Download The effect of interruption of the short posterior ciliary arteries

Reports 495
Volume 15
Number 6
dissection of eyes allowing regional flows to be
compartmentalized more accurately in the present
study. In addition, precautions were taken in the
present study to maintain blood-gases and pH
in the normal range, since variations in these can
independently alter ocular blood flow.7 The discrepancy may be due to the use of the pig in
the present study in contrast to previous studies,2' •'> n suggesting a species specific nature of
the ocular vascular response to catecholamines.
Finally, there is the possibility that vasodilation
induced by isoproterenol is secondary to stimulation of ocular metabolic rate rather than vasodilation due directly to stimulation of beta-adrenergic
receptors.1 Studies using specific antagonists of
isoproterenol are needed to confirm the existence
of beta-adrenergic receptors in the ocular vasculature.
From the Departments of Physiology and
Ophthalmology, Albany Medical College, Albany,
N. Y. 12208. Supported in part by Grant 1R02
E7000791 from the National Eye Institute. Submitted for publication Sept. 12, 1975. Reprint
requests: Dr. A. B. Malik, Department of Physiology, Albany Medical College, Albany, N. Y. 12208.
Key words: microspheres, reference sample method, pigs, regional ocular flows, isoproterenol,
norepinephrine.
REFERENCES
1. Aim, A.: Effects of norepinephrine, angiotension, dihydroergotamine, papaverine, isoproterenol, nicotinic acid, and xanthinol
nicotinate on retinal oxygen tension in cats,
Acta Ophthalmol. 50: 707, 1972.
2. Chandra, S. R., and Freidmen, E.: Choroidal
blood flow. II. The effects of autonomic
agents, Arch. Ophthalmol. 87: 67, 1972.
3. Macri, F. T.: Local ganglion-line stimulating
properties of some adrenergic amines which
affect blood vessels of the anterior segment
of the eye, INVEST. OPHTHALMOL. 13: 389,
1974.
4. Best, M., Gerstein, D., Wald, N. et al.:
Autoregulation of ocular blood flow, Arch.
Ophthalmol. 89: 143, 1973.
5. Dalske, F. H.: Pharmacologica reactivity of
isolated
6.
7.
8.
9.
ciliary arteries, INVEST. OPHTHAL-
MOL. 13:389, 1974.
Cole, D. F., and Rumble, R.: Effects of
catecholamines on circulation in the rabbit
iris, Exp. Eye Res. 9: 219, 1970.
Bill, A.: Blood circulation and fluid dynamics
in the eye, Physiol. Rev. 55: 383, 1975.
Buckberg, G. D., Luck, J. C , Payne, B. D.,
et al.: Some sources of error in measuring
regional blood flow with radioactive microspheres, J. Appl. Physiol. 31: 598, 1971.
Prince, J. H., Diesem, D., Eglitis, I., et al.:
Anatomy and Histology of the Eye and Orbit
in Domestic Animals. Springfield, 111., 1960,
Charles C Thomas, Publisher.
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10. Hayreh, S. S.: The choriocapillaries, Albrecht
Graefes. Arch. Klin. Ophthalmol. 192: 165,
1974.
The effect of interruption of the short
posterior ciliary arteries on slow axoplasmic transport and histology within
the optic nerve of the rhesus monkey.
NORMAN S. LEVY.
Tritiated leucine was injected into the vitreous
of rhesus monkey eyes to make it available for
protein synthesis by the ganglion cells. The short
posterior ciliary arteries were cut three hours later
or several weeks prior to the leucine injection. A
reduction of labeled protein within the retrolaminar optic nerve was seen in all eyes so treated.
Autoradiography revealed a diffuse reduction of
axoplasmic transport into these optic nerve heads.
There was consistent evidence of focal obstruction
of labeled protein at the interface between the
lamina scleralis and retrolaminar optic nerve.
Vacuoles appeared in the most severely affected
areas. These histologic changes were followed by
gliosis in the areas of ischemic damage. Glaucomatous cupping of the optic nerve head was not
seen within six weeks following the induced
ischemia.
