adjusting
for case mix. One possible source of variation is that some
infants were intrinsically sicker than others. A number of
scores have been developed as a measure of severity of illness,
including the Clinical Risk Index for Babies (CRIB)15 and
Score for Neonatal Acute Physiology (SNAP-PE).16 17 The
ANZNN does collect data to calculate the CRIB score but not
the SNAP-PE. However, the CRIB score includes birth weight
and gestation, which were both used in our logistic regression
model, together with highest and lowest inspired oxygen in
the first 12 hours of life, which may be influenced by care
practices. Additionally, both scores are reported to predict
hospital mortality better than they predict morbidity.18
Parry et al19 reported on a longitudinal study of mortality
among 2671 infants with birth weight ,1500 g admitted to
one of nine NICUs in the United Kingdom between 1988 and
1994. While in most years there were no significant
differences in outcome among hospitals, the apparent
performance of individual hospitals fluctuated substantially
from year to year. One interesting feature of the present study
is that when the same model was applied to 2000–1 data
most units had similar results, in particular the two NICUs
that had an excess of cases in 1998–9 still had this excess in
the later period. This does suggest the possibility that there
may be some systematic or consistent element(s) in clinical
practice in these NICUs, which may be relevant. Equally, the
one NICU with fewer than expected cases in 1998–9,
continued in this situation in 2000–1. A focus on systemic
30
20
quality improvement measures, aimed at reducing the
incidence of severe ROP in all NICUs, is likely to be more
productive than an approach that focuses solely on poorly
performing NICUs. In particular, reducing the overall network rate to the 20th centile rate is likely to prevent more
cases of severe ROP in 2 years (79) than if the two NICUs
with excess rates improved to their expected number of cases
(26 cases prevented).
One source of variation we are unable to correct for is
observer differences in the assessment of retinal changes
during indirect ophthalmoscopy. Such measurement bias is
harder to quantify for ROP than for some other neonatal
outcomes, such as intraventricular haemorrhage. At present
it is not straightforward or practical for independent
observers to audit the results of individual indirect ophthalmoscopy examinations, although the development and
availability of contact digital retinal cameras may change
this situation in the future.20 However, the NICUs in the
ANZNN are all served by experienced ophthalmologists, who
follow explicit examination standards guidelines,13 14 which
should help to minimise this source of variation.
In conclusion, we have analysed data on the incidence of
severe ROP among NICUs in the ANZNN and adjusted for
both sampling variation and case mix to explore variations in
rates among hospitals. Greater reductions in rates of severe
ROP could be achieved if the overall rate was reduced to the
20th centile than if the focus were on outliers with excess
rates. There has been much recent debate about optimal
oxygen therapy in extremely premature infants21 22 and two
recent trials23 24 have compared oxygen saturation targets
after the first few weeks of life in these infants. Other studies
have found a relation between greater variability of oxygen
levels and an increased risk of severe ROP,25 26 while
strategies to decrease some of this variability have also been
reported.27 These and other evidence based factors should be
explored in efforts to achieve network-wide improvement in
rates of severe ROP.28
ACKNOWLEDGEMENTS
Supported by the National Health and Medical Research Council of
Australia (grant 211088 to DRH-S, DAD, JMS, and NJE) and the
Centre for Perinatal Health Services Research, University of Sydney,
Sydney, Australia.
10
.....................
0
0
10
20
30
Percentage with severe ROP
Figure 3 Percentage of infants not examined for ROP by percentage
with severe ROP for 25 ANZNN NICUs (1998–9). Circle size is
proportional to number of eligible babies (those born at less than
29 weeks gestational age and surviving to 36 weeks). The NICUs are
ordered left to right on the X-axis, by increasing percentage of
unadjusted severe ROP—that is, A to Y (see table 1).
Authors’ affiliations
B A Darlow, Department of Paediatrics, Christchurch School of Medicine
and Health Sciences, Christchurch, New Zealand
J L Hutchinson, D J Henderson-Smart, D A Donoghue, Australian and
New Zealand Neonatal Network (ANZNN), Centre for Perinatal Health
Services Research, University of Sydney, NSW, Australia
J M Simpson, School of Public Health, University of Sydney, NSW,
Australia
N J Evans, Department of Neonatal Medicine, Royal Prince Alfred
Hospital, Sydney, NSW 2050, Australia
www.bjophthalmol.com
1596
APPENDIX
The ANZNN Advisory Committee and Executive (*) members:
Australia: Centre for Perinatal Health Services Research,
NSW: David Henderson-Smart*; Flinders Medical Centre, SA:
Peter Marshall; John Hunter Hospital, NSW: Chris Wake;
King Edward Memorial and Princess Margaret Hospitals,
WA: Noel French, Ron Hagan and Karen Simmer;
Launceston General Hospital, Tas: Chris Bailey; Liverpool
Health Service, NSW: Robert Guaran; Mater Mother’s
Hospital, Qld: David Tudehope; Mercy Hospital for Women,
Vic: Andrew Watkins; Monash Medical Centre, Vic: Kaye
Bawden*, Andrew Ramsden, Victor Yu; National Perinatal
Statistics Unit, NSW: Paul Lancaster*; Nepean Hospital,
NSW: Lyn Downe; Newborn Emergency Transport Service
(Vic): Michael Stewart; NSW Newborn and Paediatric
Emergency Transport Service: Andrew Berry; Perinatal
Research Centre, Qld: Paul Colditz; Royal Children’s
Hospital, Vic: Linda Johnstone, Peter McDougall; Royal
Darwin Hospital, NT: Charles Kilburn; Royal Hobart
Hospital, Tas: Peter Dargaville; Royal Hospital for Women,
NSW: Kei Lui; Royal North Shore Hospital, NSW: Jennifer
Bowen; Royal Prince Alfred Hospital, NSW: Nick Evans Royal
Women’s Hospital, Qld: David Cartwright*; Royal Women’s
Hospital, Vic: Lex Doyle, Colin Morley, Neil Roy; Sydney
Children’s Hospital, NSW: Barry Duffy; The Canberra
Hospital, ACT: Graham Reynolds; The Children’s Hospital at
Westmead, NSW: Robert Halliday; The Townsville Hospital,
Qld: John Whitehall; Western Australia Neonatal Transport
Service: Jenni Sokol; Westmead Hospital, NSW: William
Tarnow-Mordi; Women’s and Children’s Hospital, SA: Ross
Haslam; Deborah Donoghue is the ANZNN Coordinator;
New Zealand: Christchurch Women’s Hospital: Nicola
Austin; Christchurch School of Medicine: Brian Darlow*;
Dunedin Hospital: Roland Broadbent; Gisborne Hospital:
Graeme Lear; Hastings Hospital: Jenny Corban; Hutt
Hospital: Robyn Shaw; Middlemore Hospital: Lindsay
Mildenhall; National Women’s Hospital: Carl Kushell;
Nelson Hospital: Peter McIlroy; Palmerston North Hospital:
Jeff Brown; Rotorua Hospital: Stephen Bradley; Southland
Hospital: Paul Tomlinson; Taranaki Hospital: John Doran*;
Tauranga Hospital: Hugh Lees; Timaru Hospital: Philip
Morrison; University of Auckland: Jane Harding; Waikato
Hospital: David Bourchier; Wairau Hospital: Ken Dawson;
Wanganui Hospital: John Goldsmith; Wellington Women’s
Hospital: Vaughan Richardson; Whakatane Hospital: Chris
Moyes; Whangarei Hospital: Peter Jankowitz.
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2 Vyas J, Field D, Draper ES, et al. Severe retinopathy of prematurity and its
association with different rates of survival in infants of less than 1251 g birth
weight. Arch Dis Child Fetal Neonatal Ed 2000;82:F145–9.
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1597
EXTENDED REPORT
Deep lamellar keratoplasty by deep parenchyma
detachment from the corneal limbs
T Senoo, K Chiba, O Terada, J Mori, M Kusama, K Hasegawa, Y Obara
...............................................................................................................................
Br J Ophthalmol 2005;89:1597–1600. doi: 10.1136/bjo.2005.072215
See end of article for
authors’ affiliations
.......................
Correspondence to:
Tadashi Senoo, 3210293, 880 Kitakobayashi
Mibu, Tochigi/Japan,
Dokkyo University School
of Medicine, Department
of Ophthalmology,
Tochigi, Japan; senoo@
dokkyomed.ac.jp
Accepted for publication
1 July 2005
.......................
Aim: To improve the deep lamellar keratoplasty technique.
Method: For the easy and reliable perfomance of deep lamellar keratoplasty (DLKP), detachment of
Descemet’s membrane through the corneal limber flap was improved. To expose Descemet’s membrane,
the parenchyma was detached by hydrodelamination through a sclerocorneal flap made in the corneal
limbs. The parenchyma was removed after the pseudochamber between it and Descemet’s membrane was
maintained with viscoelastic material. The corneal graft was placed with a running suture. 22 eyes were
treated.
Results: Complete exposure of Descemet’s membrane was obtained in 20 of the 22 eyes (91%). The
membrane was perforated in five of the 22 eyes (23%) during surgery, and two of the 22 eyes (9%) were
converted to penetrating keratoplasty. These two eyes developed keratoconus after acute corneal hydrops.
Conclusion: Compared with the conventional procedure, this new method provides easy, reliable exposure
of Descemet’s membrane.
D
eep lamellar keratoplasty (DLKP), performed while the
corneal endothelial function is maintained, is reported
to provide a good sense of vision, comparable to that
obtained by penetrating keratoplasty (PKP). This type of
surgery is advantageous in that there is no endothelial
rejection because the host’s endothelium remains intact and
does not affect the endothelial condition of the donor cornea.
Moreover, even a stored cornea can be used.1–4 DLKP,
however, has not been used widely because the surgical
method is more complex than that for PKP. In previously
published studies, air injection and viscodelamination and
hydrodelamination between Descemet’s membrane and the
parenchyma have been used for membrane detachment,1 5
but with many of the reported techniques it is difficult to
completely expose Descemet’s membrane. To overcome this
drawback we have developed a new DLKP surgical method,
in which Descemet’s membrane is exposed quickly, completely, and safely. We here report the use of this improved
surgical procedure.
MATERIALS AND METHODS
Patients
Twenty two eyes of 21 patients (11 men, 10 women; average
age 59.1 (SD 20) years) treated by this new method were
observed for more than 6 months. The original diseases were
corneal leucoma (eight), keratoconus (six), herpetic keratitis
(three), macular dystrophy (two), granular dystrophy (one),
lattice dystrophy (one), and corneal chemical burn (one).
Sixty seven eyes of 61 patients (25 men, 36 women; average
age 57.5 (20.8) years) were operated on by the conventional
method, performed according to Sugita’s method.1 The
original diseases were corneal leucoma (25), keratoconus
(16), herpetic keratitis (eight), macular dystrophy (one),
granular dystrophy (seven), lattice dystrophy (three), corneal
chemical burn (three), Stevens-Johnson syndrome (two),
and Schneider dystrophy (two). No difference in surgical
indications was found between the two groups. Comparisons
were made between the groups on complications during and
after surgery, and on visual acuity and corneal endothelial
cell density. Patients with other associated diseases of visual
disturbance and those whose eyes were converted to PKP
were excluded from the evaluation of visual acuity. Data were
analysed statistically by the x2 and Student’s t tests.
Surgical technique
A sclerocorneal flap, as in trabeculectomy, is made at the 10–
11 o’clock position, exposing the deep parenchyma of the
corneal limbs until Schlemm’s canal is reached. Part of
Descemet’s membrane is exposed by making this flap. After
marking the range of removal of the parenchyma with a
trephine, the aqueous humour is exchanged for air through a
side port. This side port should be as close as possible to the
corneal limbs. If it is made near the corneal centre, there is
the possibility of Descemet’s membrane perforation and
hydrodelamination of the membrane from the port. Next,
Descemet’s membrane is partially separated from the posterior parenchyma by a spatula inserted through the flap. It can
be inserted into the deep corneal parenchyma without
resistance (fig 1). Descemet’s membrane then is separated
completely from the posterior parenchyma by hydrodelamination at the point of detachment by the spatula. The
membrane is pushed down by the flow of air to the anterior
chamber (fig 2). Through the flap, a viscoelastic material is
injected between the separated membrane and parenchyma.
At this point, it should be confirmed by means of the
compressed air position that the area of the separated
Descemet’s membrane is larger than the removed area
(fig 3). The corneal parenchyma then is incised with a
Barron aspiration trephine until the viscoelastic material
flows out, after which Descemet’s membrane is exposed
(figs 4, 5). After stripping off the donor Descemet’s
membrane, the full thickness donor corneal button is sutured
into the recipient bed (fig 6).
RESULTS
Table 1 compares the conventional and new methods.
Surgery took 94 (SD 25.7) minutes by the conventional
method and 59 (26.1) minutes by the new one, a statistically
significant difference (p,0.0001). Intraoperative Descemet’s
Abbreviations: DF, dispersion factor; DLKP, deep lamellar keratoplasty;
PKP, penetrating keratoplasty
www.bjophthalmol.com
1598
Figure 1 A spatula is inserted from the sclerocorneal flap, and a pocket
for hydrodelamination made in the space between Descemet’s
membrane and the parenchyma.
Figure 2 Hydrodelamination is performed through the sclerocorneal
flap. Arrows indicate the intra-anterior chamber air. In this figure,
Descemet’s membrane is pushed down by the flow of air to the anterior
chamber.
Senoo, Chiba, Terada, et al
Figure 4 Corneal perforation by a vacuum trephine. On puncture, the
viscoelastic material flows out of the perforation (arrow).
Figure 5 Descemet’s membrane is exposed completely in the
transplantation area.
Figure 6 A full thickness donor corneal button lacking Descemet’s
membrane is sutured to the recipient bed.
Figure 3 Injection of viscoelastic material. After injection, the area of
the detached Descemet’s membrane must be confirmed to be wider than
the marker of the removal area.
membrane perforation occurred in five eyes (22.7%) with the
new method and in 25 eyes (37.3%) with the conventional
one. There was no statistical difference between the two
groups (p = 0.21). Conversion to PKP during surgery
occurred for two eyes (9%) with the new method and nine
eyes (13%) with the conventional one. The two eyes, for
which there was conversion to PKP, developed keratoconus
after acute corneal hydrops. In those two cases, detachment
of Descemet’s membrane by hydrodelamination was difficult,
and forced injection of viscoelastic material between the
membrane and parenchyma caused widespread tears in the
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membrane, rendering it unrepairable. A double anterior
chamber was present after surgery in 17 (29.3%) of 58 eyes
with the conventional method and in nine (45%) of 20 eyes
with the new one. There was no significant difference
between the two groups (p = 0.16). The mean duration of
the postoperative double anterior chamber was 1.5 (2.5) days
(range 0–10 days) with our method and 4.7 (16.7) days
(range 0–120 days) with the conventional one, and there was
a significant difference between the methods (p = 0.038).
Because of a postoperative double anterior chamber in three
eyes (5.2%) PKP was performed by the conventional method
and in none by the new method. The mean postoperative,
best corrected visual acuity was 0.55 (dispersion factor (DF):
1.93, n = 45) for the conventional method and 0.73 (DF: 1.34,
Deep lamellar keratoplasty
Table 1
1599
Comparison of the conventional and new methods
Duration of surgery (minutes)
Intraoperative Descemet’s membrane perforation
Conversion to PKP (eyes)
Postoperative double anterior chamber (eyes)
Duration of the postoperative double anterior
chamber (days)
Postoperative best corrected visual acuity
Postoperative corrected visual acuity 1 month later
Endothelial cell density at 1 year (cells/mm2)
New method
Conventional method
59 (26.1)* (n = 22)
5 (22.7%) (n = 22)
2 (9%) (n = 22)
17 (29.3%) (n = 20)
1.5 (2.5)* (n = 20)
94 (25.7)* (n = 67)
25 (37.3%) (n = 67)
25 (37.3%) (n = 67)
9 (45%) (n = 58)
4.7 (16.7)* (n = 58)
0.73 (DF; 1.34)* (n = 18)
0.70 (DF: 0.30)* (n = 18)
1722.8 (665.3) (n = 18)
0.18 (DF: 2.44)* (n = 45)
0.18 (DF: 2.44)* (n = 45)
2044.8 (678.2) (n = 45)
*p,0.05 between groups.
n = 18) for the new one. The difference between the groups
was significant (p = 0.039). The mean postoperative corrected
visual acuity 1 month later was 0.18 (DF: 2.44, n = 45) for
the conventional method and 0.70 (DF: 0.30, n = 18) for the
new one, the difference between the groups being statistically
significant (p,0.0001). Mean corneal endothelial cell density
at 1 year was 2044.8 (678.2) cells/mm2 for the conventional
method and 1722.8 (665.3) cells/mm2 for the new method;
this difference between the groups was not significant
(p = 0.15).
CASE REPORT
Macular corneal dystrophy was diagnosed in a 38 year old
man, and this type of operation indicated. (The surgical
findings for this patient are shown in a paragraph under
‘‘surgical technique’’.) His preoperative visual acuity was 0.4,
and intraocular pressure 12 mm Hg. Corneal endothelial cell
density was not measured for corneal opacity. Surgery was
performed without complications in a period of 34 minutes.
Figure 7 shows photographs taken before and after surgery.
His postoperative visual acuity was 1.2, intraocular pressure
13 mm Hg, and corneal endothelial cell density 2500 cells/
mm2.
DISCUSSION
Deep lamellar keratoplasty has certain advantages over
penetrating keratoplasty for many patients who retain
endothelial function because endothelial rejection is not
expected and postoperative visual acuity is equal to that
under PKP. Furthermore, as there is no need for a graft to
obtain endothelial cells, a stored donor cornea can be used.1–4
Figure 7 Case: Corneal macular dystrophy. Preoperative visual acuity
was 0.4, postoperative visual acuity 1.2. Postoperative endothelial cell
density was 2500 cells/mm2. (A) Preoperative finding. (B) Postoperative
findings.
Although advantageous, this procedure has not been widely
used because the surgical technique is more difficult than
that for PKP. We here reported a novel method of detaching
Descemet’s membrane through a corneal limber flap, thereby
facilitating deep lamellar keratoplasty. Our surgical method,
developed from that of Sasaki et al,6 exposes Descemet’s
membrane through a limbal flap. After exposure, Descemet’s
membrane is detached by hydrodelamination. Surgeons who
have experience with DLKP and trabeculectomy can easily
understand and use this method. Our findings showed little
difference in the intraoperative complications associated with
this and the conventional method and that conversion to PKP
during surgery was infrequent, except in special cases.
Detachment of Descemet’s membrane was easy in patients
who did not have adhesion of the membrane and pathological parenchyma. In those who presumably had such
adhesion, forced injection of viscoelastic material used in
hydrodelamination caused cracking of the membrane. In
cases in which there is possible adhesion of Descemet’s
membrane to the corneal parenchyma, as when there is
keratoconus after acute corneal hydrops, the membrane must
be very carefully detached.
A postoperative double anterior chamber tended to form
with greater frequency under this new method, but it soon
disappeared because there was no remaining corneal
parenchyma unlike under the conventional method. With
this new method, a PKP reoperation caused by a postoperative double anterior chamber was needed. This is
because the corneal parenchyma had been excised completely. With the conventional method, there were three cases of
further PKP surgery.
As for mean postoperative best corrected visual acuity and
mean postoperative visual acuity 1 month after surgery, the
new method was better statistically than the conventional
one, the mean postoperative corrected visual acuity at
1 month being 0.18 for the conventional method and 0.70
for the new one. Other reports have suggested that the cause
of postoperative low visual acuity is scarring that occurs at
the interface between the donor cornea and parenchyma of
the recipient cornea.1 Because histological reconstruction can
be done early, owing to complete Descemet’s membrane
exposure by our new method, there is early visual acuity.
Melles et al reported a method used in a cataract operation
whereby Descemet’s membrane was detached by direct
injection of a viscoelastic material after making a sclerocorneal tunnel.6–9 The differences between their method and
ours are: (1) our new method uses trabeculectomy to detach
Descemet’s membrane, whereas Melles et al used an incision,
as in cataract surgery; (2) a flap is made, as in trabeculectomy, and the region directly above Descemet’s membrane is
reached under direct vision in the new method, whereas
Melles et al used air replacement in the anterior chamber and
a mirror image at the tip of a 30 gauge needle in order to be
directly above Descemet’s membrane from the corneal
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1600
parenchyma; (3) in our new method, Descemet’s membrane
is detached by hydrodelamination and viscoelastic material
used to maintain the space between the membrane and
corneal parenchyma, whereas Melles et al detached the
membrane by means of viscoelastic materials. This difference
ensures easy, reliable exposure of Descemet’s membrane for
the following reasons. With the trabeculectomy-like
approach used with the new method, the depth near
Descemet’s membrane can be confirmed under direct vision,
the procedure can be done with smaller incisions, and
surgeons with experience in trabeculectomy can easily
recognise the exposure image of Schlemm’s canal. When
there is severe corneal opacity, obtaining mirror images is
difficult by the method of Melles et al. If exposure of
Schlemm’s canal or of Descemet’s membrane can be
confirmed, subsequent surgical procedures can be done more
surely. In our experience, visual confirmation under a
microscope is essential for the success of this type of surgery,
providing a wider range of indications and reliable surgical
results. In eyes without Descemet’s membrane adhesion,
there was no difference between use of the method of Melles
et al and the new one. The former, without hydrodelamination, resulted in a faster surgical procedure. When, however,
there was adhesion between Descemet’s membrane and the
corneal parenchyma large cracks in the membrane tended to
occur when it was detached by viscoelastic material. Once the
membrane is cracked, the viscoelastic material may enter the
anterior chamber, making repair difficult. These findings
indicate that forced detachment of Descemet’s membrane by
the use of viscoelastic material should be avoided when
possible. In this respect, our new procedure is considered a
relatively safer means by which Descemet’s membrane is
detached gently by hydrodelamination. Surgery can be done
after confirming the presence or absence of Descemet’s
membrane adhesion.
A drawback of our new method that requires further study
is the sharp rise in intraocular pressure because the
membrane is exposed over the entire range of resection.
Another complication is that carelessness in suturing the
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Senoo, Chiba, Terada, et al
donor corneal flap is apt to cause cracks in the membrane.
Actually, of the three cases of Descemet’s membrane
perforation (excluding the two cases of keratoconus with
Descemet’s membrane adhesion), two were the result of
membrane cracking as a result of a rise in intraocular
pressure from the side port caused by air replacement after
membrane exposure. The other was a case of Descemet
membrane perforation by a needle during suturing of the
donor corneal flap. At present, we see to it that the graft
diameter is shortened (7.0–7.25 mm) and that a small side
port is made. Problems that occur because of the complete
exposure of Descemet’s membrane, however, need further
study.
.....................
Authors’ affiliations
T Senoo, K Chiba, O Terada, J Mori, M Kusama, Y Obara, Dokkyo
University School of Medicine, Department of Ophthalmology, Japan
K Hasegawa, International University of Health and Welfare, Japan
REFERENCES
1 Sugita J, Kondo J. Deep lamellar keratoplasty with complete removal of
pathological parenchyma for vision improvement. Br J Ophthalmol
1997;81:184–8.
2 Morrison JC, Swan KC. Full-thickness lamellar keratoplasty. A histologic study
in human eyes. Ophthalmology 1982;89:715–19.
3 Panda A, Bageshwer LM, Ray M. Deep lamellar keratoplasty versus
penetrating keratoplasty for corneal lesions. Cornea 1999;18:172–175.
4 Krumeich JH, Daniel J. Depth of lamellar keratoplasty with the guided trephine
system for transplantation of full-thickness donor sections. Ophthalmologe
1998;11:748–54.
5 Sasaki K, Shikatani Y. Exposure of Descemet membrane through corneal
limbal approach in deep lamellar keratoplasty. Jap J Clin Ophthalmol
2001;55:297–9.
6 Melles GR, Lander F, Rietveld F, et al. A new surgical technique for deep
parenchymal, anterior lamellar keratoplasty. Br J Ophthalmol
1999;83:327–33.
7 Melles GR, Remeijer L, Geerards AJ, et al. A quick surgical technique for deep
anterior lamellar keratoplasty using visco-dissection. Cornea
2000;19:427–32.
8 Melles GR, Rietveld FJ, Beekhuis WH, et al. A technique to visualize corneal
incision and lamellar dissection depth during surgery. Cornea 1999;18:80–6.
9 Melles GR, Remeijer L, Geerards AJ, et al. The future of lamellar keratoplasty.
Curr Opin Ophthalmol 1999;10:253–9.
1601
EXTENDED REPORT
Contact lenses and special back surface design after
penetrating keratoplasty to improve contact lens fit and
visual outcome
C Gruenauer-Kloevekorn, U Kloevekorn-Fischer, G I W Duncker
...............................................................................................................................
Br J Ophthalmol 2005;89:1601–1608. doi: 10.1136/bjo.2005.069500
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr Claudia GruenauerKloevekorn, Department of
Ophthalmology, MartinLuther University, ErnstGrube-Strasse 40, 06097
Halle, Germany; claudia.
gruenauer-kloevekorn@
medizin.uni-halle.de
Accepted for publication
15 June 2005
.......................
P
Aims: To describe the fitting of patients with high or irregular astigmatism following penetrating
keratoplasty with contact lenses and to answer the question whether or not contact lenses with special back
surface design can improve visual acuity in complex cases after penetrating keratoplasty.
Methods: 28 eyes were included. They were fitted with contact lenses with a special back surface that was
designed for optical rehabilitation after penetrating keratoplasty. Four different types of these lenses
(tricurve, keratoconus, reverse, oblong) were used selectively depending on abnormal eccentricity
determined by videokeratoscope. The patients were followed up for an average period of 15.5 months.
Lens tolerance and corrected visual acuity were evaluated and compared with that corrected with
spectacles.
Results: The visual acuity was significantly improved in nearly all eyes with an average increase of 3.6
lines (maximal nine lines) accompanied by good contact lens tolerance and satisfactory contact lens fit. No
noticeable complications were observed.
Conclusion: Contact lenses with special back surface design can improve visual results and lens tolerance,
and minimise problems in contact lens fitting. This is in favour of contact lenses as an alternative to surgical
procedures for correction of high or irregular astigmatism after penetrating keratoplasty. This procedure is
recommended especially in cases of patients who decline further operative interventions.
ostoperative astigmatism is the main reason for unsatisfactory visual results after grafting. Various studies
have shown that the number of grafts with (3D of
astigmatism 2 years after transplantation ranges between
27% and 34%, depending on the indication for corneal
grafting.1 2 To achieve visual benefit and binocularity there is
a strong necessity for optical or operative correction of the
postoperative high astigmatism. A number of surgical and
non-surgical approaches have been taken to reduce postoperative irregular astigmatism.
Surgical options for dealing with cylindrical error were
already developed 40 years ago.3 4
Suture adjustment or selective suture removal are the first
option to reduce the postoperative astigmatism.5–9
In regular astigmatism with a well defined steep axis
relaxing incisions may be beneficial. However, they are
limited by the common undercorrection following a suboptimal predictability.1 10–13 For relaxing incisions the spherical equivalent should not exceed 1.5 dioptres. Relaxing
incisions are followed by a flattening of the steep meridian
and a steepening of the flat meridian (coupling effect). Thus,
additional correction of the arising spherical refraction error
may be necessary.13 14
There are many approaches to correct post-keratoplasty
astigmatism using an excimer laser.15–18 In myopic astigmatism up to 6 dioptres a two step LASIK procedure can be
performed.19–23 Although excimer laser treatment of higher
degrees of astigmatism (beyond 6D) is possible, the
attempted correction of higher degrees of astigmatism may
expose the patient to a regression of astigmatism, poor
qualitative visual outcome, and even loss of several lines of
best corrected visual acuity (BCVA).19 In high astigmatism of
more than 8 dioptres and in cases of cataract the implantation of a toric posterior chamber lens through a clear cornea
incision at the steep axis should be preferred.24–26 In cases of
irregular astigmatism, caused by an irregular transplant
surface, correction by means of an excimer laser assisted by
sodium hyaluronidate may be possible.27 28
Complications, such as remaining astigmatism, much
higher incidence of grade III to IV haze formation in the
graft, and even corneal graft rejections, are mainly described
after surface ablation using different excimer lasers.15 29–31
All these surgical procedures, except for suture adjustment
or selective suture removal, should be performed 3 months
after removal of the keratoplasty sutures at the earliest and
depend on stable conditions.31
The non-surgical approaches to the management of postkeratoplasty astigmatism include spectacles and different
groups of contact lenses. Especially in those patients with
high or irregular astigmatism and anisometropia sufficient
visual rehabilitation can often not be achieved with
spectacles. In these cases contact lens fitting is a good option.
Among the different kinds of contact lenses a rigid gas
permeable lens may be the correction of choice, since this
type of lens provides good visual acuity, corrects high degrees
of regular and irregular astigmatism, has high oxygen
permeability, and, in comparison with soft contact lenses, a
lower risk for microbial keratitis (incidence 1/10 000) and for
corneal neovascularisation.32–34 Because of the special conditions after grafting, leading to changes in the corneal shape
especially at the scar between graft and host cornea, in most
cases only rigid gas permeable contact lenses with a special
back surface design can lead to optimal fit and visual
results.2 35–37
The aim of this study was to determine the special
corneal conditions after penetrating keratoplasty using
Abbreviations: BCVA, best corrected visual acuity; HOA, higher order
aberrations; RGP, rigid gas permeable; RMS, root mean square; TD,
topographic disparity
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1602
Gruenauer-Kloevekorn, Kloevekorn-Fischer, Duncker
videokeratoscopic indices and to clarify which special back
design may be used selectively and whether or not fitting of
special back surface designed contact lenses can improve
visual acuity in complex cases of high or irregular astigmatism after penetrating keratoplasty.
A
B
r3
r1
r2
r3
r1
r0
r0
PATIENTS AND METHODS
Data evaluation
A retrospective chart review was conducted of all patients
who were fitted with specially designed contact lenses
following penetrating keratoplasty between January 2000
and April 2004. The data analysis of 28 eyes in 21 patients, 13
males and eight females, included age, previous ophthalmic
history, corneal disorder, time from keratoplasty to contact
lens fitting, preoperative visual acuity, postoperative best
corrected visual acuity with spectacles, postoperative best
corrected visual acuity with contact lens, contact lens back
surface design, and follow up time. Corneal curvature,
eccentricity (e), Fourier indices, and Zernike-coefficients
were performed with the Oculus computerised videokeratoscope (Oculus, Wetzlar, Germany; Software Version 1.64).
Anterior surface of the cornea
The anterior surface of the cornea has a complex shape.38 39 To
analyse this shape statistically based on its videokeratographic colour coded map, different quantitative parameters,
such as Fourier series harmonic analysis,40 Zernike polynomials,41–45 or calculation of topographic disparity (TD)46 by
vectorial values have been developed in order to quantify the
corneal irregularity. The quantification of the irregular
astigmatism by calculation of topographic disparity is very
useful and manageable for surgical treatment of irregular
astigmatism.47 Fourier harmonic series analysis is an efficient, quantitative means of describing corneal irregular
astigmatism.48–55
Fourier series harmonic analysis
Using Fourier series harmonic analysis we can decompose
corneal topography data into a series of trigonomic functions
Table 1 Profile and classification of the patients and the
different kinds of fitted contact lenses
C
r3
r2
r1
r0
Figure 1 (A) Tricurve design. r0, central radius; r1, connecting radius
between r0 and r2; r2 peripheral radius. r2 is of 0.8 mm smaller than r0.
(B) Keratoconus design. r0, central radius; r1; r2; r3 peripheral radii. The
difference between r0 and the peripheral radius r2 is much greater than
the central radius. r3 is very flat. (C) Reverse design (tetracurve.) r0,
central radius; r1; r2; r3 peripheral radii. r2 is significantly steeper
(reverse) than the central radius r0. r3 (second reverse zone) is flatter
than r2, but steeper than r0.
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Total number
Male
Female
Binocular
Age
Indications for penetrating keratoplasty
Keratoconus
Fuchs’ dystrophy
Corneal scarring
Corneal ulcer
Herpes
Follow up time (mean)
Time between keratoplasty and first
contact lens fitting (mean)
Sutures removed?
One
Both
No
Contact lens material
Rigid gas permeable
Soft
Contact lens back surface design
Tetracurve reverse
Tricurve
Bitoric
Keratoconus design
Oblong
Contact lens front surface design
Front toric
28
13
8
7
Mean 43 years (min 20
years; max 61 years)
14
6
4
2
2
15.5 months (max
50 months; min 4 months)
42.5 months (max
336 months; min 2 months)
2
19
7
28
0
10
11
5
3
4
1
Age, follow up time, and time between keratoplasty and first contact lens
fitting are giving as means.
The group of tricurve contact lenses is subdivided into one group without
bitoric design (6) and one with bitoric design. The front toric design,
which does not influence the back design but can improve the refraction,
is part of the tricurve lenses.
Contact lenses with special back surface design after penetrating keratoplasty
and thereby quantify each component, such as the spherical
power, the regular astigmatism, asymmetry, and higher order
irregular astigmatism.41
There are 21 rings for each image. Dioptric powers on a
mire ring i, Fi(s) were transformed into trigonometric
components of the following form41 48–51:
Fi(s) = a0+c1 cos(s2a1)+c2cos2(s2a2)+c3cos3(s2a3)+…
+cncosn (s2an)
The resulting components of all rings are regrouped and
displayed in separate images, where zero order (a0) is the
spherical equivalent, first order (2c1) is the asymmetry
component (tilt or decentration), second order (2c2) is the
regular astigmatism component and third and higher orders
(c3…n) are the higher order irregularity component. Among
these, spherical equivalent power a0 and regular astigmatism
(2c2) can be corrected by a spherocylindrical lens, while the
remaining components (2c1; c3…n) cannot. 2c1 and c3…n
represent corneal irregular astigmatism.
The normal range of the Fourier indices are defined as the
mean (2 SD) in normal eyes as described at Tanabe et al,
which are 40.81–47.13D for spherical power, 0–1.04D for
regular astigmatism, 0.02–0.68D for asymmetry, and 0.05–
0.17D for higher order irregularity.50
Zernike coefficients
Optical aberrations of the human eye have a major role in the
degrading retinal image quality.41–45 56 They are typically
described in terms of wavefront error. Wavefront error is
the difference between the ideal wavefront and the actual
wavefront error of the optical system. Such deviations are
commonly classified by means of Zernike polynomials.45
Among this group, the Zernike coefficients from the third
Figure 2 (A, B) Videokeratography in a case of 0(e(0.4 (patient 6 in
table 2). There is a steep central graft and a flatter peripheral host. With
a tricurve designed lens (fig 1A) we reached an improvement of visual
acuity of three lines with a good contact lens fit. Remaining astigmatism
after the first fitting was corrected by an additional front toric design.
1603
to the sixth order describe the higher order aberrations
(HOA), which are related to symptoms such as halo, glare,
and decreased contrast sensitivity.56 57 There is a wide
individual variability in anterior corneal aberrations and this
is also influenced by age related changes.56 57
From the Zernike coefficients, we calculated the root mean
square (RMS) of higher order aberrations (HOA, square root
of the sum of the squared coefficients of orders 3 up to 6).
The normal range of the HOA RMS error is defined as the
mean (2 SD) in normal eyes as described by Wang et al, which
is 0.234–0.857 mm.56
Eccentricity
The application of mathematical equations for an ellipse is
very useful and manageable for contact lens fitting. A
common approach is to use the central radius of the ellipse
on the one hand and the eccentricity, e, the shape factor, p, or
the asphericity, Q, on the other hand.58 59 These factors
describe the changing of the radius towards the periphery.
The shape factor p and the asphericity Q are results of further
calculations using the eccentricity e.58 59
e = (1/sina) 6 !1 2 (r0/rs)2
where e = eccentricity, a = measurement angle, r0 =
central radius, rs = sagittal radius
p = 1 2 e2
where p = shape factor
Q = 2e2
where Q = asphericity.
In healthy corneas the eccentricity lies between 0.5 and
0.7.58 59
Figure 3 (A, B) Videokeratography in a case of 0(e(0.4 and
astigmatism within the graft as well as within the host (patient 11 in
table 2). In this case for optimal contact lens fit we chose a bitoric tricurve
design mostly independent of the eccentricity. We achieved an improved
visual acuity of four lines with a good contact lens fit.
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1604
Figure 4 (A, B) Videokeratography in a case of 0.7(e(0.9 (patient 14
in table 2). We fitted a keratoconus designed lens (fig 1B) and achieved an
improvement of visual acuity of two lines with a good contact lens fit.
Gruenauer-Kloevekorn, Kloevekorn-Fischer, Duncker
Figure 6 (A, B) Videokeratography in a case of extremely steep host
and high central astigmatism (patient 26 in table 2). With a reverse
designed lens (oblong; fig 1C) we reached an improvement of visual
acuity of three lines limited by a cataract. The contact lens fit was
tolerable.
A specific eccentricity is calculated especially in eyes after
penetrating keratoplasty.2 The eccentricity increases in size in
cases of a steep graft and a flat recipient’s bed. In these cases
we always find a positive eccentricity, in some cases greater
than 1. The eccentricity decreases in cases of a flat graft and a
steep recipient’s bed. In these cases the eccentricity can be
lower than 0.3 up to a negative eccentricity.
Nearly all commercially available contact lenses are
evaluated by eccentricity.
Contact lens fitting and anterior surface of the cornea
Depending on the central curvatures and the eccentricity of
the corneas we selected the back surface design of our contact
lenses. Four different kinds of special back surface contact
lenses were fitted. All contact lenses were made of rigid gas
permeable (RGP) material.
Tricurve design (fig 1A)
This is the contact lens that we fit if the eccentricity is
between 0 and 0.4. The back surface design of the contact
lens consists of three curves. The peripheral radius r2 is
0.8 mm smaller than the central radius r0; radius r1 connects
r0 and r2.
Tricurve designed lenses are the only contact lenses with
special back design that can be used in affiliation with a
torical back design.
Figure 5 (A, B) Videokeratography in a case of negative eccentricity
(patient 27 in table 2). There is a flat central graft and a steep peripheral
host. With a reverse designed lens (oblong; fig 1C) we reached an
improvement of visual acuity of seven lines with a good contact lens fit.
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Keratoconus design (fig 1B)
This kind of back surface design can be fitted if the
eccentricity is greater than 0.7. The difference between r0
Contact lenses with special back surface design after penetrating keratoplasty
and the peripheral radius r2 is bigger and the periphery r3 is
very flat.
1605
design can improve the fit of the contact lens and
additionally correct remaining astigmatism. In these cases
the role of eccentricity is minimised.
Tetracurve reverse design (fig 1C)
The second zone r2 of this design is significantly steeper than
the central radius r0. This is the derivation of the term
reverse. We have a second reverse zone r3 which is flatter
than the first reverse zone r2, but steeper than or equal to the
central radius r0. We use this contact lens in case of negative
eccentricity, especially if the patient has high central
astigmatism.
Oblong design
This is also a reverse designed lens and is comparable to the
tetracurve variation, but the periphery becomes aspheric
instead of tricurve. The eccentricity of the oblong design can
change from 20.4 to 20.9.
We also fit this kind of contact lens in cases of negative
eccentricity.
Backtoric and bitoric design
In cases of regular astigmatism from the central graft up to
the periphery of the host the choice of backtoric or bitoric
Patients
Table 1 shows the profile and the classification of the 28
patients and the kinds of contact lens fitted. In none of these
patients could we fit a contact lens with regular back surface
design because of problematical corneal conditions after
penetrating keratoplasty. The mean follow up time was
15.5 months and the time between keratoplasty and the first
contact lens fitting was 42.5 months. In 19 cases both sutures
were removed, in two cases one suture was removed, and in
seven patients the sutures were in place. In all patients the
eccentricity e was outside 0.5–0.7 and in 17 cases the
eccentricity was negative. Depending on this eccentricity
and on individual conditions we fitted 11 tricurve contact
lenses (fig 2A and B), of which five were bitoric (fig 3A and
B), three contact lenses with keratoconus design (fig 4A and
B), and 14 reverse contact lenses, of which 10 with a
tetracurve design and four an oblong design (figs 5 and 6A
and B). In addition, a front toric design was necessary in one
patient.
Table 2 Keratometric profiles of all 28 patients
Zernike
coefficients
Fourier series harmonic analysis
Spherical
power
Reg ast
3 mm
Reg ast
6 mm
Asymmetry
Higher order
irregularity
Normal range (D)50
No
simK
Axis
Tricurve design
1
26.8
43.1˚
2
24.1
25.9˚
3
23.8
143.1˚
4
27.4
140.9˚
5
28.7
11.1˚
6
21.9
159.5˚
7
25.5
9.3˚
8
25.3
160.3˚
9
24.8
158.1˚
10
23.6
169.3˚
11
28.8
11.3˚
Keratoconus design
12
21.8
141.6˚
13
27.2
39˚
14
22.9
1.7˚
Reverse design
Tetracurve reverse design
15
26.8
13˚
16
26.9
172.1˚
17
29.8
32.1˚
18
22.21
135.2˚
19
20.92
107.1˚
20
20.8
140.2˚
21
24.3
25.3˚
22
210.2
10.6˚
23
25.6
4.3˚
24
22.0
44.2˚
Oblong reverse design
25
29.4
154.1˚
26
214.8
0.4˚
27
21.0
25.9˚
28
25.0
2.3˚
HOA RMS error
Eccentricity
Normal range
(mm)56
Normal range58 59
40.81–47.13
0–1.04
0–1.04
0.02–0.68
0.05–0.17
0.234–0.857
0.5–0.7
46.86
62.69
44.33
54.11
48.14
42.75
52.09
47.86
45.04
49.34
45.78
2.31
1.37
1.97
1.92
3.92
1.77
2.26
2.55
2.16
1.42
3.48
1.42
1.97
1.22
1.77
1.97
0.34
1.77
1.27
1.27
0.92
2.50
4.40
3.16
6.91
3.79
5.62
2.50
6.53
0.97
4.27
2.69
0.81
0.45
0.30
0.25
0.38
0.66
0.31
0.24
0.37
0.29
0.50
0.37
0.80
3.47
0.57
0.84
0.97
0.28
1.33
0.41
0.48
0.48
5.38
0.35
0.1
0.25
0.29
0.39
0.01
20.77
20.73
0.0
20.9
0.28
55.17
54.28
46.73
1.32
0.55
0.24
1.77
0.66
1.37
2.36
3.43
1.72
0.51
0.23
0.48
0.65
0.41
0.35
0.89
0.75
0.82
52.34
45.28
47.52
45.28
49.41
49.34
43.24
49.93
46.09
50.76
3.43
2.60
3.57
5.25
3.97
0.76
2.02
3.38
2.12
1.27
1.42
2.07
2.02
4.53
3.66
0.34
2.02
3.43
1.62
1.02
1.87
2.60
1.97
3.97
3.79
1.72
3.92
4.01
3.34
3.88
0.28
0.29
0.67
0.19
0.15
0.21
0.49
0.23
0.23
0.29
0.62
0.54
0.70
1.08
0.96
0.29
0.50
1.24
0.60
0.66
20.74
20.32
20.89
20.35
20.02
20.22
20.02
20.12
20.17
20.07
41.58
48.98
51.22
46.47
Mean (SD)
48.66 (4.46)D
4.96
5.54
0.50
2.12
3.75
4.53
0.55
1.37
3.06
1.87
1.52
2.40
0.69
0.56
0.18
1.03
0.70
1.12
0.34
0.51
20.37
20.91
20.92
20.38
2.45 (1.41)D
1.88 (1.15)D 3.18 (1.51)D
0.39 (0.20)D
0.94 (1.06)
All values out of the normal range are in italics.
