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LABORATORY SCIENCE
A Study Investigating a Possible Link Between Lens Protein
in the Vitreous Fluid of Eyes After Uncomplicated Cataract
Surgery and Chronic Cystoid Macular Edema
Andrew M. Thompson, FRANZCO, Kaa-Sandra N. Chee, PhD, I-Ping Loh, MSc, Trevor Sherwin, PhD,
Colin R. Green, PhD, and Philip J. Polkinghorne, MD
Purpose: This study aimed to determine if the lens protein aquaporin
0 (AQP0) is present in the vitreous of pseudophakic eyes of patients
presenting with chronic cystoid macular edema (CME).
Design: A case-control study was conducted.
Methods: Ten patients undergoing therapeutic vitrectomy for chronic
CME after uncomplicated cataract surgery were enrolled in this study.
Fourteen patients with pseudophakia undergoing vitrectomy surgery for
indications other than CME acted as the comparison group.
The vitreous fluid from the 2 groups was analyzed for the presence of
the lens protein AQP0 and type II collagen (used as a positive control).
Results: Type II collagen was detected in all the vitreous samples, whereas
AQP0 was documented in 50% of eyes with chronic CME but was not
found in the vitreous of any eyes without a documented history of CME.
Conclusions: Aquaporin 0 is found in some eyes with chronic CME after
uncomplicated cataract surgery, suggesting contamination of the vitreous by
lens protein may have a role in the pathogenesis of this disorder.
Key Words: cataract, cystoid macular edema
MATERIALS AND METHODS
(Asia-Pac J Ophthalmol 2014;3: 194Y197)
C
ystoid macular edema (CME) after cataract surgery is a
well-recognized entity and is an important cause of visual
morbidity in the postoperative period.1,2 In most cases, patients
report a variable reduction in central acuity, typically occurring
approximately 1 month after surgery.3 Slit-lamp biomicroscopy
reveals a macula that is abnormally thickened, and typically,
cystic spaces are present around the fovea.4 Optical coherence
tomography (OCT) is useful in confirming the diagnosis and
has largely replaced fluorescein angiography in this regard.5
The etiology of CME after cataract surgery is poorly understood.6 Some cases are associated with complicated cataract surgery such as posterior capsule rupture or vitreous loss, but for
others, there is no clear precipitant.4,7 Fortunately, most eyes settle
spontaneously or with a variety of treatment modalities that include topical anti-inflammatory agents8 and intravitreal triamcinolone9 and bevacizumab.10 Vitrectomy surgery has been advocated
for resistant cases.11
The rationale for most of these treatment options is to restore
integrity of the blood ocular barrier, and the commonality of the
treatments suggests that the underlying pathology might be at
From the Department of Ophthalmology, Faculty of Medicine, University of
Auckland, Auckland, New Zealand.
Received for publication May 17, 2013; accepted August 30, 2013.
The authors have no funding or conflicts of interest to declare.
Reprints: Philip J. Polkinghorne, MD, Department of Ophthalmology,
37 Park Street, University of Auckland, Private Bag 92019, Auckland
1023, New Zealand. E-mail: [email protected].
Copyright * 2014 by Asia Pacific Academy of Ophthalmology
ISSN: 2162-0989
DOI: 10.1097/APO.0000000000000011
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least in part inflammatory.6 To date, however, in eyes having undergone uncomplicated cataract surgery, the latency interval before the onset of symptoms and what precipitated the presumed
inflammatory cascade has defied a suitable explanation. The observation that there is a high incidence of CME associated with
posterior capsule rupture and dropped or retained lens fragments
suggests that the passage of lens proteins into the vitreous might
be causative in at least some eyes that develop CME after uncomplicated cataract surgery. Aquaporin 0 (AQP0) is the most
abundant protein in the lens fiber cells where it forms highly selective water channels between the lens fiber cells.12 This study
investigates the relationship between the presence of the lens
protein AQP0 in the vitreous cavity, indicating that lens fragments
have passed into the vitreous after uncomplicated cataract surgery,
and the development of chronic and persistent CME in eyes undergoing vitrectomy surgery as part of their management.