Recent studies suggest that the obstruction in
axoplasmic transport seen following intraocular
pressure elevation in both rhesus and owl monkeys
is most likely ischemic in origin.1 -3 Ischemia of
the optic nerve head can be produced without intraocular pressure elevation by compromise of the
posterior ciliary arterial supply.4- 5 Under these
conditions, the quantity of slow axoplasmic transport into the optic nerve is also reduced.0 The
purpose of this study is to characterize the effects
on slow axoplasmic transport of sugical interruption of the arterial blood supply to the nerve head
over periods of varying duration.
Methods and materials. These studies were performed in twelve optic nerves of six rhesus monkeys (Macaca midatta), weighing 3 to 4 kilograms.
Each animal had an ocular examination including
tonometry, biomicroscopy, and funduscopy. Photographic documentation of the fundus was obtained
before and during the study. Electroretinography
and visually evoked responses were obtained in
some animals as previously described.6
The posterior ciliary arteries were exposed by
lateral orbitotomy under ketamine and barbiturate
anesthesia. The area at which the optic nerve
leaves the eye was observed under the microscope and the short posterior ciliary vessels surrounding the optic nerve cut at that location. In
the opposite eye of each animal, a similar ex-
Investigative Ophthalmology
June 1976
496 Reports
B
Fig. 1. The retinas from the two eyes of animal No. 47 are compared autoradiographically,
after staining with Masson-trichrome (xl60). The retinas appear normal in both eyes. The retina of the treated eye (A) shows a reduction in grain density relative to the control at four
days following interruption of the short posterior ciliary arteries.
posure was obtained but the vessels were not
severed.
At periods varying from three hours prior to
surgery to six weeks after the time of surgery,
bilateral anterior chamber paracenteses were performed to remove the aqueous. Each animal then
received from 0.15 to 0.25 ml. of leucine-3H (1
mCi. per milliliter) intraviheally into each eye.
Four days later the monkeys were deeply anesthetized and the eyes, with ample optic nerve, enucleated and placed directly in formaldehyde.
The optic nerve was excised just behind the
globe and the eye callotted horizontally. The
eyes were then washed and dehydrated in
ascending concentrations of alcohol, followed by
xylene. Xylene was replaced with paraffin, and the
tissue was vacuum infiltrated for an hour, maintaining orientation. The resultant block was
trimmed, and eight micra sections were serially
cut. In the control eye, the matched tissue was
processed identically.
The retrolaminar optic nerve was cut into 1
mm. serial sections. These were digested in 1.0
normal sodium hydroxide at 60° C. for 24 hours.
The superior half of the retina was digested in a
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similar manner. Protein determinations were made.
Appropriate standards of bovine albumin were
treated as were the experimental samples. These
samples were measured by standard techniques
using a spectronic 20 colorimeter at 500 nm,
Samples of this same tissue were counted in a
Beckman LS-100 liquid scintillation counter in
a cocktail of naphthalene, dioxane, and liquifluor
in polyethylene vials. Three 10-minute counts on
each sample were averaged and the background
subtracted. All results exceeded background levels
by at least a factor of five.
Paraffin sections eight micra thick were placed
on acid-cleaned, gelatin-chrome, alum-dipped
slides and heated for 24 hours at 60° C. These
slides were then washed serially in xylene, alcohol,
and water. The slides were dipped in Ilford K-5,
size B emulsion and placed in light-tight boxes at
4° C. for 60 days. They were developed in Kodak
D-19 for two minutes at 21° C. and stained with
Masson trichrome. Matched autoradiographic sections of the optic nerve head from each eye were
compared. The autoradiographs defined the distribution of the labeled material within the optic
nerve head and retina at the time of sacrifice.
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Number 6
Reports 497
Table I. Scintillation counts from the optic nerves of 4 monkeys four days after severing the
posterior ciliary arteries
d.p.m./mg. optic nerve protein, normalized to equivalent retinal radioactivity
P.C.A. severed optic nerve
Control optic nerve
Difference
1 mm.
behind globe
2 mm.
behind globe
3 mm.
behind globe
4 mm.
behind globe
13,824 (± 9,484)
105,488 (± 25,874)
87%
3,174 (± 1,587)
79,788 (± 13,103)
96%
2,183 (± 618)
57,490 (± 11,494)
96%
6,919 (± 3,917)
36,137 (±4,787)
Optic nerve sites at which axoplasmic transport
was altered by severing the posterior ciliary
arteries were determined and studied.