All means of the Fourier series harmonic analysis and of the HOA RMS are out of the normal range.
The irregular astigmatism within the Fourier series harmonic analysis (asymmetry and higher order irregularity) is out of the normal range in all patients.
Reg ast 3 mm, central regular astigmatism; Reg ast 6 mm, peripher regular astigmatism.
HOA RMS error, root mean square error of the higher order aberrations (third to sixth order).
www.bjophthalmol.com
1606
Gruenauer-Kloevekorn, Kloevekorn-Fischer, Duncker
Table 3 Profile of all 28 patients divided into the different contact lens groups
No
Age/sex
Diagnosis leading
to keratoplasty
Tricurve design
1
36/M
Keratoconus
2
65/F
Fuchs’ dystrophy
3
49/F
Keratoconus
4
43/M
Keratoconus
5
37/M
Herpes
6
35/M
Keratoconus
7
49/F
Fuchs’ dystrophy
8
65/F
Fuchs’ dystrophy
9
43/M
Keratoconus
10
57/M
Keratoconus
11
23/M
Herpes
Keratoconus design
12
62/F
Scarring
13
61/M
Fuchs’ dystrophy
14
52/M
Scarring
Reverse design
Tetracurve reverse design
15
41/M
Keratoconus
16
36/F
Ulcer
17
57/M
Keratoconus
18
36/M
Keratoconus
19
36/M
Keratoconus
20
17/F
Scarring
21
46/M
Scarring
22
26/F
Ulcer
23
20/F
Keratoconus
24
20/F
Keratoconus
Oblong reverse design
25
69/F
Fuchs’ dystrophy
26
69/F
Fuchs’ dystrophy
27
34/M
Keratoconus
28
52/M
Keratoconus
First CL
(months)
Central radius
Eccentricity
Spectacle VA
CL VA
17
6
336
48
26
12
60
60
12
20
4
7.98
6.08
8.95
7.16
8.23
8.06
7.37
7.88
8.69
7.92
8.27
6.88
5.66
8.12
6.18
6.79
7.72
6.58
7.02
7.81
7.29
6.81
0.35
0.1
0.25
0.29
0.39
0.01
20.77
20.73
0.0
20.9
0.28
0.5
0.02
0.2
0.2
0.5
0.3
0.6
0.5
1.0
0.8
0.6
0.7
0.05
0.5
0.8
0.9
0.6
0.9
0.9
1.25
1.0
1.0
17
60
336
6.54
7.40
7.49
6.32
6.78
7.03
0.89
0.75
0.82
0.2
0.05
0.6
0.7
0.1
0.8
8
17
14
60
17
15
15
42
10
2
7.74
8.18
8.81
6.97
7.95
6.95
8.54
7.86
8.01
6.77
6.69
7.00
7.01
6.85
6.83
6.83
7.70
6.36
7.07
6.51
20.74
20.32
20.89
20.35
20.02
20.22
20.02
20.12
20.17
20.07
0.2
0.7
0.2
0.5
0.2
0.4
0.1
0.1
0.4
0.4
0.6
0.9
0.5
1.0
0.9
0.7
0.2
0.9
0.6
0.8
18
3
34
12
9.68
8.76
7.33
7.72
7.62
6.33
7.18
7.38
20.37
20.91
20.92
20.38
0.1
0.1
0.2
0.1
0.9
0.4
0.9
0.3
Specialties
Amblyopia
mydriasis
Front toric
Bitoric CL
Bitoric CL
Bitoric CL
Bitoric CL
Bitoric CL
Amblyopia
Cataract
The selected lens design depends strongly on the eccentricity e. We fitted a tricurve design lens (fig 1) in cases of 0(e(0.4; a keratoconus design in cases of
0.7(e(0.9; a reverse design (oblong/tetracurve) in cases of negative eccentricity. If the astigmatism involves both the graft and the host, a bitoric design is
recommendable. In this case the role of eccentricity is minimised. If we found remaining astigmatism but a good fit, we selected a front toric design.
RESULTS
Table 2 summarises the keratometric data and table 3 the
data of contact lens fitting on all patients.
All patients had had an increased irregularity of the
anterior cornea. Using Fourier series harmonic analysis 16
eyes (57.2%) had spherical power out of the normal range, 27
eyes (96.4%) had central and peripheral regular astigmatism
out of the normal range and all eyes (100%) had irregular
astigmatism (asymmetry and higher order irregularity) out of
the normal range. Using Zernike coefficients eight eyes
(28.8%) had HOA RMS error out of the normal range.
In all 28 cases good visual rehabilitation was achieved with
an increase in visual acuity of up to nine lines and good
DISCUSSION
1.2
BCVA contact lenses
1.0
0.8
0.6
0.4
0.2
0
contact lens tolerance (CL tolerance without any disturbances for 6 hours/day).
In all patients it was possible to improve the visual acuity
in comparison with best corrected visual acuity with
spectacles (fig 7). The minimum improvement was one line.
In two cases of monocularity in which keratoplasty on the
second eye was planned the fitted contact lens led to less
improvement of the visual acuity, as in one patient with high
ametropia fitted with a lens to achieve binocularity. In two
cases in which vision improved only minimally the reason
was amblyopia, in another case a cataract and in a fourth
case persisting mydriasis. We were able to achieve a
satisfactory contact lens fit and we did not observe severe
contact lens complications during follow up period.
0
0.2
0.4
0.6
0.8
1.0
1.2
BCVA spectacles
Figure 7 Scatter plot of best corrected visual acuity (BCVA) with
spectacles in comparison with best corrected visual acuity with contact
lens. All spots above the line indicate improvement.
www.bjophthalmol.com
Contact lens fitting has an essential role in visual rehabilitation in cases of irregularities of the corneal surface following
penetrating keratoplasty and is an alternative to surgical
procedures, especially in patients who decline further surgical
interventions or wish transient improvement of visual acuity
before further interventions are necessary.
Nevertheless, the problematic anterior corneal surface after
penetrating keratoplasty can make it very difficult to achieve
a sufficient correlation between anterior surface of the cornea
and back surface of the contact lens and, as a result,
improved visual acuity and good contact lens tolerance.
The circumstances, that in all of the examined eyes the
irregular astigmatism was out the normal range and in 14 eyes
(50%) all Fourier indices were out of the normal range,
demonstrate the specific corneal conditions after penetrating
keratoplasty and confirm several quantitative studies on the
corneal irregular astigmatism after penetrating keratoplasty.60 61
Contact lenses with special back surface design after penetrating keratoplasty
Additionally in eight eyes (28.8%) we found an increased
HOA RMS in comparison with nontreated eyes, which is
comparable to the results of Yagci et al.62
Because of this sophisticated situation of the surfaces of
graft and host, in most of the cases only rigid gas permeable
contact lenses with special back design can solve all of these
problems.35237
Publications on the fitting of contact lenses after penetrating keratoplasty are rare and often limited to fitting of only
one special contact lens back surface design. Some authors
describe them among a group of different corneal pathologies. Several publications are small case series or clinical
opinions and none of them describe the correlation between
the corneal surface and the choice of the contact lens
design.35237
To our knowledge this is the first study to demonstrate the
use of rigid gas permeable contact lenses with different back
designs depending on the corneal conditions (eccentricity)
and to include a long term follow up of the patients of about
15 months.
We were able to fit all of our patients with contact lenses
and to achieve improvement of the visual acuity and good
contact lens tolerance. There were no severe complications in
any of the cases.
In our opinion the most important changes in the corneal
design are those towards the periphery of the cornea, which
are expressed by the eccentricity. Depending on the eccentricity, contact lenses with different back surface designs can
be chosen for fitting. Nevertheless individual conditions have
an essential role.
Contact lenses are one alternative for correction of high or
irregular astigmatism after penetrating keratoplasty that
involve minor risks and bring good visual results. In our
opinion, contact lenses with special back surface design can
minimise problems in contact lens fitting and can improve
the tolerance and the visual results. We recommend this
procedure especially in cases of poor operative prognosis, for
patients who decline further surgical interventions or for
early postoperative correction of astigmatism.
ACKNOWLEDGEMENTS
This study was supported by the European Social Fond and the
County of Sachsen-Anhalt, Germany.
.....................
Authors’ affiliations
C Gruenauer-Kloevekorn, G I W Duncker, Department of
Ophthalmology, Martin-Luther University, Halle, Germany
U Kloevekorn-Fischer, Institute of Optometry, Trothe-Optik, Halle,
Germany
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1609
EXTENDED REPORT
Lower limits of fluorescein and indocyanine green dye for
digital cSLO fluorescence angiography
A Bindewald, O Stuhrmann, F Roth, S Schmitz-Valckenberg, H-M Helb, A Wegener, N Eter,
F G Holz
...............................................................................................................................
Br J Ophthalmol 2005;89:1609–1615. doi: 10.1136/bjo.2005.070409
See end of article for
authors’ affiliations
.......................
Correspondence to:
Frank G Holz, MD,
Department of
Ophthalmology, University
of Bonn, Ernst-AbbeStrasse 2, D-53127 Bonn,
Germany; frank.holz@
ukb.uni-bonn.de
Accepted for publication
1 July 2005
.......................
F
Background: With the advent of digital confocal scanning laser ophthalmoscopy it is possible to detect low
levels of fluorescence. Here we used a novel confocal scanning laser ophthalmoscope (cSLO) to determine
lower limits of dye required for fluorescein (FL) and indocyanine green (ICG) angiography.
Methods: A cSLO (Heidelberg retina angiograph 2, Heidelberg Engineering, Dossenheim, Germany) with
an optically pumped solid state laser (488 nm) for FL and a diode laser (790 nm) for ICG angiography
(FL/ICG-A) was used. 62 FL-As were performed in 53 patients and 45 ICG-As were performed in 39
patients with neovascular age related macular degeneration. The volume and overall dye content of bolus
injections was gradually tapered (FL: 500 mg, 250 mg, 200 mg, 166 mg, 100 mg; ICG: 25 mg, 20 mg,
15 mg, 10 mg, 5 mg, 2.5 mg), while dye concentrations were kept constant at 100 mg/ml for FL and at
5 mg/ml for ICG. Images were obtained 1, 5, 15, and 30 minutes after dye injection. Image quality was
evaluated by two independent readers using standardised criteria.
Results: For amounts down to 166 mg for FL and to 5 mg for ICG, sufficient image quality was achieved
during all phases following injection. Only late phase images showed less contrast compared to typically
used dye amounts, which was irrelevant for interpretation and clinical management.
Conclusions: With the increased sensitivity of this novel cSLO system, amounts of injected dye during FL-A
can be reduced to one third for FL and to one fifth for ICG without relevant loss of image quality or
information compared to conventionally used dye levels. These amounts can be used for routine
angiography and allow relevant savings for units performing FL-A.
luorescence angiography (FL-A) with fluorescein (FL)
and indocyanine green (ICG) represents an important
diagnostic tool for evaluation of a wide spectrum of
retinal diseases.1–10 Two different imaging systems have been
introduced for FL-A. Conventional fundus cameras were
initially used by Nowotny and Alvis.11 As a result of the
pioneering work of Webb and co-workers, scanning laser
ophthalmoscopes (SLOs) have become available for routine
application.12–14 Fundus camera based systems use a bank of
capacitors that are discharged through a xenon flash tube for
FL-A. Photographs are recorded on film or digitised via a CCD
(charge coupled device) camera on a computer system. Frame
rate is limited with these systems for various reasons,
including time required for recharging the capacitors. With
the advent of cSLO and recent further technological
improvements, it is now possible to detect very low levels
of fluorescence in the eye.15–17 Compared to the flash light
used to illuminate the fundus of conventional fundus
cameras,18 cSLOs use a laser beam with adequate wavelength
for excitation of physiological or diagnostically injected
fluorophores. In confocal systems, light from a confocal
plane is detected while light from planes anterior and
posterior to the plane of interest are suppressed. Although
optical properties of the human eye still limit image
resolution, cSLO angiography allows for high contrast images
with a high horizontal resolution of the posterior pole of the
eye. Furthermore, it is possible to record images in real time
at a high frame rate and therefore to visualise detailed
dynamic processes. Because fluorescein and ICG have
different spectral characteristics, simultaneous angiographies
can be performed using SLO systems.19–23
Given the high sensitivity of cSLO systems compared to
conventional camera systems, we hypothesised that lower
amounts of FL and ICG dye would be required for routine FLA. Here we determined the lower limit of dye using stepwise
reduction in FL or ICG injected for FL-A using a novel cSLO.
PATIENTS AND METHODS
For consecutive FL angiography (A) and ICG-A, a novel cSLO
(HRA2, Heidelberg Engineering, Dossenheim, Germany) was
used. The principle of cSLO for FL-A has been described
previously.15 21 23–25 The HRA2 uses an optically pumped solid
state laser (488 nm) for FL-A and a diode laser (790 nm) for
ICG-A. Maximum retinal irradiance is approximately
2.0 mW/cm2 and therefore lies below the limits established
by the American National Standards Institute and other
international standards.26 Emission is recorded between
500 nm and 700 nm with a detection efficiency of 85% for
FL-A images, and above 810 nm with a detection efficiency of
66% for ICG recordings. A digital zoom at an angle of 30˚ was
used to obtain digital images of 7686768 pixels using the
continuous or single image acquisition mode at a line scan
frequency of 8 kHz (maximum 16 frames per second). For
digital image processing, the included software was used
(Heidelberg Eye Explorer, HEE, Heidelberg Engineering,
Dossenheim, Germany).
We performed 62 FL-As on 53 patients (20 male, 33 female;
age 76.5 (SD 7.5) years), and 45 ICG-As on 39 patients (15
male, 24 female, age 76.1 (9.0) years) seen in the retina
outpatient clinic of the department of ophthalmology,
University of Bonn. The patients all had neovascular age
Abbreviations: CCD, charge coupled device; cSLO, confocal scanning
laser ophthalmoscopy; FA, fluorescence angiography; FL, fluorescein;
FL-A, fluorescein angiography; ICG-A, indocyanine green angiography;
ICG, indocyanine green
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1610
Bindewald, Stuhrmann, Roth, et al
Figure 1 Grading system with four categories for fluorescence angiographies. (A) ‘‘Very good’’ (5 minutes), (B) ‘‘good’’ (5 minutes), (C) ‘‘poor image
quality’’ (15 minutes), (D) ‘‘not readable’’ (15 minutes).
Figure 2 Fluorescein angiography of a 79 year old pseudophakic patient with neovascular age related macular degeneration. This figure shows
results from injection of 500 mg (B, D, F, H) and 166 mg (A, C, E, G) of fluorescein dye. Note progression of disease after 6 weeks (top row) with more
leakage and haemorrhage, which is independent from the amount of administered fluorescein. (A, B) 1 minute, (C, D) 5 minutes, (E, F) 15 minutes, (G,
H) 30 minutes.
www.bjophthalmol.com
Fluorescein and ICG dye for digital cSLO FA
1611
Figure 3 Fluorescein angiography of
a 82 year old phakic patient using
166 mg fluorescein. All details of the
minimally classic choroidal
neovascularisation can be identified
during all phases of angiography. (A)
1 minute, (B) 5 minutes, (C)
15 minutes, (D) 30 minutes.
related macular degeneration (AMD). FL-As and ICG-As
were performed consecutively. The volumes and dye amounts
(mg) of the bolus injections were gradually tapered for both
FL (500 mg, 250 mg, 200 mg, 166 mg, 100 mg; fluorescein
10%, Alcon Pharma GmbH, Freiburg, Germany) and ICG
(25 mg, 20 mg, 15 mg, 10 mg, 5 mg, 2.5 mg; ICG-Pulsion,
Pulsion Medical Systems AG, Munich, Germany), while dye
concentrations were maintained at 100 mg/ml for FL and at
5 mg/ml for ICG. Patients were randomly assigned to
different volumes and dye amounts. All injections were
performed by the same injecting physician in an attempt to
achieve similar injection dynamics for all angiograms. In each
patient, 30˚ images were recorded at 1, 5, 15, and 30 minutes
after dye injection.
Inclusion criteria included media clear enough to allow
satisfactory imaging, especially absence of advanced lens
opacities, and informed written consent. Patients with
contraindications for FL or ICG injection (for example,
allergies to shellfish, penicillin, or iodine; pregnancy; known
allergies to either FL or ICG; or insufficient compliance and
Table 1 Fluorescein angiographies and dye amounts
used
No (n = 62)
FL amount
FL injected volume (100 mg/ml)
11
17
10
9
15
100
166
200
250
500
1.00
1.66
2.00
2.50
5.00
mg
mg
mg
mg
mg
ml
ml
ml
ml
ml
nystagmus) were excluded.27 The study was reviewed by the
appropriate ethics committee and performed in accordance
with the ethical standards laid down in the Declaration of
Helsinki.28
Quality of images was evaluated by two independent
readers using standardised criteria and classified as ‘‘very
good,’’ ‘‘good,’’ ‘‘poor image quality,’’ or ‘‘not readable’’
(fig 1). In case of a discrepancy, a third reader was asked to
arbitrate. The readers were not aware of the dye amounts
injected.
Statistical analyses were performed using commercially
available software (SPSS, SPSS GmbH Software, Munich,
Germany). Results at each time point of examination (1, 5,
15, and 30 minutes) were tested for interaction of the
amount of dye and image quality as quantitative variables
using x2 and linear by linear association tests.
RESULTS
We performed 62 FL-As in 53 patients, using an identical
concentration of dye in five different bolus volumes (table 1).
While 30 patients were phakic, 23 patients had undergone
cataract surgery. Four images of each FL-A (at 1, 5, 15, and
30 minutes after injection) were classified into the categories
as mentioned above by two independent readers. The two
readers graded a total of 428 FL-A and ICG-A images. While
complete agreement was achieved in 68.7% of the cases, in
30.8% there was a minor difference in grading—that is,
within one step of the grading scale. During the earlier phases
of FL-A (1 minute and 5 minutes after injection), a total of
96.7% of images after 1 minute and 85.5% of images after
5 minutes following injection were classified either as ‘‘very
good’’ or ‘‘good,’’ and none was classified as ‘‘not readable’’
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1612
Bindewald, Stuhrmann, Roth, et al
Table 2
FL
1 minute
100 mg
166 mg
200 mg
250 mg
500 mg
5 minutes
100 mg
166 mg
200 mg
250 mg
500 mg
15 minutes
100 mg
166 mg
200 mg
250 mg
500 mg
30 minutes
100 mg
166 mg
200 mg
250 mg
500 mg
Results of the evaluation of fluorescein angiographies
Very good
Good
Poor
Not
readable
No
6 (54.5%)
9 (52.9%)
8 (80%)
7 (77.8%)
11 (73.3%)
41 (66.1%)
5 (45.5%)
7 (41.2%)
2 (20%)
1 (11.1%)
4(26.7%)
19 (30.6%)
0 (0%)
1 (5.9%)
0(0%)
1 (11.1%)
0 (0%)
2 (3.2%)
0
0
0
0
0
0
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
11
17
10
9
15
62
3 (27.3%)
8 (47.1%)
7 (70%)
7 (77.8%)
10 (66.7%)
35 (56.5%)
6 (54.5%)
5 (29.4%)
3 (30%)
0 (0%)
4 (26.7)
18 (29%)
2
4
0
2
1
9
(18.3%)
(23.5%)
(0%)
(22.2%)
(6.7%)
(14.5%)
0
0
0
0
0
0
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
11
17
10
9
15
62
0 (0%)
3 (17.6%)
0 (0%)
2 (22.2%)
8 (53.3%)
13 (21%)
8 (72.7%)
8 (47.1%)
10 (100%)
5 (55.6%)
5 (33.3%)
36 (58.1%)
3 (27.3%)
5 (29.4%)
0 (0%)
2 (22.2%)
2 (13.3%)
12 (19.4%)
0
1
0
0
0
1
(0%)
(5.9%)
(0%)
(0%)
(0%)
(1.6%)
11
17
10
9
15
62
0
1
0
0
7
8
7 (63.6%)
8 (47.1%)
7 (70%)
7 (77.8%)
6 (40%)
35 (56.5%)
3 (27.3%)
6 (35.3%)
1 (10%)
0 (0%)
2 (13.3%)
12 (19.4%)
1
2
2
2
0
7
(9.1%)
(11.8%)
(20%)
(22.2%)
(0%)
(11.3%)
11
17
10
9
15
62
(0%)
(5.9%)
(0%)
(0%)
(46.7%)
(12.9%)
Figure 4 Indocyanine green (ICG)
angiography of a 79 year old
pseudophakic patient using the full
amount of 25 mg of dye. (A) 1 minute,
(B) 5 minutes, (C) 15 minutes, (D)
30 minutes.
www.bjophthalmol.com
Fluorescein and ICG dye for digital cSLO FA
1613
Figure 5 Indocyanine green (ICG)
angiography of a 73 year old phakic
patient using 5 mg of dye. Only the late
phase images show somewhat less
contrast. (A) 1 minute, (B) 5 minutes,
(C) 15 minutes, (D) 30 minutes.
(table 2). Statistical analysis did not reveal a significant
difference between different amounts of FL during the early
phases (linear by linear association = 0.291 for 1 minute and
0.114 for 5 minutes) (table 2). At 15 minutes after FL bolus
injection, only one image (166 mg FL) was classified as ‘‘not
readable.’’ For all volumes and dye amounts 21% of FL-A
images were classified as ‘‘very good’’ and 58.1% as ‘‘good’’;
higher amounts of FL led to an overall better classification
(linear by linear association = 0.004). At 30 minutes after FL
bolus injection, no image obtained with 500 mg fluorescein
was classified as ‘‘not readable.’’ The rates for ‘‘not readable’’
late phase fluorescein angiograms are given in table 2.
Statistical analysis indicated overall better image quality
using higher amounts of FL (linear by linear association = 0.002) (table 2) (figs 2 and 3).
For the ICG dye, we performed 45 ICG-As in 39 patients,
using identical dye concentrations in different volumes
Table 3 Indocyanine green angiographies and dye
amounts used
No
(n = 45)
ICG
amount
ICG injected
volume
(5 mg/ml)
5
13
8
8
5
6
2.5 mg
5 mg
10 mg
15 mg
20 mg
25 mg
0.50
1.00
2.00
3.00
4.00
5.00
ml
ml
ml
ml
ml
ml
(table 3). Of the group, 19 patients were phakic and 20
patients were pseudophakic. Four frames of each ICG-A (1, 5,
15, and 30 minutes after injection) were evaluated. A
proportion of 71.1% of images taken 1 minute after injection
were rated as ‘‘very good’’ (table 4). At 5 minutes after
injection, ICG-A with 2.5 mg and 5 mg of fluorescence dye
led to single images with ‘‘poor’’ image quality; in general,
images were significantly better with higher amounts of dye
(linear by linear association = 0.09 for 1 minute and 0.05 for
5 minutes) (table 5). The late phase images overall had
slightly worse ratings with lower levels of dye (linear by
linear association = 0.00 for 15 minutes and 0.00 for 30 minutes) (table 5). Out of all 15 minute frames, only one image
(20%) taken with 2.5 mg and two images (15.3%) taken with
5 mg dye were classified as ‘‘not readable.‘‘ Proportions for
30 minutes shots classified as ‘‘not readable’’ were 60%
(three out of five) in the 2.5 mg group and 30.8% (four out of
13) in the 5 mg group. At higher amounts, none of the
images was classified as ‘‘not readable’’ (table 4) (figs 1 and 4).
DISCUSSION
With the advent of cSLO, low levels of fluorescence in the
human eye can be recorded. Recommendations regarding the
amount of dye to be injected for FL-A (500 mg) and ICG-A
(25 mg) are based on conventional camera systems that were
introduced decades ago. With a stepwise reduction of dye
volumes injected while maintaining the same concentrations,
the current study indicates that for routine clinical purposes,
166 mg of fluorescein and 5 mg of ICG are sufficient when
using a new cSLO system.
The use of only one third of the FL-A and one fifth of the
ICG-A conventionally used dye amounts, respectively, allows
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1614
Bindewald, Stuhrmann, Roth, et al
Table 4
ICG
1 minute
2.5 mg
5 mg
10 mg
15 mg
20 mg
25 mg
5 minutes
2.5 mg
5 mg
10 mg
15 mg
20 mg
25 mg
15 minutes
2.5 mg
5 mg
10 mg
15 mg
20 mg
25 mg
30 minutes
2.5 mg
5 mg
10 mg
15 mg
20 mg
25 mg
Results of the evaluation of indocyanine green (ICG) angiographies
Very good
Good
Poor
Not
readable
Total
2 (40.0%)
7 (53.8%)
5 (62.5%)
8(100%)
5 (100%)
5(83.3%)
32 (71.1%)
3 (60.0%)
6 (46.2%)
3 (37.5%)
0 (0%)
0 (0%)
1 (16.7%)
13 (28.9%)
0
0
0
0
0
0
0
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
0
0
0
0
0
0
0
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
5
13
8
8
5
6
45
1 (20%)
4 (30.8%)
4 (50.0%)
6 (75%)
3 (60%)
5 (83.3%)
23 (51.1%)
3 (60%)
8 (61.5%)
4 (50%)
2 (25%)
2 (40%)
1 (16.7%)
20 (44.4%)
1
1
0
0
0
0
2
(20%)
(7.7%)
(0%)
(0%)
(0%)
(0%)
(4.4%)
0
0
0
0
0
0
0
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
(0%)
5
13
8
8
5
6
45
0
0
1
1
4
3
9
(0%)
(0%)
(12.5%)
(12.5%)
(80%)
(50%)
(20%)
1 (20%)
6 (46.2%)
4 (50%)
5 (62.5%)
1 (20%)
3 (50%)
20 (44.4%)
3 (60%)
5 (38.5%)
3 (37.5%)
2 (25%)
0 (0%)
0 (0%)
13 (28.9%)
1
2
0
0
0
0
3
(20%)
(15.4%)
(0%)
(0%)
(0%)
(0%)
(6.7%)
5
13
8
8
5
6
45
0 (0%)
0 (0%)
1 (12.5%)
2 (25%)
4 (80%)
3 (50%)
10 (22.2%)
0 (0%)
3 (23.1)
5 (62.5%)
5 (62.5%)
1 (20%)
1 (16.7%)
15 (33.3%)
2 (40%)
6 (46.2%)
2 (25%)
1 (12.5%)
0 (0%)
2 (33.3)
13 (28.9%)
3
4
0
0
0
0
7
(60%)
(30.8%)
(0%)
(0%)
(0%)
(0%)
(15.6%)
5
13
8
8
5
6
45
for relevant savings in times of cost constraints for all health
systems. These savings would especially apply to high volume
medical retina departments. Furthermore, there is evidence
to suggest that side effects of fluorescein, such as nausea, are
dose dependent,27 and it would be expected that using lower
dye amounts would reduce the incidence of these side effects.
Therefore, it appears prudent to use minimally necessary dye
amounts for fluorescence angiography.
The analyses of both FL-A and ICG-A images indicated that
image quality diminishes in late phases, especially with a
reduction in dye amounts, which was the expected outcome.
Lower amounts of dye below the threshold mentioned above
would not be sufficient to obtain enough information from
the angiographic examinations. Minor reductions in contrast
and resolution appear irrelevant for the clinical management
of patients in a routine setting. For example, as long as the
borders of a classic choroidal neovascularisation (CNV) are
clearly delineated in the early phase and leakage of dye is
identified in later frames, the physician can determine his or
her therapeutic strategy (for example, photodynamic therapy). In addition, other factors may be more important for
Table 5 Statistical analysis of ICG-A for evaluation of
the relation between dye amount and image quality using
different amounts of dye
Time
(minutes)
Linear by linear
association
(,0.05 significant)
1
5
15
30
0.09
0.05
0.00
0.00
www.bjophthalmol.com
interpreting angiographic findings than optimal image
resolution.29
For special purposes—for example, identification of miniscule structures visualised during angiography such as flow
in the capillary perifoveal network or recordings for illustrations, the use of conventional dye amounts may be
considered to achieve optimal resolution. Again, such use
does not appear to be necessary for routine angiographies.
Lower dye amounts can also be used for simultaneous FLA and ICG-A using the cSLO system. We have shown
previously that both FL and ICG dye can be mixed in one
syringe and injected as bolus with subsequent simultaneous
recordings.17 30
Various limitations have to be considered when interpreting this study. We only investigated patients with various
manifestations of AMD; however, we assume that these
findings would be comparable in the presence of other retinal
pathologies. Furthermore, only patients with relatively clear
media were examined. Advanced lens opacities may impair
fluorescence image quality by absorption both in the
excitation and absorption spectra of the fluorescent dyes.
Therefore, lower amounts of dye may be disadvantageous in
eyes with advanced cataract, and it may be prudent to use
standard amounts under such circumstances. The relatively
small number of subjects in each subgroup represents a
limitation of the study and needs to be considered when
interpreting the data. However, since there was overall
relatively little variability within the subgroups, we would
assume that larger numbers would in essence not yield other
results. Finally, the classification of image quality is obviously
a subjective evaluation; however, there is no objective means
available to accomplish more accurate ratings.
In summary, this new cSLO (HRA2) allows for detection of
low levels of fluorescence. We have shown that it is possible
to use amounts of fluorescein and/or ICG dyes for routine
Fluorescein and ICG dye for digital cSLO FA
fluorescence angiography that are lower than those previously used for conventional camera based systems. This
finding also allows for relevant savings in expenses.
.....................
Authors’ affiliations
A Bindewald, O Stuhrmann, F Roth, S Schmitz-Valckenberg, H-M Helb,
A Wegener, N Eter, F G Holz, Department of Ophthalmology, University
of Bonn, Ernst-Abbe-Strasse 2, D-53127 Bonn, Germany
Competing interests: none declared
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www.bjophthalmol.com
1616
EXTENDED REPORT
Systemic carboplatin for retinoblastoma: change in tumour
size over time
D H Abramson, S D Lawrence, K L Beaverson, T C Lee, I S Rollins, I J Dunkel
...............................................................................................................................
Br J Ophthalmol 2005;89:1616–1619. doi: 10.1136/bjo.2005.072033
See end of article for
authors’ affiliations
.......................
Correspondence to:
David H Abramson, MD,
70 East 66th Street, New
York, NY 10021, USA;
[email protected]
Accepted for publication
1 August 2005
.......................
P
Background/aim: Chemotherapy for intraocular retinoblastoma is used to shrink individual retinal
tumours to a size amenable to focal treatments. Quantitative data regarding retinal tumour response
following treatment with primary systemic carboplatin are reported.
Methods: Changes in area and largest basal diameter of tumours that were exposed to carboplatin, had
no concomitant focal treatment, and had digital funduscopic photography performed before and after
treatment, were measured. Response was evaluated.
Results: 36 tumours were measured following one treatment: 34/36 (94.4%) responded, with a 37.1%
mean decrease in area (median = 37.0%; range 4.0%–76.7%). Mean reduction in basal diameter was
21.3% (med = 21.0%; 27.9%–52.5%). 20 tumours were treated with a second cycle: 15/20 (75.0%)
responded. Mean decrease in area was 17.8% (med = 15.3%; 27.0%–49.7%). The mean cumulative
decrease in area after two treatments was 55.1% (med = 56.2%; 33.0%–74.5%). Mean cumulative
reduction in basal diameter was 33.6% (med = 33.6%; 10.9%–53.2%). 12 tumours were treated with a
third cycle: 3/12 (25.0%) responded, 8/12 were stable, and one progressed. Mean decrease in area was
5.4% (med = 7.2%; 217.7%–20.6%). Cumulative decrease in area after three treatments was 58.1%
(med = 57.3%; 34.8%–77.2%). Mean cumulative reduction in basal diameter was 38.8% (med = 38.2%;
19.1%–54.1%).
Conclusions: Carboplatin caused measurable shrinkage of retinoblastoma tumours. Response was
greatest following the initial treatment and decreased with subsequent treatments.
rimary chemotherapy is often used to treat intraocular
retinoblastoma, but when used alone, it is rarely
curative. For select eyes, chemotherapy may be used to
shrink retinal tumours (chemoreduction) to a size cured by
the application of focal techniques such as cryotherapy,
brachytherapy, and laser photocoagulation. The protocol
most commonly used to achieve chemoreduction is a
combination of vincristine, etoposide, and carboplatin
(VEC). Ciclosporin may be added to the regimen with the
goal of decreasing multidrug resistance.1 One’s choice of
drugs, dosage and treatment schedule must weigh patient/
tumour response against the risks and/or complications of
treatment. Known risks of chemotherapy treatment exist;
including, haematological (myelosuppression) and gastrointestinal distress and infection.2 Furthermore, eyes remain
at risk for new retinal tumour development following
treatment with chemotherapy.3 Known complications of
chemoreduction plus focal therapies also exist; including,
rhegmatogenous retinal detachment in the setting of
chemoreduction plus cryotherapy,4 and focal iris atrophy,
peripheral focal lens opacity, retinal traction, retinal vascular
occlusion, and transient localised serous retinal detachment
in the setting of thermotherapy.5 The development of
additional cancers has been reported following treatment
with chemotherapy. Secondary myelodysplastic syndrome/
acute myelogenous leukaemia is a rare, but devastating, side
effect of a number of chemotherapeutic agents, but has been
particularly associated with etoposide.6 Known patient/
tumour response data to chemoreduction techniques are
most often defined by outcome variables associated with
ocular and patient survival, as well as avoidance of external
beam radiotherapy (EBR). The most consistent correlate for
success is extent of intraocular disease; Reese-Ellsworth
group (RE) I-III eyes usually avoid EBR and enucleation,
while RE group IV-V eyes have lower success rates.2 7 Several
www.bjophthalmol.com
studies have examined the effects of systemic chemotherapy
on intraocular retinoblastoma in the setting of concomitant
focal therapies.8–13 These studies accurately reflect common
clinical practice, but when reporting outcome data, their
methodology does not allow for the evaluation of any isolated
effect of chemotherapy on tumour morphology.
A few studies have attempted to rigorously quantify the
change in retinal tumour size following primary treatment
with chemotherapy by means of serial measurements,
pretreatment and post-treatment.14–16 These published studies
focus on response after two and three drug regimens and
measure tumour dimensions by various methods. All report
notable shrinkage after a single cycle of chemotherapy, with
decreased continued reduction following successive doses.
We are unaware of any study to date that has quantified
reduction in tumour size as a function of a single agent,
systemic chemotherapy, in the absence of additional treatment(s).
PATIENTS AND METHODS
We performed a retrospective study of retinoblastomas that
met the following criteria: (1) they were identified in patients
diagnosed with intraocular bilateral or unilateral retinoblastoma, (2) they were exposed to primary carboplatin only, and
(3) they had adequate digital fundus photographs (RETCAM) taken before and after exposure to systemic carboplatin. Thirty six tumours in 27 eyes of 21 patients who were
treated 1994 to March 2004 at the Ophthalmic Oncology
Center of New York Presbyterian Hospital (NYPH) and
Memorial Sloan-Kettering Cancer Center (MSKCC) fulfilled
Abbreviations: CBC, complete blood count; CTT, chemothermotherapy;
dd, disc diameter; EBR, external beam radiotherapy; RE, ReeseEllsworth; TTT, transpupillary thermotherapy; VEC, vincristine,
etoposide, and carboplatin
Retinal tumour changes after carboplatin
1617
Variable
Number of tumours
Table 1 Patient and tumour demographics.
No (%)
Sex
Male
Female
Mean age at diagnosis (months) (range)
Disease involvement*
Bilateral
Unilateral
Tumour location
Macular
Macular-equator
Equator-ora serrata
12 (57%)
9 (43%)
9 (0.75–28.5)
15 (71%)
6 (29%)
8 (22%0
22 (61%)
6 (17%)
*At diagnosis and follow up (mean follow up = months).
the inclusion criteria. This study protocol was approved by
NYPH-Weill Cornell Medical College’s institutional review
board (Protocol 0404-277).
Collected patient data included sex, age at diagnosis,
disease laterality, location of tumour(s), and all subsequent
treatments. Tumour location was classified according to its
zone: macular (between the superior and inferior temporal
arterioles), macular-equator (area of the retina between the
macular zone and the equator), and equator-ora serrata
(anterior to the equator). Tumours were excluded from
analysis if they received any treatment before administration
of systemic carboplatin. Tumours were measured using RETCAM imaging software that calculated area from a tracing of
the outer margins of the base of the tumour. Two observers
conducted independent area measurements of each tumour
and the results were averaged into one final data set. The
largest basal diameter of each tumour was measured from
the RET-CAM images at baseline and following each cycle of
chemotherapy. Tumours were censored from the study when
additional therapeutic modalities were applied.
We defined tumour response in two ways: (1) the
percentage reduction in area and percentage reduction in
largest basal diameter were calculated for each treatment
(average follow up 3–4 weeks), and (2) based on area
measurements, a tumour was described, per treatment, as
responsive (.10% reduction in area), progressive (.10%
increase in area), or stable (,10% decrease or increase in
area).
The diagnosis of retinoblastoma was made on the basis of
dilated fundus examination under anaesthesia using indirect
ophthalmoscopy, scleral depression, and RET-CAM imaging.
Intravenous (IV) carboplatin was administered within days
to weeks of diagnosis, at a dose of 18.7 mg/kg for children
under 12 kg in weight and 560 mg/m2 for children 12 kg or
more. All patients receiving IV carboplatin had a history and
physical, complete blood count (CBC), and serum chemistry
performed in order to evaluate systemic effects of the drug.
Ophthalmological examinations were performed at 3–4 week
intervals (average).
Statistical methods
All statistical analyses were performed using SAS (version
8.1, Cary, NC, USA). Percentage reduction in tumour area
following an initial cycle of carboplatin treatment was
compared with tumour response after a second and third
cycle by means of a two tailed, Student’s t test (alpha = 0.05).
Percentage reduction in tumour area (per treatment) was
then considered as a function of initial size of tumour using
the initial largest basal diameter (0–4 disc diameters versus
.4 disc diameters, where 1 disc diameter (dd) = 1.5 mm),
also using the Student’s t test (alpha = 0.05). Finally,
percentage reduction in tumour area, per treatment, was
stratified by tumour location/zone and analysed by ANOVA.
RESULTS
Thirty six tumours in 27 eyes of 21 patients were evaluated
for response following 1–3 cycles of carboplatin. Table 1
summarises the patient demographics.
At baseline, 16 tumours measured 0–4 dd (44%), 19
tumours measured 4–10 dd (53%), and one tumour was more
than 10 dd (3%; fig 1). Following initial carboplatin
treatment (mean follow up 22.0 days; 12–41), 34/36 tumours
(94.4%) showed a response to treatment and 2/36 (5.6%)
were stable. No tumours progressed. The mean reduction in
area was 37.1% (median 37.0%; 4.0%–76.7%) and the mean
reduction in largest basal diameter was 21.3% (median
21.0%; 27.9%–52.5%). Tumour response following initial
carboplatin treatment is stratified in figure 2.
Twenty of the original 36 tumours received a second cycle
of carboplatin (mean follow up 26.1 days; range 20–35): 15
70
Per treatment
Cumulative
Basal diameter
44%
40
3%
>10
Initial basal diameter (disc diameters)
Figure 1 Initial size of tumour at the time of first treatment with systemic
carboplatin. The largest basal diameter for each tumour (1 disc
diameter = 1.5 mm) was measured using RET-CAM imaging.
39%
37% 37%
34%
30
21%
20
4–10
58%
55%
50
53%
0–4
>50
25–50
Figure 2 Percentage reduction in tumour area after one initial
treatment with systemic carboplatin, stratified.
% reduction
Number of tumors
<25
Percentage reduction in tumor area
60
20
18
16
14
12
10
8
6
4
2
0
18
16
14
12
10
8
6
4
2
0
18%
10
0
5%
1
2
3
Carboplatin treatment
Figure 3 Mean reduction in tumour size. RET-CAM imaging software
was used to measure tumour area and largest basal diameter, before
and after each cycle of systemic carboplatin.
www.bjophthalmol.com
1618
Abramson, Lawrence, Beaverson, et al
DISCUSSION
Figure 4 RET-CAM images of two solitary retinoblastomas: before and
after treatment with primary carboplatin.
(75.0%) were responsive to treatment and five (25.0%) were
stable. No tumours progressed. Mean reduction in area for
the second treatment alone was 17.8% (median 15.3%; range
27.0%–49.7%). The mean cumulative reduction in area after
two treatments for the 20 tumours was 55.1% (median
56.2%; 33.0%–74.5%), and the mean cumulative reduction in
largest basal diameter was 33.6% (median 33.6%; 10.9%–
53.2%).
Twelve tumours received a third cycle of carboplatin (mean
follow up 21.8 days; 20–37): three tumours (25%) were
responsive, eight tumours (67%) were stable, and one
tumour showed progression. Mean reduction in area for
the third treatment alone was 5.4% (median 7.2%; 217.7%–
20.6%). The mean cumulative reduction in area after
three treatments was 58.1% (median 57.3%; 34.8%–77.2%),
and the mean cumulative reduction in largest basal diameter
was 38.8% (median 38.2%; 19.1%–54.1%). Figure 3 summarises the treatment specific and cumulative tumour
responses.
The mean cumulative reduction in tumour area after both
two and three cycles of systemic carboplatin was significantly
greater than that achieved after one treatment (p,0.01).
Three cycles of carboplatin, however, did not result in a
significantly greater response than two treatments (p.0.05).
Percentage reduction in tumour area was not statistically
related to initial tumour size (0–4 dd versus 4–10 dd;
p = 0.30). Percentage reduction in tumour area was also not
statistically related to tumour location (p.0.05). Tumour
location and percentage reduction were compared following
one cycle of carboplatin: macular versus macular-equator
tumours (p = 0.10), macular versus equator-ora serrata
(p = 0.69), and macular-equator versus ora-serrata
(p = 0.27).
A proportion of the tumours that qualified for this study
were in patients with intraocular retinoblastoma rigorously
followed under a treatment protocol designed to evaluate
patient and ocular survival after carboplatin chemotherapy.
The observed side effects were minimal: mild blood count
suppression was noted but was not clinically significant. No
hearing loss specifically attributed to carboplatin treatment
was observed, nor renal and/or hepatic toxicity (Dunkel,
unpublished data, 2005).
Figure 4 shows RET-CAM images of two representative
solitary tumours, each before and after treatment with
primary carboplatin.
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Previous studies have evaluated the response of intraocular
retinoblastoma to chemoreduction using a variety of outcome
variables and methodologies. The purpose of this study is to
add to the existing literature measurements of tumour
response after carboplatin chemotherapy. We chose to record
the morphology of tumours in two dimensions before and
after therapy, and felt this to be an accurate representation of
response. Ocular and patient survival, and/or effectiveness of
additional treatments for retinoblastoma were not assessed
in this study.