Ten eyes of 10 patients with chronic CME after uncomplicated cataract surgery were enrolled in this study. To date, CME
had not responded to combinations of topical and systemic therapies, and the patients were offered vitrectomy as a therapeutic
option (Table 1). All patients had undergone cataract surgery
within the preceding 24 months using standard phacoemulsification
techniques and all had intact posterior capsules. Patients were critically examined for clinical evidence of complicated cataract surgery, and the operative records were reviewed. No patient had
undergone previous vitrectomy surgery or received intravitreal
therapy. Patients were excluded if they had a history of diabetes,
hypertension, retinal vascular occlusive disease, or uveitis.
The comparison group consisted of 14 pseudophakic eyes
of 14 patients without a history of CME undergoing vitrectomy
surgery for other macular pathology, namely, symptomatic
epiretinal membranes and macular holes. None of the eyes from
the comparison group had preoperative evidence of complicated
surgery and all had intact posterior capsules. These patients had
not received intravitreal therapy and had not undergone previous vitrectomy surgery.
Diagnostic Criteria
The diagnosis of CME was made clinically and was defined
as a reduction in central best corrected visual acuity in the perioperative period in the presence of a thickened macula and/or
perifoveal cysts visible on slit-lamp biomicroscopy. Each patient had their diagnosis confirmed by a Stratus OCT scan of
their macula (version 4; Carl Zeiss Meditec, Inc., Oberkochen,
Germany). Radial line scanning was used to determine the presence or absence of an epiretinal membrane, confirm the macular
edema (defined by a macular thickness of at least 300 Km), and
note the appearance of intraretinal cysts. Fluorescein angiography
was not included as part of the investigation.
Asia-Pacific Journal of Ophthalmology
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Volume 3, Number 3, May/June 2014
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Volume 3, Number 3, May/June 2014
Lens Protein and Chronic Cystoid Macular Edema
TABLE 1. Demographic and Clinical Features of Patients With Chronic Cystoid Macular Edema After Uncomplicated Cataract
Surgery
Patient No
(Sex)
1 (F)
2 (M)
3 (F)
4 (M)
5 (F)
6 (F)
7 (M)
9 (M)
12 (M)
13 (M)
Age,
Laterality
y
Right
Left
Left
Right
Right
Left
Right
Right
Right
Left
Interval Between
Cataract and
Vitrectomy, mo
AQP0
Signal
Presenting
Acuity
Central Macular
Thickness at
Presentation, Km
Final
Acuity
Central Macular
Thickness at
Follow-up, Km
7
9
13
7
6
17
7
3
20
24
Absent
Absent
Strong
Absent
Strong
Absent
Absent
Strong
Weak
Weak
6/12
6/30
6/36
CF
6/36
6/60
6/12
6/12
6/36
6/60
559
485
486
600
390
560
559
324
624
450
6/7.5
6/12
6/9
6/48
6/9
6/9
6/7.5
6/7.5
6/24
6/48
324
323
290
489
256
290
324
256
520
330
70
75
85
78
68
64
70
70
76
63
CF, count fingers; F, female; M, male.
Sample Collection
Three-port 25-gauge transconjunctival sutureless pars plana
vitrectomies were performed under regional anesthesia by an experienced vitreoretinal surgeon using the Alcon Accurus Surgical
System (Alcon Laboratories, Inc, Fort Worth, Tex). The Oculus
BIOM noncontact wide-angle viewing system (Oculus Asia Inc,
Hong Kong) ensured adequate visualization during vitrectomy.
A pure sample of vitreous was collected at the commencement of vitrectomy. The technique required the 25-gauge cutter
not to have been primed and the ocular infusion be initiated with
air at a standard setting.13 The vitreous sample was collected into a
sterile test tube coupled to the aspiration line. Once the 1Y to
2YmL sample was collected, the test tube was removed from the
aspiration line, the infusion was changed to a balanced salt solution, and the vitrectomy was completed.