Shields et al measured change in size of 54 tumours in 31
eyes following a 2 month regimen (two cycles) of triple agent
chemotherapy (VEC).14 A 29% mean reduction in basal
diameter and 40% mean reduction in thickness was reported
after one cycle. A second cycle yielded a 35% cumulative
reduction in basal diameter and 49% in thickness. Each
tumour was measured via echography and estimates by
indirect ophthalmoscopy. Digital imaging was not used in
reporting the data. The response rate to treatment was 100%:
approximately 50% of the tumours classified achieved a
complete response after 2 months and the remaining 50%
achieved a partial response. No progressive disease was seen.
In their analysis, the authors noted that larger tumours
demonstrated a more dramatic response to treatment, but the
percentage shrinkage was nearly identical between tumours
.8 mm thick versus tumours ,8 mm thick. Furthermore,
they reported that in some patients, reduction in tumour size
correlated with the location of the intraocular tumour:
reduction correlated with increased distance of the tumour
from the optic disc and foveola.
Sussman et al utilised RET-CAM imaging and echography
to evaluate the effects of chemotherapy (VEC plus or minus
cyclosporin) on RE group IV–V retinoblastomas. Their study
did not purport to isolate the effects of systemic chemotherapy alone—patients received concurrent transpupillary diode
laser and/or cryotherapy. The study objective was to compare
the time course and extent of tumour reduction associated
with this treatment regimen versus EBR in treating advanced
intraocular retinoblastoma. Only the largest tumour in an eye
was evaluated. The authors concluded that reduction in
tumour volume was greatest in the first 2 months of therapy
(68% reduction from baseline after 1 month) and disappeared by 12 months.15
Finally, Demirci et al studied 10 eyes with intraocular
retinoblastoma that had been enucleated after receiving an
average of four cycles of VEC. Nine of the eyes were RE
groups IV–V, and all 10 eyes had been enucleated for tumour
recurrence as subretinal seeds and/or vitreous seeds (seven)
or for vitreous haemorrhage (three). A 24% mean reduction
of basal diameter and 34% in thickness of the main tumour in
each eye was observed from pretreatment baseline to the
time of enucleation.16
Of the 36 tumours we measured following exposure to
carboplatin (1–3 cycles), 94% responded. The remaining
tumours were stable. One tumour progressed following the
third cycle of carboplatin. These results are comparable with
reported tumour regression following chemothermotherapy
(CTT); whereby 96% of tumours responded after 1–6 cycles.
CTT consists of transpupillary thermotherapy (TTT) delivered
to a tumour shortly after IV administration of carboplatin
(plus or minus etoposide), with repeated TTT 8 days later.17
One cycle of carboplatin alone produced a 37.1% mean
reduction in tumour area (fig 4A and 4B). We found a
continued reduction (17.8%) in area after a second cycle. All
tumours subjected to two cycles of carboplatin showed
substantial cumulative regression (mean reduction in area
55.1%). When we looked at tumour response in terms of
percentage reduction of the largest basal diameter, as other
Retinal tumour changes after carboplatin
studies have done, we found a 21.3% mean decrease after one
cycle of carboplatin. Two and three cycles of chemotherapy
resulted in a cumulative reduction in largest basal diameter
of 33.6% and 38.8%, respectively. While variations in
methodology limit comparison between individual studies,
our results suggest that tumour response to treatment with a
single agent (carboplatin) is comparable to that achieved in
studies evaluating treatment with multiple agents.
Tumour response appeared to level off after two cycles of
carboplatin. It is possible that maximal benefit of systemic
chemotherapy is achieved during the first two cycles of
treatment and that the residual mass represents treated
tumour. It is also possible that our study selected for resistant
tumours by focally treating tumours that responded more
favourably to systemic carboplatin. Regardless, our observation is consistent with tumour reduction following EBR; it
too appears to plateau, although the time to the plateau
following EBR appears to be greater.15
Gombos et al, in an attempt to identify factors predictive of
outcome, described tumour associated features of retinoblastomas successfully responding to triple agent chemotherapy
(VEC). Their results suggested that small tumours (,2 mm
in basal diameter) were potentially less responsive to
systemic chemotherapy.18 Furthermore, tumours in the
macular region were more likely to be managed by systemic
chemotherapy alone than tumours in the periphery; perhaps
because disproportionate choroidal blood flow to the macular
region facilitated greater drug delivery to centrally located
tumours . Our study classified tumours on the basis of largest
basal diameter in disc diameters (0–4 dd, 4–10 dd, and .10
dd); size ranges reflecting the dimensions used for RE
classification. Our analysis did not reveal a significant
relation between initial tumour size and tumour response
to treatment. However, there was a noticeable trend for each
cycle of chemotherapy: larger tumours showed a greater
percentage reduction in tumour area than smaller tumours
for each treatment. Our analysis also did not reveal a
correlation between the location of a given tumour and its
percentage reduction in area following treatment with
carboplatin. Given the information from previous studies by
others, it is likely that our study was underpowered to detect
differences in the percentage reduction across location.
Our results demonstrate quantifiable tumour chemoreduction following treatment with carboplatin. It appears
comparable to the reported tumour response data following
treatment with multi-agent chemotherapy; in terms of
percentage reduction in largest basal diameter, percentage
reduction in area, response rate, and early timing of
maximum response. Our data suggest that, perhaps for some
tumours, there is not a direct relation between the additional
exposure to multiple chemotherapeutic agents and additional
tumour reduction. We did not assess response to carboplatin
treatment in terms of eye survival and/or eye event free
survival, choosing not to introduce such variables as which
focal technique is applied following chemoreduction, how
often and how long it is applied, and the actual techniques
used in applying the focal technique (which vary from centre
to centre). Knowing that carboplatin alone will not cure the
tumours despite a good initial response, complete analysis of
these variables is necessary in order to (1) evaluate the
overall success of a chemoreduction and focal therapy
1619
strategy for curing intraocular retinoblastoma, and (2) fully
disclose to patients information regarding the risks and
benefits of treatment choices.
.....................
Authors’ affiliations
D H Abramson, K L Beaverson, T C Lee, I S Rollins, Ophthalmic
Oncology Service, Department of Surgery, Memorial Sloan-Kettering
Cancer Center, New York, NY, USA
D H Abramson, S D Lawrence, T C Lee, Department of Ophthalmology,
New York Presbyterian Hospital-Weill Cornell Medical College, New
York, NY, USA
K L Beaverson, Department of Medicine, Memorial Sloan-Kettering
Cancer Center, New York, NY, USA
D H Abramson, I J Dunkel, Department of Pediatrics, Memorial SloanKettering Cancer Center, New York, NY, USA
Supported in part by the Fund for Ophthalmic Knowledge, New York,
NY, USA.
Competing interests: none declared
REFERENCES
1 Chan HS, DeBoer G, Thiessen JJ, et al. Combining cyclosporine with
chemotherapy controls intraocular retinoblastoma without requiring radiation.
Clin Cancer Res 1996;2:1499–508.
2 Rodriguez-Galindo C, Wilson MW, Haik BG, et al. Treatment of intraocular
retinoblastoma with vincristine and carboplatin. J Clin Oncol
2003;21:2019–25.
3 Lee TC, Hayashi NI, Dunkel IJ, et al. New retinoblastoma tumor formation in
children initially treated with systemic carboplatin. Ophthalmol
2003;110:10:1989–94.
4 Anagnoste SR, Scott IU, Murray TG, et al. Rhegmatogenous retinal
detachment in retinoblastoma patients undergoing chemoreduction and
cryotherapy. Am J Ophthalmol 2000;129:817–19.
5 Shields CL, Santos JA, Diniz W, et al. Thermotherapy for retinoblastoma. Arch
Ophthalmol 1999;117:885–93.
6 Smith MA, Rubinstein L, Anderson JR, et al. Secondary leukemia or
myelodysplastic syndrome after treatment with epipodophyllotoxins. J Clin
Oncol 1999;17:569–77.
7 Beck MN, Balmer A, Dessing C, et al. First-line chemotherapy with local
treatment can prevent external-beam irradiation and enucleation in low-stage
intraocular retinoblastoma. J Clin Oncol 2000;18:2881–7.
8 Kingston JE, Hungerford JL, Madreperla SA, et al. Results of combined
chemotherapy and radiotherapy for advanced intraocular retinoblastoma.
Arch Ophthalmol 1996;114:1339–43.
9 Gallie BL, Budning A, DeBoer G, et al. Chemotherapy with focal therapy can
cure intraocular retinoblastoma without radiotherapy. Arch Ophthalmol
1996;114:1321–8.
10 Friedman DL, Himelstein B, Shields CL, et al. Chemoreduction and local
ophthalmic therapy for intraocular retinoblastoma. J Clin Oncol
2000;18:12–17.
11 Murphree AL, Villablanca JG, Deegan III WF, et al. Chemotherapy plus local
treatment in the management of intraocular retinoblastoma. Arch Ophthalmol
1996;114:1348–56.
12 Shields CL, Shields JA, Needle M, et al. Combined chemoreduction and
adjuvant treatment for intraocular retinoblastoma. Ophthalmology
1997;104:2101–11.
13 Lumbroso L, Doz F, Levy C, et al. Diode laser thermotherapy and
chemothermotherapy in the treatment of retinoblastoma. J Fr Ophtalmol.
2003;26: 2, 154–9.
14 Shields CL, De Potter P, Himelstein BP, et al. Chemoreduction in the initial
management of intraocular retinoblastoma. Arch Ophthalmol
1996;114:1330–8.
15 Sussman DA, Escalona-Benz E, Benz MS, et al. Comparison of retinoblastoma
reduction for chemotherapy vs external beam radiotherapy. Arch Ophthalmol
2003;121:979–84.
16 Demirci H, Eagle RC, Shields CL, et al. Histopathologic findings in eyes with
retinoblastoma treated only with chemoreduction. Arch Ophthalmol
2003;121:1125–31.
17 Lumbroso L, Doz F, Urbieta M, et al. Chemothermotherapy in the
management of retinoblastoma. Ophthalmology 2002;109:1130–6.
18 Gombos DS, Kelly A, Coen PG, et al. Retinoblastoma treated with primary
chemotherapy alone: the significance of tumour size, location, and age.
Br J Ophthalmol 2002;86:80–3.
www.bjophthalmol.com
1620
EXTENDED REPORT
Unilateral electronegative ERG of non-vascular aetiology
A G Robson, E C Richardson, A H C Koh, C E Pavesio, P G Hykin, A Calcagni, E M Graham,
G E Holder
...............................................................................................................................
Br J Ophthalmol 2005;89:1620–1626. doi: 10.1136/bjo.2005.071357
See end of article for
authors’ affiliations
.......................
Correspondence to:
Dr G E Holder,
Electrophysiology,
Moorfields Eye Hospital,
162 City Road, London
EC1V 2PD, UK; graham.
[email protected]
Accepted for publication
2 August 2005
.......................
Background: Full field and pattern electroretinograms (ERG, PERG) are performed to assess generalised
retinal function and macular function, respectively. An (electro) negative full field ERG usually describes an
ISCEV standard maximal response in which the b-wave is smaller than a normal or minimally reduced awave and indicates dysfunction that is post-phototransduction. The most common cause of a unilateral
negative ERG is central retinal artery occlusion (CRAO) or birdshot chorioretinopathy (BCR). This study
examines the clinical and electrophysiological features of patients with unilateral negative ERG who do not
have CRAO or BCR.
Methods: 12 patients were ascertained with a unilateral negative ERG in whom a vascular aetiology and
BCR were excluded. Most presented with symptoms of central retinal dysfunction. In 11 of the 12 patients
additional long duration photopic stimuli were used to test cone system ON and OFF responses.
Results: All 12 patients had unilateral electronegative bright flash full field ERGs indicating total or relative
preservation of rod photoreceptor function, but dysfunction post-phototransduction. Seven of these patients
had non-specific inflammatory changes in the eye with the negative ERG. Six patients, including five with
inflammatory signs, had involvement of the cone ON response with complete preservation of cone OFF
responses. A further three patients showed evidence of cone ON response abnormality with less severe
OFF response involvement.
Conclusion: The ERGs in this heterogeneous group of patients predominantly showed post-phototransduction involvement of the ON pathways. Sparing of the cone OFF response was often observed. The majority
of patients had signs of previous inflammation and it is speculated that these highly unusual unilateral
changes may be mediated via an autoimmune mechanism.
A
n electronegative electroretinogram (ERG) describes a
full field ERG in which the b-wave is smaller than the
normal or minimally attenuated a-wave. It is usually
best seen in the rod dominated maximal ERG following
stimulation with a bright flash under scotopic conditions,1–3
although sometimes manifest in cone derived ERGs recorded
under photopic conditions as a reduced b:a ratio.4 5 A selectively reduced b-wave indicates impaired retinal function
which is post-phototransduction, usually post-receptoral, and
may be associated with a number of congenital and acquired
conditions such as X linked juvenile retinoschisis,6 7
Congenital stationary night blindness (CSNB),3 8 9 central
retinal artery occlusion (CRAO),10–12 birdshot chorioretinopathy (BCR),13–15 melanoma associated retinopathy
(MAR),16 17 retinal toxicity,2 18 19 and a number of other less
common disorders (see table 1).
Unilateral electronegative ERGs are commonly associated
with CRAO10–12 and may occur in some patients with
BCR.3 15 20 A previous retrospective review of 2640 patients
from this unit21 revealed 128 with negative ERGs (table 1).
There were 25 undiagnosed cases, of which seven had
unilateral electronegative ERGs. As an extension of that
audit, the present study examines in detail the clinical and
electrophysiological features of 12 patients with unilateral
electronegative ERG in whom a vascular aetiology and BCR
have been excluded. These patients have been reviewed in an
attempt to ascertain common features and elucidate underlying mechanisms.
MATERIALS AND METHODS
Twelve patients form the basis of this report. Seven had
previously been identified as having unilateral electronegative ERGs as part of an earlier study.21 The clinical notes were
www.bjophthalmol.com
Table 1
Causes of electronegative ERG (after Koh et al21)
Number of
patients
X linked juvenile retinoschisis
Congenital stationary night blindness
Central retinal artery occlusion
Birdshot chorioretinopathy
Toxic retinopathy (quinine, vincristine, vigabatrin)
Melanoma associated retinopathy
Batten disease
Unspecified inflammatory retinopathy
Photoreceptor dystrophy
Undiagnosed
Total
19
17
13
7
5
4
1
3
34*
25
128
*Additional abnormal a-wave reduction.
examined and any relevant follow up data and investigation
results were reviewed. A vascular aetiology and birdshot
chorioretinopathy were excluded in all patients on this basis.
Full field ERGs were performed using extended testing
protocols incorporating the ISCEV (International Society for
Clinical Electrophysiology of Vision) minimum standard22 in
order to assess generalised retinal function.1 2 The minimum
protocol incorporates the rod specific and standard bright
flash ERGs, both recorded after a minimum of 20 minutes
Abbreviations: ANA, antinuclear antibody; BCR, birdshot
chorioretinopathy; CAR, carcinoma associated retinopathy; CSNB,
congenital stationary night blindness; CRAO, central retinal artery
occlusion; EOG, electro-oculogram; ERG, electroretinograms; FFA,
fluorescein angiography; ISCEV, International Society for Clinical
Electrophysiology of Vision; MAR, melanoma associated retinopathy;
PERG, pattern electroretinogram; RAPD, relative afferent pupillary defect
Non-vascular unilateral negative ERG
1621
dark adaptation, and the photopic 30 Hz flicker and transient
photopic ERGs, both recorded after a standard period and
intensity of light adaptation. A stimulus 0.6 log units greater
than the ISCEV standard flash was also used, better to
demonstrate the a-wave, as suggested in the recent revision
of the ISCEV standard for ERG.22 Pupils were dilated before
full field ERG testing using tropicamide (1%) and phenylephrine hydrochloride (2.5%). ISCEV standard pattern ERG
(PERG) was performed23 before mydriasis and the P50
component, partly originating in structures anterior to the
retinal ganglion cells and driven by the macular photoreceptors, was used to assess macular function.3 In most cases,
ISCEV standard electro-oculogram (EOG) was also performed24 in order to assess the function of the retinal
pigment epithelium/photoreceptor interface.2 25 Long duration ON-OFF ERGs were used to assess post-receptoral cone
ON and OFF pathways, predominantly arising in relation to
depolarising and hyperpolarising bipolar cell function.17 26
Table 2 Summary of clinical findings in all patients
Visual
acuity
RAPD
Signs of
inflammation
RE: Juxtafoveal
telangectasia
6/9
–
–
RE: small nasal
scotoma
LE: Juxtafoveal
hypertrophic laser scar
6/12
–
RE: normal
6/5
LE: normal
6/5
RE: normal
6/12
+
–
LE: small nasal
scotoma relating to
laser scar
RE: mild temporal
deficit
LE: mild inferior
temporal loss
RE: Superionasal
deficit
LE: normal
RE: small juxtaoveal
cyst.
LE: inferior chorioretinal
atrophy, attenuated
arterioles, venous
bleeding, granular
fovea. Vitreous cells
RE: normal
LE: cupped disc
6/9
6/5
+
–
–
6/6
6/6
+
RE: pale disc
6/18
+
LE: normal
6/12
RE: CMO, snowballs
6/9
–
LE: normal
RE: swollen disc
multiple foci of atrophy
LE: normal
RE: punched out scars
at posterior pole.
6/6
6/12
+
RE: vitritis
6/5
6/6
–
–
LE: punched out scars
at posterior pole and
subfoveal CNV.
RE: swollen disc,
chorioretinal scars,
CMO
6/60
LE: swollen disc,
chorioretinal scars.
RE: normal
6/5
6/5
–
–
LE: normal
RE: CMO. Inferior
chorioretinal lesions,
snowballs, haze
LE: chorioretinal lesions
and scar related to
laser treatment for
CNV, snowballs
6/5
6/6
–
–
RE: cells+
Case Sex
Age
Fundi at presentation*
1
58
2
F
M
62
3
F
46
4
M
33
5
6
M
M
59
40
7
F
30
8
F
25
9
10
F
M
42
31
11
M
45
12
F
54
–
–
6/60
6/60
6/6
–
LE: vitreous cells
Field defect
Presenting
symptoms
Brief clinical history
LE:
metamorphopsia
and loss of
colour vision
Breast carcinoma and
mastectomy 1 year after
presentation. Sudden loss of
colour vision in RE 6 years
later.
RE: colour vision
loss
History of cutaneous
melanoma.
RE: intermittent
visual loss
Chicken pox 7 months prior
to symptoms.
LE: dense central
scotoma
LE: sudden visual
loss
–
–
LE: Inferior
RE: vitreous cells
RE: central+
LE: faded colour
vision
LE: visual
disturbance
LE: cells+, flare +, LE: central
vitreous cells +
RE: uveitis and
RE: Constricted
scleritis
12 year history of panuveitis. 2–3 years after
presentation reported colour
vision loss RE. 4 years later
LE capsulotomy.
RE: floaters and
reduced vision
RE photopsia 2 years after
presentation and RE
nyctalopia 5 months later.
RE: enlarged blind
spot
RE: sudden
visual loss
`
–
LE: reduced vision One month after presentation
developed photopsia RE.
Initially diagnosed with PIC.
RE: vitreous cells
RE: enlarged blind
spot
RE: reduced
vision
–
LE: enlarged blind
spot
–
–
–
LE: cells
RE: small central
defect
4 years after presentation
had vitreous cells and vessel
sheathing bilaterally. RE
developed ERM. LE CMO
and photopsia.
LE: reduced vision
and red
desaturation
RE: nyctalopia
10 year history of bilateral
intermittent uveitis with 4
month exacerbation RE.LE.
LE: small defect on
left related to laser
scar
RE, right eye; LE, left eye; CMO, cystoid macular oedema; CNV, choroidal neovascularisation, ERM: epiretinal membrane; PIC, punctate inner choroidopathy.
*Eyes with negative ERG in bold.
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1622
Robson, Richardson, Koh, et al
Table 3 Summary of electrophysiology and colour vision testing
1
2
3
4
5
6
7
8
9
10
11
12
Eye
Pattern
ERG P50
Scotopic
rod
ERG
Scotopic
maximal
ERG
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
RE
LE
N
A
A
N
A
N
N
A
N
A
A
A
A
N
A
N
N
A
A(now N)
A
N
A
A
A
N
A
A
N
A
N
N
A
N
A
N
A
A
N
A
N
N
A
A
A
N
A
A+
A
N
A (-ve)
A (-ve)
N
A (-ve)
N
N
A (-ve)
N
A (-ve)
N
A (-ve)
A (-ve)
N
A (-ve)
N
A
A (-ve)
A
A (-ve)
N
A (-ve)
A(-ve)
N
OPs
Photopic
transient
ERG
Photopic
30 Hz
ERG
ON
b-wave
OFF
d-wave
EOG
CCS
N
A
A
N
A
N
N
A
N
A
N
A
A
N
A
N
N
A
A
A
N
A
A
N
N
A
A
N
A
N
N
A
N
A (-ve)
N
A (low b:a)
A
N
A
N
N
A
A
A
N
A
A
A
N
A
A
N
A
N
N
A
N
A
N
A
A
N
A
N
N
A
A
A
N
A
A
A
n/p
n/p
A++
N
A+
N
N
A+
N
A++
n/p
A(-ve)
A(-ve)
N
A(-ve)
N
N
A
A
A(-ve)
N
A(-ve)
A(-ve)
N
n/p
n/p
A+
N
A(mild)
N
N
A
N
A+
n/p
A+
N
n/r
N
N
N
N
A (mild)
N
N
A+
N
N
N
N
n/p
n/p
N
N
N
A
N
A
n/p
n/p
N
N
A
N
N
N
A
A
N
N
N
N
N
A
A
N
A
N
N
A
N
A
A
A
A
N
A
N
N
A
A
N
n/p
n/p
Eyes with electronegative ERGs in bold. Ops, oscillatory potentials; EOG, electro-oculogram; CCS, colour contrast sensitivity. A: abnormal. A+: very abnormal.
A++: extremely abnormal. N: normal. n/p: not performed. n/r: not reliable. -ve: electronegative (relatively normal a-wave that is larger than the b-wave). Low
b:a: low b-wave to a-wave ratio
The LED stimulator has been previously described.27 In brief,
the duration of the amber stimulus was 150 ms or 200 ms.
Stimulus luminance was 560 cd/m2 with a green background
of 160 cd/m2, suitable to suppress rod function.28
Colour contrast thresholds along isoluminant protan,
deutan, and tritan axes were determined psychophysically
in all patients using the Arden colour contrast sensitivity
system.29
RESULTS
Group findings
The clinical and electrophysiological findings in the 12
patients are summarised in tables 2, 3, and 4. Within this
group, seven had clinical signs of inflammation. These signs
ranged from fundus scarring in the absence of vitritis (case 9)
to panuveitis (case 8). Abnormality of visual fields was also a
Table 4
common finding, seen in 10/12 cases. This was, however,
variable in its extent.
Maximal scotopic ERGs from all patients showed an
electronegative ERG in one eye, consistent with postphototransduction dysfunction affecting the rod system.
Full field ERGs were additionally abnormal in the other eye
of three cases, indicating dysfunction at the level of the
photoreceptors (cases 9 and 10) or confined to the cone
system (case 12, see below). Fellow eyes of nine patients had
normal full field ERGs. The cone ON responses were affected
more than the OFF responses in 9/11 patients, including 6/7
patients with signs of inflammatory disease. Inflammatory
signs were confined to the eye with an electronegative ERG in
three patients (cases 4, 7, and 8) or occurred bilaterally but
more severely in the eye with an electronegative ERG (cases 9
and 12). In one case there was evidence of bilateral
ERG and clinical follow up
Case
No of
ERGs*
ERG changes
Clinical changes since initial ERG
1
2
3
3 (50)
1
3 (63)
Stable
–
Stable
Sudden loss of colour vision in RE 6 years later.
Not available
VA fell from 6/12 to 6/36 over 5 years. Developed
transient episodes of loss of vision. Fundus remains
normal
4
5
9 (69)
3 (22)
6
1
7
8
2 (10)
5 (24)
9
10
11
12
1
8 (67)
2 (10)
1
See text
Mild deterioration in cone Mild deterioration in colour contrast sensitivity over 2
ERGs and PERG
years
–
Persistent inflammation, sudden reduction in VA LE
from 6/12 to 6/36 after 1 year associated with
episode of vitritis without CMO. Vitritis settled with
steroids but VA unchanged 1 year later
See text
Stable
Right lens opacity developed over 5 years, otherwise
stable
–
Stable since last tested 3 years ago
See text
Stable
Stable over 10 months
–
–
*The period between first and last ERGs are shown as months in parentheses. VA, Snellen visual acuity.
www.bjophthalmol.com
Non-vascular unilateral negative ERG
1623
a recurrence of inflammation in the affected eye (case 12, see
detailed history below).
Four illustrative cases are presented in more detail.
Case 4
Figure 1 Fundus autofluorescence images of case 4 were initially
normal bilaterally (A) but 2 years later revealed a low density area in the
left eye (B).
inflammatory eye disease associated with bilateral but
asymmetric ERG abnormalities (case 10).
Pattern ERG P50 components were reduced unilaterally in
all eyes with an electronegative ERG and were reduced
bilaterally in three cases, consistent with macular dysfunction. Colour contrast thresholds were elevated in the affected
eye of 9/10 patients, bilaterally in one (case 6).
Retinal function was monitored in eight patients over
periods of between 10 months and 69 months, using serial
ERGs. There was mild but progressive photopic ERG
deterioration in case 4 (see below) and mild non-specific
worsening of full field ERGs in cases 5 and 10 (table 4 and
below). Five patients showed no significant electrophysiological change between recordings. In total, 7/10 patients
showed evidence of clinical or subjective deterioration since
ERGs were initially performed (table 4): in three of these
cases abnormalities related to the development of cataract
(case 8) or mild to moderate inflammatory changes (cases 6
and 7, see below). One patient with a history of bilateral
chronic uveitis developed unilateral night blindness following
Rod
400 µV
Case 4
400 µV
200 µV
200 µV
0V
0S
Max
0V
100 mS
0S
100 mS
200 µV
30 Hz
Case 7
400 µV
200 µV
200 µV
0V
0S
0S
400 µV
400 µV
Normal 200 µV
200 µV
0V
0S
100 mS
0V
0S
100 µV
50 µV
50 µV
0V
0S
0V
0S
Photopic
0V
50 mS
0S
50 mS
2 µV
25 µV
0V
0V
–2 µV
–25 µV
–4 µV
0S
2 µV
100 µV
0V
0V
0S
100 mS
0V
–25 µV
100 mS
–50 µV
0S
200 µV
200 µV
4 µV
150 µV
150 µV
50 µV
2 µV
100 µV
100 µV
25 µV
0V
50 µV
0V
50 µV
–2 µV
–25 µV
0V
0S
0V
50 mS
0S
50 mS
–4 µV
0S
200 mS
25 µV
–4 µV
0S
–50 µV
0S
50 µV
–2 µV
50 mS
ON–/OFF–
50 µV
150 µV
50 µV
50 mS
PERG
4 µV
4 µV
200 µV
50 µV
100 mS
200 µV
150 µV
100 µV
100 mS
A 30 year old woman presented with right acuity loss and
floaters. Right VA was 6/9. There was cystoid macular
oedema and vitreous snowballs on the right, and a diagnosis
of intermediate uveitis was made. There was tritan threshold
elevation
on
colour
contrast
sensitivity
testing.
Investigations, including HLA-A29, were negative. She
developed photopsia 2 years later, with right visual field
constriction, and was re-referred. FFA added no new
information. There was no RAPD. She developed nyctalopia
on the right 5 months later.
100 µV
150 µV
0V
100 mS
Case 7
150 µV
200 µV
400 µV
A 33 year old man presented with sudden painless loss of left
visual acuity. His VA was 6/60 and he had some very mild
peripheral inferior chorioretinal atrophy with a few vitreous
cells. There was a left relative afferent pupillary defect
(RAPD). Fields showed a dense left central scotoma. Colour
contrast thresholds were profoundly elevated on the left,
normal on the right. Antinuclear antibody (ANA) was weakly
positive but all other tests for lupus were negative. Oral
immunosuppression with steroids and azathioprine had no
effect. Fluorescein angiography (FFA) showed mild leakage
from one inferonasal vessel. Autofluorescence imaging
showed no definite abnormality but 2 years later showed
central hypofluorescence (fig 1).
ERGs were normal on the right (not shown). On the left,
the scotopic rod ERG was subnormal and the maximal ERG
was mildly electronegative, indicating post-phototransduction or post-receptoral rod system dysfunction (fig 2).
Photopic ERG a-wave and b-wave amplitudes and 30 Hz
flicker ERGs were severely reduced and showed mild to
moderate delay and the ON b-wave and OFF d-waves were
reduced in keeping with generalised cone system dysfunction. The PERG on the left was undetectable, consistent with
severe macular dysfunction. Serial electrophysiological studies revealed mild but progressive deterioration of photopic
full field ERGs on the left over a 5 year period. The patient
remained clinically stable.
100 mS
–50 µV
0S
200 mS
200 mS
Figure 2 Electrophysiological findings in the left eye of case 4 and right eye of case 7 (see text for details). ON-OFF ERGs were elicited using stimulus
duration 120 ms (case 4) and 200 ms (case 7 and normal). For clarity, blink artefacts occurring after the ERGs have been replaced by broken lines.
www.bjophthalmol.com
1624
Robson, Richardson, Koh, et al
Figure 3 Fundus photographs of case 10 showing chorioretinal
scarring in the periphery.
The right eye ERGs (fig 2) showed an electronegative
maximal response indicating dysfunction at a level that is
post-phototransduction or post-receptoral. The 30 Hz flicker
ERG was reduced and delayed with a flattened trough
between adjacent peaks; single flash photopic ERG shows a
broadened, bifid a-wave with a sharply rising b-wave. This
photopic ERG appearance is often associated with loss of ON
pathway function, but preservation of OFF pathway function,
and this was confirmed by the use of photopic ON and OFF
response recording. There was significant macular involvement shown by the reduced PERG. There was no evidence of
dysfunction on the left (not shown). Repeat testing
10 months later revealed no significant change. There has
been a recurrence of low grade uveitis since electrophysiology
was last performed.
Case 10
A 31 year old man presented with a 2 year history of
decreased acuity in his right eye. VA was 6/60 right, 6/5 left.
There was bilateral disc swelling with occasional vitreous
cells on the right, and chorioretinal scars (fig 3). There was
Rod
Max
400 µV
400 µV
RE
200 µV
0S
100 mS
400 µV
200 µV
100 mS
0V
0S
0S
0V
100 mS
200 µV
0V
100 mS
0S
100 mS
0S
0S
0S
100 mS
–50 µV
0S
200 µV
4 µV
150 µV
50 µV
2 µV
100 µV
25 µV
0V
0V
50 µV
–2 µV
–25 µV
0S
–4 µV
50 mS
0S
200 µV
150 µV
150 µV
100 µV
100 µV
0V
50 µV
50 µV
–2 µV
0S
100 mS
4 µV
0S
200 mS
25 µV
0V
–25 µV
–4 µV
50 mS
–50 µV
0S
200 mS
50 µV
2 µV
0V
50 mS
–25 µV
–4 µV
50 mS
200 µV
0V
0S
0V
–2 µV
0V
50 mS
25 µV
0V
0V
ON–/OFF–
50 µV
2 µV
50 µV
50 mS
PERG
4 µV
100 µV
50 µV
400 µV
200 µV
0V
0S
150 µV
100 µV
0V
400 µV
N
150 µV
150 µV
200 µV
0V
0S
100 mS
Photopic
200 µV
200 µV
400 µV
LE
0S
A 54 year old woman presented with a 3 month history of
unilateral night blindness. There was a 10 year history of
chronic bilateral intermediate uveitis. There had been a
recent flare up of uveitis with right CMO, successfully treated
with post-sub-Tenon’s injection of steroids. Visual acuities
were 6/6 bilaterally. There were inactive chorioretinal lesions
and snowballs scattered across the inferior periphery of both
30 Hz
50 µV
0V
0V
Case 12
200 µV
100 µV
200 µV
no RAPD. Fields showed marked enlargement of the blind
spot bilaterally. Magnetic resonance imaging scan was
normal. Colour vision testing with Ishihara plates gave scores
of 1/17 and 17/17 (right and left); colour contrast thresholds
were profoundly elevated on the right along protan, deutan
and tritan axes; thresholds along protan and deutan axes
were normal on the left with mild elevation along the tritan
axis.
ERG studies on the right initially revealed rod ERG and
maximal ERG a-wave reduction, delayed 30 Hz ERGs and
delayed and reduced photopic transient ERGs. The findings
indicate generalised retinal dysfunction affecting the rod and
cone systems, primarily at the level of the photoreceptors
with involvement of cone ON and OFF responses (fig 4). The
PERG was initially present but of markedly reduced
amplitude, suggesting moderately severe right macular
dysfunction.
Full field ERGs from the left eye (fig 4) showed ON
pathway involvement in both rod and cone systems (electronegative maximal response and cone ON b-wave loss). The
PERG was consistent with macular dysfunction, less severe
than on the right. The EOG revealed generalised dysfunction
affecting the photoreceptor/RPE interface bilaterally, worse
on the right (not shown).
Four years after presentation there were vitreous cells and
vessel sheathing bilaterally. The right eye developed an
epiretinal membrane, the left eye developed mild macular
oedema. There were increasing areas of peripapillary atrophy
bilaterally. Photopsia was reported on the left, without
significant acuity reduction. Over this period there was slight
deterioration in full field ERGs; PERG also deteriorated but
left visual acuity remained normal. Following treatment with
prednisolone and Diamox, full field ERGs remained stable
and the left PERG normalised.
100 mS
–50 µV
0S
200 mS
Figure 4 Electrophysiological findings in case 10 (see text for details). ON-OFF ERGs were elicited using stimulus duration 200 ms. For clarity, blink
artefacts occurring after the ERGs have been replaced by broken lines.
www.bjophthalmol.com
Non-vascular unilateral negative ERG
Rod
200 µV
200 µV
LE
100 mS
400 µV
400 µV
200 µV
200 µV
0V
0S
N
0S
100 mS
0V
100 mS
0S
30 Hz
100 mS
Photopic
200 µV
200 µV
150 µV
150 µV
100 µV
100 µV
0V
0S
0S
200 µV
150 µV
150 µV
100 µV
100 µV
50 µV
50 µV
50 mS
0V
0S
200 µV
200 µV
400 µV
400 µV
150 µV
150 µV
200 µV
200 µV
100 µV
100 µV
50 µV
50 µV
0V
0V
0S
100 mS
0S
100 mS
0V
0S
50 mS
0V
0S
ON–/OFF–
50 µV
25 µV
0V
0V
–25 µV
0V
50 mS
200 µV
0V
0S
PERG
4 µV
50 µV
50 µV
0V
0V
0S
Max
400 µV
400 µV
RE
1625
50 mS
–4 µV
0S
100 mS
–50 µV
0S
200 mS
50 µV
4 µV
25 µV
0V
0V
–25 µV
–4 µV
50 mS
0S
100 mS
4 µV
–50 µV
0S
200 mS
50 µV
25 µV
0V
0V
–25 µV
50 mS
–4 µV
0S
100 mS
–50 µV
0S
200 mS
Figure 5 Electrophysiological findings in case 12 (see text for details). ON-OFF ERGs were elicited using stimulus duration 200 ms. For clarity, blink
artefacts occurring after the ERGs have been replaced by broken lines.
eyes. In addition, the left eye showed significant vitritis,
vascular sheathing, and a peripapillary chorioretinal scar as a
result of a choroidal neovascular membrane.
The 30 Hz flicker ERGs were reasonably symmetrical,
markedly delayed but without significant amplitude reduction, in keeping with non-specific bilateral inflammatory
disease (Holder 2001), but only the night blind right eye had
an electronegative ERG and selective reduction in the ON bwave (fig 5). Reduced PERGs were consistent with bilateral
macular dysfunction.
DISCUSSION
A unilateral ‘‘negative’’ ERG, not related to occlusive vascular
disease or birdshot chorioretinopathy, is uncommon. Twelve
such patients are described who were ascertained as an
extension of a large retrospective study into electronegative
ERGs, in which seven such cases were identified.21 The
patients presented with a variety of signs and symptoms.
Posterior segment inflammatory changes were evident in
seven of the 12 cases and in six of these cases inflammatory
signs or symptoms were either confined to the eye with an
electronegative ERG or were more severe in this eye. In one
patient (case 10) there were bilateral inflammatory changes
associated with an electronegative ERG in one eye. There
were ERG abnormalities in the other eye indicating generalised retinal dysfunction affecting rod and cone systems
(fig 4), primarily at the level of the photoreceptors, associated
with a longer history, and chorioretinal scarring (fig 3).
Selective involvement of the retinal ON bipolar pathway was
present in the majority of cases.
The clinical presentation of the patients in our series was
diverse. Most presented with loss of acuity, blurred or
distorted vision, or altered colour vision. Although difficulty
in dim lighting was admitted by some patients on direct
questioning, it was not usually a presenting symptom.
However, one patient with a 10 year history of recurrent
intermediate uveitis developed unilateral problems with
night vision 3 months before ERG testing (case 12).
Previous reports have appeared of patients with acquired
unilateral night blindness, normal fundi, raised rod thresholds and unilateral negative ERGs,30 including one case in
which ON-OFF ERGs were additionally performed.31 32
Similar findings were present in case 7, who also developed
unilateral night blindness. The negative ERG, reduced ON bwave and other unilateral electrophysiological abnormalities
in these patients are qualitatively identical to those seen
(bilaterally) in ‘‘complete’’ X linked CSNB3 8 and MAR.3 16 17 31
Similar bilateral changes can occasionally occur in association with carcinoma associated retinopathy (CAR; Holder,
unpublished observations), although CAR more commonly
affects photoreceptor function and rarely gives a negative
ERG. Both CAR and MAR are paraneoplastic retinopathies in
which antibodies produced in response to a tumour antigen
cross react with elements in the retina. The characteristic ONOFF ERG abnormalities of MAR can be induced in monkeys
by intravitreal injection of IgG from a patient with MAR.33
Immunohistochemistry using sera from MAR patients shows
selective labelling of rod bipolar cells in human retina34 and
there is reduced density of the bipolar cell layer in
postmortem specimens from MAR patients.35 It is of interest
that although one patient in the present series had a history
of cutaneous melanoma, the findings in that patient involved
both ON and OFF pathways and were not in keeping with
MAR (case 2).
Autoimmune retinopathy has been postulated as a cause
for severe visual loss and photopsia in two patients with no
signs of ocular inflammation or systemic malignancy.36 Both
patients had evidence of a familial immune regulation defect
and in one of these cases ERGs were bilaterally electronegative but more severely abnormal in one eye. Both
patients’ sera had antiretinal antibodies that localised to
the inner plexiform layer. The progression of the visual loss
was much slower than that in CAR and was asymmetrical.
Anterior segment inflammation has been previously
described in a patient with acquired unilateral nyctalopia,
photopsia, and visual loss with no history of malignancy.37
That patient also had a unilateral negative ERG with selective
involvement of the ON response (similar to our cases 7 and
12).
The mechanisms underlying the functional changes in
these patients are not established but the high incidence of
previous inflammatory disease in affected eyes is noted, as is
the predilection for ON response b-wave loss and OFF
response sparing. Five of seven patients with evidence of
inflammatory disease had selective or predominant dysfunction of the cone ON response. This association between
www.bjophthalmol.com
1626
functional disruption of the ON response and a history of
unilateral or asymmetrical ocular inflammation possibly
involves an autoimmune reaction. The target site of the
autoimmune reaction in the retina remains unclear but, as in
MAR, is post-transduction and may be the bipolar cell or its
connections. Antibodies may be directed towards antigens,
disrupting signalling through the bipolar cell layer, or
inflammatory changes may cause damage with subsequent
exposure of antigens leading to secondary antibody production.37
In summary, a unilateral negative ERG in the absence of an
occlusive vascular aetiology or BCR is an unusual finding and
often associated with sparing of inner retinal OFF response
function. Patients present with diverse symptoms and signs
but the high incidence of previous inflammatory disease
raises the possibility that the electrophysiological changes are
mediated by autoimmune mechanisms. Additional studies,
testing serum against retinal tissue, may assist in confirming
a possible autoimmune aetiology.
ACKNOWLEDGEMENTS
The Foundation Fighting Blindness (AGR).
.....................
Authors’ affiliations
A G Robson, A H C Koh, A Calcagni, G E Holder, Electrophysiology,
Moorfields Eye Hospital, London, UK
E C Richardson, C E Pavesio, P G Hykin, Medical Retina Service,
Moorfields Eye Hospital, London, UK
E M Graham, Ophthalmology, St Thomas’s Hospital, Lambeth Palace
Road, London, UK
REFERENCES
1 Heckinlively JR, Arden GB. Principles and practice of clinical
electrophysiology of vision. St Louis: Mosby Year Book, 1991.
2 Fishman GA, Birch DG, Holder GE, et al. Electrophysiologic testing in
disorders of the retina, optic nerve and visual pathway. 2nd ed.
Ophthalmology Monographs No 2. San Francisco: The Foundation of the
American American Academy of Ophthalmology, 2001.
3 Holder GE. Pattern ERG and an integrated approach to visual pathway
diagnosis. Prog Retin Eye Res 2001;20:531–61.
4 Miyake Y, Sushiroyama N, Horiguchi M, et al. Bull’s eye maculopathy and the
negative electroretinogram. Retina 1989;9:210–15.
5 Kellner U, Foerster MH. Cone dystrophies with negative photopic
electroretinogram. Br J Ophthalmol 1993;77:404–9.
6 Kellner U, Brummer S, Foerstert MH, et al. X-linked congenital retinoschisis.
Graefes Arch Clin Exp Ophthalmol 1990;228:432–7.
7 Bradshaw K, George N, Moore A, et al. Mutations of the XLRS1 gene cause
abnormalities of photoreceptor as well as inner retinal responses of the ERG.
Doc Ophthalmol 1999;98:153–73.
8 Miyake Y, Yagasaki K, Horiguchi M, et al. Congenital stationary night
blindness with negative electroretinogram. Arch Ophthalmol
1986;104:1013–20.
9 Noble KG, Carr RE, Siegel IM, et al. Autosomal dominant congenital
stationary night blindness and normal fundus with with an electronegative
ERG. Am J Ophthalmol 1990;109:44–8.
www.bjophthalmol.com
Robson, Richardson, Koh, et al
10 Henkes HE. Electroretinography in circulatory disturbances of the retina:
electroretinogram in cases of occlusion of the central retinal artery or one of its
branches. Arch Ophthalmol 1954;52:42–53.
11 Karpe G, Ucherman A. The clinical electroretinogram. VII: The
electroretinogram in circulatory disturbances of the retina. Acta Ophthalmol
1955;33:493–516.
12 Yotsukura J, Adach-Usami E. Correlation of electroretinographic changes
with visual prognosis in central retinal artery occlusion. Ophthalmologica
1993;207:13–18.
13 Priem HA, De Rouck, De Laey J, et al. Electrophysiologic studies in birdshot
chorioretinopathy. Am J Ophthalmol 1988;106:430–6.
14 Hirose T, Katsumi O, Pruett RC, et al. Retinal function in birdshot
retinochoroidopathy. Acta Ophthalmol 1991;69:327–37.
15 Holder GE, Robson AG, Pavesio C, et al. Electrophysiological characterisation
and monitoring in the management of birdshot chorioretinopathy.
Br J Ophthalmol 2005;89:709–18.
16 Berson EL, Lessell S. Paraneoplastic night blindness with malignant melanoma.
Am J Ophthalmol 1988;106:307–11.
17 Alexander KR, Fishman GA, Peachey NS, et al. ON response defect in
paraneoplastic night blindness with cutaneous malignant melanoma. Invest
Ophthalmol Vis Sci 1992;33:477–83.
18 Bacon P, Spalton DJ, Smith SE. Blindness from quinine toxicity. Br J Ophthalmol
1988;72:219–224.
19 Treichel JL, Murray TG, Lewandowski MF, et al. Retinal toxicity in methanol
poisoning. Retina 2004;24:309–12.
20 Kaplan HJ, Aaberg TM. Birdshot retinochoroidopathy. Am J Ophthalmol
1980;90:773–782.
21 Koh AH, Hogg CR, Holder GE. The incidence of negative ERG in clinical
practice. Doc Ophthalmol 2001;102:19–30.
22 Marmor MF, Holder GE, Seeliger MW, et al. Standard for clinical
electroretinography (2004 update). Doc Ophthalmol 2004;108:107–14.
23 Bach M, Hawlina M, Holder GE, et al. Standard for pattern
electroretinography. Doc Ophthalmol 2000;101:11–18.
24 Marmor MF, Zrenner E. Standard for clinical electro-oculography. Doc
Ophthalmol 1993;85:115–24.
25 Arden GB, Kelsey JH. Changes produced by light in the standing potential of
the human eye. J Physiol 1962;161:189–204.
26 Sieving PA. Photopic ON and OFF-pathway abnormalities in retinal
dystrophies. Trans Am Ophthalmol Soc 1993;91:701–73.
27 Spileers W, Falcao-Rewis F, Smithj R, et al. The human ERG evoked by a
Ganzfield stimulator powered by red and green light emitting diodes. Clin Vis
Sci 1993;8:21–38.
28 Calcagni A, Hogg CR, Spackman L, et al. Characterisation of the normal
photopic electroretinographic responses to long duration stimuli evoked by a
light emitting diode stimulator. Clin Neurophysiol 2001;112:1964–6.
29 Arden GB, Gunduz K, Perry S. Colour vision testing with a computer graphics
system. Clin Vis Sci 1988;2:303–20.
30 Ayaki M, Oguchi Y, Matsuhashi M. Unilateral night blindness with a normal
fundus. Am J Ophthalmol 1984;98:630–2.
31 Fishman GA, Alexander KR, Milam AH, et al. Acquired unilateral night
blindness associated with negative electroretinogram waveform.
Ophthalmology 1996;103:96–104.
32 Barnes CS, Brigell MG, Alexander KR. ON-pathway dysfunction in a patient
with acquired unilateral night blindness. Retina 1998;18:531–8.
33 Lei B, Bush RA, Milam AH, et al. Human melanoma-associated retinopathy
(MAR) antibodies alter the retinal ON-response of the monkey ERG in vivo.
Invest Ophthalmol Vis Sci 2000;41:262–6.
34 Milam AH, Saari JC, Jacobson SG, et al. Autoantibodies against retinal
bipolar cells in cutaneous melanoma-associated retinopathy. Invest
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35 Gittinger JW, Smith TW. Cutaneous melanoma-associated paraneoplastic
retinopathy: histopathologic observations. Am J Ophthalmol
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36 Mizener JB, Kimura AE, Adamus G, et al. Autoimmune retinopathy in the
absence of cancer. Am J Ophthalmol 1997;123:607–8.
37 Murayama K, Kawabata H, Adachi-Usami E. An electrophysiological followup study on acquired unilateral nyctalopia. Eye 1999;13:629–34.
1627
SCIENTIFIC REPORT
Increased expression of vascular endothelial growth factor
associated with accumulation of lipids in Bruch’s membrane
of LDL receptor knockout mice
M Rudolf, B Winkler, Z Aherrahou, L C Doehring, P Kaczmarek, U Schmidt-Erfurth
...............................................................................................................................
Br J Ophthalmol 2005;89:1627–1630. doi: 10.1136/bjo.2005.071183
Aim: To investigate the pathogenesis of age related macular
degeneration (ARM) with respect to lipid accumulation within
Bruch’s membrane (BrM) in a knockout model with low
density lipoprotein (LDL) receptor deficiency.
Methods: LDL receptor deficient mice and C57BL/6 controls
were fed a standard diet or a high fat (HF) diet. Plasma total
cholesterol (pTC) was determined. Eyes were examined by
transmission electron microscopy. Immunohistochemical
staining for VEGF was performed.
Results: pTC were highest in LDL receptor deficient mice after
HF diet and elevated after standard diet compared to controls
with and without HF diet. While BrM of controls did not
exhibit any visible changes, membrane bound translucent
particles were seen in all BrM of knockout mice. The amount
of these particles was substantially increased and membranes were thickened after HF diet. VEGF staining was
positive in knockout mice only and was located in retinal
pigment epithelial cells, the outer plexiform layer, and
photoreceptor inner segments. Most intensive VEGF expression was documented after HF diet.
Conclusion: LDL receptor deficient mice exhibit an accumulation of lipid particles in BrM which is further increased after
fat intake. VEGF expression is found in the outer retinal
layers of LDL receptor deficient mice and appears to correlate
with the amount of lipid particles present in BrM.
A
ge related maculopathy (ARM) is the most frequent
cause of severe visual impairment in the elderly in
industrial countries. In these patients, most dramatic
irreversible vision loss occurs as a result of subretinal
choroidal neovascularisation (CNV).1
The pathogenesis of ARM is poorly understood. Lesions
that involve retinal pigment epithelium (RPE) and Bruch’s
membrane (BrM) are the most prominent clinical and
histopathological features of ARM.2–4 Recent findings highlight a role for lipids. In normal human retina, neutral lipids
accumulate in BrM throughout life.5 In some epidemiological
studies atherosclerosis has been associated with ARM, but a
correlation was not consistently found in all surveys.1 6–8 Also
risk factors for atherosclerosis, such as advanced age,
hypercholesterolaemia, smoking, high intake of saturated
fat, and decreased oestrogen exposure, have been associated
with ARM in other studies.1 7 8
In this study we used a well established atherosclerotic
murine model to evaluate changes in BrM and associated
biological effects caused by elevated plasma total cholesterol
(pTC) levels.
LDL (low density lipoprotein) receptor deficient mice
(C57BL/6J background) are not able to incorporate plasma
cholesterol into body cells sufficiently, resulting in increased
pTC even under standard diet. Our controls were C57BL/6J
mice without any receptor deficiency. pTC levels were
furthermore modified by different diets varying in fat
content. Using this setting we already observed distinctive
lipid rich degenerations of BrM.9
Vascular endothelial growth factor (VEGF) was identified
as one of the major stimuli in experimental CNV.10 We
performed immunohistochemical staining for VEGF in
normal and knockout animals to identify its expression and
distribution patterns and its eventual correlation with BrM
changes.
MATERIALS AND METHODS
The use of animals was in accordance with the ARVO
statement for the use of animals in ophthalmic and vision
research. Ethics committee institutional review board
approval was obtained.
Female wild type C57BL/6J and LDL receptor deficient
C57BL/6J-LDL-r(2/2) mice were perchased (Jackson
Laboratories, Bar Harbor, ME, USA).
Animals were maintained in plastic cages with even lightdark cycle with permanently free access to water and food. At
the age of 2 months, regular chow (12% of calories as fat)
was changed in one half of C57BL/6J (n = 6) and C57BL/6JLDL-r(–/–) (n = 6) population to high fat (HF), high
cholesterol diet western style (TD 90221, Harlan-Teklad,
Madison, WI, USA) containing 30% of calories as fat. All
other C57BL/6J (n = 6) and C57BL/6J-LDL-r(–/–) (n = 6)
continued their regular chow diet. Both diets contained
comparable mineral and vitamin mixes.
Plasma cholesterol levels
Heparins blood was obtained by retro-orbital bleeding from
anaesthetised mice before sacrifice. pTC and triglyceride
concentrations were measured using standard colorimetric
assays purchased from Sigma (Deisenhofen, Germany) and
Roche Diagnostics (Hamburg, Germany).
Tissue preparation
At the age of 4 months all animals were anaesthetised with
isoflurane and sacrificed by cervical dislocation. Eyes were
enucleated immediately. Whole eyes were fixed in 2.5%
glutaraldehyde in 0.1 M cacodylate-0.2 M sodium phosphate
buffer, postfixed in 1% osmium tetroxide, stained with 1%
uranyl acetate, and embedded in epoxy resin (Epon 812).
Abbreviations: ARM, age related macular degeneration; BrM, Bruch’s
membrane; CNV, choroidal neovascularisation; HF, high fat; IPS, inner
photoreceptor segments; LDL, low density lipoprotein; OPL, outer
plexiform layer; pTC, plasma total cholesterol; RPE, retinal pigment
epithelium; TEM, transmission electron microscopy; VEGF, vascular
endothelial growth factor
www.bjophthalmol.com
1628
Rudolf, Winkler, Aherrahou, et al
Table 1
Plasma lipid levels in C57BL/6 and LDL receptor deficient mice
Plasma total
LDL+VLDL
Mouse
Diet
Cholesterol
Triglycerides
Cholesterol
C57BL/6J
C57BL/6J
C57BL/6 ldlr2/2
C57BL/6 ldlr2/2
chow
high fat
chow
high fat
82.0 (19.5)
119.2 (36.5)
189.0 (37)
498.3 (134.3)
29.2 (6.0)
36.0 (3.1)
72.7 (20.6)
137.8 (44.8)
28.0 (15.9)
109.0 (44.3)
125.7 (29.3)
443.8 (124.7)
Data are expressed in mean (SD) mg/dl, LDL, low density lipoprotein; VLDL, very low density lipoprotein; n = 6 in
each group.
Eyes were sectioned in 1 mm on an ultramicrotome. The
tissue sections were stained with 2% toluidine blue O and
examined under light microscope.
Transmission electron microscopy
After determining areas of interest, thin sections of approximately 50–90 nm were cut, collected on copper grids and
stained with 4% uranyl acetate and lead citrate.
Subsequently, the sections were evaluated by transmission
electron microscopy (TEM, Carl Zeiss EM9, Oberkochen,
Germany) and photographed.
Immunohistochemical analysis
Tissue sections were stained using the streptavidin-biotin
method with staining agent AEG (red) and Mayer’s
haemalaun for nucleus staining (blue). The purified G143850 antibody (Mouse IgG2a, PharMingen, San Diego, USA)
has been used to immunoprecipitate native VEGF (1 g mAb/
50 ml lysate) and to identify three VEGF isoforms (165, 189,
206aa).
Statistical analysis
Mean plasma cholesterol levels were calculated. The
Wilcoxon rank sum test was performed to determine
statistically significant differences in cholesterol levels
according to diet and LDL receptor deficiency.
Figure 2 TEM: 4 months old LDL receptor deficient mice (C57Bl/6JLDL-r(–/–)) after 2 months of high fat diet. Bruch’s membrane (BrM) is
substantially thickened with beginning condensation of collagenous and
elastic fibres. The number of membrane bound translucent vesicles is
increased with additional deposits of non-membrane bound particles.
The non-membrane bound electron lucent particles were round and
occasionally confluent. (arrows) Retinal pigment epithelium (RPE),
melanosomes (MS); scale: white bar = 0.5 mm.
RESULTS
pTC levels
pTC levels were highest in LDL receptor deficient mice after
HF diet (p = 0.0121) and significantly elevated after chow
diet (p = 0.032) compared to control mice with (p = 0.024)
and without HF diet (table 1).
Bruch’s membrane changes in TEM
Eleven of 12 control animals did not exhibit any visible
changes in BrM by TEM even after HF diet (fig 1A). In
contrast, membrane bound translucent particles were seen in
membranes of all 12 LDL receptor deficient mice (+1 control
after HF) (fig1B).
Additionally, in all six knockout mice following HF diet
membranes were substantially thickened with a beginning
condensation of collagenous and elastic fibres. In this group
the number of translucent vesicles was also increased with
additional deposits of non-membrane bound particles (fig 2)
which were round, occasionally confluent, and scattered
throughout both collagenous layers.
Figure 1 TEM: (A) 4 months old control mice (C57Bl/6J) after
2 months of high fat diet. Homogeneous Bruch’s membrane (BrM) with
regular structure, retinal pigment epithelium (RPE), melanosomes (MS).
(B) 4 months old LDL receptor deficient mice (C57Bl/6J-LDL-r(–/–)) after
chow diet, Bruch’s membrane with regular architecture but with basal
membrane bound translucent vesicles (arrows); scale: white bar
= 0.5 mm.
www.bjophthalmol.com
VEGF immunohistology
No significant VEGF expression was detected in all 12 control
mice irrespectively of diet (+1 knockout after chow) (fig 3A).
However, positive staining for VEGF was found in 11 of
12 LDL receptor deficient mice and was predominantly
located in basal RPE, the outer plexiform layer and
photoreceptor inner segments (fig 3B). Highest intensity in
VEGF associated with lipids in Bruch’s membrane
Figure 3 VEGF immunohistology: (A) 4 months old control mice
(C57Bl/6J) after chow diet, no changes were observed after VEGF
antibody incubation. VEGF staining was negative. (B) 4 months old LDL
receptor deficient mice (C57Bl/6J-LDL-r(–/–)) after 2 months of high fat
diet. VEGF staining is positive (intense red) in retinal pigment epithelium
(RPE), outer plexiform layer (OPL), inner photoreceptor segments (IPS).
VEGF expression was documented in all six LDL receptor
deficient mice following HF diet.
DISCUSSION
The LDL receptor knockout model serves as an established
model for atherosclerotic pathomechanisms because of the
opportunity to induce elevated pTC levels.
In our study pTC was significantly increased by LDL
receptor deficiency as well as HF diet. LDL receptor deficient
mice after HF diet showed the highest average pTC, which
was 6.0 times more than in C57Bl/6J control mice consuming
standard chow diet.
Furthermore, we demonstrated a structural degeneration
of BrM with thickening and accumulation of membrane and
non-membrane bound translucent particles in LDL receptor
deficient mice correlating with significantly elevated pTC
levels. The layer arrangement was affected as well. These
degenerations were most prominent in LDL receptor deficient
mice after HF diet. Hypercholesterolaemia as an atherosclerotic risk factor seems to predispose to these structural
alterations in BrM.
The observed changes in murine BrM resemble those seen
in human eyes of aged donors and donors with ARM.11–13
Electron lucent droplets in adult human eyes are also
scattered throughout BrM and form in elderly eyes a discrete
layer external to the RPE basal lamina.4 5 Membrane bounded
vesicles are the principal component of basal linear deposits
and large drusen. The presence of such lipid deposits is
associated with early and late ARM.4 There is evidence in
humans that the observed translucent vesicular changes are
not vesicles, but solid neutral lipid rich particles extracted by
regular tissue processing for TEM.3 14 In histological studies
such particles bind oil red O and contain esterified cholesterol
and triglyceride.5 14 The electron lucent droplets in basal
linear deposits resemble extracellular material found in
atheromatous plaques.15 In general, human BrM seems to
undergo ageing processes similar to the arterial intima layer
1629
and other connective tissues for which plasma lipoproteins
are the known source of extracellular cholesterol.3 15
BrM is a connective tissue composed of five layers. It is
responsible for many transport and support functions
between RPE and choriocapillaris and is therefore essential
for the health of the entire retina. Accumulation of lipids in
BrM is believed to alter its diffusion characteristics and
potentially compromises the metabolic exchange between
choroid and retina affecting photoreceptor function.1
Expression of VEGF was detectable exclusively in LDL
receptor deficient mice. Most intensive VEGF staining was
found in LDL receptor deficient mice following HF diet
corresponding to the increased amount of vesicles in this
group. VEGF was detected in RPE, photoreceptor inner
segments, and the outer plexiform layer.
VEGF is an angiogenic growth factor which is known to be
a major stimulator for ocular neovascularisation.1 2 The two
smaller VEGF isoforms 165 and 121 are secreted proteins and
act as diffusible agents while the two larger isoforms 189 and
VEGF 206 remain cell associated. Predominantly, the RPE is
known to secret VEGF supporting paracrine and autocrine
functions.1 A major pathomechanism for increased VEGF
expression in the pathogenesis of CNV is not clearly
identified yet.2 Unlike retinal neovascularisation, it is not
apparent that general hypoxia is an important factor for
upregulation of VEGF for CNV development.2 16–19 Cumulative
oxidative stress caused by reactive oxygen intermediates
occurring as byproducts of cellular metabolism and photochemical reactions as well as advanced glycation end
products may have a role.16 17 20 Compromised diffusion via
lipid rich, hydrophobic BrM may cause increased oxidative
stress by accumulating metabolic end products.1 20 It is
beyond the scope of this pilot study to determine the cause
of more intensive VEGF expression. However, an increased
VEGF expression in relation to advanced alterations of BrM
caused by elevated plasma cholesterol levels was clearly
observed. In our model no spontaneous CNV was observed,
but no substantial breaks in BrM either, which is a supposed
prerequisite. Increased thickness and hydrophobicity of BrM
by lipids may prevent VEGF from reaching the choriocapillaris and stimulating CNV.1 A break in BrM by, for example,
advanced degeneration definitively would overcome this
barrier and CNV may develop. These observations appear to
bridge the gap between hypercholesterolaemia and BrM
changes as well present in parts of a possible progression
mechanism of early ARM to neovascular stages.
CONCLUSION
Atherosclerosis and ARM appear to be complex diseases with
many environmental and genetic factors. In part, they share
common risk factors and common animal models can be
used to learn more about these age related degenerative
diseases.
.....................
Authors’ affiliations
M Rudolf, Department of Ophthalmology, University of SchleswigHolstein, Campus Kiel, Germany
B Winkler, Department of Ophthalmology, University of SchleswigHolstein, Campus Luebeck, Germany
Z Aherrahou, L C Doehring, P Kaczmarek, Atherosclerotic Study Group,
Department of Medicine, University of Luebeck, Germany
U Schmidt-Erfurth, Department of Ophthalmology, University of Vienna,
Austria
Competing interests: none declared
Correspondence to: Dr med Martin Rudolf, University of SchleswigHolstein, Campus Kiel, Department of Ophthalmology, HegewischStrasse 2, 24105 Kiel, Germany; [email protected]
Accepted for publication 1 July 2005
www.bjophthalmol.com
1630
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1 Ambati J, Balamurali K, Ambati K, et al. Age-related macular degeneration:
etiology, pathogenesis, and therapeutic stragegies. Surv Ophthalmol
2003;48:257–92.
2 Campochiaro RA. Retinal and choroidal neovascularization. J Cell Physiol
2000;184:301–10.
3 Curcio AC, Millican CL, Bailey T, et al. Accumulation of cholesterol
with age in human Bruch’s membrane. Invest Ophthalmol Vis Sci
2001;42:265–73.
4 Curcio AC, Millican CL. Basal linear deposits and large drusen are specific for
early age-related maculopathy. Arch Ophthalmol 1999;117:329–39.
5 Pauleikoff D, Harper CA, Marshall J, et al. Aging changes in Bruch’s
membrane: a histochemical and morphological study. Ophthalmol
1990;97:171–8.
6 Vingerling JR, Dielemans I, Bots ML, et al. Age-related macular degeneration
is associated with atherosclerosis. The Rotterdam Study. Am J Epidemiol
1995;142:404–9.
7 Snow K, Seddon J. Do age-related macular degeneration and
cardiovascular disease share common antecedents? Opthalmic Epidemiol
1999;6:125–43.
8 The Eye Disease Case Control Study Group. Risk factors for neovascular agerelated macular degeneration. Arch Ophthalmol 1992;110:1701–1708.
9 Rudolf M, Ivandic B, Winkler J, et al. Akkumulation von Lipidpartikeln in der
Bruchmembran von LDL-Rezeptor-defizienten Mäusen als Modell für die
Altersbezogene Makuladegeneration. Ophthalmol 2004;101:715–19.
10 Kwak N, Okamoto N, Wood JM, et al. VEGF is major stimulator in
model of choroidal neovaskularisation. Invest Ophthalmol Vis Sci
2000;41:3158–64.
www.bjophthalmol.com
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11 Sheraidah GS, Steinmetz R, Maguire J, et al. Correlation between lipids
extracted from Bruch’s membrane and age. Ophthalmology
1993;100:47–51.
12 Green WR, Enger C. Age-related macular degeneration histopathologic
studies. Ophthalmology 1993;100:1519–35.
13 Dithmar S, Curcio AC, Le HA, et al. Ultrastructural changes in Bruch’s
membrane of apolipoprotein E-deficient mice. Invest Ophthalmol Vis Sci
2000;41:2035–42.
14 Holz FG, Sheraidah G, Pauleikoff D, et al. Analysis of lipid deposits extracted
form human macular and peripheral Bruch’s membrane. Arch Ophthalmol
1994;112:402–6.
15 Bocan TM, Schifani TA, Gyuton JR. Ultrastructure of human aortic fibrolipid
lesion: formation of the atherosclerotic lipid-rich core. Am J Pathol
1986;123:413–24.
16 Kuroki M, Voest EE, Amano S, et al. Reactive oxygen intermediates increase
vascular endothelial growth factor expression in vitro and in vivo. J Clin Invest
1996;98:1667–75.
17 Lu M, Kuroki M, Amano S, et al. Advanced glycation end products increase
retinal vascular endothelial growth factor expression. J Clin Invest
1998;101:1219–24.
18 Wada M, Ogata N, Otsuji T, et al. Expression of VEGF and its receptor (KDR/
flk-1) mRNA in experimental choroidal neovascularization. Curr Eye Res
1999;18:203–13.
19 Stone J, Itin A, Alon T, et al. Development of retinal vasculature is mediated by
hypoxia-induced VEGF expression by neuroglia. J Neurosci
1995;15:4738–47.
20 Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the
pathogenesis of age-related macular degeneration. Surv Ophthalmol
2000;45:115–34.
1631
SCIENTIFIC REPORT
Advanced glycation end products can induce glial reaction
and neuronal degeneration in retinal explants
A Lecleire-Collet, L H Tessier, P Massin, V Forster, G Brasseur, J A Sahel, S Picaud
...............................................................................................................................
Br J Ophthalmol 2005;89:1631–1633. doi: 10.1136/bjo.2005.079491
Background/aims: Neuronal degeneration has been
reported to occur in diabetic retinopathy before the onset
of detectable microvascular abnormalities. To investigate
whether advanced glycation end products (AGE) could be
directly responsible for retinal neurodegeneration, retinal
explants were incubated with glycated bovine serum albumin
(BSA).
Methods: Retinal explants obtained from non-diabetic adult
rats were incubated 4 days with or without 200 mg/ml
glycated BSA. Neural apoptosis was quantified by terminal
dUTP nick end labelling (TUNEL) binding and immunostaining with anti-cleaved caspase-3 antibody. Expression of glial
fibrillary acidic protein (GFAP) was localised by immunofluorescence.
Results: TUNEL and cleaved caspase-3 positive cells
increased significantly by 2.2-fold and 2.5-fold in retinal
explants incubated in glycated BSA (p,0.05), respectively.
The ganglion cell layer was the most sensitive retinal layer to
the glycated BSA. Neuronal degeneration was confirmed by
the increased GFAP labelling in Müller glial cells from retinal
explants treated with glycated BSA.
Conclusion: These results suggest that AGE could induce
retinal neurodegeneration in the absence of blood perfusion.
Cells in the ganglion cell layer appeared to be the most
sensitive as in diabetic retinopathy and its animal models.
AGE toxicity could therefore contribute to the early
pathological mechanisms of diabetic retinopathy.
N
eural cell depletion was observed decades ago in
diabetic patients.1 Although diabetic retinopathy (DR)
is classically considered a retinal microangiopathy,
loss of colour vision, of contrast sensitivity, electroretinogram
abnormalities, and neuronal apoptosis were detected in
diabetic patients before capillary closure and microaneurysms, suggesting that DR could have a neuropathic component that may occur early in the disease.2 3 These
observations have been confirmed by animal studies showing
that neuronal apoptosis and glial reaction could occur after
only 1 month of experimental diabetes in streptozotocin
induced diabetic rat.3 4
The mechanisms by which diabetes induces neuroglial
reactions remain unknown. These neuroglial reactions are
unlikely to be the result of ischaemic necrosis since they
occur very early in diabetic rats before the onset of any
known vascular occlusion.3 4 However, neural cell death due
to increased vascular permeability cannot be ruled out, since
some increase in permeability can occur after 2–4 weeks of
experimental diabetes in rats.3 4 The advanced glycated end
products (AGE) can provide the early molecular pathogenic
mechanisms responsible for neuronal apoptosis and neuroglial reactions. Their involvement in the development of DR is
supported by the prevention of DR by blockers of AGE
formation in animal models.5 6 The potential retinal toxicity
of AGE was consistent with apoptosis of cultured cortical
neurons7 and with their accumulation in the retina of
diabetic patients.8
The present study was performed to assess whether AGE
can directly trigger neuronal apoptosis in the retina,
independently of blood vessel circulation. AGE were applied
to retinal explants, as they both preserve the retinal cellular
architecture and limit the influence of the blood vasculature.
MATERIALS AND METHODS
Male non-diabetic Long Evans rats (Charles River
Laboratories, France) aged 7 weeks were used for this study.
Animal studies conformed to the principles of laboratory
animals (NIH publication no 85–23, revised 1985), and the
French law on animal protection.
Retinal explants were obtained according to the protocol
described by Pinzon-Duarte et al.9 The choroid was gently
peeled away from the retinal pigment epithelium (RPE),
leaving the RPE attached to the neurosensory retina. The
retina was transferred onto a polycarbonate membrane
(Transwell, Corning, Netherlands) and cultured in
Dulbecco’s Modified Eagle Medium containing 10% fetal calf
serum and 10 mg/ml gentamicin. For each animal, one retina
was used as a control retinal explant and the other retina was
incubated in 200 mg/ml glycated BSA (Sigma Chemical Co, St
Louis, MO, USA, 95% purity) for 4 days, a period providing
the lowest variability when inducing apoptosis in such ex
vivo retina.10 This glycated BSA concentration was selected
because similar concentrations (250 mg/ml; 100 mg/ml) were
used previously to demonstrate AGE neurotoxicity7 and AGE
role in increased retinal expression of vascular endothelium
growth factor (VEGF).11 Furthermore, circulating AGE
ranged in diabetic patients between 1–120 mg/ml, which
was considered equivalent to 4–480 mg/ml glycated BSA by
the authors of the study.12 No albumin was added to the
control condition because the culture medium already
contains albumin (2700 mg/ml) from the fetal calf serum
such that the addition of glycated BSA represented less than
8% increase of its concentration.
The retinal tissue was fixed and processed for immunohistochemistry and terminal dUTP nick end labellingfluorescein-isothiocyanate (TUNEL) labelling, as previously
described.10 13 Retinal explant sections were labelled with
anti-cleaved active caspase-3 antibody (Cell Signalling
Technology, Herts, UK, 1:100), anti-vimentin antibody
(Chemicon International, Temecula, CA, USA, 1:200),
Abbreviations: AGE, advanced glycation end products; BSA, bovine
serum albumin; DAPI, diamidino-phenyl-indole; DR, diabetic
retinopathy; GCL, ganglion cell layer; GFAP, glial fibrillary acidic
protein; GLAST, L-glutamate/L-aspartate transporter antibody; INL, inner
nuclear layer; ONL, outer nuclear layer; OPL, outer plexiform layer;
RAGE, AGE receptor; RPE, retinal pigment epithelium; TUNEL, terminal
dUTP nick end labelling; VEGF, vascular endothelium growth factor
www.bjophthalmol.com
1632
Lecleire-Collet, Tessier, Massin, et al
(250 mm long) from the retinal explant section. The number
of cells positive for the TUNEL staining or the cleaved
caspase-3 immunolabelling was normalised to the DAPI
labelled retinal cell nuclei. Data were statistically analysed
with the Mann-Whitney rank sum test.
RESULTS
AGE induced retinal cell apoptosis
The treatment with glycated BSA for 4 days induced a 2.1fold increase in the number of TUNEL positive cells (glycated
BSA: 14.44% (SD0.62%), n = 7; control: 6.63% (0.46%), n
= 7, p ,0.001), which was statistically significant in all
nuclear retinal layers (fig 1A, B, E). Small groups of adjacent
TUNEL positive cells were furthermore observed in the inner
part of the outer nuclear layer (ONL) in four retinal explants
treated with glycated BSA (n = 7) (fig 1B), whereas these
characteristics were not observed in control retinal explants
(fig 1A).
To confirm the retinal toxicity elicited by glycated BSA,
retinal explant sections were labelled with an earlier marker
of apoptosis, the anti-cleaved active caspase-3 antibody
(fig 1C, D). The treatment with glycated BSA induced a
2.4-fold increase in active caspase-3 positive cells (glycated
BSA: 8.59% (0.39%), n = 7; control: 3.48% (0.44%), n = 7, p
,0.001), statistically significant in all nuclear retinal layers
(fig 1F). These data suggest that a glycated protein can
induce neuronal apoptosis in the retina as early as 4 days of
incubation.
AGE induced glial reaction
Neuronal apoptosis is classically associated to a glial reaction
which is demonstrated by a local increase in GFAP expression
in the retina at the site of the lesion.13 In control retinal
explants, GFAP was restricted to the innermost retinal layer,
Figure 1 Retinal cell apoptosis induced by glycated BSA. Terminal
dUTP nick end labelling (TUNEL-FITC) of cryostat sections from control
retinal explants (A) or explants treated with glycated BSA (B).
Immunolabelling for the active cleaved caspase-3 on cryostat sections
from control retinal explants (C) and explants treated with glycated BSA
(D). Note that small groups of adjacent TUNEL positive cells can be
observed in the inner part of the outer nuclear layer in the retinal explant
treated with glycated BSA (B) while these features are not found in the
control retinal explant (A). (E) Quantification of the TUNEL positive
retinal cells in control retinal explants (n = 7) and explants treated with
glycated BSA (n = 7). (F) Quantification of cleaved active caspase-3
positive cells in control retinal explants (n = 7) and explants treated with
glycated BSA (n = 7). In all retinal layers, the number of TUNEL and
caspase-3 positive cells were significantly greater in retinal explants
treated with glycated BSA than in control explants (p,0.001). GCL,
ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer;
OPL, outer plexiform layer; ONL, outer nuclear layer; PRL, photoreceptor
layer, RPE, retinal pigment epithelium. Errors bars indicate SEM. Scale
bars represent 50 mm.
anti-glial fibrillary acidic protein (GFAP) antibody (Dako,
Trappes, France, 1:50), anti-L-glutamate/L-aspartate transporter antibody (GLAST) (Chemicon, 1:200), and antiglutamine synthetase antibody (Chemicon, 1:400). Nuclear
labelling was achieved by incubating the sections in
diamidino-phenyl-indole (DAPI) solution (Sigma, solution
stock: 500 ng/ml concentration, 1:400).
Cells were counted under the 406 objective on the
microscope (Olympus, Melville, NY, USA) in five visual fields
www.bjophthalmol.com
Figure 2 Glial reaction induced by the glycated BSA. GFAP
immunostaining in cryostat sections of control retinal explants (A, C) and
explants treated with glycated BSA (B, D). In control retinal explants (A,
C), GFAP staining is localised to the innermost retinal layer, where
astrocytes are located, and to the outer plexiform layer (OPL). In
contrast, in retinal explants treated with glycated BSA (B, D), Müller cell
processes are extensively GFAP positive from their end tips at the inner
limiting membrane to their opposite end at the outer limiting membrane.
This increased GFAP staining is observed on the whole retinal explant
section. GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner
nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; PRL,
photoreceptor layer, RPE, retinal pigment epithelium. Scale bars
represent 50 mm.
AGE induced neuroglial reactions
where astrocytes are located, and to the outer plexiform layer
(OPL). Limited GFAP positive Müller cell processes were
occasionally observed (fig 2A, C). In contrast, in retinal
explants treated with glycated BSA, all Müller cell processes
were extensively GFAP positive from their end tip at the inner
limiting membrane to their opposite end at the outer limiting
membrane. The GFAP staining was not locally restricted but
extended throughout the entire retinal explant sections
(fig 2B, D). No change in GLAST and glutamine synthetase
was detected following the AGE treatment (data not shown).
The increase in GFAP expression showed that a treatment
with a glycated protein can induce a glial reaction.
DISCUSSION
Electrophysiological measurements in diabetic patients and
animal models generally have located the earlier deficits to
the inner retina but some series have indicated visual
dysfunction in the outer retina and RPE.2 Histologically,
although neuronal apoptosis has been prominent in the
ganglion cell layer (GCL),3 the cell loss also affected the inner
nuclear layer (INL) and has been occasionally reported in the
ONL.14 In our retinal explants treated with glycated BSA,
neuronal apoptosis was more evenly distributed in all retinal
layers, but was predominant in the GCL and ONL. These
observations were consistent with those obtained after
incubation of retinal cultures with the AGE precursor
glyoxal.15 However, apoptotic cells accounted for only 14%
in the 4 day glycated BSA incubation with a maximum of
20% in the GCL, whereas Reber et al15 reported up to 50%
retinal cell death after only 9 hour glyoxal incubation with a
similar higher sensitivity of the GCL. The difference may be
the result of the different mechanisms involved, as glyoxal
cannot only generate AGE but also reactive species, whereas
in our experiment, cell toxicity can only result from the
incubated glycated BSA. Our results indicated further that
retinal cell death can occur by activation of caspase-3,
although they do not exclude the contribution of other death
pathways like caspase independent mechanisms.16
In diabetic patients and animal models, Müller glial cells
are also affected as indicated by their increased GFAP
expression.3 4 Under our experimental conditions, Müller
glial cells also exhibited a major upregulation of GFAP
expression after 4 days of incubation in glycated BSA. This
Müller cell change is likely to result from the neuronal
apoptosis induced by glycated BSA. However, we cannot
exclude that the Müller cell change could result from a direct
effect of glycated BSA on these cells. The presence of a large
mass of TUNEL positive materials may further indicate that
glial cells have impaired phagocytic abilities, as previously
reported for resident peritoneal macrophages in streptozotocin induced diabetic animals17 or in vitro following AGE
incubation.18
Neuroglial alterations are early events in the development
of DR. Our study using retinal explants suggest that
neuroglial lesions could result from AGE diffusion into the
retinal tissue independently of major vascular perturbations
like occlusions. Our study further underlines that AGE do not
necessarily require to be synthesised into the retinal tissue to
induce neuroglial reactions, as incubation of the retinal tissue
with glycated BSA is sufficient to induce these lesions. Unless
synthesised in retinal cells like Müller cells that participate in
the retinal blood barrier and stock glucose,3 AGE need to
cross the retinal-blood barrier in order to affect retinal cells.
Breakdown of the retinal-blood barrier can be induced by
AGE themselves, or by other molecules released during
diabetic conditions, like VEGF, which synthesis is stimulated
by AGE.6 11 The required diffusion of AGE from the
1633
circulation would suggest that an increase in permeability
of the retinal-blood barrier occurs before the neuroglial
reaction in diabetic retinopathy. The molecular pathways of
AGE induced neuroglial reactions are not known but could be
related to AGE receptors like RAGE receptors, that are located
in the inner retina in humans and rats19 or the galectin-3
receptor that is located in Müller cells in rats.20 Further
studies will investigate the molecular mechanisms of AGE
induced neural apoptosis in the retina.
.....................
Authors’ affiliations
A Lecleire-Collet, L H Tessier, V Forster, J A Sahel, S Picaud, INSERMU592, Laboratory of Retinal Cellular and Molecular Physiopathology,
Paris, France
A Lecleire-Collet, G Brasseur, Department of Ophthalmology, Rouen
University Hospital Charles Nicolle, Rouen, France
P Massin, Department of Ophthalmology, Lariboisière Hospital, Paris,
France
Correspondence to: Dr Serge Picaud, INSERM-U592, Laboratory of
Retinal Cellular and Molecular Physiopathology, Bâtiment Kourilsky, 184
rue du Faubourg Saint-Antoine, 75571 Paris cedex 12, France;
[email protected]
Accepted for publication 1 August 2005
REFERENCES
1 Bloodworth JM Jr. Diabetic microangiopathy. Diabetes 1963;12:99–114.
2 Shirao Y, Kawasaki K. Electrical responses from diabetic retina. Prog Retin
Eye Res 1998;17:59–76.
3 Barber AJ. A new view of diabetic retinopathy: a neurodegenerative disease
of the eye. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:283–90.
4 Rungger-Brändle E, Dosso AA, Leuenberger PM. Glial reactivity, an early
feature of diabetic retinopathy. Invest Ophthalmol Vis Sci 2000;41:1971–80.
5 Stitt A, Gardiner TA, Alderson NL, et al. The AGE inhibitor pyridoxamine
inhibits development of retinopathy in experimental diabetes. Diabetes
2002;51:2826–32.
6 Stitt AW. The role of advanced glycation in the pathogenesis of diabetic
retinopathy. Exp Mol Pathol 2003;75:95–108.
7 Takeuchi M, Bucala R, Suzuki T, et al. Neurotoxicity of advanced glycation
end-products for cultured cortical neurons. J Neuropathol Exp Neurol
2000;59:1094–105.
8 Hammes HP, Alt A, Niwa T, et al. Differential accumulation of advanced
glycation end products in the course of diabetic retinopathy. Diabetologia
1999;42:728–36.
9 Pinzon-Duarte G, Kohler K, Arango-Gonzales B, et al. Cell differentiation,
synaptogenesis, and influence of the retinal pigment epithelium in a rat
neonatal organotypic retinal culture. Vision Res 2000;40:3455–65.
10 Vallazza-Deschamps G, Cia D, Gong J, et al. Excessive activation of cyclic
nucleotide-gated channels contributes to neuronal degeneration of
photoreceptors. Eur J Neurosci 2005 (in press).
11 Lu M, Kuroki M, Amano S, et al. Advanced glycation end products increase
retinal vascular endothelial growth factor expression. J Clin Invest
1998;101:1219–24.
12 Ono Y, Aoki S, Ohnishi K, et al. Increased serum levels of advanced glycation
end-products and diabetic complications. Diabetes Res Clin Pract
1998;41:131–7.
13 Picaud S, Peichl L, Franceschini N. Dye-induced photolesion in the
mammalian retina: glial and neuronal reactions. J Neurosci Res
1993;35:629–42.
14 Park SH, Park JW, Park SJ, et al. Apoptotic death of photoreceptors in the
streptozotocin-induced diabetic rat retina. Diabetologia 2003;46:1260–8.
15 Reber F, Geffarth R, Kasper M, et al. Graded sensitiveness of the various
retinal neuron populations on the glyoxal-mediated formation of advanced
glycation end products and ways of protection. Graefes Arch Clin Exp
Ophthalmol 2003;241:213–25.
16 Donovan M, Carmody RJ, Cotter TG. Light-induced photoreceptor apoptosis
in vivo requires neuronal nitric-oxide synthase and guanylate cyclase activity
and is caspase-3-independent. J Biol Chem 2001;276:23000–8.
17 Duncan JL, LaVail MM, Yasumura D, et al. An RCS-like retinal dystrophy
phenotype in Mer knockout mice. Invest Ophthalmol Vis Sci 2003;44:826–38.
18 Liu BF, Miyata S, Kojima H, et al. Low phagocytic activity of resident
peritoneal macrophages in diabetic mice: relevance to the formation of
advanced glycation end products. Diabetes 1999;48:2074–82.
19 Soulis T, Thallas V, Youssef S, et al. Advanced glycation end-products and
their receptors co-localise in rat organs susceptible to diabetic microvascular
injury. Diabetologia 1997;40:619–28.
20 Uehara F, Ohba N, Ozawa M. Isolation and characterization of galectins in
the mammalian retina. Invest Ophthalmol Vis Sci 2001;42:2164–72.
www.bjophthalmol.com
1634
SCIENTIFIC REPORT
5,10-Methylenetetrahydrofolate reductase C677T gene
polymorphism in Behçet’s patients with or without ocular
involvement
Y Özkul, C Evereklioglu, M Borlu, S Taheri, M Calis, M Dündar, Ö Ilhan
...............................................................................................................................
Br J Ophthalmol 2005;89:1634–1637. doi: 10.1136/bjo.2005.076836
Background: Increased serum levels of homocysteine (Hcy)
have been reported in patients with Behçet’s disease (BD)
with an established risk factor for vascular involvement.
Recently, the authors demonstrated that elevated Hcy levels
are associated with ocular involvement in such patients. On
the other hand, elevated levels of Hcy can result from genetic
errors. Indeed, a mutation in the 5,10-methylenetetrahydrofolate reductase (MTHFR C677T) gene influences Hcy
metabolism and, therefore, MTHFR C677T polymorphism
provokes hyperhomocysteinaemia.
Aim: To investigate the possible genetic factor for the
elevation of plasma Hcy level in patients with BD by
examining gene interaction with the MTHFR C677T polymorphism, a crucial factor of the Hcy metabolism. In
addition, the authors aimed to evaluate if there is an
association between the C677T polymorphism and the
presence of ocular involvement in such patients.
Method: A total of 59 patients with BD (25 men, 34 women)
with a mean age of 34.9 years and 42 age and sex matched
healthy control subjects (19 men, 23 women; mean age
32.2) were included in this investigation. MTHFR gene
polymorphism was investigated by the polymerase chain
reaction (PCR) and restriction fragment length polymorphism
(RFLP) of a genomic DNA fragment at nucleotide 677 in all
subjects in both groups. The genetic equilibrium is assumed
for the gene frequencies of the MTHFR polymorphism in both
samples.
Results: The genotype of the MTHFR gene differed between
the Behçet’s patients and control subjects (TT: 11.9 v 2.4%;
CT: 55.9 v 61.9%; CC: 32.2 v 35.7 %). TT homozygous
genotype was more frequently in BD patients than the
controls, though the difference was not significant
(p = 0.063). In BD patients with ocular involvement, however,
the frequencies of MTHFR TT homogenetic type (27.8%) were
significantly and statistically higher than those in BD patients
without ocular involvement (4.9%, p = 0.022, odds
ratio = 7.5), or the controls (2.4%, p = 0.003, odds
ratio = 20.0). TT homozygous genotype was associated with
an increased risk for ocular involvement.