To remove surgical intervention as a possible confounder,
vitreous from a postmortem donor eye was made available at the
New Zealand National Eye Bank. The donor eye was phakic
with no known ophthalmic disease and had not received any
ophthalmic treatment or undergone any surgery. Vitreous from
this eye was collected, frozen, and stored until analyzed.
Analysis
Each of the 24 vitreous samples collected at surgery were
labeled with the patient’s national health number and immediately
transferred to the laboratory, frozen, and stored at j20-C until
analyzed. The vitreous from the postmortem eye was stored in an
identical environment.
The laboratory personnel did not have access to the patients’ clinical history and were not privy to intraoperative
findings.
Samples
Each sample, including the postmortem vial, was slowly
thawed to room temperature and centrifuged at 14,500 rpm for
90 minutes. The pellet was extracted and resuspended in phosphate buffer and spun for additional 60 minutes.
Western Blot Analysis
A bicinchoninic acid assay (Thermo Fisher Scientific,
Rockford, Ill) was performed on both the pellet and the supernatant fractions. Approximately 30 Kg per lane of proteins was first
separated on a 15% sodium dodecyl sulphate-polyacrylamide
gel (120 V until dye front reached the base of the gel) and
then transferred onto nitrocellulose membranes (Hybond C;
Amersham Life Sciences, Arlington Heights, Ill) by electrophoresis for 90 minutes at 170 mA. Proteins were stained [1%
Ponceau and 1% acetic acid in MilliQ H2O (Millipore, Billerica,
MA)] to confirm transfer and integrity of proteins, washed in
MilliQ H2O, and then membranes were incubated overnight
at 4-C in blocking solution [1% bovine serum albumin and
0.1% Tween 20 (Sigma, St. Louis, MO) in tris-buffered saline;
2 mmol/L Tris-HCl, 140 mmol/L NaCl, pH 7.6]. After this,
membranes were incubated for 2 hours with primary antibodies diluted in tris-buffered saline (AQP0 [Jomar Biosciences
FIGURE 1. Gel demonstrating the presence of the specific lens protein AQP0 (*) in vitreous samples from patients with chronic CME after
uncomplicated cataract surgery. Deidentified vitreous samples from patients after uncomplicated surgery were loaded onto a
polyacrylamide gel in random order. Detection of AQP0 (lens specific protein) as a 28 kiloDaltons band was only seen in lanes marked
with an asterisk, whereas type II collagen (vitreous-specific protein) was identified in all samples. Later, unmasking of the samples
revealed that all samples containing AQP0 came from patients who developed CME.
* 2014 Asia Pacific Academy of Ophthalmology
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Asia-Pacific Journal of Ophthalmology
Thompson et al
Catalogue AQP01-A], 1:500 dilution; type II collagen
[Chemicon MAB 38887, clone 6B3], 1:1000 dilution). Antibody
labeling was visualized using chemiluminescence as per the
manufacturer’s instructions (ECL; Amersham Life Sciences).
Type II collagen (present in the vitreous) was used as a positive
control marker for the samples.
RESULTS
The mean age of the 10 patients undergoing vitrectomy for
CME was 71.4 years (range, 63Y85 years) and 72 years (range,
56Y85 years) for the 14 patients undergoing vitrectomy for pathologies unrelated to CME. The mean interval between cataract
surgery and vitrectomy in eyes with CME was 10.6 months
(range, 3Y24 months) and 15.0 months (range, 11.6Y20.0 months)
for the controls. On clinical examination, all intraocular lenses
were of the posterior chamber type and were in the capsular
bag. None of the eyes had undergone neodymium-doped yttrium aluminum garnet laser treatment for posterior capsular
opacification. No eye with CME had evidence of an epiretinal
membrane on any of the OCT scans.
None of the vitreous samples from the 14 controls contained
AQP0. Immunoprobing did detect the positive control type II
collagen protein in these controls though, as well as in the vitreous
from the 10 eyes with CME. Vitreous from the donor eye also
contained type II collagen but AQP0 was absent.