Conclusion: Elevated serum levels of Hcy seem to be a
result of C677T polymorphism of the MTHFR gene, with
increased TT individuals over CC and CT genotype BD
patients. Although no association was shown between
the MTHFR reductase C677T polymorphism and the
increased risk of oral aphtahe or genital ulcers, a mutation
in this gene was associated with an increased risk of
ocular involvement, suggesting genetic instability with a
potential initiation of Hcy lowering therapy in this patient
group.
www.bjophthalmol.com
M
ethylenetetrahydrofolate reductase (MTHFR) is a
crucial enzyme that regulates the metabolism of
homocysteine (Hcy) and methionine by catalysing
the reduction of 5,10-methylene THF to 5-methyl THF, the
methyl donor for methionine synthesis from Hcy.1 Genetic
polymorphisms (mutation) of the C677T MTHFR are associated
with reduced enzyme activity and, therefore, cause impaired
remethylation of Hcy to methionine with subsequent hyperhomocysteinaemia.2 Hyperhomocysteinaemia, in turn, describes a
mild to moderate elevation of Hcy in blood or serum, resulting
in a cascade of cytokine activation and lipid peroxidation with
vascular endothelial injury, prothrombotic surface, atherothrombogenesis, thromboembolism, and systemic and retinal
vascular occlusive disease.3 4
Behçet’s disease (BD), first described in 1937 by a
dermatologist Dr Hulusi Behçet from Istanbul, as a triad of
symptom complex (oral aphthae, genital ulcers, hypopyon
uveitis), is a chronic, relapsing, multisystemic idiopathic
inflammatory disease characterised by an occlusive vasculitis.5–9 This unique disorder is endemically higher, particularly
in Turkey and Japan with a prevalence between 8/10 000 and
42/10 000, the population derived historically from the
ancient Silk Road.10 11 It occurs more commonly in men than
in women and affects primarily subjects between the second
and fourth decades of life, with a more aggressive course in
young male adults.4 The leading cause of chronic morbidity is
high especially with ophthalmic inflammation, which can
eventually result in blindness. Although various aetiopathogenic mechanisms have been suggested, the management of
the disease with severe organ involvement is still unsatisfactory.12
Hcy is suggested to be a new risk factor in the
hypercoagulability state and in thrombotic complications of
BD patients. Indeed, we have recently demonstrated that
serum levels of Hcy are increased and correlated with ocular
disease.13 This novel finding was supported by various
independent investigations with genetic implications.14–16
Because hyperhomocysteinaemia is assumed to be an
independent and correctable risk factor for venous thrombosis in such patients, such recent evidence and our results on
ocular BD gave us the unique opportunity to test further the
hypothesis that polymorphisms of the MTHFR C677T gene
may be the underlying variant for the demonstrated
hyperhomocysteinaemia. Therefore, this study evaluated the
association between the MTHFR C677T polymorphism and
BD and the significance of ocular BD in relation to the gene
polymorphism.
Abbreviations: BD, Behçet’s disease; Hcy, homocysteine; MTHFR,
methylenetetrahydrofolate reductase; PCR, polymerase chain reaction;
RFLP, restriction fragment length polymorphism
5,10-MTHFR C677T gene polymorphism in Behçet’s disease
1635
MATERIALS AND METHODS
RESULTS
The local ethics committee of the Erciyes University Faculty
of Medicine approved the initial research proposal. A total of
59 Turkish patients with BD (25 men, 34 women) with a
mean age of 34.9 (SD 10.1) years and 42 age and sex matched
healthy controls (19 men, 23 women; mean age 32.2 (8.6))
from a similar ethnic background were included in the
present investigation. All patients were diagnosed according
to the criteria of the International Study Group for Behçet’s
Disease.17 After all patients and control subjects gave their
informed consent to participation in this study, MTHFR gene
polymorphism was investigated by the polymerase chain
reaction-restriction fragment length polymorphism (PCRRFLP) of a genomic DNA fragment in all subjects in both
groups. The genetic equilibrium is assumed for the gene
frequencies of the MTHFR polymorphism in both samples.
The details of the ocular BD patients were obtained from
case notes and ocular examinations were performed by the
experienced ophthalmologists using a standard procedure. In
particular, evidence for retinal vascular occlusion was sought.
BD patients with an end stage ocular disease (phthisical or
completely blind) were assumed to have retinal vasoocclusive disease if the posterior segment could not be
visualised.
The most frequent clinical symptoms in BD patients were oral
apthea (100%), genital ulcers (91.5%), arthralgia (67%),
papulopustular eruptions (62.7%), erythema nodosum
(49.2%), ocular disease (30.5%), neurological findings
(11.8%), and gastrointestinal symptoms (8.4%). No arterial
or venous vascular disease was detected. A positive pathergy
reaction was observed in 18 patients (30.5%). Thirteen of of
18 patients with ocular disease had panuveitis with occlusive
vasculitis and the remaining five had only anterior uveitis.
There were three possibilities of genotypes, including TT,
CT, and CC, about base variation of MTHFR gene at locus
677. The genotype of the MTHFR gene differed between the
Behçet’s patients and control subjects (TT 11.9 v 2.4%; CT
55.9 v 6 %; CC 32.2 v 35.7%). The distributions of the MTHFR
genotypes in patients and controls are shown in table 1.
Overall, the C677T polymorphism of the MTHFR gene was
not significantly different in frequency in patients with BD
and controls (67.8% v 64.3%; p = 0.678, odds ratio = 1.276).
Although the frequency of TT homogenetic type was higher
in BD patients than the controls (11.9 v 1.9%), the difference
was not significant (p = 0.063; odds ratio = 7); the frequency
of CT allele was not different in patients and controls either
(p = 0.702). Similarly, the CT genotype was not significantly
different in ocular BD patients compared with the non-ocular
BD patients or controls (p = 0.718 and p = 0.264, respectively).
However, the frequencies of TT genotype were significantly
higher in ocular BD patients (27.8%) than those in nonocular BD patients (4.9%) (OR: 7.5, CI: 1.29 to 43.43,
p = 0.022) or controls (OR: 20.0, CI: 2.14 to 186.3,
p = 0.003; fig 1). The presence of the TT allele appeared to
have a strong association with the development of ocular
disease in Behçet’s patients. Family history was elicited in
four patients (6%), one of them had homozygous TT and the
others had CT genotype. No correlation or relation was found
between the MTHFR polymorphism with erythema nodosum
(p = 0.646), papulopustular lesions (p = 0.949), arthralgia
(p = 0.728), neurological involvement (p = 0.986), gastrointestinal symptoms (p = 0.423), and positive family history
(p = 0.182).
Genetic evaluation/MTHFR genotyping
Genomic DNA was isolated, using standard methods.18 The
DNA’s sites were analysed by PCR based RFLP methods as
described previously.19 PCR was performed in a Perkin Elmer
9600 and the profile consisted of an initial melting step of
2 minutes at 94˚C; followed by 35 cycles of 30 seconds at
94˚C, 30 seconds at 61˚C, and 30 seconds at 72˚C; and a final
elongation step of 7 minutes at 72˚C. PCR primers (5’-TGA
AGG AGA AGG TGT CTG CGG GA-3’ and 5’-AGG ACG GTG
CGG TGA GAG TG-3’) were used to amplify a portion of the
MTHFR gene from 100 ng of genomic DNA in a 50 ml
reaction containing 5 ml of 10X PCR buffer, 0.15 mM dNTP,
1.5 mM MgCl2, 0.6 pM each primers, and two units of Taq
DNA polymerase.
After amplification, the 198 bp PCR product was digested
with Hinf I in a 20 ml reaction solution containing 10 ml of
PCR product, 2 ml of 10X buffer, and five units of Hinf I at
37˚C overnight.
The digestion products were separated on 3% agarose gels,
and fragments stained with the ethidium bromide were
photographed on a ultraviolet transilluminator. Wild type
(CC) individuals were identified by only a 198 bp fragment,
heterozygotes (CT) by both the 175/23 bp, and homozygote
variants (TT) by the 175 bp.19
Statistical analysis
Results were given as the mean (standard deviation, SD). The
software SPSS for Windows version 10.0 was used to perform
statistical analysis. The x2 test was used to analyse
differences between the patients and controls. Odds ratio
(OR) and their 95% confidence intervals (CI) were used to
estimate the risk for ocular involvement. A multiple logistic
regression model was used to identify risk factors for ocular
involvement in patients with BD.
Table 1
DISCUSSION
An activated haemostatic system with arterial and venous
occlusive process or thrombus formation has been demonstrated during the course of BD.20 21 Prothrombin gene
G20210A mutations and increased levels of a mutant blood
clotting factor of G1691A (factor V Leiden), especially in
ocular BD, further supported this thrombotic tendency,
indicating a systemic prothrombotic (hypercoagulable) state
with endothelial cell activation in such patients.22–26
Although genetic thrombotic defects, impaired coagulation, defective fibrinolysis, and endothelial injury or dysfunction with many other immunoinflammatory molecules have
all been proposed as contributors,27–32 the underlying cause of
such a thrombotic state in BD still remains to be identified.
Recent studies have reported that the elevated levels of
homocysteine are related to arterial and venous thromboembolism.33–35 These studies suggest that homocysteine may only
The distributions of MTHFR genotypes in patients and controls
Groups
No
CC
CT
BD patients
BD patients with ocular involvement
BD patients without ocular involvement
Control subjects
59
18
41
42
19 (32.2%)
4 (22.2%)
15 (36.6%)
15 (37.5%)
33
18
24
26
TT
(55.9%)
(50.0%)
(58.5%)
(61.9%)
7
5
2
1
(11.9%)
(27.8%)
(4.9%)
(2.4%)
www.bjophthalmol.com
1636
Ö zkul, Evereklioglu, Borlu, et al
Figure 1 Gel photography of 5,10methylenetetrahydrofolate reductase
C677T gene polymorphism in ocular
Behçet’s disease. First and last columns
(BM) = size markers; column 2–3 =
homozygote normal; column 4–5 =
heterozygote; column 6–7 =
homozygote mutant; column 8 =
undigested PCR product.
exert an effect on vascular risk in synergy with other risk
factors. In addition, we have reported that serum levels of
Hcy are increased and correlated with ocular disease in
Behçet’s patients.13 Moreover, C677T polymorphism in
MTHFR gene may lead to hyperhomocysteinaemia in such
patients.36 37 Heterozygous or homozygous individuals for
mutations with an alanine to valine substitution have
reduced enzyme activity and thermolability, resulting in
elevated plasma Hcy caused by suboptimal intake of folate.
This study aimed to investigate the MTHFR C677 polymorphism in patients with BD and evaluate if there was an
association with ocular involvement.
We found equal frequencies of MTHFR C677T polymorphism overall in patients and healthy controls. Although the
frequency of the TT homozygous genotype was higher than in
the controls, the difference was statistically not different. On
the other hand, TT homozygous genotype was significantly
more frequent in patients with ocular BD. Therefore, it is
possible to speculate that the MTHFR C677TT genotype and
related hyperhomocysteinaemia might further increase the
risk of ocular vascular involvement and related complications
during the course of BD.
In the normal population, the frequency of MTHFR C677T
polymorphism may differ from the country to country, and
this mutation is not the unique factor that regulates the
homocysteine levels.2 38 Indeed, it has been hypothesised that
the region linked to the MTHFR polymorphism is involved in
folate binding and that the enzyme may be stabilised in the
presence of sufficient levels of folate; the World Health
Organization has proposed a lower limit of 13.6 nmol/l for
folate concentrations.39 40 It is plausible to speculate that the
combination of the genetic defect and inadequate folate
intake may cause elevated Hcy concentrations, and this
elevation of Hcy could be corrected with folic acid supplements. However, such a speculation is open to further
research.
Since hyperhomocysteinaemia could be caused by deficiencies of nutritional factors, dietary supplements of folic
acid and vitamin B12 might reduce the elevated plasma Hcy
levels. Moreover, another common polymorphism (1298A–C;
glutamate to alanine) has recently been reported as an
association with hyperhomocysteinaemia, but this polymorphism was suggested to increase homocysteine only in
individuals who carried the bp677 variant.38
In conclusion, this study further supports our previous
studies and demonstrates for the first time that the MTHFR
C677T polymorphism (TT genotype, but not CT genotype)
may represent as a genetic risk factor for BD, particularly for
ocular vascular events. However, before definitive conclusions can be reached, long term, large scale interventional
studies assessing both Hcy levels and folate status with
related gene polymorphism in BD are needed. Similarly,
clinical trials, especially in high risk populations, await
further investigation before a novel therapeutic approach is
formulated for this unique groups of patients.
www.bjophthalmol.com
ACKNOWLEDGEMENTS
The authors have no proprietary or commercial interest in any
concept or product discussed in this article.
.....................
Authors’ affiliations
Y Özkul, M Dündar, S Taheri, Department of Medical Genetics, Erciyes
University Medical Faculty, Kayseri, Turkey
C Evereklioglu, Ö Ilhan, Department of Ophthalmology, Erciyes
University Medical Faculty, Kayseri, Turkey
M Borlu, Department of Dermatology, Erciyes University Medical
Faculty, Kayseri, Turkey
M Calis, Departments of Physical Medicine and Rehabilitation, Erciyes
University Medical Faculty, Kayseri, Turkey
Correspondence to: Dr Cem Evereklioglu, Sivas Cad Cebeci Apt A–
Blok, 175/15, TR–38020, Kayseri, Turkey; [email protected]
Accepted for publication 8 August 2005
REFERENCES
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Br J Ophthalmol 2002;86:653–7.
14 Canataroglu A, Tanriverdi K, Inal T, et al. Methylenetetrahydrofolate
reductase gene C677T mutation and plasma homocysteine level in Behçet’s
disease. Rheumatol Int 2003;23:236–40.
15 Ateş A, Aydintug O, Ölmez U, et al. Serum homocysteine level is higher in
Behçet’s disease with vascular involvement. Rheumatol Int 2005;25:42–4.
16 Yeşilova Z, Pay S, Öktenli C, et al. Hyperhomocysteinemia in patients with
Behçet’s disease: is it due to inflammation or therapy? Rheumatol Int 2005
(in press).
17 International Study Group for Behçet’s Disease. Criteria for diagnosis of
Behçet’s disease. Lancet 1990;335:1078–80.
18 Blin N, Stafford DW. A general method for isolation of high molecular weigth
DNA from Eukaryotes. Nucleic Acids Res 1976;3:2303–8.
5,10-MTHFR C677T gene polymorphism in Behçet’s disease
19 Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular
disease:a common mutation in methylenetetrahydrofolate reductase. Nat
Genet 1995;10:111–3.
20 Haznedaroglu IC, Özcebe O, Çelik I, et al. Haemostatic markers of
procoagulant imbalance in Behçet’s disease. Eur J Haematol
1996;57:107–8.
21 Espinosa G, Font J, Tassies D, et al. Vascular involvement in Behçet’s disease:
relation with thrombophilic factors, coagulation activation, and
thrombomodulin. Am J Med 2002;112:37–43.
22 Gül A, Aslantaş AB, Tekinay T, et al. Procoagulant mutations and venous
thrombosis in Behçet’s disease. Rheumatology 1999;38:1298–9.
23 Verity DH, Vaughan RW, Madanat W, et al. Factor V Leiden mutation is
associated with ocular involvement in Behçet disease. Am J Ophthalmol
1999;128:352–6.
24 Kiraz S, Ertenli I, Haznedaroglu IC, et al. Pathological haemostasis and
‘‘prothrombotic state’’ in Behçet’s disease. Thromb Res 2002;105:125–33.
25 Batioglu F, Atmaca LS, Karabulut HG, et al. Factor V Leiden and prothrombin
gene G20210A mutations in ocular Behçet’s disease. Acta Ophthalmol Scand
2003;81:283–5.
26 Probst K, Fijnheer R, Rothova A. Endothelial cell activation and
hypercoagulability in ocular Behçet’s disease. Am J Ophthalmol
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27 Evereklioglu C, Er H, Türköz Y, Çekmen M. Serum levels of TNF–a, sIL–2R, IL–
6, and IL–8 are increased and associated with elevated lipid peroxidation in
patients with Behçet’s disease. Mediators Inflamm 2002;11:87–93.
28 Evereklioglu C, Inalöz HS, Kirtak N, et al. Serum leptin concentration is
increased in patients with Behçet’s syndrome and is correlated with disease
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29 Evereklioglu C, Türköz Y, Er H, et al. Increased nitric oxide production in
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Behçet’s disease. Mediators Inflamm 2003;12:255–6.
31 Erkiliç K, Evereklioglu C, Çekmen M, et al. Adenosine deaminase enzyme
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original contributions/clinical and laboratory investigations. Mediators
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www.bjophthalmol.com
1638
EXTENDED REPORT
Gremlin gene expression in bovine retinal pericytes
exposed to elevated glucose
R Kane, L Stevenson, C Godson, A W Stitt, C O’Brien
...............................................................................................................................
Br J Ophthalmol 2005;89:1638–1642. doi: 10.1136/bjo.2005.069591
See end of article for
authors’ affiliations
.......................
Correspondence to:
Professor Colm O’Brien,
Institute of Ophthalmology,
60 Eccles Street, Dublin 7,
Ireland; [email protected]
Accepted for publication
10 July 2005
.......................
D
Aim: To assess the influence of high extracellular glucose on the expression of the bone morphogenetic
protein (BMP) antagonist, gremlin, in cultured bovine retinal pericytes (BRPC).
Methods: BRPC were cultured under conditions of 5 mM and 30 mM D-glucose for 7 days and total RNA
was isolated. Gremlin mRNA levels were correlated, by RT-PCR, with other genes implicated in the
pathogenesis of diabetic retinopathy and the signalling pathways in high glucose induced gremlin
expression were probed using physiological inhibitors. Gremlin expression was also examined in the
retina of streptozotocin induced diabetic mice.
Results: High glucose stimulated a striking increase in BRPC gremlin mRNA levels in parallel with increases
in mRNA for the growth factors vascular endothelial growth factor (VEGF), transforming growth factor b
(TGFb), and connective tissue growth factor (CTGF) and changes in other genes including fibronectin and
plasminogen activator inhibitor-1 (PAI-1). High glucose triggered gremlin expression was modulated by
anti-TGFb antibody, by the uncoupler of oxidative phosphorylation, CCCP, and by inhibition of MAPkinase (MAPK) activation. Striking gremlin expression was observed in the outer retina of diabetic mice
and also at the level of the vascular wall.
Conclusions: Gremlin gene expression is induced in BRPC in response to elevated glucose and in the retina
of the streptozotocin induced diabetic mouse. Its expression is modulated by hyperglycaemic induction of
the MAPK, reactive oxygen species, and TGFb pathways, all of which are reported to have a role in
diabetic fibrotic disease. This implicates a role for gremlin in the pathogenesis of diabetic retinopathy.
iabetic retinopathy is one of the most frequent causes
of new blindness in the working age population.1 There
is a strong and consistent relation between hyperglycaemia and the incidence and progression of diabetic
retinopathy. Clinical studies have reported that the normalisation of glycaemia control can prevent diabetic microangiopathies and possibly cardiovascular complications.2 3
Several mechanisms exist by which hyperglycaemia results
in retinal damage, including increased polyol pathway,4
activation of protein kinase C (PKC),5 increased nonenzymatic glycation,6 and generation of reactive oxygen
species (ROS) by oxidative stress.7 Furthermore, other
mediators, including growth factors such as vascular
endothelial growth factor (VEGF), platelet derived growth
factor (PDGF), and transforming growth factor b (TGFb)
contribute to the pathological manifestations of diabetic
retinopathy, including basement membrane thickening,
vessel occlusion and retinal hypoxia, which induces neovascularisation (reviewed by Cai and Boulton8). New vessels and
connective tissue grow on the surface of the retina or optic
nerve head and into the vitreous. Visual loss at this time
results from vitreous haemorrhage or fluid exudation from
fragile new vessels.9
Retinal pericytes are smooth muscle-like cells with
attenuated processes enveloping the abluminal surface of
microvessels and sharing a common basement membrane
with the underlying endothelium (reviewed by Diaz-Flores et
al10). Pericytes express a smooth muscle actin (a-SMA) and
have been implicated to have a contractile function,11 thus
regulating blood flow. They are proposed to regulate
microvascular angiogenesis and synthesise components of
the vascular basement membrane.12 13 Pericytes have been
demonstrated to be involved in the regulation of endothelial
cell number and morphology and microvessel architecture.14
www.bjophthalmol.com
One of the first histological features of diabetic retinopathy is
the loss of retinal pericytes.15
Gremlin is a member of the differential screening-selected
gene aberrative in the neuroblastoma (DAN) family of bone
morphogenetic protein (BMP) antagonists.16 The protein is
highly conserved through evolution and contains a cysteine rich
region, a cysteine knot, which is also shared by members of the
TGFb family, PDGF family, nerve growth factor, and other
secreted proteins.17 Gremlin exists as both secreted and cell
associated forms. It can be post-translationally modified by
glycosylation and phosphorylation.18 Gremlin influences diverse
processes in growth, differentiation, and development19 20
Gremlin has been demonstrated to antagonise the activities of BMP-2, BMP-4, and BMP-7. It does this by direct
binding to and heterodimerisation with the BMP. This then
results in these BMP ligands failing to bind their receptors,
which are members of the TGFb receptor superfamily.16 19
Increased expression of gremlin has recently been demonstrated in several models of diabetic nephropathy,21 22
pointing to a role for gremlin in diabetic fibrotic disease. In
this study, we explore gremlin expression in a model of
diabetic eye disease, using BRPC cultured in high glucose
levels. We also examine in vivo gremlin expression in the
retina of C57 mice with streptozotocin induced diabetes.
Abbreviations: a-SMA, a smooth muscle actin; BMP, bone
morphogenetic protein; BRPC, bovine retinal pericytes; CCCP, carbonyl
cyanide m-chlorophenylhydrazone; CTGF, connective tissue growth
factor; DAN, differential screening-selected gene aberrative in the
neuroblastoma; EST, expressed sequence tag; MAPK, MAP-kinase; PAI1, plasminogen activator inhibitor-1; PDGF, platelet derived growth
factor; PKC, protein kinase C; ROS, reactive oxygen species; RT-PCR,
reverse transcription-polymerase chain reaction; TGFb, transforming
growth factor b; VEGF, vascular endothelial growth factor
Gremlin gene expression in bovine retinal pericytes
1639
METHODS
Cell culture
Bovine retinal pericytes (BRPC) were cultured in MCDB
131 (Invitrogen) supplemented with 2 mM L-glutamine and
5% fetal bovine serum (passages 5–7), and maintained in
medium containing either 5 mM or 30 mM D-glucose for
7 days. Culture medium was replenished three times during
this period to maintain glucose levels in the desired range. For
the low/high glucose experiments (n = 3) BRPC were cultured
in 5 mM glucose, 30 mM glucose, or 5 mM glucose and 25 mM
mannitol (as an osmolarity control) for 7 days. To examine
regulation of gremlin expression BRPC were cultured in 30 mM
glucose plus 10 mM PD 98059 (Calbiochem), or 10 mM GF
109203X (Calbiochem), or 500 nM CCCP (Sigma), or 1 mg/ml
aTGFb1 antibody (R&D Systems) for 7 days. PD 98059 is a
selective inhibitor of MEK23 that acts by inhibiting activation of
MAPK and subsequent phosphorylation of MAK substrates. GF
109203X is a selective PKC inhibitor.24 CCCP (carbonyl cyanide
m-chlorophenylhydrazone) is an uncoupler of oxidative phosphorylation.25 aTGFb1 antibody neutralises the bioactivity of
TGFb1.26 BRPC were also cultured in 5 mM glucose with 10 ng/
ml TGFb1 or 10 ng/ml TGFb2.
RNA extraction and reverse transcription-polymerase
chain reaction
RNA was extracted from BRPC using Trizol (Invitrogen)
according to the manufacturers’ instructions. RT-PCR
(reverse transcription-polymerase chain reaction) was performed as follows: 2 mg of total RNA was treated with DNaseI
(Invitrogen), reverse transcription was carried out using
random primers and Superscript II (Invitrogen) using the
manufacturer’s protocol. Limited cycle PCR was carried out
using the following primers: VEGF; sense 59-GGA TCA AAC
CTC ACC AAA GC-39, antisense 59-CAG GAT TGT TCT GTC
GAT GG-39, CTGF, sense 59-GAA AGG CAA AAA GTG CAT
CC-39, antisense 59-CTT GTG CCA CTG AAA TCA CG-39,
TGFb1, sense 59-TGA TGT CAC CGG AGT TGT GC-39,
antisense 59-TCC AGG CTC CAA ATG TAG GG-39,
Fibronectin, sense 59-CAC TGC CCA CTC CTA CAA CC-39,
antisense 59-ATG GAT CCC AAT CCA AAT CC-39, PAI-1,
sense 59-TGA CCA CAG GAG GAG AAA CC-39, antisense 59GGT GTT TGA TCT GGG AAG G-39. Bovine gremlin was
amplified using the primers sense 59-CCT GAA GCG GGA
CTG GTG YAA RAC-39, antisense 59-TCG ATG GAT ATG CAA
CG-39, where Y = C+T, R = A+G. As a control for equal
amounts of RNA, either 18S rRNA or the housekeeping gene
GAPDH was amplified. 18S, sense 59-GTG GAG CGA TTT GTC
TGG TT-39, antisense 59-CGC TGA GCC AGT CAG TGT AG-39.
GAPDH, sense 59- GGT GAT GCT GGT GCT GAG TA –39,
antisense, 59- GTC TTC TGG GTG GAG TGA T –39.
To confirm the correct PCR product was amplified PCR
products were subcloned into the vector pCRII-TOPO
(Invitrogen). Subcloned cDNAs were isolated by colony PCR
amplification. Sequencing was performed using an automated ABI 3310 DNA sequencing system. Sequence reactions
were carried out with the ABI prism big dye terminator cycle
sequencing ready reaction kit (Perkin-Elmer). The sequences
obtained were compared against GenBank and expressed
sequence tag (EST) databases using BLAST searches.27
Animal model and immunohistochemistry
The animal model is as described by Cox et al.28 Briefly, male
c57Bl/6 mice (20–25 g at 5–6 weeks old) were randomly
assigned to non-diabetic control or diabetic groups. Diabetes
was induced by a single intraperitoneal injection of streptozotocin (Sigma) at 180 mg/kg bodyweight. Control animals
received an equivalent dose of the drug vehicle (citrate buffer
at pH 4.6). The mice were caged individually and allowed food
and water ad libitum. Blood glucose levels were measured
–
5 mM 25 mM 30 mM
glucose mannitol glucose
VEGF
389 bp VEGF165
250 bp VEGF121
25 cycles
CTGF
391 bp
22 cycles
TGFβ1
405 bp
30 cycles
Fibronectin
413 bp
25 cycles
PAI-1
410 bp
22 cycles
GAPDH
299 bp
25 cycles
Figure 1 Glucose regulated gene expression in BRPC. RT-PCR analyses
of quantitatively standardised total RNA samples from BRPC cultured in
5 mM glucose, 5 mM glucose, and 25 mM mannitol (as an osmolarity
control), or 30 mM glucose for 7 days. SDW was used as a negative
control instead of cDNA and is indicated by -. 18S ribosomal RNA was
used as a control for equal input amounts of RNA.
fortnightly. Diabetic animals with blood glucose levels between
20 mM and 30 mM were included in the study. Groups of 8–10
animals were taken for each experimental and control group
and the experiment was carried out three times. All animals
were sacrificed after 8 weeks’ duration of diabetes.
An anti-gremlin antibody was generated by Fusion
Antibodies (Belfast). Sections of mouse eyes were de-waxed
and rehydrated in PBS. The sections were then subjected to
antigen retrieval for 20 minutes in citrate buffer (pH 6.0) in a
pressure cooker. After washing in PBS, the sections were
blocked with 5% normal goat serum, 1% BSA, 0.01% TritonX100 and then incubated in primary antibody to gremlin at
1:100 dilution overnight at 4˚C. Controls were perfomed
using primary antibody exclusion and rabbit non-immune
serum.
Before
detection
using
the
anti-rabbit
Envision+System (Dako Ltd.), endogenous peroxidase activity was quenched in 3% hydrogen peroxide. After allowing
diaminobenzidine reaction product to develop, the sections
were then washed extensively, counterstained with haematoxylin, and mounted with Glycermount (Dako Ltd).
RESULTS
Glucose induced growth factor gene expression in
BRPC
The expression of growth factor genes suggested to be
involved in the pathogenesis of diabetic retinopathy, such
as VEGF, TGFb, and CTGF29–31 were examined. Figure 1 shows
the results of RT-PCR for glucose induced gene expression in
BRPC. All three growth factor genes examined, VEGF, CTGF,
and TGFb1 were expressed. Two alternatively spliced forms of
bovine VEGF were expressed; VEGF 164 (orthologue of
human VEGF 165) and VEGF 120 (orthologue of human
www.bjophthalmol.com
1640
Kane, Stevenson, Godson, et al
A
–
5 mM 25 mM 30 mM
glucose mannitol glucose
A
30 mM
5 mM
Gremlin
282 bp
30 cycles
GAPDH
299 bp
25 cycles
PD
GF
CCCP
αTGFb
Gremlin
35 cycles
18S
12 cycles
Control
B
B
Man 30 mM
TGFβ1
TGFβ2
Gremlin
35 cycles
18S
10 cycles
Figure 3 Regulation of bovine gremlin expression. (A) RT-PCR analyses
of quantitatively standardised total RNA samples from BRPC cultured in
5 mM glucose, 5 mM glucose and 25 mM mannitol (as an osmolarity
control), 30 mM glucose or 30 mM glucose plus 10 mM PD 98059, or
10 mM GF 109203X, or 500 nM CCCP, or 1 mg/ml aTGFb1 antibody
for 7 days. 18S ribosomal RNA was used as a control for equal input
amounts of RNA. (B) RT-PCR analyses of quantitatively standardised total
RNA samples from BRPC cultured in 5 mM glucose (control), 5 mM
glucose and 10 ng/ml TGFb1, and 5 mM glucose and 10 ng/ml
TGFb2. 18S ribosomal RNA was used as a control for equal input
amounts of RNA.
Figure 2 Gremlin is upregulated by glucose in BRPC. (A) RT-PCR
analyses of quantitatively standardised total RNA samples from BRPC
cultured in 5 mM glucose, 5 mM glucose, and 25 mM mannitol (as an
osmolarity control), or 30 mM glucose for 7 days. SDW was used as a
negative control instead of cDNA and is indicated by -. 18S ribosomal
RNA was used as a control for equal input amounts of RNA. (B) The
bovine Gremlin PCR product was subcloned in to the vector pCR2.1TOPO (Invitrogen) and sequenced in both directions. The sequence was
aligned in a pairwise alignment using ‘‘BLAST 2 sequences’’34 with its
human homologue (accession number AF110137).
VEGF 121). Expression levels of all three growth factor genes
increased under conditions of elevated glucose. Basement
membrane thickening and increased contractility and coagulation are associated with diabetic retinopathy.5 32 33 The
expression of genes associated with these processes was also
examined by RT-PCR. Expression of both fibronectin and
PAI-1 increased (fig 1).
Gremlin expression in BRPC
Figure 2A shows increased expression of gremlin in BRPC
exposed to 30 mM glucose for 7 days. The gremlin PCR product
obtained was subcloned and sequenced. The sequence of bovine
gremlin was determined. Following pairwise using ‘‘BLAST 2
sequences’’34 with its human homologue (accession number
AF110137) the sequence showed 92% identity to its human
homologue, over the amplified region (fig 2B). This sequence of
bovine Gremlin was translated and aligned with a translation of
the human mRNA sequence. At the protein level the homology
is 98% identity, data not shown. Western blotting for gremlin
(data not shown) demonstrates that gremlin is both cell
associated and secreted.
Regulation of bovine gremlin expression
Regulation of gremlin expression was examined. BRPCs were
cultured in 30 mM glucose plus inhibitors of signalling
pathways. Inhibition of the MEK signalling pathway by PD
98059, uncoupling of oxidative phosphorylation by CCCP, or
TGFb signalling, using aTGFb1 antibody all modulate
gremlin expression. Inhibition of protein kinase C signalling
www.bjophthalmol.com
by GF109203X had no effect on gremlin expression. Gremlin
expression was measured by RT-PCR (fig 3A). Culturing
BRPC in 30 mM glucose and aTGFb1 antibody abolishes
gremlin expression. Culturing BRPC in 30 mM glucose and
PD 98059 or CCCP reduces gremlin expression, therefore
implying the MAPK pathway and hyperglycaemia induced
ROS in regulation of gremlin expression in this model. To
determine if gremlin was directly regulated by TGFb in BRPC,
the cells were cultured in 5 mM glucose and 10 ng/ml TGFb1
or 10 ng/ml TGFb2. Again gremlin expression was examined
by RT-PCR and was found to increase with both TGFb1 and
TGFb2 stimulation (fig 3B).
Gremlin is expressed in the retina of diabetic mice
Gremlin immunoreactivity was localised to the nerve fibre
layer, ganglion cell layer and inner plexiform layers in the
retina of both non-diabetic (fig 4A), and diabetic mice
(fig 4B). The diabetic animals also demonstrate gremlin
immunoreactivity in the outer retina (fig 4B), and also at the
level of the vascular wall (arrow)—especially noticeable in
the large retinal vessels (fig 4C).
DISCUSSION
Within the retina, pericytes provide vascular stability, exert
control over endothelial cell proliferation and morphology,
and microvessel architecture.14
Multiple growth factors are involved in the regulation of
the retinal vasulature, and are also involved in the
pathogenesis of diabetic retinopathy. We have demonstrated
in this study the increased expression of the profibrotic
growth factors, CTGF and TGFb1, and the angiogenic factor
VEGF in retinal pericytes exposed to high concentrations
(30 mM) of extracellular glucose. CTGF is a novel, cysteine
rich secreted protein, which is implicated in fibrotic disorders
and has been associated with proliferative retinopathies.35 36
More recently, other studies show increased expression of
CTGF in the diabetic retina37 and demonstrate the expression
of CTGF in pericytes and point to a role for CTGF in diabetic
retinopathy.38 High glucose induced CTGF expression has
Gremlin gene expression in bovine retinal pericytes
1641
Figure 4 Immunohistochemistry of
gremlin in retina of diabetic and nondiabetic mice. The nerve fibre layer
(NFL), ganglion cell layer (GCL), and
inner plexiform layers (IPL) of both nondiabetic (A) and diabetic (B) mice show
strong immunoreactivity. Diabetic
animals demonstrate gremlin
immunoreactivity in the outer retina (B)
and also at the level of the vascular
endothelium (arrow), especially
noticeable in the large retinal vessels
(C). Primary antibody omission controls
show no apparent deposition of DAB
reaction product (D). Original
magnifications: 6200 (A, B, and D).
6400 (C). The outer nuclear layer
(ONL) and photoreceptors (PR) are also
labelled.
been demonstrated in other cell types such as mesangial
cells,22 where it may contribute to increased fibrosis by
inhibiting matrix breakdown39 and mediating TGFb induced
matrix deposition.40 TGFb1, is a member of a superfamily of
multifunctional cytokines, which regulate multiple cellular
functions including growth, differentiation, and death.
TGFb1 is a potent inducer of extracellular matrix proteins
and inhibits matrix degradation.41 TGFb produced in cocultures of endothelial cells and pericytes has been demonstrated to inhibit retinal endothelial cell proliferation.42 We
have demonstrated here increased TGFb transcription in
pericytes in response to elevated glucose. VEGF, a potent
angiogenic factor, is synthesised by pericytes and is capable of
stimulating endothelial cells to proliferate, and may synergistically act with other growth factors to enhance this
effect.43 VEGF has been demonstrated to increase CTGF levels
in retinal capillary cells mediated primarily by PI3-kinase
activation.31 In retinal pigmented epithelial cells, 30 mM
glucose also induces VEGF expression.44 We have demonstrated in this study direct induction by high glucose of these
growth factors, VEGF, CTGF and TGFb, thus demonstrating
that concomitant overexpression of growth factors by retinal
pericytes occurs in response to elevated glucose levels and
may contribute to the pathogenesis of diabetic retinopathy.
Hyperglycaemia induces basement membrane thickening
in the diabetic retina and this may contribute to the closure
of capillaries.8 Increased coagulation and contractility are also
associated with diabetic retinopathy. We have demonstrated
here that when exposed to elevated levels of glucose, retinal
pericytes increase fibronectin and PAI-1 expression.
Increased CTGF levels may also contribute to increased
matrix deposition.39 40 Pericytes may also contribute to the
process of basement membrane thickening by secreting
fibronectin. Fibronectin mRNA elevation has previously
been demonstrated in pericytes in response to 22 mM
glucose,9 and we demonstrate increases following exposure
of pericytes to 30 mM glucose.
There exists an emerging paradigm that patterns of
developmental gene programs reappear in the context of a
disease process.45 This may attempt to repair or regenerate
tissue. It is also possible that this execution of a developmental
program may contribute to the disease process. One of these
developmental genes is gremlin. Increased expression of
gremlin has been recently demonstrated in models of fibrotic
disease processes, most notably diabetic nephropathy.22 We
have also demonstrated in this study that increased gremlin
expression is associated with retinal pericytes exposed to high
extracellular glucose. We have shown that gremlin expression
in retinal pericytes can be abolished by culturing pericytes with
aTGFb1 antibody, and induced by stimulation with either
TGFb1 or TGFb2, therefore implying this signalling pathway in
gremlin expression.
Gremlin gene expression is induced in response to high
glucose in this system. Its expression is modulated by the
MAPK, ROS, and TGFb pathways, all of which are reported to
have a role in the pathogenesis of diabetic retinopathy
(reviewed by Brownlee46). The concomitant increased expression of multiple profibrotic genes and gremlin in retinal
pericytes exposed to 30 mM glucose, and regulation of
gremlin expression by the profibrotic cytokine, TGFb may
imply a role for gremlin in fibrogenesis. Gremlin is a 184
amino acid protein and a member of the cysteine knot
superfamily. The protein is highly conserved during evolution
and is present in soluble and cell associated forms. It belongs
to a novel family of bone morphogenetic protein (BMP)
antagonists that includes the head inducing factor Cerberus
and the tumour suppressor DAN. These proteins have
important roles in limb development and neural crest cell
differentiation. Gremlin expression can be induced in
mesangial cells in response to elevated glucose, TGF-b, and
cyclic mechanical strain.21 A pathogenic role may be
attributed to gremlin in the context of diabetic nephropathy
as overexpression of gremlin induces transdifferentiation of
cultured tubular epithelial cells to a more fibroblast-like
phenotype.47 We have observed in this study that gremlin
protein expression in BRPC is both cell associated and
secreted. As a cell associated protein it may function to alter
gene expression and signalling pathways, as demonstrated by
Chen et al48 in tumour derived cell lines. Gremlin also
functions as a secreted protein and in addition we have
shown in this study that gremlin is expressed in the retinas of
both non-diabetic and diabetic mice, and that expression
www.bjophthalmol.com
1642
increases in the outer retina and the vascular wall of diabetic
animals. Mathura et al49 demonstrated high expression of
BMPs in the adult outer retina, more specifically the RPE,
and suggest that both BMP-2 and BMP-4 may serve as
negative growth regulators in the retina. The expression of
gremlin in the vascular endothelium is significant as this may
contribute to proliferation of the vascular endothelium.
Trousse et al50 demonstrate how BMP-4 mediates apoptosis
in the retina, and this may be antagonised by Noggin,
another member of the BMP antagonist family. As gremlin is
a known antagonist of BMPs, it may have a role in
proliferation by antagonising the antiproliferative effects of
BMP in the retina. Therefore, modulation of BMP expression
may have a role in proliferative retinopathies.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge funding from the Programme for
Research in Third Level Institutes (PRTLI), Ireland.
.....................
Authors’ affiliations
R Kane, C Godson, School of Medicine and Medical Science University,
College Dublin, Republic of Ireland
C O’Brien, Department of Ophthalmology, Mater Misericordiae
Hospital, Dublin, Republic of Ireland
R Kane, C Godson, C O’Brien, Conway Institute for Biomolecular and
Biomedical Research, University College Dublin and the Dublin
Molecular Medicine Centre, Republic of Ireland
L Stevenson, A W Stitt, Ophthalmology and Vision Science, Queen’s
University Belfast, Institute of Clinical Science, Royal Victoria Hospital,
Belfast, UK
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14 Hellstrom M, Gerhardt H, Kalen M, et al. Lack of pericytes leads to endothelial
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1643
EXTENDED REPORT
Expression of FGFR-2 and FGFR-3 in the normal human
fetal orbit
S H Khan, J A Britto, R D Evans, K K Nischal
...............................................................................................................................
Br J Ophthalmol 2005;89:1643–1645. doi: 10.1136/bjo.2005.075978
See end of article for
authors’ affiliations
.......................
Correspondence to:
Mr K K Nischal,
Department of Paediatric
Ophthalmology, Great
Ormond Street Hospital for
Children, Great Ormond
Street, London WC1N
3JH, UK; kkn@btinternet.
com
Accepted for publication
1 August 2005
.......................
M
Aims: To demonstrate the expression patterns of two fibroblast growth factor receptors
(FGFR-2 and FGFR-3) in the normal human fetal orbit.
Methods: 6 mm orbital slide sections were prepared from 12 week old human fetal material obtained
within established ethical guidelines. Radioactive in situ hybridisation techniques were used to demonstrate
the expression patterns of FGFR-2 and FGFR-3 within these sections. Only one foetus had appropriate
orbital sections taken.
Results: FGFR-2 was expressed within the extraocular muscles (EOMs) and the optic nerve sheath and to a
lesser degree within the orbital periosteal margins and the cranial sutures. FGFR-3 was expressed a lot
within the periosteal margins and cranial sutures but not within either the EOMs or the optic nerve sheath.
Conclusions: FGFR-2 and FGFR-3 are differentially expressed within different orbital components. FGFR-2
gene mutations may be responsible for craniosynostotic syndromes such as Crouzon, Pfeiffer, and Apert,
while those in the FGFR-3 gene may cause isolated unicoronal synostosis. EOMs may be histologically
abnormal in cases of Apert, Pfeiffer, and Crouzon syndromes but not isolated unicoronal synostosis. The
pattern of expression of FGFR-2 in the normal human fetal orbit may explain some of the EOM histological
findings seen in some cases of Apert, Pfeiffer, and Crouzon syndromes.
utations in the fibroblast growth factor receptor
(FGFR) genes are known to result in craniosynostosis
which is the abnormal premature fusion of one or
more of the cranial sutures.1 The craniosynostoses may be
syndromic or non-syndromic: mutations in FGFR-2 are most
commonly responsible for the autosomal dominant syndromic craniosynostoses (such as Crouzon, Apert, and Pfeiffer),
while the non-syndromic craniosynostoses may occur as a
result of mutations in other genes including FGFR-3, which
is known to be responsible for some cases of unicoronal
synostosis.2
All the craniosynostotic syndromes have the common
feature of craniosynostosis but vary in their combination of
other anomalies of bone differentiation, which mainly affect
their hands and feet3; for example, in Apert syndrome there is
typically syndactyly while in Pfeiffer syndrome there is an
anomalous broad thumb.
The position of the rectus muscles in patients with
craniosynostoses is often exocyclorotated.4 This is thought
to be due to a mechanical exocyclorotation of the orbits
secondary to premature cranial synostosis4 and may be seen
in non-syndromic and syndromic cases.
However, anomalies of extraocular muscle (EOM) structure have only been described in the syndromic craniosynostoses and not in the non-syndromic unicoronal, bicoronal,
sagittal, or metopic craniosynostoses.5–8
Captuo and Lingua5 reported abnormal bifid medial rectus
and anomalous insertions of both horizontal rectus muscles
in a patient with Crouzon syndrome. Absence and/or
hypoplasia of EOMs has also been shown to occur with
increased frequency in Crouzon syndrome.6 Margolis et al7
described structural alterations in extraocular muscle fibres
as seen by light and electron microscopy in a patient with
Apert syndrome, while Greenberg and Pollard8 have
described both muscle hypoplasia and also absence of
multiple EOMs in patients with both Pfeiffer and Apert
syndromes. Findings of a fibrous band in lieu of rectus
muscle are not a surprise when operating on these patients
and have necessitated novel approaches to the treatment of
their eye movement disorders.9
We studied the expression patterns of FGFR-2 and FGFR-3
in a normal human fetal orbit in an effort to explain the
above clinical findings.
METHODS
We looked at the expression of FGFR-2 and FGFR-3 in a
12 week old human fetal orbit as per methodology previously
reported.10
Human fetuses of 12 weeks of age were collected under the
aegis of the Developmental Biology Tissue Resource maintained at the Institute of Child Health and University College,
London. The fetuses were obtained within established ethical
guidelines, from the social termination of pregnancy by
RU486 or surgical methods. None was terminated for reasons
of fetal abnormality but no formal karyotyping was
performed. The fetal age was determined by the magnified
assessment of external morphology. All fetuses were fixed in
4% paraformaldehyde, in phosphate buffered saline at 4˚C for
2–5 days, then rinsed in phosphate buffered saline, and
passed through increasing concentrations of phosphate
buffered saline/ethanol solutions until storage at 4˚C in 70%
ethanol. The specimens were then dehydrated through an
alcohol series, cleared in Histoclear (RA Lamb, East Sussex,
UK), embedded in paraffin wax, and serially sectioned at
6 mm onto TESPA stubbed slides. Only one fetus had the
appropriate sections taken through the orbits.
Slides were examined using an Olympus BH2 microscope,
and images were captured electronically using a Kontron
ProgRes3012 digital camera, version 2 of the associated
software, and stored as Adobe Photoshop version 5.0 files.
Abbreviations: EOMs, extraocular muscles; FGFR, fibroblast growth
factor receptors; ICP, intracranial pressure
www.bjophthalmol.com
1644
Khan, Britto, Evans, et al
Figure 1 Expression of FGFR2—dark brown indicates positive stain
(arrows)—predominantly within muscles and optic nerve sheath. LR,
lateral rectus; SR, superior rectus; MR, medial rectus; SO, superior
oblique; ON, optic nerve.
Figure 2 Expression of FGFR3—dark brown indicates positive stain
(arrows)—periosteal margins and sutures but not in muscle or optic
nerve sheath. LR, lateral rectus; SR, superior rectus; MR, medial rectus;
SO, superior oblique; ON, optic nerve.
RESULTS
activation), resulting in uncontrolled growth and differentiation of its target tissues.2 This in turn may also lead to the
production of the excessive fibrous tissue seen in rectus
muscle analysis from such patients.11 Anecdotally, these
muscles feel gristly and inelastic when manipulated during
operation.
Light microscopy of muscles in Apert syndrome shows cells
in varying stages of degeneration scattered among a majority
of normal looking muscle fibres; other muscle fibres are
described as enlarged and hyalinised.7 Electron microscopy
further shows these hyalinised fibres to have a loss of
myofibrillar organisation and to contain large clusters of
mitochondria, which in themselves display vacuolation,
disruption, and fragmentation of cristae. A variety of nuclear
abnormalities are also described, with subsarcolemmal
inclusions being seen frequently.
It is interesting to note that similar mitochondrial
abnormalities have been described in both myopathic and
neurogenic diseases where altered movements are a feature,
while granular inclusions similar to the subsarcolemmal
inclusions have previously been described in myotonic
dystrophy and myasthenic muscles.
The fact that FGFR-3 mutation related conditions such as
Muenke’s have not been described as having structurally
abnormal or absent EOMs is consistent with our finding of a
lack of expression of FGFR-3 in the EOMs.
Our results also demonstrate positive expression of FGFR-2
but not FGFR-3 in the optic nerve sheath. Absence of optic
disc swelling in the presence of confirmed raised intracranial
pressure has been well described in the syndromic craniosynostoses and may be seen in up to 50% of cases with
confirmed raised intracranial pressure (ICP).12 13
Although the absence of disc swelling in the presence of
raised ICP in these patients may be attributed to the presence
of optic atrophy in some cases, this is not likely to be true in
every case. The Schmidt-Manz transport theory describes
optic disc swelling in raised ICP to occur as a result of
cerebrospinal fluid being forced into intravaginal spaces of
the optic nerve sheath within the lamina cribrosa, resulting
in oedema and incarceration of the optic nerve head.14 In
cases where there is a lack of optic disc swelling, it suggests
that there must be an obstruction to the communication
between the cranial cavity and the subvaginal spaces of the
optic nerve sheath to prevent development of the oedema.15
Based on our findings, we speculate that the lack of optic disc
swelling in the presence of raised ICP in cases of syndromic
craniosynostosis may be the result of abnormal fibrous tissue
in the optic nerve sheath and/or the lamina cribrosa.
Unfortunately, at present no optic nerve sheath histology
is available in a patient with FGFR2 mutation related
FGFR-2 expression was positively demonstrated within the
extraocular muscles and also within the optic nerve sheath. It
was also demonstrated to a lesser degree within the
periosteal margins of the orbit and within the cranial sutures
(see fig 1).
FGFR-3 was not demonstrated within either the extraocular muscles or the optic nerve sheath, but was more
positively expressed than FGFR-2 within the periosteal
margins and cranial sutures (see fig 2).
DISCUSSION
Fibroblast growth factors (FGF) are a family of structurally
similar mitogenic factors that exert their key effects within
the body via their roles in the growth and differentiation of
various tissues of mesenchymal and neuroectodermal origin.2
They are also important for chemotaxis, angiogenesis, and
cell apoptosis. The key members of this family are acidic FGF
(FGF-1) and basic FGF (FGF-2), which exert their effects by
interacting with membrane spanning tyrosine-kinase receptors. There are four types of high affinity receptors, simply
known as FGFR 1-4. Binding by fibroblast growth factors to
these receptors causes receptor transphorylation and activation of their kinase domains.
FGFR-2 and FGFR 3 staining in a normal human orbit has
not previously been reported to the best of our knowledge.
Although the findings are in only one fetus it is nevertheless
valuable information and perhaps a first step to phenotypegenotype correlation.
Apert, Crouzon, and Jackson-Weiss syndromes result from
FGFR-2 gene mutations. When Pfeiffer syndrome is the
result of an FGFR-2 mutation, it is not possible to distinguish
between it, Crouzon or Jackson-Weiss even at a molecular
level, while Apert syndrome can be seen to be an allelic
variation of these syndromes.11 Pfeiffer syndrome may also
result from mutations in either FGFR-1 (chromosome 8) or
FGFR-2 (chromosome 10).
The FGFR-3 gene is found on chromosome 4 and
mutations here may result in Muenke’s syndrome2 which is
also known as non-syndromic coronal synostosis, involving
one or both coronal sutures. Owing to the incomplete
penetrance of this anomaly, it is suggested that all cases of
coronal synstosis should be assessed for this FGFR3 mutation.
Our results demonstrate positive expression of FGFR-2 but
not FGFR-3 in the EOMs. This may explain the abnormal
EOM structure described in the literature to be found in
patients with syndromic craniosynostoses such as Apert,
Pfeiffer, and Crouzon. FGFR-2 mutation is known to cause a
loss of regulation/gain of function defect (ligand independent
www.bjophthalmol.com
FGFR expression in normal human fetal orbit
syndromic craniosynostosis to be able to determine whether
fibrous changes do occur in the sheath and lamina cribrosa.
Therefore, whether the expression of FGFR-2 in optic nerve
sheath might explain the high incidence of absence of
papilloedema seen in syndromic craniosynostoses despite
documented raised ICP, remains purely speculative at
present.
In summary, we have shown expression patterns of FGFR2 in the normal human fetal orbit, contrasting with
expression patterns of FGFR-3, which provide a reasonable
platform for explaining some of the clinical and histopathological features of EOMs in patients with syndromic
craniosynostosis.
.....................
Authors’ affiliations
S H Khan, K K Nischal, Department of Paediatric Ophthalmology, Great
Ormond Street Hospital for Children, Great Ormond Street, London
WC1N 3JH, UK
S H Khan, K K Nischal, Visual Sciences Unit, Institute of Child Health, 30
Guilford Street, London WC1N 1EH, UK
J A Britto, R D Evans, K K Nischal, Craniofacial Unit, Great Ormond
Street Hospital for Children, Great Ormond Street, London WC1N 3JH,
UK
Competing interests: none declared
REFERENCES
1 Khan SH, Nischal KK, Dean F, et al. Visual outcomes and amblyogenic risk
factors in the syndromic craniosynostoses—a review of 141 cases.
Br J Ophthalmol 2003;87:999–1003.
1645
2 Vajo Z, Francomano CA, Wilkin D. The molecular and genetic basis of
fibroblast growth factor receptor 3 disorders: The achondroplasia family of
skeletal dysplasias, Muenke craniosynostosis, and Crouzon syndrome with
acanthosis nigricans. Endocrine Rev 2000;21:23–39.
3 De Moerlooze L, Dickson C. Skeletal disorders associated with fibroblast
growth factor receptor mutations. Curr Opin Genet Develop 1997;7:378–85.
4 Cheng H, et al. Dissociated eye movements in craniosynostosis: a hypothesis
revived. Br J Ophthalmol 1993;77:563–8.
5 Captuo A, Lingua R. Abberrant muscle insertions in Crouzon’s disease.
J Pediatr Ophthalmol Strabismus 1980;17:239–41.
6 Diamond GR, Katowitz JA, Whittaker LA, et al. Variations in extraocular
muscle number and structure in craniofacial dysostosis. Am J Ophthalmol
1980;90:416–18.
7 Margolis S, Pachter B, Breinin G. Structural alterations of extraocular muscle
associated with Apert’s syndrome. Br J Ophthalmol 1977;61:683–9.
8 Greenberg M, Pollard Z. Absence of multiple extraocular muscles in
craniosynostosis. J AAPOS 1998;2:307–9.
9 Rattigan S, Nischal KK. Foster-type modification of the Knapp procedure for
anomalous superior rectus muscles in syndromic craniosynostoses. J AAPOS
2003;7:279–82.
10 Britto JA, Chan JC, Evans RD, et al. Differential expression of fibroblast
growth factor receptors in human digital development suggests common
pathogenesis in complex acrosyndactyly and craniosynostosis. Plast Reconstr
Surg 2001;107:1331–8.
11 Steinberger D, Reinhartz T, Unsold R, et al. FGFR2 mutation in clinically
nonclassifiable autosomal dominant craniosynostosis with pronounced
phenotypic variation. Am J Med Genet 1996;66:81–6.
12 Tuite GF, Chong WK, Evanson J, et al. The effectiveness of papilledema as an
indicator of raised intracranial pressure in children with craniosynostosis.
Neurosurgery 1996;38:272–8.
13 Liasis A, Thompson DA, Hayward R, et al. Sustained raised
intracranial pressure implicated only by pattern reversal visual evoked
potentials after cranial vault expansion surgery. Pediatr Neurosurg
2003;39:75–80.
14 Somani S, Mackeen LD, Morad Y, et al. Assessment of extraocular muscles
position and anatomy by 3-dimensional ultrasonography: a trial in
craniosynostosis patients. J AAPOS 2003;7:54–9.
15 Hayreh SS. Pathogenesis of oedema of the optic disc—the mechanical theory.
Thesis 1965;Chapter 2:16–21.
www.bjophthalmol.com
1646
PERSPECTIVE
Hypertensive retinopathy revisited: some answers, more
questions
A Grosso, F Veglio, M Porta, F M Grignolo, T Y Wong
...............................................................................................................................
Br J Ophthalmol 2005;89:1646–1654. doi: 10.1136/bjo.2005.072546
Hypertension is associated with cardiovascular risk and
systemic target organ damage. Retinopathy is considered
one of the indicators of target organ damage. This review
focuses on recent studies on hypertensive retinopathy and
their implications for clinical care. Early recognition of
hypertensive retinopathy signs remains an important step
in the risk stratification of hypertensive patients.
...........................................................................
H
ypertension is a worldwide problem that
affects up to 50 million people in the
United States and approximately one
billion worldwide, and is the single most
important modifiable risk factor for stroke.1–3
Even milder degrees of blood pressure elevation
pose increased risk for cardiovascular events.
Unfortunately, hypertension awareness, treatment, and control remain less than optimal.4 5
Hypertension acts as a silent killer many years
before overt end organ damage is clinically
apparent. Hence, the importance of refining risk
stratification strategies to ensure reliable detection of hypertension related end organ damage
before it becomes symptomatic.
The retina provides a window to study the
human circulation. Retinal arterioles can be
visualised easily and non-invasively and share
similar anatomical and physiological properties
with cerebral and coronary microcirculation.6–10
DETECTION OF HYPERTENSIVE
RETINOPATHY
See end of article for
authors’ affiliations
.......................
Correspondence to:
Andrea Grosso, MD,
Department of Clinical
Physiopathology,
Ophthalmology Section,
Turin University, Via
Juvarra, 19, 10122 Turin,
Italy; [email protected]
Accepted for publication
1 July 2005
.......................
www.bjophthalmol.com
Poorly controlled systemic hypertension causes
damage to the retinal microcirculation, so that
recognition of hypertensive retinopathy may be
important in cardiovascular risk stratification of
hypertensive patients.11 However, there is no
widely accepted classification or definition of
hypertensive retinopathy. Various international
management guidelines are not consistent in this
respect. For example, the risk stratification table
(table 1) from the European Society of
Hypertension-European Society of Cardiology
Guidelines (ESH-ESC 2003)12 indicates that
hypertensive retinopathy grades III and IV (as
defined from table 2) are associated clinical
conditions, while the Joint National Committee
on Prevention, Detection, Evaluation, and
Treatment of High Blood Pressure (JNC VII) in
the United States indicates generically retinopathy (without mention of grade) as target organ
damage.13 Additionally, the WHO International
Society of Hypertension (WHO-ISH) 2003 statement14 and the British Hypertension Society 2004
Guidelines (BHS IV)15 consider retinopathy as
target organ damage, although again only for
grades III and IV.
There are a number of considerations that may
militate against systematic retinal examination
in patients with hypertension. These include
vague definitions and heterogeneous classifications of hypertensive retinopathy, making severity staging a largely arbitrary process, as well as
the lack of well defined prognostic value for
either systemic outcomes or visual impairment.
EPIDEMIOLOGY
Several recent studies have shown that retinal
microvascular changes can be reliably documented by retinal photographs.16–23 In general, reproducibility from photographs has been found to
be excellent for well defined retinopathy signs
(kappa values ranged from 0.80 to 0.99 for
microaneurysms and retinal haemorrhages) and
fair to moderate for other more subtle retinal
arteriolar lesions (0.40–0.79 for arteriolar narrowing and arteriovenous nicking).24
Furthermore, these studies suggest that generalised arteriolar narrowing could be estimated
from an assessment of retinal vessel diameters
on photographs by use of imaging software. The
development of specific software packages have
made it possible to objectively measure the
arteriole to venule ratio (AVR) in selected
standardised portions of the retina.16 17 This
technique appears to have substantial reproducibility (intraclass correlation coefficient ranged
from 0.80–0.99).17 20–23
On the basis of retinal photography, retinal
microvascular signs are common in adults
40 years of age and older, even in those without
history of diabetes and hypertension. Both
prevalence and incidence of between 2–15% have
been reported for various retinal microvascular
lesions.19–25
WHAT RETINAL SIGNS ARE CLINICALLY
USEFUL TO CLINICIANS FOR RISK
ASSESSMENT?
Data from population based studies indicate that
certain signs of hypertensive retinopathy (table 3)
are associated with increased cardiovascular risk,
Abbreviations: ABPM, ambulatory blood pressure
monitoring; AION, anterior ischaemic optic neuropathy;
AMD, age related maculopathy; AVR, arteriole to venule
ratio; BP, blood pressure; CHD, coronary heart disease;
CVD, cardiovascular disease; IMT, intima-media
thickness; LVH, left ventricular hypertrophy; MRI,
magnetic resonance imaging; RAO, retinal arterial
occlusion; RVO, retinal vein occlusion; WCH, white coat
hypertension
Hypertensive retinopathy
1647
Table 1 Different prognostic classification of hypertensive retinopathy, according to the
European Society of Hypertension-European Society of Cardiology (ESH-ESC) 2003
Guidelines, the JNC 7 Report, the British Hypertension Society (BHS) IV 2004 Guidelines,
and the World Health Organization–International Society of Hypertension (WHO/ISH)
2003 statement on diagnosis and treatment of hypertension
ESH-ESC 2003 guidelines
12
JNC 7 report 2003
Associated clinical conditions
Advanced retinopathy: haemorrhages or
exudates, papilloedema
13
Target organ damage
Retinopathy
independently of other risk factors.23 26–28 Generalised and
focal retinal arteriolar narrowing has been shown to predict
the risk of hypertension in normotensive people.29–31
Generalised arteriolar narrowing (fig 1), focal arteriolar
narrowing, arteriovenous nicking (fig 2), opacity (copper
wire) of arteriolar wall, or a combination of these (mild grade
of retinopathy) have been associated with a mild increase
(odds ratio greater than 1 but less than 2) of incident clinical
stroke, coronary heart disease, and death. The Atherosclerosis
Risk in Communities Study showed that generalised arteriolar narrowing of the retinal arterioles was associated with
subsequent coronary heart disease in women (relative risk,
2.2; 95 confidence interval 1.0 to 4.6) but not in men (relative
risk, 1.1; 95 confidence interval 0.7 to 1.8).32 Furthermore, in
the ARIC Study generalised arteriolar narrowing of the
retinal arterioles was found to be independently associated
with increased risk for type 2 diabetes (odds ratio, 1.71; 95
confidence interval 1.13 to 2.57).33
Haemorrhages (blot, dot, or flame shaped), microaneurysms, cottonwool spots, hard exudates (fig 3), or a combination of these signs (moderate grade of retinopathy) are more
strongly associated (odds ratio of 2 or greater) with risk of
incident clinical stroke,34 presence and severity of magnetic
resonance imaging (MRI) defined cerebral white matter
lesions and cerebral atrophy defined on MRI,35 reduced
cognitive performance on standardised neuropsychological
tests,36 37 and death from cardiovascular causes.28 The ARIC
Study reported that people with microaneurysms, retinal
haemorrhages, and soft exudates were two to three times
more likely to develop an incident clinical stroke over 3 years
than people without these retinal lesions, independently of
blood pressure, diabetes, cigarette smoking, elevated lipid
levels, and other risk factors.34 Furthermore, there was a
multiplicative interaction between the presence of retinal
microvascular changes and white matter lesions on the risk
of stroke. The 5 year relative risk of stroke among participants who had both hypertensive retinopathy and cerebral
lesions on MRI, compared with those who had neither of
these findings, was 18.1 (confidence interval, 5.9 to 55.4);
WHO/ISH 2003 statement14 and
15
BHS IV 2004 guidelines
Target organ damage
Hypertensive retinopathy grade III
or IV
Figure 1 Mild hypertensive retinopathy. Photograph shows generalised
retinal arteriolar narrowing.
Figure 2 Mild hypertensive retinopathy. Photograph shows
arteriovenous nicking (white arrow).
among participants who had white matter lesions only, the
relative risk of stroke was 3.4 (confidence interval, 1.5 to
7.7).35
In a nested case-control study in patients with age related
eye diseases in Wisconsin (the Beaver Dam Eye Study) the
presence of retinal microaneurysms, retinal haemorrhages,
and retinal arteriolar narrowing was associated with a high
Table 2 The Keith, Wagener, and Barker117 hypertensive retinopathy classification
(grade I–IV), based on the level of severity of the retinal findings
Grade
Classification
Symptoms
Grade I (mild hypertension)
Mild generalised retinal arteriolar narrowing or
sclerosis
Definite focal narrowing and arteriovenous
crossings. Moderate to marked sclerosis of the
retinal arterioles. Exaggerated arterial light reflex
Retinal haemorrhages, exudates and cotton wool
spots. Sclerosis and spastic lesions of retinal
arterioles
Severe grade III and papilloedema
No symptoms
Grade II (more marked hypertension
retinopathy)
Grade III (mild angiospastic
retinopathy)
Grade IV
Asymptomatic
Symptomatic
Reduced survival
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Grosso, Veglio, Porta, et al
Table 3
Classification of hypertensive retinopathy by Wong and Mitchell26
Grades
Description
No retinopathy
Mild retinopathy
(retinal arteriolar
signs only)
(figs 1, 2 )
Systemic associations
No detectable retinal signs
One or more of the following arteriolar
signs:
N Generalised arteriolar narrowing
N Focal arteriolar narrowing
N Arteriovenous nicking
N Arteriolar wall opacity (silver wiring)
Moderate retinopathy One or more of the following retinal signs:
(fig 3)
N Haemorrhage (blot, dot, or flame
shaped)
N Microaneurysm
N Cottonwool spot
N Hard exudates
Malignant
Moderate retinopathy plus optic disc
retinopathy (fig 5)
swelling`
None
Modest* association with risk of clinical
stroke, subclinical stroke, coronary heart
disease, and mortality.
Strong association with risk of clinical
stroke, subclinical stroke, cognitive decline,
and cardiovascular mortality
Strong association with mortality
*Modest: risk and odds ratios of .1 but ,2. Strong: risk and odds ratios of .2. `Anterior ischaemic optic
neuropathy, characterised by unilateral optic disc swelling, visual loss, and sectoral visual field loss, should be
excluded neuropathy, characterised by unilateral optic disc swelling, visual loss, and sectorial visual field loss,
should be excluded.
10 year risk of stroke mortality.28 In the Cardiovascular
Health Study, people with similar signs of retinopathy were
twice as likely to have a history of stroke as those who did not
have these signs (odds ratio, 2.0; confidence interval, 1.1 to
3.6).22 Other population based studies reported that the risk
of fatal and non-fatal stroke are two to three times as high in
people do not have these signs, independently of cardiovascular risk factors.28 38
WHY ARE SPECIFIC RETINAL SIGNS ASSOCIATED
WITH DIFFERENT CARDIOVASCULAR
COMPLICATIONS?
Population based studies reported that mild and moderate
grades of retinopathy correlate with different main outcome
measures.18 23 26 28 32–35 This requires a plausible interpretation.
It is possible that different manifestations of hypertensive
retinopathy do not originate from the same pathogenic
mechanism, and therefore predispose to different levels of
cardiovascular risk. An alternative explanation is that, from a
quantitative point of view, a higher degree of generalised
vascular damage might bring together more severe retinal
findings and more frequent main outcome measures.
Findings from the ARIC, Blue Mountains Eye, and Beaver
Dam Eye studies23 26 27 28 indicate that the pathogenesis of
retinal arteriolar changes (focal narrowing, generalised
arteriolar narrowing, and arteriovenous nicking) is distinct
from that of more severe signs of hypertensive retinopathy
(microaneurysms, haemorrhages, hard exudates, and cottonwool spots).
According to histopathological studies,39 generalised retinal
arteriolar narrowing and arteriovenous nicking seem to be
related to chronically high blood pressure. In the ARIC Study,
independently of blood pressure, generalised arteriolar
narrowing was also related to systemic markers of inflammation, whereas arteriovenous nicking was related to
markers of inflammation and endothelial dysfunction and
may reflect persistent structural damage from these processes.23 27 28 40–42 Endothelial function of the retinal vasculature is impaired in early essential hypertension.43 The role of
nitric oxide in the maintenance of choroidal and retinal flow
has been recently verified.44–50 L-NMMA reaction of retinal
capillary flow is impaired in hypertensive patients43 and in
patients with type 1 diabetes a reduced response of choroidal
flow to L-NMMA has also been observed.51 Additionally,
arteriolar narrowing and arteriovenous nicking were inconsistently associated with diabetes, glucose, and glycosylated
haemoglobin.28
In contrast, hypertensive retinopathy was strong and
consistently associated with diabetes, its duration, and its
severity. In the ARIC Study hypertensive retinopathy was
related to concurrent but not past blood pressure values.29
Microaneurysms, retinal haemorrhages, and soft exudates
are most commonly seen when there is a breakdown of the
blood-retinal barrier.
Thus, a possible explanation for these data is that mild
hypertensive retinopathy reflects cardiovascular disease
(CVD) risk in relation to chronic effects of elevated blood
pressure, whereas moderate grade of hypertensive retinopathy reflects CVD risk in relation to diabetes, glycaemia, and
to recently diagnosed, more severe hypertension.28 52
Furthermore, the prognostic significance of specific retinal
vascular abnormalities may vary with age. The fact that
arteriovenous nicking was almost twice as frequent in
younger people (6.5%) than older people (3.3%) who died
of CVD causes is consistent with such a hypothesis.28
HYPERTENSION AND DIABETES
Figure 3 Moderate hypertensive retinopathy. Photograph shows
cottonwool spots and retinal haemorrhages.
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Diabetes and hypertension are both vascular risk factors and
may share similar pathophysiological mechanisms. Both
conditions are also linked by the metabolic syndrome.53 54
The prevalence of diabetes among patients with hypertension
is high,55 and type 2 diabetes may remain unrecognised for
years before being diagnosed.56 When diabetes is associated
with hypertension, cardiovascular risk rises exponentially
and retinopathy becomes more severe and rapidly progressive. In turn, more tight control of blood pressure in
Hypertensive retinopathy
hypertensive diabetic people was shown to prevent cardiovascular events as well as deterioration of both retinopathy
and visual acuity.57–59 Among the various pathophysiological
mechanisms, endothelial dysfunction has been implicated in
the pathogenesis of the metabolic syndrome and points to a
link between diabetes and hypertension.60 61 It was observed51
that systemic and ocular haemodynamic reactivity to NOsynthase inhibition is reduced in patients with long standing
insulin dependent diabetes mellitus, compared with healthy
control subjects The blunted response of retinal capillary flow
to L-NMMA observed43 in young hypertensive patients with
essential hypertension indicates a reduced contribution of
nitric oxide to the maintenance of retinal perfusion.43 Therapy
with AT1 receptor blocker candesartan celexitil restored both
the contribution of nitric oxide to the maintenance of retinal
perfusion and nitric oxide dependent vasodilation in the
retinal vasculature of patients with arterial hypertension.43
Other mechanisms linking diabetes and hypertension are
inflammatory processes and overt atherosclerosis.62 63
DO RETINAL SIGNS CORRELATE WITH
HYPERTENSION SEVERITY?
A correlation between retinal lesions, as detected by direct
ophthalmoscopy, and left ventricular hypertrophy, as defined
by echocardiography, was suggested64 but the study was
limited by the imprecision of clinical ophthalmoscopy in
quantifying retinal arteriolar narrowing and by the rather
small sample size. Some studies have linked renal dysfunction with retinal vascular changes,65–68 but the relation of
early retinal vessel vascular changes and risk of cardiovascular complications is not well understood.
Recent findings from a clinical study69 showed no
significant relation between retinal microvascular changes
(diffuse arteriolar narrowing, arteriovenous crossings),
detected by qualitative examination of the fundus, and
prognostically validated markers of target organ damage,
such as 24 hour ambulatory blood pressure monitoring,
24 hour urine collection for microalbuminuria, echocardiography, carotid ultrasonography in early stages of untreated
essential hypertension. Early retinal alterations were extremely frequent in this cohort of relatively young untreated
subjects with recently diagnosed grade 1 or 2 hypertension.
Furthermore, the prevalence of retinal microvascular
abnormalities was much higher than that of left ventricular
hypertrophy, carotid wall alterations, and microalbuminuria.69–71 Patients with arteriovenous crossings did not have
more cardiac, carotid, and renal alterations compared with
those without this retinal pattern. The distribution of retinal
microvascular changes was similar in lower, intermediate, or
higher tertiles of left ventricular mass.
THE ROLE OF SYSTEMIC HYPERTENSION AS A RISK
FACTOR FOR OTHER EYE DISEASES SUCH AS
GLAUCOMA OR AGE RELATED MACULAR
DEGENERATION
In addition to hypertensive retinopathy, elevated blood
pressure is a risk factor for many ocular conditions. These
include anterior ischaemic optic neuropathy, retinal vein
occlusion, retinal arteriolar emboli and, possibly, age related
maculopathy (AMD) and glaucoma.72 With regard to AMD,
the Framingham Study73 reported an association of AMD
with systemic hypertension, a relation that increased with
the duration of the hypertension.73 However, other studies
have not found consistent relations. No such correlation was
found for the development of the neovascularisation in the
studies by Bressler,74 the Eye Disease Case Control Study
Group,75 and the Beaver Dam Eye Study.76 More recently, the
Macular Photocoagulation Study77 found a relative risk of 1.7
for the development of choroidal neovascularisation in
1649
patients with definite systemic hypertension. Over 5 years,
the incidence of choroidal neovascularisation was 49%
among patients with definite hypertension versus 33% in
patients without definite hypertension. The authors stressed
the importance of high blood pressure on the prognosis of the
fellow eye. With regards to glaucoma, a population based
study78 showed a modest positive association of primary open
angle glaucoma with systolic and diastolic blood pressure. In
another study,79 however, no correlation was showed in the
prevalence of arterial hypertension in primary and secondary
open angle glaucoma.
Are retinal examinations more useful in specific
subgroups of populations?
There is strong evidence that identifying and targeting
subsets of hypertensive patients at highest risk improves
the cost effectiveness of antihypertensive treatment.80
Subjects with white coat hypertension (WCH) or masked
hypertension—that is, the phenomenon of consistently
elevated clinic blood pressure levels but normal 24 hours
ambulatory blood pressure monitoring,81 may represent an
intermediate group between healthy people and sustained
hypertensives as far as target organ damage and cardiovascular risk is concerned. Prevalence of this condition is quite
variable, depending of the selection groups, suggesting a
range between 12%–30%, being more common in the elderly82
and among females.82 Previous studies have suggested that
WCH is associated with end organ damage.82 83
The presence of hypertensive retinopathy in WCH may
suggest an indication to antihypertensive therapy.
Evidence is increasing that even mild blood pressure (BP)
elevation can have an adverse effect on vascular structure
and function in asymptomatic young people. High BP in
childhood had been considered a risk factor for hypertension
in early adulthood. The retinal examination is recommended
to identify retinal vascular changes in young patients with
co-morbid risk factors and BP 90th–94th percentile and in all
patients with BP >95th percentile.84–89 A previous study
reported a prevalence of 41% for the arteriolar narrowing and
of 8% for arteriovenous nicking, as defined by retinal
photographs, in a cohort of 97 children and adolescents with
essential hypertension.90 Further longitudinal studies will be
necessary to determine how these retinal vascular signs
progress over time in juveniles with essential hypertension
and whether the abnormalities are of prognostic importance.91 Finally, it is unclear whether retinal examination
would confer a greater benefit in women and black people.26
WHAT SHOULD CLINICIANS DO WITH THE CURRENT
EVIDENCE?
On the basis of the available data we propose a flow chart
about the supplemental risk assessment by the ophthalmoscopic consultation in hypertensive subjects (fig 4). The
strongest evidence of the usefulness of hypertensive retinopathy for risk stratification is based on its association with
stroke (tables 3–5).26 34 35 In the presence of equivocal signs
(borderline or inconsistent hypertension or WCH with no
other evidence of target organ damage) or visual symptoms,
an examination by the ophthalmologist may be useful. The
presence of retinopathy may be an indication for initiating
antihypertensive treatment.12 13 26 For hypertensive patients
grade 212 without overt target organ damage ophthalmological referral may also be useful. The presence of retinopathy
may be an indication for more aggressive intervention on
associated cardiovascular risk factors and co-morbidities and
has an important practical impact for treatment decisions
(for example, antihypertensive and anti-platelet aggregation)
and for close follow up. Furthermore, for some patients,
ophthalmic consultation may be useful to rule out diabetic
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1650
Grosso, Veglio, Porta, et al
Patient with hypertension
Medical history, examination, appropriate laboratory investigations, and management
Grade 1 hypertension
Grade 2 hypertension
Grade 3 hypertension
Malignant retinopathy
• Routine care
• Lifestyle measures
• Monitoring of blood
pressure
• Routine care
• Lifestyle measures
• Monitoring of blood
pressure
Compelling indication to the
ophthalmological referral for
the evaluation and treatment
of retinal vascular
complications (RVO, RAO,
non-arteritic AION)
• Urgent hypertension
treatment
Consider ophthalmology
referral for patients with
diabetes and with visual
symptoms
Diabetic retinopathy
and vascular
complications
Consider ophthalmology
referral for certain patients
(eg, patients with white
coat hypertension with no
other evidence of target
organ damage, patients
with visual symptoms or
headache)
Mild
retinopathy
More vigilant
monitoring of
cardiovascular risk
Figure 4
Moderate
retinopathy
• Treatment of diabetic
and hypertensive
retinal complications
• Further cardiac
evaluation to improve
cardiovascular and
cerebrovascular risk
stratification in the
presence of moderate
retinopathy
• Aggressive approach to the risk reduction in
presence of moderate signs of retinopathy
• Possible indication for hypertension treatment
Flow chart: supplemental risk assessment by retinal examination.
retinopathy, retinal vein occlusion, anterior ischaemic optic
neuropathy, or retinal arterial occlusion.44 47 26 For all grade 3
hypertensive patients12 there are compelling indications for
an ophthalmological referral (fig 5) for evaluation and
treatment of retinal vascular complications.92 In WCH the
ophthalmological referral may be indicated when there is no
other evidence of target organ damage. In the presence of
both mild and moderate (table 3) retinal signs, pharmacological treatment may be warranted. In the presence of
moderate retinal signs (table 3) ophthalmologists may refer
people for further cardiac evaluation to improve the
cerebrovascular risk stratification.93
LIMITATIONS OF AVAILABLE DATA
Epidemiological studies provide additional insight that
arteriolar constriction and narrowing may have a critical role
in the development of hypertension. However, caution must
be applied to the interpretation of these data.
Firstly, raised intraocular pressure may affect retinal
arteriolar calibre. The ARIC Study did not include an
assessment of IOP.94–96
Secondly, photographs were not synchronised with the
cardiac cycle and vessel diameter may vary because of
pulsatility. A variation of 2% to 17% has been described.97 98
However, because photography was independent of any
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Figure 5 Malignant hypertensive retinopathy. Photograph shows
multiple cotton wool spots, retinal haemorrhages, optic disc swelling,
and macular star.
subject characteristics, this variation, at most, would have
caused random misclassification. The optimal conditions for
taking measurements, with reference to posture,99 blood
pressure,100 and autonomic nervous system,101 also need to be
standardised. Furthermore, pregnancy induces modifications
on the vascular dynamics.102
Thirdly, the overall prevalence of retinopathy signs in some
recently reported studies may be too high.69 70 It is unclear
Hypertensive retinopathy
1651
Table 4 Mild hypertensive retinopathy (retinal arteriolar signs only)
Sign
Diagnosis
Histopathology correlations
Clinical correlations
Future research
Generalised
arteriolar
narrowing
N Qualitative examination of
retinal photographs69 70
N Computer assisted fundus
image analysis and AVR
calculation in selected
standardised portions of
the retina16 17
N Vasoconstrictive phase:
vasospasm and an increase
6
in retinal arteriolar tone
N Sclerotic phase: intimal
thickening, hyperplasia of
the tunica media, and
hyaline degeneration in the
subsequent sclerotic stage6
N Risk of hypertension (odds ratio,
1.62; CI 95% 1.21 to 2.18)29–31
N Risk of stroke (relative risk, 1.24,
34 35
CI 95% 0.66 to 2.31)
N Risk of CHD in women (relative
32
risk, 1.37; CI 95% 1.08 to 1.72)
N Risk of diabetes mellitus (odds
ratio, 1.71, CI 95% 1.13 to 2.57)33
N Relation to stiffness of the
carotid arteries116
Risk of cognitive
3
impairment:modest association
Clinical validation of the AVR
Clinical significance
(a) CVD evaluation in presence
of retinal mcrovascular lesions
(b) Potential value of
specifically targeting the
microcirculation in the
treatment of hypertension
Prevention
(c) Role of retinal photography
for CVD risk stratification
Focal arteriolar
narrowing
N Constricted area of two
thirds or less the width of
proximal and distal vessel
17
segments
N Areas of localised
vasoconstriction evaluated
on the disc and within K
17
DD of its margin zone
N Risk of any stroke (relative risk
crude, 1.57, CI 95%, 1.0 to
2.45)34 35
N Risk of cognitive
impairment:modest association36
N Prevention
Usefulness of focal arteriolar
narrowing evaluation in the
cerebrovascular risk
stratification
Arteriovenous
nicking
N Present if seen in at least one of
the temporal quadrants; definite
if the venous blood column was
tapered on both sides of its
crossing under an arteriole;
17
EDTRS standard photograph 9
Sclerotic phase: intimal
thickening, hyperplasia of
the tunica media, and
hyaline degeneration in the
subsequent sclerotic stage6
N Risk of any stroke (relative risk,
2.21, CI 95%, 1.44 to 3.38)34 35
N Risk of cognitive
36
impairment:modest association
N Prevention
Usefulness of arteriovenous
nicking evaluation in the
cerebrovascular risk
stratification in hypertensives
how these changes were defined and how signs were
classified. Moreover, the terminology used is neither consistent nor comparable with data from other population
based studies. The recommendations issued by ESH-ESC
200312 do not take in account recent data from epidemiological studies, but are derived only from the conclusion of one
clinical study.70 Fourthly, there are no reliable clinical data to
relate signs of retinopathy with other prognostically validated
markers of target organ damage, such as intima-media
thickness (IMT), left ventricular hypertrophy (LVH), and
ABPM. Reclassification of cardiovascular risk recently proposed,69 taking into account the evaluation of the arteriovenous nicking, is biased by the cross sectional design of the
study. Finally, from a methodological viewpoint, retinal
photographs in those large population based studies were
evaluated in standardised settings, which are typical of
clinical research but may not be transferred easily to everyday
practice.
WHAT ARE FUTURE RESEARCH QUESTIONS?
Researchers should develop a common and standardised
photographic classification of the retinal signs similar to
diabetic retinopathy.
Secondly, the ARIC study offers insights that support the
hypothesis that microvascular disease may have a more
prominent role in the development of myocardial ischaemia
and coronary heart disease (CHD) in women. However, this
and other population base studies are prevented from making
a more definite conclusion because they assessed retinal
microvasculature, not the coronary or cerebral microcirculation. In addition, data from population base studies do not
necessary imply a cause (generalised arteriolar narrowing)
Table 5 Moderate hypertensive retinopathy
Sign
Diagnosis
Histopathology correlations
Clinical correlations
Microaneurysm
Present v absent
Exudative phase: disruption of
blood-barrier, degeneration of
vascular smooth muscle and
endothelial cell necrosis
leading to blood and lipid
exudation and ischaemia6
N Risk of any stroke (relative risk , 6.11; Prevention
34
CI 95% 3.72 to 10.05)
N Is retinal photography useful in the
measurement of stroke risk?
N Risk of cognitive impairment :strong
36
association
Retinal
N Blot
haemorrhage (blot, N Flame shaped
dot, or
N Both
flame,shaped)
N Exudative phase
N Risk of any stroke (relative risk, 6.44;
34
CI 95% 3.61 to 11.49)
N Risk of any stroke (relative risk, 6.38;
CI 95% 2.97 to 13.73)34
N Risk of cognitive impairment:strong
association36
Prevention
(a) Is retinal photography useful in the
measurement of stroke risk?
(b) Cognitive impairment may be
amenable to treatment and prevented
strategies targeted at vascular diseases
Soft exudates
Ischaemia of the nerve fibre
layer
N Risk of any stroke (relative risk 7.80,
CI 95% 4.07 to 14.96)34
N Risk of cognitive impairment:strong
36
association
Prevention
N It is important to replicate some of these
findings in other populations to assess the
associations of retinal microvascular
disease to different stroke subtypes and to
other clinical and subclinical cerebral
disorders with a supposed microvascular
aetiology
Present v absent
Future research
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1652
and effect (incident CHD and stroke) relation. Thus, it is
unclear why the association of generalised arteriolar narrowing was not associated with incident CHD in men. Further
investigation is required to support the hypothesis that
microvascular disease has a more prominent role in development of myocardial ischaemia and CHD in women.103
Other unmeasured factors (for example, use of vasodilator
medications, diurnal and nocturnal fluctuations of blood
pressure) associated with generalised arteriolar narrowing of
retinal arterioles might have caused incident CHD or the
stroke.103
Thirdly, the ARIC Investigators have shown that generalised retinal arteriolar narrowing may precede the onset of
diabetes mellitus in middle aged people and may even have a
role in its initial development. However, the authors have
only shown a short term association between generalised
arteriolar narrowing of the retinal arterioles and incident
diabetes. Further studies are required to determine whether
longer term associations do exist.
Fourthly, there has not been a consistent demonstration
that these retinal signs have independent predictive value
and that the addition of retinal photography may help to
optimise global risk evaluation in primary hypertension and
modify the therapeutic decisions.26
Finally, population based studies suggest that narrowed
arterioles are associated with the development of hypertension and therefore that small vessel disease may be a target
for antihypertensive treatment.104–107 Thus, there is a need to
evaluate whether specific therapy focused on the retinal
microcirculation can reverse108 change in retinopathy or
reduce retinal microvascular damage,43 109 and, if so, whether
this approach will also result in a reduced cardiovascular risk.
NEW WAYS TO DETECT HYPERTENSIVE
MICROVASCULAR DAMAGE: GENERALISED
ARTERIOLAR NARROWING AS AN EXAMPLE OF
FUTURE TECHNOLOGY
A quantitative way of assessing one of the microvascular
changes—generalised arteriolar narrowing in the retina—has
been developed and used in population based studies.16 19–22
The photographs were digitised and the diameters of
individual arterioles and venules coursing through a zone
located K–1 disc diameter from the optic disc margin were
measured with a dedicated software and summarised as an
arteriole-venule ratio (AVR). Use of the ratio was introduced
to counter several potential problems. Firstly, it introduces
some adjustment for the wide range of vessel diameter in the
normal population. Secondly, by virtue of being a ratio it
offers some protection against several potential problems: (a)
variable magnification caused by differences in refractive
error among individuals, (b) apparent broadening of vessel
calibre as a result of poor photographic focus or ocular media
clarity, and (c) differences among graders regarding the
precise determination of the vessel edge.
It remains unclear what exactly the separate arteriolar and
venular diameters contributed to the AVR and what kind of
vascular disease this ratio precisely reflects. In the ARIC,
Beaver, and Blue Mountains Eye Study the authors attributed
a lower AVR to generalised arteriolar narrowing. Data from
the Rotterdam Study indicate that the AVR does not reflect
only generalised arteriolar narrowing but also a separate
contribution from venular diameters. The authors hypothesise other pathogenic mechanisms related to the disruption of
the endothelial surface layer and to inflammatory processes.110–112
Findings from the Wisconsin Epidemiologic Study of
Diabetic Retinopathy113 114 showed that in eyes with nonproliferative diabetic retinopathy, measurement of venous
dilation may add prognostic information independently of
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Grosso, Veglio, Porta, et al
the severity scale. Wider retinal venular diameters have been
suggested to reflect hyperperfusion resulting from both
hyperglycaemia and retinal hypoxia.115 Thus, in future
research, more attention should be paid to the role of venules
in vascular disease.