Aquaporin 0 was, however, strongly detected in 3 of the
vitreous samples from eyes with chronic and persistent CME,
indicating the presence of lens fiber material. Weak signals were
detected in another 2 eyes with CME (Fig. 1). There was no
relationship between the strength of AQP0 signal and the severity of CME as documented with OCT (Table 1).
DISCUSSION
Most patients who develop symptomatic CME after uncomplicated cataract surgery have a good functional outcome,
but small subset of eyes may have persistent edema, which is
unresponsive to topical anti-inflammatory medications.10 This
study demonstrated that detectable levels of the specific lens
protein AQP0 are present in the vitreous of at least some patients with chronic and persistent CME after uncomplicated
cataract surgery. The absence of this protein in the control group
suggests that this protein is not normally found in pseudophakic
eyes without a history of CME. Similarly, no AQP0 was
detected in the vitreous sample from the postmortem donor.
That sample was from a phakic eye and suggests that leaching
of lens protein does not occur through an intact lens capsule.
The observation that not all eyes with chronic persisting CME
had AQP0 in the vitreous would support the notion that either
other pathways are important in the pathogenesis of CME,
which AQP0 was cleared from the vitreous cavity before vitrectomy surgery, or that fewer or smaller lens fragments in those
samples were simply not detectable in our Western blot analysis.
Alternatively, it is possible that the sample of vitreous studied,
approximately one third to one half of the total volume, did not
contain AQP0 and so simply reflects a sampling error. Conceivably too, any lens fragments that were in the vitreous may
have already settled on the retinal surface. Thus, 50% of the
samples that resulted positive in the test for lens protein could
be an underestimate.
Aquaporin 0 is the most abundant lens membrane protein
that is present in both nucleus and cortex of the human crystalline
lens.14 It is a specific lens protein and, therefore, is a useful marker
for detecting the presence of crystalline lens material. The prime
function of AQP0 is to regulate the transfer of water and small
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neutral solutes across the lens cell membranes and is thought to
have a role in the maintenance of lens transparency.15
Lens fragments have been postulated to enter the retrolenticular space after uncomplicated cataract surgery,16 but morphologic evidence has been lacking to date.17 This discrepancy
may partly be due to the vitreous sampling technique and/or an
inability to identify small lens particles with a cytopathologic
approach. In the current study, AQP0 was identified using standard Western blot analysis, which enables low levels of protein to
be detected in larger volumes of soluble liquids. The use of control vitreous including that from a nonsurgical donor eye ensures
the validity of this experimental approach. Furthermore, documenting type II collagen in all the vitreous samples would imply that the experimental protocol used in this study did not lead to
false negatives.
The passage of small lens fragments into the vitreous
during or after uneventful cataract surgery may be facilitated by
a number of mechanisms. These include a pressure differential
between the anterior chamber and posterior segment, size of the
anterior capsulotomy, and integrity of the lens zonules. Conversely, the barrier effect of viscoelasticity and the anterior
vitreous may limit the posterior migration of lens proteins and
so mitigate this event. Irrespective of which mechanism(s) is
operating, this study demonstrates that crystalline lens fragments can enter the vitreous during or after uncomplicated
cataract surgery.
When macroscopic particles or fragments of lens material
enter the vitreous cavity during cataract surgery complicated by
posterior capsule rupture, the risk of developing CME is approximately 25%.18 Wilkinson et al19 reported the appearance
of macrophages in the vitreous of eyes undergoing vitrectomy
for retained lens material after only a few days and suggested
that vitrectomy surgery should be performed early while the
inflammatory response is in its early stages. However, Merani
et al20 did not find any correlation between CME and timing of
the vitrectomy. This discrepancy, together with the obvious conundrum that not all eyes with retained lens fragments develop
CME, suggests that other mechanisms might be involved.
Fragments may also not always settle onto the retina or may
remain trapped within the vitreous.
The relevance of detecting lens protein AQP0 in the vitreous of eyes with chronic and persistent CME highlights the
possibility that improvements in surgical technique might decrease the rate of CME after uncomplicated cataract surgery.
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"The brain is wider than the sky."
V Emily Dickinson
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