CONCLUSION
In conclusion, hypertensive retinopathy remains a recognised
manifestation of target organ damage in hypertensive
patients.118 Digital retinal photography aimed at the automated measurement of retinal arteriolar diameter is useful in
research on the microvascular contributions to clinical
cardiovascular disease. In the future, a retinal examination
might acquire a specific indication to predict (that is,
consider CVD evaluation in presence of retinal microvascular
lesions) and prevent (that is, role of retinal photography for
CVD risk stratification) metabolic and/or cardiovascular
events in the general population, even in the absence of
overt hypertension or diabetes.
.....................
Authors’ affiliations
A Grosso, F M Grignolo, Department of Clinical Physiopathology,
Ophthalmology Section Turin University, Italy
F Veglio, Department of Medicine and Experimental Oncology, Turin
University, Italy
M Porta, Department of Internal Medicine, Turin University, Italy
T Y Wong, Centre for Eye Research Australia, University of Melbourne,
Australia, and Singapore Eye Research Institute, National University of
Singapore, Singapore
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1655
PERSPECTIVE
Impression cytology of the ocular surface
R Singh, A Joseph, T Umapathy, N L Tint, H S Dua
...............................................................................................................................
Br J Ophthalmol 2005;89:1655–1659. doi: 10.1136/bjo.2005.073916
Impression cytology refers to the application of a
cellulose acetate filter to the ocular surface to remove the
superficial layers of the ocular surface epithelium. These
cells can then be subjected to histological,
immunohistological, or molecular analysis. Proper
technique is essential as the number of cells sampled can
vary considerably. Generally two to three layers of cells are
removed in one application but deeper cells can be
accessed by repeat application over the same site.
Applications for impression cytology include diagnosing a
wide range of ocular surface disorders, documenting
sequential changes in the conjunctival and corneal surface
over time, staging conjunctival squamous metaplasia, and
monitoring effects of treatment. It is also a useful
investigational tool for analysing ocular surface disease
with immunostaining and DNA analysis. It is non-invasive,
relatively easy to perform, and yields reliable information
about the area sampled with minimal discomfort to the
patient. Major ophthalmic centres should develop and
introduce this technique into routine clinical practice. This is
best achieved with a team approach including the
ophthalmologist, pathologist, microbiologist, and the
immunologist.
...........................................................................
I
See end of article for
authors’ affiliations
.......................
Correspondence to:
Harminder S Dua, Division
of Ophthalmology, B
Floor, Eye ENT Centre,
University Hospital,
Queens Medical Centre,
Nottingham NG7 2UH,
UK; harminder.dua@
nottingham.ac.uk
Accepted for publication
28 July 2005
.......................
mpression cytology refers to the application
of cellulose acetate filter to the ocular surface
to remove the superficial layers of the ocular
surface epithelium. The cells thus removed can
be subjected to histological, immunohistological, or molecular analysis. Egbert et al first
described this minimally invasive method of
studying conjunctival goblet cells in 1977.1
Since then the technique has been used to
evaluate several ocular surface disorders and
modifications to the original technique have
been introduced. It is non-invasive, easy to
perform, and yields reliable information about
the area sampled with minimal discomfort to the
patient. This makes it a valuable tool in the
understanding of ocular surface disorders.
Applications of impression cytology include the
aetiological diagnosis of various ocular surface
disorders, documenting sequential changes in
the conjunctival and corneal surface over time,
monitoring effects of treatment and staging
conjunctival squamous metaplasia, and as an
investigational tool for analysing ocular
surface disease with immunostaining and DNA
analysis.2
IMPRESSION CYTOLOGY TECHNIQUE
Egbert et al used Millipore filters to collect
conjunctival specimens, which were then air
dried and stained with periodic acid Schiff (PAS)
and haematoxylin.1 Tseng modified the method
of collection of specimens and stained them with
a combination of PAS and Papanicolaou stains.2
Maskin and Bodé have described a technique
with which conjunctival epithelial cells acquired
by impression cytology can be studied by electron
microscopy.3 Pure nitrocellulose membranes and
Biopore membrane devices have been used to
enable immunocytochemical staining.4 5
SPECIMEN COLLECTION
The type of filter paper used and the technique of
cell collection depend on the purpose for which
the specimen is collected. The size of the filter
paper pores affects the consistency of epithelial
cells collected and the resolution of cell detail.
Larger pore sizes collect cells better, but the cell
detail is less well preserved. Treatment of the
filter paper with surfactant also reduces cell pick
up. Most authors use surfactant free filter paper
of a pore size between 0.22 mm and 0.44 mm.6
Tseng’s modified method of specimen collection uses cellulose acetate filter paper from
Millipore, which is trimmed into a 5 mm strip
with one square end and one tapering end. The
asymmetrical shape with a pointed tip facilitates
grabbing and transferring the paper to the
desired area with blunt smooth edged forceps.2
We use a 13 mm diameter Millipore paper
divided in two ‘‘D’’-shaped halves (fig 1). The
end of the paper to be applied to the nasal side is
clipped for orientation. One drop of local
anaesthetic is instilled into the eye and excessive
tear fluid and medication are wiped away. The
paper is applied on the conjunctiva or cornea or
both together, straddling the limbus. The area to
be sampled depends on the underlying pathology. The filter paper is smoothed onto the ocular
surface by applying gentle pressure with a
Goldmann tonometer headpiece held between
finger and thumb. The smooth flat surface of the
headpiece allows uniform pressure to be applied
over the surface area of the paper. The paper is
allowed to remain in contact with the eye for
approximately 5–10 seconds and then peeled off
with a forceps. During the period of contact it is
important that the lids are held away from the
paper and it is not allowed to be wetted by tear
fluid that may at times appear as a result of
stimulation of lacrimation. If the paper gets
Abbreviations: OSSN, ocular surface squamous
neoplasia; PAS, periodic acid Schiff; RT PCR, reverse
transcriptase polymerase chain reaction; TDC, total dye
content
www.bjophthalmol.com
1656
Singh, Joseph, Umapathy, et al
a
0.22–0.45 µm pore size
filter paper disc cut in
halves
b
No 1 filter paper before using it for the first time. Scott’s tap
water substitute consists of 1 g sodium bicarbonate and 5 g
magnesium sulfate, anhydrous or 10 g magnesium sulfate,
crystalline in 500 ml of tap water. The pH of this solution is
8.02 and plays a significant part in determining the blue
colour of the nuclei. Modified orange G is made of 10 ml
orange G, 10% total dye content (TDC) aqueous solution
combined with 490 ml of 95% ethyl alcohol and 0.075 g
phosphotungstic acid. Modified eosin Y consists of 350 ml of
95% ethyl alcohol, 125 ml absolute methyl alcohol, 10 ml
glacial acetic acid, 0.18 g light green SF yellowish, 5 ml 3%
TDC aqueous solution, 10 ml eosin Y 20% TDC aqueous
solution, and 1 g phosphotungstic acid.
SPECIAL TECHNIQUES
Dry area to be sampled. Apply paper with a notch to mark side.
Straight edge towards limbus (a) or corneal centre (b). Press firmly
with Goldmann tonometer head. Peel off paper with forceps and
place in appropriate fixative solution
Figure 1 Schematic representation of impression cytology procedure
as followed by the authors, showing cut filter paper disc into halves (left),
and application of resulting D-shaped segments (clipped for orientation)
on bulbar conjunctiva and cornea using Goldmann tonometer head, to
apply even pressure on filter paper (right).
unduly wet, the yield of cells will be poor. The paper is
immediately transferred into a well of a 24 well plate
containing fixative solution. It may be necessary to mark
the back of the paper before applying it on to the ocular
surface so that the surface to be stained later can be easily
identified.
SPECIMEN STAINING
Papanicolaou or haematoxylin and PAS stains are the
commonly used stains for routine histological staining of
impression cytology specimens. The filter paper with the
specimen is fixed for approximately 10 minutes in a solution
containing glacial acetic acid, formaldehyde, and ethyl
alcohol in a 1:1:20 volume ratio.2 A 24 well culture plate or
a 24 well Teflon sample holder is used to hold the specimens
during fixation and staining. The specimens are rehydrated in
70% ethyl alcohol and then placed successively in periodic
acid Schiff reagent, sodium metasulfite, Gill’s haematoxylin,
and Scott’s tap water substitute for 2 minutes each, rinsing in
two changes of tap water in between each step.7 This is
followed by dehydration in two changes of 95% ethyl alcohol,
staining with modified orange G for 2 minutes, rinsing in
95% ethyl alcohol for 3 minutes, and staining with modified
eosin Y for 2 minutes, again rinsing in 95% ethyl alcohol for
5–10 minutes, before dehydration in absolute alcohol for
5 minutes. Throughout the staining the cell side of the filter
paper must be completely soaked with staining solution. For
each destaining or rinsing, the holder is either dipped 10
times or suspended in a large jar with continuous magnetic
stirring so that there is no need for constant monitoring.2
After the final destaining step, xylene is used to make the
filter paper transparent. Before mounting, the filter paper is
placed with the epithelial cells facing up. The completed
slides are examined by light microscopy.
PREPARATION OF STAINING SOLUTIONS
Gill et al have described the detailed preparation of each
solution.7 Gill’s haematoxylin is prepared by combining
365 ml of distilled water, 125 ml of ethylene glycol, 1 g of
anhydrous haematoxylin, 0.1 g of sodium iodate, 8.8 g of
aluminium sulphate, and 10 ml of glacial acetic acid. The
chemicals are stirred for 1 hour on a magnetic mixer at room
temperature. The final solution is filtered through Whatman
www.bjophthalmol.com
Special staining techniques have been devised for studying
the specimens by electron microscopy3 and immunocytochemistry.4 5 For electron microscopy, the specimen on
cellulose acetate paper is fixed in 4% phosphate buffered
formaldehyde with 1% glutaraldehyde and ruthenium red
dye, post fixed in buffered osmium fixative, dehydrated, and
embedded in resin.3 Immunocytochemical staining of impression cytology specimens collected on conventional cellulose
acetate membranes results in high levels of background
staining. Use of xylene to chemically clear the cellulose
acetate filter destroys the cell surface antigen. To overcome
this problem Krenzer and Freddo developed a method of
collecting the specimen on a pure nitrocellulose membrane
which was then fixed with a spray fixative, transferred on to
a poly-L-lysine coated glass slide, and dried. The slide was
then placed in acetone for 1 hour with continuous agitation
to dissolve the filter membrane, washed for 5 minutes in tap
water, and subjected to cellulose digestion for 2 hours at 37˚C
to remove residual membrane material before proceeding to
immunocytochemical staining.4 Thiel et al used mounted
Biopore membranes (Millicell-CM 0.4 mm PICM 012550,
Millipore Corp, Bedford, MA, USA) for the immunopathological diagnosis of superficial viral infections. The Biopore
membrane is fully transparent in the wet state and allows a
detailed cytological examination by light and fluorescence
transmission microscopy.5
MICROSCOPY AND CLINICAL APPLICATIONS
An impression cytology usually removes only 1–3 cell layers
and does not yield the same information as a flat mount or
cross section preparation of the ocular surface. It is therefore
ideal for studying the surface epithelium rather than the
basal epithelium or the basement membrane (fig 2A).
However, using multiple impressions of the same area (in
vivo or in vitro cadaver eye) we were able to demonstrate the
morphology of the basal limbal epithelium. The limbal cells
are smaller, more densely packed, and have a greater nucleus
to cytoplasm ratio compared to adjacent corneal and
conjunctival cells (fig 2B). Morphologically Egbert first used
this method to determine the density of goblet cells in
different areas of the conjunctiva.1 He found the greatest
density of goblet cells in the nasal palpebral conjunctiva, with
decreasing densities in the temporal and palpebral conjunctiva, bulbar conjunctiva near the fornices, and the bulbar
conjunctiva near the limbus. His results were similar to those
obtained by studies of whole mounts of conjunctiva. Adams
studied the morphology of normal human conjunctival
mucus using impression cytology and described granules,
strands, and structureless patterns of mucus on different
parts of the conjunctiva.8
Impression cytology has also been used in the evaluation
of ocular surface diseases such as keratoconjunctivitis
sicca,9 10 vitamin A deficiency,11 cicatricial pemphigoid,12
atopic disease,13 superior limbic keratoconjunctivitis and
Impression cytology
1657
Figure 2 (A) Impression cytology of normal corneal surface showing corneal epithelial cells. Normal cells are flat with a prominent nucleus. The
nuclear cytoplasmic ratio is low (6100, periodic acid Schiff staining). (B) Impression cytology of normal transition zone from cornea to limbus (640,
periodic acid Schiff staining). The limbal epithelial cells are small, densely packed with a high nuclear cytoplasmic ratio. The limbal zone is clearly
demarcated from the adjacent corneal epithelial cells.
mucopolysaccharidoses,3 vernal keratoconjunctivitis,14 and
the effect on these of various therapies. Tseng classified
conjunctival squamous metaplasia into six stages according
to the presence or absence of goblet cells and goblet cell
density, morphological changes of the nucleus, nucleuscytoplasm ratios, metachromatic changes of cytoplasmic
colour, and emergence of keratinisation.2 Nelson graded
conjunctival impression cytology specimens (grades 0–3,
table 1) based on the appearance of the epithelial cells and
the numbers of goblet cells.15 A specimen of small, round
epithelial cells with large nuclei and more than 500 goblet
cells/mm2 was considered grade 0, whereas another of large
polygonal epithelial cells with small nuclei and less than 100
goblet cells/mm2 was considered grade 3. All specimens that
were grade 2 or more were abnormal. The findings of grades 2
and 3 on the interpalpebral conjunctiva and grades 0 and 1
on the inferior palpebral ocular surface in the absence of
inflammatory cells suggest a diagnosis of keratoconjunctivitis
sicca, whereas grades 2 and 3 on both the bulbar and
palpebral conjunctiva suggest an intrinsic ocular surface
disease such as ocular cicatricial pemphigoid, StevensJohnson syndrome, or severe chemical burns.15 The presence
of inflammatory cells suggests that the disease is active.
Marner studied impression cytology specimens of conjunctival epithelial cells of patients with keratoconjunctivitis sicca
and described a snake-like appearance of nuclear chromatin
in clusters of abnormal cells from the upper bulbar
conjunctiva (fig 3).9 Prabhasawat and Tseng used impression
cytology to demonstrate normal conjunctival epithelial cells
and goblet cells following ocular surface reconstruction by
preserved amniotic membrane.16 Modifications of the impression cytology technique were made to study cytokeratin
expression in bulbar conjunctiva by using pure nitrocellulose
membranes and immunocytochemical staining.4 The conjunctiva was found to demonstrate a unique cytokeratin
expression pattern containing cytokeratins characteristic of
non-keratinised, stratified epithelia (K4 and K13) as well as
others more typical of a simple differentiation pattern (K8
and K19), a glandular differentiation pattern (K7), or both.4
This technique was later used in the preoperative diagnosis of
seborrhoeic keratosis of the conjunctiva simulating a
malignant melanoma.17 Thiel et al used a biopore membrane
device to collect ocular surface specimens and apply
immunopathological methods to diagnose superficial viral
infections like herpes simplex virus, varicella zoster virus, and
adenovirus.5
More recently impression cytology has been used to
demonstrate conjunctival metaplasia as a result of the use
of topical antiglaucoma drugs.18 19 Free radical production
was found in patients on long term antiglaucoma treatment
and contact lens wearers by investigations on impression
cytology specimens.20 Immunohistochemistry on impression
cytology specimens has been used to compare the efficacy of
drugs and to determine the mechanism of action of topical
agents in vernal conjunctivitis.21 Impression cytology has also
helped in evaluation of ocular surface changes after excimer
laser phototherapeutic keratectomy in patients with corneal
dystrophies and other corneal pathology.22 It has enabled
documentation of limbal cell deficiency in patients with
Figure 3 Impression cytology of the conjunctival surface showing
snake-like chromatin in keratoconjunctivits sicca (6100, periodic acid
Schiff staining).
Table 1 Nelson’s classification for squamous
metaplasia
Grade Features
0
1–2
3
.500 goblet cells/mm2
Small, round epithelial cells with large nuclei
100–500 goblet cells/mm2
,100 goblet cells/mm2
Large, polygonal epithelial cells with small nuclei
Grade 2 or more = abnormal.
Figure 4 Impression cytology of the conjunctivalised corneal surface in
limbal stem cell deficiency showing reddish pink goblet cells (6100,
periodic acid Schiff staining).
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1658
Singh, Joseph, Umapathy, et al
1
2
3
4
5
6
7
8
300 bp
200 bp
100 bp
Figure 5 rtPCR image of cDNA extracted from cells obtained by
impression cytology from cornea (lanes 3, 5, 7) and conjunctiva (lanes
2, 4, 6). Primers for human b defensins 1 and 2 were used. (Reproduced
by courtesy of Masahel Al-Abed, Larry A Donoso Laboratory for Eye
Research, Division of Ophthalmology, University of Nottingham).
ocular burns and other surface disorders by demonstrating
the presence of goblet cells on the corneal surface (fig 4).23
Limbal stem cell deficiency has also been assessed using
immunoperoxidase staining for cytokeratins: K3 for corneal,
and K 19 for conjunctival phenotype. The limitation to this
method was inadequate sampling of cells in 58% of cases
(defined as less than 50% cellularity in specimen). Western
blotting has been used to demonstrate increased expression
of epidermal growth factor receptors ErbB2 and ErbB3 in
patients with keratoconjunctivitis sicca.24
Cells harvested from the cornea by impression cytology
have been used for investigation of DNA polymorphisms
using the polymerase chain reaction (fig 5).25 MUC 7 gene
has been demonstrated in conjunctival cells obtained by
impression cytology detecting elevated mRNA levels using
semi-quantitative reverse transcriptase polymerase chain
reaction (RT PCR).26 RT-PCR techniques have been used to
show that defensin h-BD2 is preferentially expressed in
moderate dry eye patients, and demonstrated that it can be
induced in cultured conjunctival cells by exposing them to
inflammatory cytokines.27
Pisella et al have used flow cytometry on conjunctival
impression cytology specimens in dry eye and acne rosacea
patients and found upregulation of HLA DR and ICAM-1
expression compared to control samples, supporting an
inflammatory basis for their ocular surface disease.28 They
also found a reduction in M-1 positive staining cells (goblet
cell stain), and suggested inflammation may be responsible.
Cytokines IL-6, IL-8, IL-10 have been found to be elevated in
patients on chronic antiglaucoma medications using flow
cytometry.29
Impression cytology has been used widely as a noninvasive method for conjunctival biopsy for suspected ocular
surface squamous neoplasia (OSSN).30 31 Using biopore
membrane for specimen collection an 80% correlation was
found between impression cytology diagnosis and histopathology specimens obtained from incisional biopsy (fig 6).
Keratinising malignancies offer the highest chance of false
negatives because of paucity of cells in the specimen and
should be kept in mind in such cases. Detailed cytomorphology of OSSN using impression cytology has been described.32
Mitomycin C (MMC) has gained acceptance for the
treatment of OSSN especially in cases of recurrence or
extensive disease where excision may jeopardise limbal stem
cell function. McKelvie et al have followed patients after
treatment with MMC for OSSN and, using impression
cytology, demonstrated eradication of malignant cells,
primarily by apoptosis, and a small amount of necrosis
accompanied by inflammatory cells.33 Normal cells undergo
cytoplasmic enlargement and vacuolisation, and nuclear
enlargement, but maintained a normal nuclear to cytoplasmic ratio. These changes persisted for a variable time
following treatment, but resolved eventually. Impression
cytology has been studied in pigmented conjunctival lesions
and predicted conjunctival melanocytic malignancy in 73% of
cases studied.34
Impression cytology has also been used to diagnose
acanthomoeba keratitis in three patients by visualisation of
cysts and trophozoites taken from the superficial cornea from
patients with clinically suspicious infections.35
While there are numerous clinical and research applications of impression cytology, it has not yet become a routine
diagnostic tool in most clinics because it is relatively
cumbersome and time consuming for both the clinician and
pathologist. However, the ability to obtain multiple samples
of the ocular surface at one sitting with minimal discomfort
to the patient makes it an ideal method of investigating
ocular surface disorders when the diagnosis is not clinically
obvious or when the clinical diagnosis needs to be substantiated and documented. It is also a handy research tool.
We recommend that major ophthalmic centres should
develop and introduce this technique into routine clinical
practice. For this to be achieved a team approach including
the ophthalmologist, pathologist, microbiologist, and the
immunologist is essential.
.....................
Authors’ affiliations
R Singh, A Joseph, T Umapathy, N L Tint, H S Dua, Division of
Ophthalmology and Visual Sciences, Larry A Donoso Laboratory for Eye
Research, University Hospital, Queen’s Medical Centre, Nottingham
NG7 2UH, UK
Figure 6 Impression cytology of the ocular surface showing dysplastic
squamous cells with increased nucleus:cytoplasmic ratio, hyperchromatic
nuclei, irregular nuclear membranes, and prominent nucleoli (6250
magnification, Papanicolaou stain). (Reproduced by courtesy of Dr
Ushma Samaraweera, Department of Pathology, Prince of Wales
Hospital, Sydney, Australia).
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2 Tseng SCG. Staging of conjunctival squamous metaplasia by impression
cytology. Ophthalmology 1985;92:728–33.
3 Maskin SL, Bodé DD. Electron microscopy of impression-acquired conjunctival
epithelial cells. Ophthalmology 1986;93:1518–23.
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4 Krenzer KL, Freddo TF. Cytokeratin expression in normal human bulbar
conjunctiva obtained by impression cytology. Invest Ophthalmol Vis Sci
1997;38:142–52.
5 Thiel MA, Bossart W, Bernauer W. Improved impression cytology techniques
for the immunopathological diagnosis of superficial viral infections.
Br J Ophthalmol 1997;81:984–8.
6 Vadrevu VLD, Fullard RJ. Enhancements to the conjunctival impression
cytology technique and examples of applications in a clinico-biochemical
study of dry eye. CLAO J 1994;20:59–63.
7 Gill GW, Frost JK, Miller KA. A new formula for a half-oxidised haematoxylin
solution that neither over stains nor requires differentiation. Acta Cytol
1974;18:300–11.
8 Adams AD. The morphology of human conjunctival mucus. Arch Ophthalmol
1979;97:730–4.
9 Marner K. Snake-like appearance of nuclear chromatin in conjunctival
epithelial cells from patients with keratoconjunctivitis sicca. Acta Ophthalmol
(Copenh) 1980;58:849–53.
10 Nelson JD, Wright JC. Conjunctival goblet cell densities in ocular surface
disease. Arch Ophthalmol 1984;102:1049–51.
11 Wittpenn JR, Tseng SC, Sommer A. Detection of early xerophthalmia by
impression cytology. Arch Ophthalmol 1996;104:237–9.
12 Nelson JD. Ocular surface impression using a cellulose acetate filter material.
Arch Ophthalmol 1982;27:67–9.
13 Dogru M, Katakami C, Nakagawa N, et al. Impression cytology in atopic
dermatitis. Ophthalmology 1998;105:1478–84.
14 Aragona P, Romeo G, Puzzolo D, et al. Impression cytology of the
conjunctival epithelium in patients with vernal conjunctivitis. Eye
1996;10:82–5.
15 Nelson DJ. Impression cytology. Cornea 1988;7:71–81.
16 Prabhasawat P, Tseng SCG. Impression cytology study of epithelial
phenotype of ocular surface reconstructed by preserved human amniotic
membrane. Arch Ophthalmol, 115:1360–7.
17 Tseng SH, Chen YT, Huang FC, et al. Seborrheic keratosis of the conjunctiva
simulating a malignant melanoma. An immunocytochemical study with
impression cytology. Ophthalmology 1999;106:1516–20.
18 Turacli E, Budak K, Kaur A, et al. The effects of long-term topical glaucoma
medication on conjunctival impression cytology. Int Ophthalmol
1997;21:27–33.
19 Arici MK, Arici DS, Topalkara A, et al. Adverse effects of topical antiglaucoma
drugs on the ocular surface. Clin Experiment Ophthalmol 2000;28:113–17.
20 Debbasch C, Pisella PJ, Rat P, et al. Evaluation of free radical production by
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Avunduk AM, Avunduk MC, Kapicioglu Z, et al. Mechanisms and comparison
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Dogru M, Katakmi C, Miyashita M, et al. Ocular surface changes after
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Shimazaki J, Yang H, Tsubota K. Amniotic membrane transplantation for
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Liu Z, Carvajal M, Carothers CA, et al. Increased expression of the type 1
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Williams KA, Brereton HM, Aggarwal R, et al. Use of DNA polymorphisms
and the polymerase chain reaction to examine the survival of a human limbal
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McKelvie PA, Daniell M. Impression cytology following mitomycin C therapy
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Paridaens ADA, McCatney ACE, Curlin OM, et al. Impression cytology of
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Br J Ophthalmol 2005;89:1660–1671
PostScript
..............................................................................................
LETTERS
Ultrahigh resolution optical
coherence tomography of
birdshot retinochoroidopathy
Birdshot retinochoroidopathy is a rare
inflammatory eye disease with typical clinical
presentation and strong association with the
HLA-A29 allele. Characteristic appearances
on fluorescein angiogram (FA), indocyanine
green (ICG) angiography, and electroretinogram (ERG) have been described.1 However,
histopathology of the disease has been rare.2 3
The following case is an example of birdshot
retinochoroidopathy imaged with ultrahigh
resolution optical coherence tomography
(UHR-OCT), capable of 3 mm axial resolution.4 UHR-OCT is able to clearly delineate
individual intraretinal layers (fig 1).
Figure 1 Ultrahigh resolution optical coherence tomography image from a normal eye. The
intraretinal layers are labelled: NFL, nerve fibre layer, GCL, ganglion cell layer, IPL, inner plexiform
layer, INL, inner nuclear layer, OPL, outer plexiform layer, ONL, outer nuclear layer, IS/OS,
photoreceptor inner/outer segment junction, RPE, retinal pigment epithelium.
Case report
A 64 year old man presented to the New
England Eye Center (NEEC) for progressive
visual deterioration despite cataract surgery
in the left eye 2 years earlier. The patient’s
major complaints were difficulty seeing at
night and difficulty driving. Best corrected
visual acuity (BCVA) was 20/50 right eye and
20/60 left eye. Anterior eye examination
revealed mild cells and flare in both eyes, a
moderate cataract in the right eye, and a
posterior chamber intraocular lens in the left.
Dilated fundus examination revealed mild
vitritis bilaterally. The optic discs appeared
slightly pale and the retinal vasculature was
narrowed. Fundus appearance was consistent
with the diagnosis of birdshot retinochoroidopathy (fig 2A). FA and ICG angiography
were also consistent with this diagnosis
(fig 2B). Six mm radial macular OCT3 scans
showed bilateral epiretinal membranes
(ERM), with mild thickening in the left eye.
The patient subsequently tested positive for
the HLA-A29 antigen. Over the next
6 months, the patient was treated for macular oedema with intravitreal Kenalog injections in both eyes, and the macular oedema
subsided.
UHR-OCT images were obtained 6 months
later (fig 3), at which time BCVA remained
stable. Repeat fundus examination and OCT3
imaging revealed an ERM with no macular
oedema and normal retinal thickness in both
eyes. UHR-OCT images additionally showed
photoreceptor atrophy in several areas of
both eyes. RPE degeneration was present
underneath areas of photoreceptor involvement. The inner retinal layers were difficult
to delineate, probably because of anatomical
disorganisation of these layers.
Comment
This case represents a fairly severe case of
birdshot retinochoroidopathy. In a review by
Gasch et al, epiretinal membrane was the
second most common complication of birdshot retinochoroidopathy next to macular
oedema,1 which our patient also had on
initial presentation. ERG findings have
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Figure 2 (A) Colour photograph of the left eye. (B) Fluorescein angiogram of the left eye,
2 minutes after injection. The image shows two small haemorrhages originating from the superior
arcade, multiple hyperfluorescent spots, and central cystoid macular oedema.
shown Mueller and bipolar cell involvement
early in the disease, while photoreceptors are
affected later.1 The UHR-OCT images presented here show disorganisation of inner
retinal layers as well as photoreceptor and
RPE atrophy. Choroidal ischaemia, suggested
by ICG angiography, may be the cause of RPE
and photoreceptor degeneration.
We found two histolopathological reports
of birdshot retinochoroidopathy. One case
was a blind phthisical patient.2 The other was
a more typical yet mild case, which showed
lymphocytic infiltration around the choroidal
and retinal vasculature with minimal retinal
disturbance.3 Serial UHR-OCT imaging of
patients could help in understanding and
following progression of macular involvement in this disease.
A J Witkin, J S Duker
New England Eye Center, Tufts-New England Medical
Center, Tufts University, Boston, MA, USA
T H Ko, J G Fujimoto
Department of Electrical Engineering and Computer
Science and Research Laboratory of Electronics,
Massachusetts Institute of Technology, Cambridge,
MA, USA
J S Schuman
UPMC Eye Center, Department of Ophthalmology,
University of Pittsburgh School of Medicine,
Pittsburgh, PA, USA
Correspondence to: Jay S Duker, MD,
Ophthalmology Department, Tufts-New England
Medical Center, 750 Washington Street, Boston, MA
02111, USA; [email protected]
doi: 10.1136/bjo.2005.076570
Accepted for publication 29 July 2005
Supported in part by NIH contracts RO1-EY1128916, R01-EY13178, and P30-EY13078, NSF contract
ECS-0119452, Air Force Office of Scientific Research
contract F49620-98-1-0139, Medical Free Electron
Laser Program contract F49620-01-1-0186 and by
Carl Zeiss Meditec.
JGF and JSS receive royalties from intellectual
property licensed by MIT to Carl Zeiss Meditec. JGF
and JSS receive research support from Carl Zeiss
Meditec.
References
1 Gasch AT, Smith JA, Whitcup SM. Birdshot
retinochoroidopathy. Br J Ophthalmol
1999;83:241–9.
PostScript
1661
Figure 3 (A) Horizontal ultrahigh resolution OCT (UHR-OCT) image through the right macula.
Notable are an epiretinal membrane (ERM) (yellow arrows), and an area of thinning of the outer
nuclear layer (ONL) with underlying absence of the photoreceptor inner/outer segment junction
(IS/OS) (red asterisk). Retinal pigment epithelium (RPE) disruption is also seen as an increase in
choroidal signal backscattering. Other retinal layers are also labelled as in figure 1. (B) Horizontal
UHR-OCT image through the left macula. ERM is present (yellow arrows). Thinning of the ONL and
disruption of the photoreceptor IS/OS junction is present outside of the fovea (red asterisks). RPE
disruption is also present in these areas. The inner retinal layers are not clearly delineated.
2 Nussenblatt RB, Mittal KK, Ryan S, et al. Birdshot
retinochoroidopathy associated with HLA-A29
antigen and immune responsiveness to retinal Santigen. Am J Ophthalmol 1982;94:147–58.
3 Gaudio PA, Kaye DB, Crawford JB.
Histopathology of birdshot retinochoroidopathy.
Br J Ophthalmol 2002;86:1439–41.
4 Drexler W, Morgner U, Ghanta RK, et al.
Ultrahigh-resolution ophthalmic optical coherence
tomography. Nat Med 2001;7:502–7.
and pain. There was corneal inflammation
with flare in the anterior chamber, anterior
uveitis with cellular deposits on the corneal
endothelium (keratic precipitates) but without posterior synechiae and grade B3 vitritis.
A focus of chorioretinitis was just visible
in the macular area. The blood neutrophil
count was high (14.49 white cells/6106/l
with 13.48 neutrophils/6106/l), while serology showed high levels of anti-toxoplasma
IgG (543 IU/ml) with an IgM index of 53.73.
Anti-toxoplasma therapy was attempted by
administration of sulfadiazine, pyrimethamine, and folinic acid for 2 days, followed
by prednisone. Despite this treatment, the
ocular inflammation worsened and led to loss
of vision and ocular divergence. A clinical
examination revealed posterior synechiae and
aggravation of the vitritis and B echography
showed retinal detachment (fig 1A).
Surgery was performed, which comprised
pars plana vitrectomy after phacoemulsification, with ablation of the incompletely
detached posterior hyaloid. The retina was
reattached with silicone oil. The inferior
retina appeared necrotic with a focus of
inflammatory chorioretinitis in the macular
area. Twelve months after surgery, the eye
was no longer painful but vision was limited
to perception of hand movements with ocular
divergence (fig 1B). A fundus of the right eye
revealed retraction of the inferior retina and
extended gliosis of the macula (fig 1C).
The severity of the clinical manifestations
prompted an evaluation of the patient’s
immunocompetence, which appear to be
normal, except that the amount of HLA class
I molecules expressed on the plasma membrane of the lymphocytes was reduced 20fold (figs 2 and 3). The parents were
unrelated, but shared an identical HLA
haplotype, so the patient and his brother
were HLA homozygous (HLA-A*24; B*14;
Cw*08; DRB1*13; DQB1*06). TAP genes,
located in the HLA genetic region, were
characterised, and a stop mutation in the
TAP1 was identified at codon 522 (sequence
AAS55412.1 in GenBank), because of a C to T
substitution.
The patient did not display pulmonary
involvement, contrary to his elder brother
who displayed a bronchial obstruction unresponsive to inhaled bronchodilators, a bacterial
colonisation of the lower airways associated to
asthma-like symptoms, but no bronchiectasies.
Unilateral necrotising
toxoplasmic retinochoroiditis as
the main clinical manifestation of
a peptide transporter (TAP)
deficiency
Congenital HLA class I deficiency is a rare
disease frequently resulting in chronic inflammation of the respiratory tract, and/or skin
granulomas.1 2 The deficiency may be unnoticed for decades, so pathological outcome is
relatively unpredictable.3 We here describe a
14 year old patient with a severe ocular
toxoplasmosis who is HLA class I deficient, as
a result of a homozygous mutation in the gene
encoding one of the two subunits of the
peptide transporter associated with antigen
processing (TAP). We propose that such a
defect should be investigated in patients with
severe ocular toxoplasmosis without acquired
immunodeficiency.
Case report
At the time of referral, the patient did not
have any particular medical history except an
exaggerated reaction to an intradermal tuberculin test 1 year earlier. His right eye displayed a strong reduction of acuity with
anterior and posterior inflammatory lesions
Figure 1 Analysis of lesions before and after surgery. (A) B echography before operation
demonstrates total retinal detachment wit a grade D vitreoretinal proliferation. (B) 12 months after
surgery, circumferential synechiae are noted with capsule opacification and corneal opacities. (C)
Posterior pole is not easily recognisable. Nevertheless, a white scar is distinguishable.
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1662
PostScript
Acknowledgements
10 000
We thank José Sahel and Claude Bénichou for
critical reading of the manuscript. This work was
supported by ARMESA, INSERM, and the
Etablissement Français du Sang-Alsace.
IgG
Bw6
W6/32
1000
A Parissiadis, A Dormoy
100
Laboratoire d’Histocompatibilité, EFS-Alsace,
Strasbourg, France
10
D Fricker, D Hanau, H de la Salle
INSERM U. 725, EFS-Alsace, Strasbourg, France
1
S1
S2
M
F
Figure 2 HLA class I deficiency of the patient
and his brother. Expression of HLA class I
molecules on the plasma membrane of
lymphocytes. Peripheral blood mononuclear
cells were isolated from the patient and his
brother (S1, S2) and their parents (M, F) and
stained with the mAb W6/32 (pan-anti-HLA
class I), or 126.39 (anti-HLA-Bw6), or a control
IgG1 mAb. Mean fluorescence intensities of the
staining were quantified in the gated
lymphocyte subpopulations.
Comment
The presence of anti-toxoplasma IgM suggests that the infection was recent and is
compatible with a primary ocular infection.
The particular titre of anti-toxoplasma IgG
suggests that these antibodies might have
had a role in the immunological defence, as
has been observed for viral infections.4
Remarkably, during the pathology, more
than 40% of the T cells of the patient were cd,
which can be explained by the infection,
known to induce the expansion of this T cell
subset.5 6 After recovery, this number
decreased twofold.
These observations suggest that TAP deficiency should now be considered as a potential
cause of unexplained exacerbated pathology in
response to intracellular parasites, notably T
gondii. In our patient, special follow up including prophylactic antibiotherapy is required, in
order to avoid infection of the other eye.
J-P Cazenave
INSERM U. 311, EFS-Alsace, Strasbourg, France
P Lenoble
Service d’Ophtalmologie, Centre Hospitalier de
Mulhouse, Mulhouse, France
L Donato
Pneumologie Pédiatrique, Hôpital Universitaire de
Strasbourg, Strasbourg, France
Correspondence to: Henri de la Salle, PhD, INSERM
U.725, EFS-Alsace, 10, rue Spielmann, 67065
Strasbourg Cedex, France;
[email protected]
doi: 10.1136/bjo.2005.078097
Accepted for publication 1 August 2005
References
1 Gadola SD, Moins-Teisserenc HT, Trowsdale J, et
al. TAP deficiency syndrome. Clin Exp Immunol
2000;121:173–8.
2 De la Salle H, Donato L, Hanau D, et al. Peptide
Transporter Defects in HLA Class I Deficiency. In:
Ochs HD, Smith CIE, Puck JM, eds. Primary
immunodeficiency diseases. 2nd ed. New York:
Oxford University Press, 2005.
3 De la Salle H, Saulquin X, Mansour I, et al.
Asymptomatic deficiency in the peptide
transporter associated to antigen processing
(TAP). Clin Exp Immunol 2002;128:525–31.
4 Donato L, de la Salle H, Hanau D, et al.
Association of HLA class I antigen deficiency
related to a TAP2 gene mutation with familial
bronchiectasis. J Pediatr 1995;127:895–900.
5 De Paoli P, Basaglia G, Gennari D, et al.
Phenotypic profile and functional characteristics
of human gamma and delta T cells during acute
toxoplasmosis. J Clin Microbiol
1992;30:729–31.
6 Scalise F, Gerli R, Castellucci G, et al.
Lymphocytes bearing the gamma delta T-cell
receptor in acute toxoplasmosis. Immunology
1992;76:668–70.
Scleral contact lenses are not
optically inferior to corneal
lenses
Figure 3 HLA class I deficiency of the patient
and his brother. Identification of a mutation in
the TAP1 gene. Genomic DNA from S1 and F
was amplified using the polymerase chain
reaction technique and the amplified fragments
were sequenced. The sequence was read on the
reverse complementary non-coding strand and
showed the presence of an A in the
homozygous mutated genome, at the position
where the superposition of an A and a G,
depicted as an N on the electropherogram, was
observed in the hemizygous genome of F.
www.bjophthalmol.com
In the 1950s, chronic corneal hypoxia and its
attendant complications were associated with
scleral contact lenses (ScCLs) compared to
corneal contact lenses (CCLs).1 Changes in
mechanical design to improve transfer of
oxygenated tears between the corneal-tear
film interface were partially successful, but
accompanied by increased complexity of lens
fitting and design.2 These included fenestrations, slots, truncations, and channels. Apart
from being time consuming and technically
difficult to manufacture, these modifications
were almost invariably associated with trapping of air bubbles behind the ScCL, resulting
in reduced vision and localised corneal
desiccation. Without a sealed tear film, the
ScCL rested progressively more on the corneal
apex and limbus—that is, settling back,
which caused corneal erosions, scarring, and
hypoxia.
The advent of fluorocarbon silicone/acrylate
co-polymer ScCLs resulted in greater utility
because of high gas permeability.3 One major
criticism of ScCLs has been the suboptimal
visual acuity achieved when compared to
CCLs.3 In this study we compared the best
corrected visual acuity (BCVA) in patients with
RGP ScCLs who failed a trial of CCLs.
Method and results
The case notes of 15 patients prescribed
ScCLs were reviewed over an 18 month
period. The reasons for discontinuing CCL
use included discomfort, excessive mobility,
poor fit, short wearing times, and subjective
lens intolerance. There were 18 eyes in 15
patients whose average age was 37 years (18–
80). There were eight males and seven
females.
The BCVA varied according to the preexisting pathology. These were post-penetrating keratoplasty (seven); keratoconus (six),
and herpetic scarring (two). Mean astigmatism was 9.7D (3.5–18D). CCL average BCVA
was 6/18, but with ScCLs was 6/9, of which
eight (44%) achieved 6/5, p = 0.1; x2 test.
The greatest improvement occurred in the
keratoconus group (BCVA 6/18 with CCLs; to
6/9–6/5 with ScCLs); followed by the keratoplasty group (with ScCL 6/9 in five cases and
6/18 in two cases from pre-existing corneal
scarring). In all cases the scleral lenses were
well tolerated. No complications were noted.
Comment
The relatively close apposition of the cornea
to a CCL provides a stable refractive interface.
In a normal cornea, the centre is assumed to
be spherical and regular so that a singly curved
CCL can be made based on keratometry
readings. In corneas with highly abnormal
topography such as high astigmatism, severe
flattening, apical protrusion, thinning, and
scarring, the nature of the refractive interface
between the cornea, precorneal tear film, and
contact lens is altered because the above
assumptions no longer hold true.
ScCLs vault the cornea, which eliminates
the need for close alignment to the cornea.
This compensates for very abnormal corneas
giving good BCVA that can be difficult to
achieve with CCLs.4 As the power of CCLs
increases, positional stability and accuracy of
fit decreases. High power CCLs tend to be
bulkier, thicker, and with a larger diameter
that alters the centre of gravity. These CCLs
tend to sag or droop with axis mislocation so
vision is through the peripheral lens and not
the optic zone. Induced prismatic effects
cause reduced vision, lens intolerance, and
discomfort. This is exacerbated by edge
sensation from high edge lift. Lens instability
with excessive frictional mobility on the
cornea also increases the potential for erosions, scarring, and intolerance. ScCLs retain
positional stability and tend not to be
associated with the aforementioned problems.
Based on the findings of this study, we
think the use of ScCLs should not be
prejudiced because of the perception that
they are optically inferior to CCLs. The optical
and therapeutic benefit of ScCLs should not
be underestimated. They can have an important role in management of patients where
surgery is undesirable or high risk.
A Salam, B Melia
Hull and East Yorkshire Eye Hospital, Fountain Street,
Anlaby Road, Hull HU3 2JZ, UK
PostScript
1663
A J Singh
Department of Ophthalmology, Leeds General
Infirmary, Clarendon Wing, Belmont Grove, Leeds,
UK
Correspondence to: A J Singh, Department of
Ophthalmology, Leeds General Infirmary, Clarendon
Wing, Belmont Grove, Leeds LS2 9NS, UK;
[email protected]
doi: 10.1136/bjo.2005.074377
Accepted for publication 24 July 2005
References
1 Tan DT, Pullum KW, Buckley RJ. Medical
applications of scleral contact lenses: 1. A
retrospective analysis of 343 cases. Cornea
1995;14:121–9.
2 Tan DT, Pullum KW, Buckley RJ. Medical
applications of scleral contact lenses: 2. Gaspermeable scleral contact lenses. Cornea
1995;14:130–7.
3 Pullum KW, Buckley RJ. A study of 530 patients
referred for rigid gas permeable scleral contact
lens assessment. Cornea 1997;16:612–22.
4 Schein OD, Rosenthal P, Ducharme C. A gaspermeable scleral contact lens for visual
rehabilitation. Am J Ophthalmol
1990 15;109:318–22.
Optometric referrals: towards a
two way flow of information?
ommunity optometrists in the United
Kingdom carry out 17.2 million primary
eyecare examinations per annum,1 which
result in at least 0.5 million referrals to the
hospital eye service.2 3 Optometrists only
infrequently receive a reply to these referrals,4 5 possibly because 69% are handwritten
on GOS18 forms,6 which can lack legibility6
and details.5 Most optometrist initiated referrals take place via general practitioners
(GPs), who are increasingly likely to forward
the optometrist’s letter.6 We improved our
referrals and audited the replies.
Methods
The Institute of Optometry set minimum
criteria for referrals in 2004 by typing letters
on headed notepaper, including the practitioner’s name, enclosing a second copy for
the GP to forward to the ophthalmologist,
and including text which explicitly requested
a reply.
In February–April 2005 we audited referrals from 2004. Handwritten emergency
referrals, which make up less than 1% of
referrals, were not included.
Results
There were 181 referrals following 7164 eye
examinations, 51% were female, and the
reasons for referral are given in figure 1.
The provisional diagnoses for the ‘‘other’’
category include a wide range of conditions,
all with a prevalence of less than 3%.
A reply was requested in 95% of letters, but
was received for only 23 (13%). There was no
relation between the likelihood of reply and
the reason for referral (p = 0.37).
Comment
The institute’s referral rate (2.5%) is lower
than previously reported for optometrists.2 3
This might reflect better facilities for monitoring (for example, ocular hypertension) or
managing (for example, dry eye) conditions
and an ethos that encourages management
when appropriate.
Although our results are only for one centre
it is disappointing that, despite taking steps to
Cataract
26%
Other
37%
Strabismus
3%
Glaucoma
22%
Diabetic eye
disease
4%
Maculopathy
8%
Figure 1 Reasons for referral.
encourage a reply, this is still rarely forthcoming. The proportion of referrals that receive
replies is so low (13%) that we think it unlikely
to be attributable to patients failing to consult
the GP or ophthalmologist after referral. For
the few ophthalmologists who do reply this is
not an onerous task: they instruct their
secretary to copy the reply to the GP to the
optometrist. The NHS code of practice on
confidentiality notes that explicit consent is
not usually required for information disclosures that support the delivery of the patient’s
care.7 Even if it is thought necessary to obtain
consent, there is no good reason why the
ophthalmologist should not obtain this.4 The
issue of consent does not seem to be the main
reason for lack of replies: a study found that
ophthalmologists actually replied to a higher
proportion of referrals when the optometrists
had not obtained consent than when they had.4
Replying to referrals helps optometrists
provide continuing patient care and avoids
possible errors from the optometrist relying on
the patient’s recollection of the ophthalmologist’s findings. Feedback also contributes to
optometrists’ professional development and
helps to ensure that inappropriate referrals
are minimised in the future.8
Now that direct referral from primary care
optometrists to secondary care ophthalmology units is becoming more commonplace,9
we hope that a two way flow of information
will become the norm.
B J W Evans, D E Harle, B Cocco
Institute of Optometry, 56–62 Newington Causeway,
London SE1 6DS, UK
Correspondence to: Professor Bruce Evans, Institute of
Optometry, 56–62 Newington Causeway, London
SE1 6DS, UK; [email protected]
doi: 10.1136/bjo.2005.075531
Accepted for publication 24 July 2005
References
1 Department of Health. Sight tests volume and
workforce survey 2003–2004. www.dh.gov.uk/
assetRoot/04/08/65/64/04086564.pdf 2004,
accessed 11 April 2005.
2 Hobley AJ, Woodward EG, Port MJ.
Retrospective study of optometric referrals.
Ophthalmic Physiol Opt 1992;12:395–9.
3 Port MJ. Referrals and notifications by
optometrists within the UK: 1988 survey.
Ophthalmic Physiol Opt 1989;9:31–5.
4 Whittaker KW, Ikram K, Anderson DF, et al. Noncommunication between ophthalmologists and
optometrists. J R Soc Med 1999;92:247–8.
5 Lash SC. Assessment of information included on
the GOS 18 referral form used by optometrists.
Ophthalmic Physiol Opt 2003;23:21–3.
6 Pooley JE, Frost EC. Optometrists’ referrals to the
hospital eye service. Ophthal Physiol Opt
1999;19:S16–S24.
7 Department of Health. Confidentiality: NHS Code
of Practice. www.publications.doh.gov.uk/ipu/
confiden/protect/copv3.pdf 2003, accessed 11
April 2005.
8 Ingram DV, Culham LE. Ophthalmologists and
optometrists—interesting times? Br J Ophthalmol
2001;85:769–70.
9 Department of Health. Delivering quality in primary
care: General ophthalmic service practitioners.
www.dh.gov.uk/assetRoot/04/10/60/33/
04106033.pdf 2005, accessed 11 April 2005.
Retinal changes in juvenile X
linked retinoschisis using three
dimensional optical coherence
tomography
Juvenile X linked retinoschisis is a congenital
X linked recessive retinal disorder, the characteristic funduscopic findings of which are a
silver-grey retinal reflex, foveal retinoschisis,
and peripheral retinoschisis. Electroretinograms (ERGs) typically record a reduced bwave amplitude with relative preservation of
the a-wave amplitude. Visual acuity (VA)
usually deteriorates slowly until the patient is
about 20 years of age, stabilises around
0.2,0.5, and sometimes deteriorates further
because of macular degeneration.1–4
Podoleanu and associates developed a
novel integration of scanning laser ophthalmoscopy (SLO) and optic coherence tomography (OCT)—three dimensional optical
coherence tomography (3-D OCT).5 Using
transverse scanning, typical for SLO, the
instrument simultaneously produces SLO
and interferometric OCT images.6 We can
obtain both cross sectional scans (B-scans) as
with conventional OCT and transverse scans
(C-scans) using 3-D OCT. This is the first
report of 3-D OCT findings in juvenile X
linked retinoschisis.
Case report
A 7 year old boy presented with VA of 0.5 and
0.6 in the right and left eyes, respectively.
Funduscopy showed a silver-grey retinal reflex
and cartwheel-like macular degeneration bilaterally. Peripheral retinoschisis was absent.
ERGs were recorded and dark adaptation
testing was performed. Single flash ERG
showed decreased b-wave amplitude, which
was consistent with the diagnosis. Dark adaptation revealed a decreased curve overall.
The B-scan findings of 3-D OCT (fig 1)
showed the retina split into four distinct
planes. Two wide hyporeflective spaces split
the retina. Anteroposterior or oblique linear
columns were seen across the superficial
wide hyporeflective space, forming a bridge
that was not found in the fovea. These
columns are considered to be Muller cells by
OCT and histological studies.3 7 8
There was a large cystoid space in the fovea
connected to the superficial wide hyporeflective parafoveal space. A deeper wide hyporeflective space was in the parafoveal retina but
disappeared in the fovea. Small cystoid
spaces in the superficial parafoveal retina
split the retina. Retinal cleavage involving the
fovea was found in the outer plexiform layer.
Superficial retinal cleavage was most likely in
the nerve fibre layer or the ganglion cell layer.
The deep retinal cleavage was in or just
around the outer nuclear layer. C-scan findings of 3-D OCT showed the extent of the
cleavage planes and the hyporeflective spaces
(fig 2). Of particular note, the C-scans
www.bjophthalmol.com
1664
PostScript
doi: 10.1136/bjo.2005.075648
Line M
Line N
Line P
Accepted for publication 29 July 2005
Competing interests: none declared
References
Figure 1 B-scans of 3-D OCT. Two wide
hyporeflective spaces split the retina.
Anteroposterior or oblique linear columns form
a bridge across a superficial wide
hyporeflective space. In the same layer, there is
a large cystoid space in the fovea (line N). This
layer is probably the outer plexiform layer.
Deeper cleavage is seen in the parafoveal area
but not in the fovea (line P). This layer is
probably the outer nuclear layer. Small cystoid
spaces (arrowhead) are seen in the superficial
parafoveal retina that split the retina (line M).
This layer is probably the nerve fibre layer or
the ganglion cell layer.
showed many columns in a large space
(schisis). This is in contrast with the B-scans
that showed the spaces between the columns
to be cystic spaces. The C-scans provided a
better understanding of this pathology.
Comment
Recently, conventional OCT findings of foveal
schisis were reported1 7 to be in the outer
plexiform layer and adjacent nuclear layers.
Histopathologically, foveal schisis was reported
to occur in the outer plexiform layer,2 although
peripheral retinoschisis was found in the nerve
fibre layer and ganglion cell layer.3
3-D OCT demonstrated that schisis can
occur in any retinal layers in juvenile X linked
retinoschisis. We obtained cross sectional and
transverse images of the retinoschisis with
near histological precision that showed the
details of the inner retinal structures and the
extent of the schisis. 3-D OCT is useful to
evaluate, non-invasively, the retinal pathology and follow patients with juvenile X
linked retinoschisis.
Y Minami, S Ishiko, Y Takai, Y Kato,
H Kagokawa, A Takamiya, T Nagaoka,
R Kinouchi, A Yoshida
Department of Ophthalmology, Asahikawa Medical
College, Asahikawa, Japan
Correspondence to: Satoshi Ishiko, Department of
Ophthalmology, Asahikawa Medical College, 2-1
Midorigaoka Higashi, Asahikawa, 078-8316, Japan;
[email protected]
1 Ozdemir H, Karacorlu S, Karacorl K. Optical
coherence tomography findings in familial foveal
retinoschisis. Am J Ophthalmol
2004;137:179–81.
2 Ando A, Takahashi K, Sho K, et al.
Histopathological findings of X-linked retinoschisis
with neovascular glaucoma. Graefes Arch Clin
Exp Ophthalmol 2000;238:1–7.
3 Condon GP, Brownstein S, Wang NS, et al.
Congenital hereditary (juvenile X-linked)
retinoschisis. Arch Ophthalmol
1986;104:576–83.
4 Forsis H, Krause U, Helve J, et al. Visual acuity in
183 cases of X-chromosomal retinoschisis.
Can J Ophthalmol 1973;8:385–95.
5 Podoleanu AG, Seeger M, Dobre GM, et al.
Transversal and longitudinal images from the
retina of living eye using low coherence
reflectometry. J Biomed Optics 1998;3:12–20.
6 Yannuzzi LA, Ober MD, Slakter JS, et al.
Ophthalmic fundus imaging. Today and beyond.
Am J Ophthalmol 2004;137:511–24.
7 Ikeda F, Takahashi K, Kishi S. Optical coherence
tomographic features of juvenile retinoschisis.
Ringan 1998;52:1497–82.
8 Kirsch LS, Brownstein S, de Wolff-Rouendaal D. A
histopathological, ultrastructural and
immunohistochemical study of congenital
hereditary retinoschisis. Can J Ophthalmol
1996;31:301–10.
Linezolid induced toxic optic
neuropathy
Linezolid is a new oxazolidinone antibiotic
with activity against many important pathogens
including
methicillin
resistant
Staphylococcus
and
penicillin
resistant
1
Streptococcus. We report a case of toxic optic
neuropathy from chronic treatment with
linezolid. Correct diagnosis and discontinuation of the drug resulted in significant
recovery of vision.
Case report
A 56 year old man presented with bilateral,
progressive decline in visual acuity for 6–
8 months. Medical history included chronic
diabetes mellitus, below the knee amputation
of the right leg, hypertension, sinusitis with
nasal allergies, and asthma. Several years
earlier he had fractured his left ankle and
developed osteomyelitis from methicillin
resistant Staphylococcus. He had received linezolid 600 mg by mouth twice a day for
12 months, then once daily for 44 months.
Other medications included rosiglitazone,
metoprolol, rifampin, furosemide, lisinopril,
amlodipine, insulin, and vitamin B complex/
folic acid for assistance with wound healing.
He was a non-smoker and consumed less
than one unit of alcohol per week.
Best corrected visual acuities were 20/400
in both eyes with eccentric fixation. Ishihara
colour plates were 1/8 right eye and 3/8 left
eye. No relative afferent pupillary defect was
present.
Intraocular
pressures
were
16 mm Hg in both eyes. 1+ nuclear sclerosis
was present in both eyes. Fundus examination revealed temporal optic nerve pallor with
a corresponding temporal nerve fibre layer
defect more evident in the right eye (fig 1)
and a normal macula in both eyes. Humphrey
visual field testing (full field 120 point
screen) revealed central scotomas in both
eyes (fig 2). Fluorescein angiography revealed
a normal macula without staining of the
peripapillary region in both eyes (not shown).
Optical coherence tomography (Stratus OCT,
Carl Zeiss Ophthalmic Systems Inc,
Humphrey Division, Dublin, CA, USA) of
the fovea revealed a central foveal thickness
of 205 (SD 5) mm right eye and 216 (4) mm
left eye and a normal macular volume
(7.28 mm3 right eye; 6.94 mm3 left eye).
Retinal nerve fibre layer thickness analysis
by OCT revealed a normal 360˚ average
measurement (79.55 mm right eye, 80.57 mm
left eye) with no significant change in
thickness detected in the temporal quadrant
(64 mm right eye and 58 mm left eye). Full
field scotopic and photopic electroretinography demonstrated a normal amplitude and
latency in both eyes, as expected given the
small central scotoma.
The patient was diagnosed with bilateral
optic neuropathy; chronic use of linezolid
was suspected as the cause. Linezolid was
discontinued and the patient noted subjective
visual improvement within several weeks.
Three months later his vision improved to 20/
40 in both eyes with resolution of the central
scotomas (fig 2). There were 8/15 and 13/15
central fixation losses in the right and left eye
respectively initially; this improved to 4/15
right eye and 2/16 left eye 3 months after
discontinuing therapy.
Comment
Figure 2 C-scans of 3-D OCT. C-scans M, N, and P correspond to the same depth of the B-scans
(fig 1) in lines M, N, and P. *The location of the fovea. (M) A large cystic space is seen in the fovea
and the retina, which includes the small cystic spaces. The small spaces found in the B-scan are
confirmed in the C-scan. (N) This space is equivalent to a superficial schisis and shows the space in
the fovea and the columns around it. In B-scan images, the spaces between the columns are
hypothesised to be cystic space; however, in C-scan images, these spaces are not cystic, and many
columns can be seen in a large space (schisis). (P) This is equivalent to the deeper schisis and shows
the hyperreflective area (no schisis) in the fovea and the large space (schisis) around the fovea.
www.bjophthalmol.com
Toxic optic neuropathy has been associated
with numerous compounds, including exposure to ethylene glycol, methanol, isoniazid,
ethambutol, and fluoroquinolone, and deficiency of vitamin B12, folate, and thiamine.2 3
Linezolid was approved by the FDA, based on
studies employing 28 days of administration.1
There have been five cases of optic neuropathy associated with prolonged use of linezolid3–6 and more than 20 cases of peripheral
neuropathy including one individual who
developed both sequelae.6–9 In previous optic
neuropathy cases the duration of treatment
ranged from 5–10 months at a dose of
600 mg once or twice per day.3–6 All cases
were bilateral with initial vision decreased
from 20/60 to counting fingers both eyes.3–6
Discontinuation of therapy resulted in
improvement of the optic neuropathy with
significant improvement in visual acuity
within 1–8 months in all patients, although
PostScript
1665
susceptible to mitochondrial disruption.2
Mitochondrial dysfunction is the cause of
Leber’s hereditary optic neuropathy, chloramphenicol induced bone marrow suppression, and optic neuropathy due to ethambutol
and a variety of antibiotics.2 11 12 It is likely
that the development of linezolid associated
optic neuropathy, manifest by the development of central scotomas and temporal optic
nerve pathology, may be the result of a
similar mechanism.
It is important for ophthalmologists to
perform a complete review of systems and
elicit a history of prescription and nonprescription medication use. Awareness of
the potential for linezolid induced optic
neuropathy is important since drug withdrawal can lead to visual recovery.
K Kulkarni, L V Del Priore
Department of Ophthalmology, Columbia University,
635 West 165th Street, New York, NY 10032, USA
K Kulkarni
University of Medicine and Dentistry of New Jersey,
Robert Wood Johnson Medical School, 675 Hoes
Lane, Piscataway, NJ 08854, USA
Figure 1 Top, photograph suggests subtle temporal pallor to the optic nerve in the right eye with a
nerve fibre layer defect temporally (arrow, upper left); left eye appears normal (arrow, upper right)
with no evident nerve fibre layer defect. Bottom, 3 months after discontinuing linezolid there is
temporal pallor and nerve fibre layer defects in the right eye (lower left), again with no definite
changes in the left eye (lower right).
residual deficits in central acuity or visual
evoked response may persist.3–6
Oxazolidinones inhibit bacterial protein
synthesis by binding to the 70S ribosomal
initiation complex.10 In nutritional optic
neuropathies, paracentral scotomas develop
from disruption in mitochondrial function in
retinal ganglion cells,8 which are more
Figure 2 Humphrey full field 120. Top left and right, left eye and right eye, respectively, demonstrating
central scotomas at presentation. Bottom left and right, left eye and right eye, respectively, showing
improvement in the central scotomas 3 months after discontinuation of linezolid.
Correspondence to: Lucian V Del Priore, MD, PhD,
Robert L Burch III Scholar, Department of
Ophthalmology, Columbia University, 635 West
165th Street, New York, NY 10032, USA; ldelpriore@
yahoo.com
doi: 10.1136/bjo.2005.074237
Accepted for publication 1 August 2005
References
1 Birmingham MC, Rayner CR, Meagher AK, et al.
Linezolid for the treatment of multidrug-resistant,
gram-positive infections: experience from a
compassionate-use program. Clin Infect Dis
2003;36:159–68.
2 Carelli V, Ross-Cisneros FN, Sadun AA.
Mitochondrial dysfunction as a cause of optic
neuropathies. Prog Retin Eye Res
2004;23:53–89.
3 Lee E, Burger S, Shah J, et al. Linezolid-associated
toxic optic neuropathy: a report of 2 cases. Clin
Infect Dis 2003;37:1389–91.
4 Frippiat F, Bergiers C, Michel C, et al. Severe
bilateral optic neuritis associated with prolonged
linezolid therapy. J Antimicrob Chemother
2004;53:1114–15.
5 Lewis KE, Ebden P, Wooster SL, et al. Multisystem infection with Nocardia farcinica—
therapy with linezolid and minocycline. J Infect
2003;46:199–202.
6 Corallo CE, Paull AE. Linezolid-induced
neuropathy. Med J Aust 2002;177:332.
7 Bressler AM, Zimmer SM, Gilmore JL, et al.
Peripheral neuropathy associated with
prolonged use of linezolid. Lancet Infect Dis
2004;4:528–31.
8 Rho JP, Sia IG, Crum BA, et al. Linezolidassociated peripheral neuropathy. Mayo Clin
Proc 2004;79:927–30.
9 Legout L, Senneville E, Gomel JJ, et al. Linezolidinduced neuropathy. Clin Infect Dis
2004;38:767–8.
10 Shinabarger DL, Marotti KR, Murray RW, et al.
Mechanism of action of oxazolidinones: effects
of linezolid and eperezolid on translation
reactions. Antimicrob Agents Chemother
1997;41:2132–6.
11 Skinnider LF, Ghadially FN. Chloramphenicolinduced mitochondrial and ultrastructural
changes in hemopoietic cells. Arch Pathol Lab
Med 1976;100:601–5.
12 Bernstein WB, Trotta RF, Rector JT, et al.
Mechanisms for linezolid-induced anemia and
thrombocytopenia. Ann Pharmacother
2003;37:517–20.
www.bjophthalmol.com
1666
PostScript
Delayed progressive visual loss
following wrapping of bilateral
clinoidal aneurysms: recovery of
vision and improvement in
neuroimaging during
corticosteroid treatment
Reinforcement with muscle, cotton, fibrin
glue, or some other material is an alternative
to clipping in some intracranial aneurysms1;
the surgeon must balance the need to create
local inflammation (to reinforce the arterial
wall) with the risk that the inflammation will
spread and damage adjacent structures.
Wrapping of clinoidal aneurysms, in particular,
rarely may produce delayed and severe visual
loss or ocular motor dysfunction.2–7 The clinical
course and potential outcome of damage to the
visual pathway, ocular motor tracts, or both
remains controversial, as does the optimum
management when visual loss occurs. We
present the case of a patient who developed
severe bilateral visual loss and neuroimaging
evidence of inflammation in the paraclinoid
and suprasellar regions 2 months after wrapping of bilateral clinoidal aneurysms with
cotton and fibrin glue, but who recovered
visual function and whose neuroimaging
appearance improved after treatment with
systemic corticosteroids.
Case report
A 61 year old woman underwent magnetic
resonance imaging (MRI) and angiography
after experiencing a minor stroke. The studies
revealed aneurysms of the clinoidal portion of
both internal carotid arteries. Endovascular
treatment was unsuccessful. Accordingly,
craniotomy was performed. As neither aneurysm could be clipped, both were wrapped
with cotton gauze saturated with fibrin glue.
The patient did well postoperatively until
2 months after surgery, when she noted
blurred vision in the right eye. An incomplete
left homonymous hemianopia associated
with a mild right optic neuropathy was
found, and MRI showed a thickened, nodular, enhancing area in the paraclinoid and
suprasellar regions with involvement of both
optic nerves and the optic chiasm.
Observation was elected, but the patient
developed a severe headache with worsening
visual loss over the next 6 weeks. Repeat MRI
showed an increase in the extent of the area
of the enhancing process (fig 1A), and the
patient was admitted to hospital.
At admission, visual acuity was 1/400
temporally in the right eye and 20/40 in the
left eye. Colour vision was markedly diminished in both eyes. Kinetic perimetry showed
an incomplete, incongruous left homonymous hemianopia (fig 2A). There was no
relative afferent pupillary defect. Extraocular
motility was normal, as were corneal and
facial sensation. The right optic disc was
minimally pale; the left optic disc appeared
normal. Lumbar puncture showed normal
cerebrospinal fluid glucose and protein levels;
there were 14 mononuclear white blood cells.
Complete blood count and serum chemistries
were normal. An acute infectious aetiology
was determined to be unlikely, and the
patient was treated with intravenous dexamethasone 10 mg every 4 hours. Within
48 hours, visual acuity had improved to 20/
200 in the right eye and 20/30 in the left eye,
and the visual field of the right eye had
expanded temporally. Intravenous dexamethasone was continued at 6 mg every
www.bjophthalmol.com
Figure 1 T1 weighted coronal MRI of head with gadolinium contrast (A) obtained at the time of
admission to hospital. An enhancing lesion surrounds the right internal carotid artery (ICA) and is
adjacent to a smaller enhancing lesion abutting the left ICA. The process extends into the right
temporal lobe. (B) 2 months after initiation of steroid therapy. Marked reduction in both the size
and extent of the lesions as well as the degree of contrast enhancement is noted.
4 hours for 2 days, and then reduced to
4 mg every 4 hours. After 7 days of treatment, the patient’s visual acuity had
improved to 20/40 in the right eye and to
20/20 in the left eye, with further expansion
of the peripheral visual field of the right eye.
Repeat MRI revealed marked reduction in the
size and enhancement of the basal process.
The patient was discharged home on a
2 week tapering oral dose of dexamethasone.
Four weeks after discharge, the patient had
visual acuity of 20/20 with slightly diminished colour vision in each eye. An incongruous, left homonymous hemianopia
remained (fig 2B–C), but as this visual field
deficit was scotomatous rather than absolute,
Figure 2 Kinetic perimetry with incongruous left homonymous hemianopia (A) at the time of
admission and (B) performed after 4 weeks of steroid therapy, with marked expansion of the
peripheral visual field of each eye. Static perimetry results (Humphrey 24-2, SITA Standard), both
eyes, demonstrate the scotomatous nature of the residual visual field defects (C) after 4 weeks of
steroid therapy and (D) 2 years after steroid treatment for vision loss.
PostScript
1667
the patient had been able to return to driving
and was now able to perform all of the
activities of daily living. MRI 6 weeks after
discharge showed no evidence of enhancement or mass effect in the paraclinoid or
suprasellar region (fig 1B). Two years after
discharge and without further treatment, the
patient remains well with stable vision and
visual fields (fig 2D).
Comment
Reinforcement of unclippable intracranial
aneurysms with autologous or alloplastic
materials was proposed over 80 years ago,
with subsequent studies showing that only a
subset of these materials produce the desired
local effect.1 Unfortunately, some patients in
whom this treatment is used develop visual
loss, occasionally several months or years
after the surgery.6–9 Although both ischaemia
and infection are thought to be inciting
factors in some cases,10 most cases appear to
result from an inflammatory reaction to the
material used to wrap the aneurysm.6 7 The
reason that the material incites such a
reaction is unknown.
High quality MRI permits recognition of
the inflammatory process that usually is
found in cases of vision loss.3 4 Unlike in
recent reports,2–4 6 our patient presented with
a markedly enhancing bilateral process that,
after corticosteroid treatment, diminished
greatly in both size and degree of contrast
enhancement, providing an anatomical correlate with the functional improvement
demonstrated clinically. In our patient, therapy was initiated approximately 2 months
after the visual loss ensued, as was treatment
in the other cases where no diminution of the
inflammatory mass was seen. Thus, as noted
by others,6 it seems clear that some patients
recover spontaneously, some improve with
steroid treatment, some improve with surgery, and some do not improve regardless of
treatment.
To date, there has been no reported
demonstration by MRI of size reduction of
the inflammatory mass after medical therapy
alone. We demonstrate here that cotton
associated inflammation may respond dramatically to anti-inflammatory therapy both
clinically and by neuroimaging. Furthermore,
this case suggests that wrapping of intracranial aneurysms with cotton or cotton products reinforced with fibrin glue is justified
when no suitable alternative exists, and that
treatment with corticosteroids should be
pursued aggressively should visual loss and
imaging evidence of postoperative inflammation result, as non-surgical treatment may
result in both anatomical and functional
improvement. Should this treatment be
unsuccessful, a neurosurgeon should be
prepared to explore the patient and attempt
debridement of the inflammatory process.
P S Subramanian, N R Miller
Department of Ophthalmology, The Johns Hopkins
Hospital, 600 N Wolfe Street, Baltimore, MD 21287,
USA
V Renard, R J Tamargo, N R Miller
Department of Neurosurgery, The Johns Hopkins
Hospital, 600 N Wolfe Street, Baltimore, MD 21287,
USA
Correspondence to: Prem S Subramanian, MD, PhD,
Ophthalmology Service, Walter Reed Army Medical
Center, 6900 Georgia Ave NW, Washington DC
20307-5001, USA;
[email protected]
doi: 10.1136/bjo.2005.078626
doi: 10.1136/bjo.2005.082602
Accepted for publication 8 August 2005
Accepted for publication 8 September 2005
Competing interets: none declared
References
1 Choudhari KA. Wrapping and coating of
cerebral aneurysms: history, evolution and
surgical management after a re-bleed.
Br J Neurosurg 2004;18:259–67.
2 Berger C, Hartmann M, Wildemann B.
Progressive visual loss due to a muslinoma—
report of a case and review of the literature.
Eur J Neurol 2003;10:153–8.
3 Brochert A, Reynolds T, Baker R. MRI in a case of
muslin-induced granuloma. Neuroradiology
2003;45:82–4.
4 Bhatti MT, Holder CA, Newman NJ, et al. MR
characteristics of muslin-induced optic
neuropathy: report of two cases and review of the
literature. Am J Neuroradiol 2000;21:346–52.
5 McFadzean RM, Hadley DM, McIlwaine GG.
Optochiasmal arachnoiditis following muslin
wrapping of ruptured anterior communicating
artery aneurysms. J Neurosurg 1991;75:393–6.
6 Goldsberry DH, Ross IB, Dhillon G, et al. Visual
dysfunction caused by gauze wrapping of an
intracranial aneurysm. J Neuroophthalmol
2004;24:42–5.
7 Repka MX, Miller NR, Penix JO, et al. Optic
neuropathy from the use of intracranial muslin.
J Clin Neuroophthalmol 1984;4:147–50.
8 Felsberg GJ, Tien RD, Haplea S, et al. Muslininduced optic arachnoiditis (‘‘gauzoma’’):
findings on CT and MR. J Comput Assist Tomogr
1993;17:485–7.
9 Prabhu SS, Keogh AJ, Parekh HC, et al.
Optochiasmal arachnoiditis induced by muslin
wrapping of intracranial aneurysms. A report of
two cases and a review of the literature.
Br J Neurosurg 1994;8:471–6.
10 Kirollos RW, Tyagi AK, Marks PV, et al. Muslin
induced granuloma following wrapping of
intracranial aneurysms: the role of infection as an
additional precipitating factor. Report of two
cases and review of the literature. Acta Neurochir
(Wien) 1997;139:411–15.
MAILBOX
Visual loss may be due to silicone
oil tamponade effect rather than
silicone oil removal
We read with great interest the article by
Cazabon et al.1
In all the three patients it would have been
better to compare the visual acuity just before
the silicone oil removal than immediate
visual acuity after initial vitrectomy, because
contact of the eye with the silicone oil could
also be responsible for visual loss as it was
known to cause optic nerve damage, as
described in earlier reports.2
Earlier, Newsom et al also3 reported unexplained sudden visual loss following silicone
oil removal in seven patients. They also
observed only electrophysiological abnormalities.
Maybe the unexplained visual loss could be
the result of optic nerve damage and diffuse
gangilion cell dysfuntion caused by a silicone
oil tamponade effect on the eye rather than
the procedure of silicone oil removal itself.
P K Rani, R Raman, P Bhende, T Sharma
Vitreoretinal Services, Sankara Nethralaya, 18
College Road, Chennai, 60006, India
Correspondence to: Dr Padmaja Kumari Rani,
Vitreoretinal Services, Sankara Nethralaya, 18
College Road, Chennai, 60006, India;
[email protected]
References
1 Cazabon S, Groenewald C, Pearce IA, et al.
Visual loss following removal of intraocular
silicone oil. Br J Ophthalmol 2005;89:799–802.
2 Budde M, Cursiefen C, Holbach LM, et al. Silicone
oil-associated optic nerve degeneration.
Am J Ophthalmol 2001;131:392–4.
3 Newsom RS, Johnston R, Sullivan PM, et al.
Sudden visual loss after removal of silicone oil.
Retina 2004;24:871–7.
Visual loss after silicone oil
removal
We read with interest the paper by Cazabon et
al1 on the important emerging problem of
sudden visual loss after removal of silicone
oil. We have seen a similar pattern of visual loss
in our own patients, typically in the macula on
detachments associated with giant retinal
tears. We have identified 12 cases in two units
(St Thomas’s, London, and Sunderland Eye
Infirmary), but five of these clearly had onset of
visual loss before oil removal (onset between
1 month and 5 months after oil insertion).2
Results of investigations were very similar to
those reported by Cazabon et al. In four of five
pattern ERG was suggestive of macular dysfunction. The timing of onset of visual loss
obviously alters the potential aetiology, which
as stated is unknown.
In their paper, information on acuity for
cases 2 and 3, between 1 week after oil
insertion and oil removal is not provided. Did
these cases have visual loss preceding oil
removal? Developing cataract can obviously
hinder interpretation of acuity measurements.
In our cases the symptoms described did not fit
with cataract (scotoma, red desaturation) and
persisted if any cataract was removed.
We have seen a further case since this
report, a 46 year old woman with a giant
retinal tear and macula-on retinal detachment affecting the right eye. Acuity reduced
during the period of tamponade from 6/6 at
2 weeks after oil insertion to 6/36+1, which
did not recover after oil removal. She reported
a central negative scotoma. Electrophysiology
suggested macular dysfunction.
We have speculated that phototoxicity may
have a role, as oil transmits light more in the
blue spectrum than aqueous.3 The fat soluble
macular pigments, lutein and zeaxanthin, are
thought to protect the macula from photooxidative damage. Silicone oil has previously
been reported to dissolve fat soluble elements
from the retina.4
We measured the macular pigment optical
density (MPOD) in this case using a modified
confocal scanning laser ophthalmoscope and
two wavelength autofluorescence technique
3 weeks after oil removal. The results showed
a substantially reduced MPOD in the eye that
had silicone oil compared to the fellow eye.
Although the peak MPOD, at the foveal
centre of both eyes was similar (0.47 right
versus 0.52 left), the MPOD at K degree,
1 degree, and 2 degrees eccentricity from the
foveal centre was markedly lower in the eye
that had silicone oil (0.12, 0.06, 0.02 respectively versus 0.40, 0.22, 0.07).
Although MPOD varies greatly between
individuals, there is usually high interocular
symmetry in normal eyes.5 Further work is
required to determine whether or not this
relates to the visual loss and whether
www.bjophthalmol.com
1668
PostScript
therapeutic supplementation may reduce the
risk of visual loss.
E N Herbert, S H M Liew, T H Williamson
Department of Ophthalmology, St Thomas’s Hospital,
Lambeth Palace Road, London SE1 7EH, UK
Correspondence to: Mr Edward N Herbert,
Department of Ophthalmology, St Thomas’s Hospital,
Lambeth Palace Road, London SE1 7EH, UK;
[email protected]
doi: 10.1136/bjo.2005.082610
Accepted for publication 8 September 2005
References
1 Cazabon S, Groenewald C, Pearce IA, et al.
Visual loss following removal of intraocular
silicone oil. Br J Ophthalmol 2005;89:799–802.
2 Herbert EN, Habib M, Steel D, et al. Central
scotoma associated with intraocular silicone oil
tamponade develops before oil removal. Graefes
Arch Clin Exp Ophthalmol DOI 10.1007/
s00417-005-0076-6.
3 Azzolini C, Docchio F, Brancato R, et al.
Interactions between light and vitreous fluid
substitutes. Arch Ophthalmol
1992;110:1468–71.
4 Refojo MF, Leong FL, Chung H, et al. Extraction of
retinol and cholesterol by intraocular silicone oils.
Ophthalmology 1998;95:614–18.
5 Bone RA, Sparrock JM. Comparison of macular
pigment densities in human eyes. Vis Res
1971;11:1057–64.
patients and with an optic neuropathy in one.
In this paper only the macular function is
commented on, the 30 Hz cone flicker being
presented, and it is therefore difficult to
compare data without the full ISCEV data.3 4
It is not clear how the pattern visually evoked
potential (VEP) can be ‘‘normal’’ in case 1,
with a visual acuity of 6/36 and an abnormal
pattern electroretinogram (PERG); even in
macular disease with this level of visual
acuity and an abnormal PERG, the pattern
VEP is invariably abnormal.3
A recent report of optic neuropathy
induced by silicone oil may perhaps explain
our findings in one case.5 However, all the
other cases reported so far seem to point to a
new as yet unexplained phenomenon of
sudden visual loss following silicone oil
removal. Photoreceptor apoptosis, triggered
by rapid change in vitreous potassium concentrations, is an attractive theory, but more
work is required to elucidate this phenomenon further. In the meantime we advocate a
cautious approach to silicone oil in patients
with macular-on detachments.
R S B Newsom, R Johnston, P Sullivan,
B Aylward, G Holder, Z Gregor
Southampton Eye Unit, UK
Correspondence to: Richard S B Newsom,
Southampton Eye Unit, UK;
[email protected]
doi: 10.1136/bjo.2005.082644
Visual loss following silicone oil
removal
We congratulate Cazabon et al on their recent,
well illustrated, report.1 Their cases reflect a
similar group of seven patients we recently
observed at Moorfields Eye Hospital.2 They
were relatively young, 19–57 years old, had
macula-on, or ‘‘just off’’ retinal detachments.
Five of seven had giant retinal tears and the
others multiple posterior tears with retinal
detachment. Following vitrectomy and oil
insertion, vision was good and then fell when
the silicone oil was removed. The oil was in
place for between 105–220 days; three
patients had combined cataract surgery with
oil removal.
One difference between the reports is that
vision in our group fell immediately following
oil removal, whereas in Liverpool patients
reported visual loss at 1 week. Visual loss
could be severe, some lost vision to counting
fingers with a relative afferent papillary
defect, and all lost vision without macular
signs, optical coherence tomographic, or
angiographic changes.
The interpretation of electrophysiological
changes is different from that in our paper,
where macular dysfunction was associated
with generalised retinal dysfunction in some
www.bjophthalmol.com
Accepted for publication 8 September 2005
References
1 Cazabon S, Groenewald C, Pearce IA, et al.
Visual loss following removal of intraocular
silicone oil. Br J Ophthalmol 2005;89:799–802.
2 Newsom RS, Johnston R, Sullivan PM, et al.
Sudden visual loss after removal of silicone oil.
Retina 2004;24:871–7.
3 Fishman GA, Birch DG, Holder GE, et al.
Electrophysiologic testing in disorders of the
retina, optic nerve, and visual pathway. 2nd ed.
Ophthalmology monograph 2. San Francisco:
The Foundation of the American Academy of
Ophthalmology, 2001.
4 Marmor MF, Hood DC, Keating D, et al.
Guidelines for basic multifocal
electroretinography (mfERG). Doc Ophthalmol
2003;106:105–15.
5 Eckle D, Kampik A, Hintschich C, et al. Visual field
defect in association with chiasmal migration of
intraocular silicone oil. Br J Ophthalmol
2005;89:918–20.
CORRECTION
doi: 10.1136/bjo.2005.63123corr1
The paper titled, Intermittent extropia
increasing with near fixation: a ‘‘soft’’ sign
of neurological disease (Br J Ophthalmol
2005:89;1120–2) has been reprinted in this
issue due to an error in the final paragraph,
which has now been corrected.
NOTICES
World Ophthalmology Congress
2006 – Brazil
The World Ophthalmology Congress (which
is replacing the International Congress of
Ophthalmology) is meeting in February 2006
in Brazil.
For further information on the congress and
committees, scientific program and coordinators of different areas are available at the
congress website www.ophthalmology2006.
com.br
Vision 2020
The latest issue of Community Eye Health (No
54) assesses the progress of Vision 2020 at
the district level. For further information
please contact: Journal of Community Eye
Health, International Resource Centre,
International Centre for Eye Health,
Department of Infectious and Tropical
Diseases, London School of Hygiene and
Tropical Medicine, Keppel Street, London
WC1E 7HT, UK (tel: +44 (0)20 7612 7964;
email: [email protected]; online edition: www.jceh.co.uk). Annual subscription
(4 issues) UK £28/US$45. Free to developing
country applicants.
19th International Society for
Geographical & Epidemiological
Ophthalmology Congress
The 19th ISGEO congress will be held in Sao
Paulo, Brazil on 18-19 February, 2006, just
prior to the ICO. Abstract submission and
registration forms can be obtained by emailing Dr Paul Courtright (pcourtright@kcco.
net, url: ) or by accessing the ISGEO website
at www.kcco.net/isgeo.
Medical Contact Lens & Ocular
surface Association Winter
Symposium
The MCLOSA Winter Symposium will be held
at the Western Eye Hospital, Marylebone
Road, London NW1 on Friday 18 November
2005. For further information please visit
the MCLOSA website at www.mclosa.org.uk/
annualmtg.html.
‘‘When we see a flash of lightning, it is
because the lightning is emitting light,
which might have to travel several kilometers toward us before reaching our eyes.
Ancient philosophers wondered how the
speed of light affected the act of seeing. If
light travels at a finite speed, then it would
take some time to reach us, so by the time
we see the lightning it may no longer
actually exist. Alternatively, if light travels
infinitely fast then the light would reach
our eyes instantaneously, and we would
see the lightning strike as it’s happening.
Deciding which scenario was correct
seemed beyond the wit of the ancients.’’
(Sing, Simon. Big Bang. The Origin of the
Universe. New York: Harper Collins,
2004:87)
Numerous studies have evaluated the
ability of confocal scanning laser ophthalmoscopy to discriminate between healthy
optic nerves and those with established
glaucoma. In a prospective study the
Confocal Scanning Laser Ophthalmoscopy
Ancillary Study Group documented that
several baseline topographic optic measurements alone or combined with baseline clinical and demographic factors were
significantly associated with the development of primary open angle glaucoma. The
authors suggest longer follow up is
required to evaluate the true predicted
accuracy of this technique. (Arch
Ophthalmol 2005;123:1188–97)
Recently, three international groups published the DNA sequence of parasites that
cause Chagas’ disease, African sleeping
sickness, and leishmanisis. These deadly
ailments kill 125 000 people every year
and they also compromise blood banks.
Treatments derived from the DNA sequencing work are still several years away but
this work represents new hope in the
battle against these diseases. (Sci Am
2005;293:29–30)
The pathophysiology of eating disorders is
incompletely understood although certain
www.bjophthalmol.com
psychological traits have been identified.
Recently,
investigators
from
the
Karolinska Institute in Stockholm have
suggested that abnormal levels of autoantibodies against hormones called melanocortins are a crucial part of the cause of
these two diseases. Melanocortins are
small protein molecules that carry messages between nerve cells in the brain.
They are involved in regulating a variety of
complex behaviours including food intake.
(Proc Natl Acad Sci USA 2005; September 29
(epub ahead of print))
In a controversial paper published in 2001
in Nature it was reported that genetically
modified corn ended up where none
should have been in the Mexican state of
Oaxaca. The following year the journal
retracted the paper because of insufficient
evidence, but subsequent Mexican government studies backed the initial report.
Now a report analysing over 150 000 seeds
from 870 maize plants in 125 fields in
Oaxaca suggest that transgenic maize has
not survived in these fields. The question
of whether transgenic varieties of maize
may survive in other environments is
unanswered. (Proc Natl Acad Sci USA
2005;August 10:928–34)
Stroke in the young patient (under the age
of 50) has usually been seen as a poor
prognostic sign. However, in a recent study
young people who had ischaemic strokes
but no obvious risk factors did not appear
to require long term secondary prevention.
After 6 years the risk of a second vascular
event was approximately 2% when no risk
factors existed compared to 67% when five
traditional risk factors could be identified.
(Neurology 2005;65:609–11)
The practice of homeopathic medicine
continues despite a lack of scientific
evidence of its efficacy. Indeed, a metaanalysis of 110 randomised double blind
trials of homeopathy with 110 trials of
conventional medicine suggested that the
reported beneficial effects in the trials of
homeopathy are unlikely to be specific and
are most probably compatible with placebo
effects. (Lancet 2005;366:726–32)
Recent reports suggest that physicians
often do not comply fully with published
guidelines. In a retrospective analysis of
patients enrolled in a large managed care
organisation investigators found that a
large number of individuals thought to
require treatment for glaucoma or suspected glaucoma are falling out of care and
are being monitored at rates lower than
expected from recommendation of public
guidelines. Prospective studies are needed
to confirm these findings and to determine
the reasons for low rates of effective care
being provided to glaucoma patients.
(Ophthalmology 2005;112:1494–9)
High endogenous concentrations of oestrogen are a known risk factor for breast
cancer. Impairment of oestrogen synthesis
induced by chronic stress may explain a
lower incidence of breast cancer in women
with high stress. In a study of more than
6000 women participating in the
Copenhagen City heart study investigators
found a significant reduction in the risk of
breast cancer in women with self reported
high levels of stress. They emphasise,
however, that impairment of normal body
function should not be considered a
healthy response and accumulative health
consequences of stress may be disadvantageous. (BMJ 2005;331:548–50)
An amazing number, almost half, of all
medicines prescribed today have a striking
common feature. At a molecular level they
act on the same type of target—the Gprotein coupled receptors (GPCRs). This
group of receptors appears to be extremely
versatile with a response to a large number
of neurotransmitters. Researchers have
isolated at least 650 GPCR genes, about
330 of which might be blueprints for
receptors worth targeting in future drug
development. (Sci Am 2005;293:51–7)