Results. In eight eyes, from four animals, tritiated leucine was injected into the vitreous two to
three hours prior to surgery. This should have permitted sufficient time for the synthesis of ganglion
cell protein to be completed7 before the induction
of alterations in blood dynamics. A four-day interval between the surgical procedure and the time
of sacrifice was employed, so that slow axoplasmic
transport to the optic nerve was well underway.
Intraocular pressure fell to 6 to 8 mm. Hg in the
treated eyes vs. 16 to 20 mm. Hg in the control
eyes.5- °
Edema of the macula and optic nerve was apparent one week following surgery. Some pallor of
the nerve head itself was present. The retinas of
the treated eyes appeared similar in histologic appearance to the controls, except for areas of intraretinal and subretinal hemorrhage seen in two
of the surgically treated eyes. There was a significant difference in the specific activity of the
retinal labeled protein in each of the surgically
treated eyes, ranging from 29 to 90 per cent less
than the control. Autoradiographic reduction in
the grain density was seen in the retina of the
eye in which the short posterior ciliary arteries had
been severed (Fig. 1).
However, this reduction in specific retinal activity was small relative to the much larger reduction in the egress of labeled axoplasmic protein
from the treated eyes. In Table I, the disintegrations per minute (d.p.m.) per milligram of optic
nerve protein have been normalized to reflect
equivalent retinal specific activity, so that only the
differences in protein transport into the optic
nerve are expressed. After this normalization, axoplasmic transport into the surgically treated nerves
was at least 80 per cent less in every animal
studied. This difference is consistent with the reduction noted in our previous study, three and six weeks
after severing the short posterior ciliary arteries.(;
Autoradiography of the optic nerve heads from
the two eyes of each animal were compared (Fig.
2). The most prominent change was diffuse reduction in the grain density in the optic nerve
heads of the treated eyes. At the posterior margin
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of the lamina scleralis of the optic nerve head,
labeled axoplasm appeared to be obstructed in the
treated eyes only (Fig. 2). Vacuolization in the
prelaminar optic nerve was noted in the most
severely damaged nerve sections.
After six weeks, the histologic picture at the
optic nerve showed proliferation of glial tissue
with replacement of some axonal bundles (Fig. 3).
The point of maximum cup change six weeks following the procedure is seen. Although there is
some enlargement of the cup, the picture contrasts
markedly with glaucomatous cupping.3
Discussion. In this study, ischemic effects upon
axoplasmic transport were separated from pressure
effects by severing the short posterior ciliary arteries about the optic nerve head. The slow transport
of axoplasmic protein into the optic nerve was
then measured. The histology of the nerve head
under these conditions was also studied. After
normalizing to achieve equal retinal specific protein activity, ischemia produced in excess of 80
per cent decrease in the amount of labeled protein entering the retrolaminar optic nerve. There
was a diminution in the density of grains seen
autoradiographically in the prelaminar and laminar
optic nerve head. In addition, focal abnormalities
in transport at the level of the lamina scleralis
were seen after severing the posterior ciliary vessels. Since the slow component of axoplasmic
transport has a rate of 1.5 mm. per 24 hours,R only
ganglion cell somata within 6 mm. of the optic
nerve head were contributing to the observed
transport four days after injection of the labeled
precursor. Data from more distant cells were not
measured in this study.
The faster moving components of axoplasmic
protein are known to be dependent upon focal
high energy phosphate supplies at each point
along the axon for continued transport. The same
is believed true of the slow component. Prompt
recovery of axoplasmic transport will occur after
up to 1.5 hours of anoxia.0 Recovery of transport
is delayed with anoxia up to four hours, and is
irreversibly blocked after six hours of anoxia.
These observations on fast axoplasmic transport
may help to explain the reduction in transport
observed in this study following the induction of
ischemia.
Investigative Ophthalmology
June 1976
498 Reports
B
Fig. 2. This Masson-trichrome stained autoradiograph (xl60) with insets (x400) shows the
optic nerve heads from both eyes of animal No. 47. At the posterior margin of the lamina
scleralis in the eye with induced surgical ischemia of the short posterior ciliary arteries (A),
labeled axoplasm appears to be obstructed (arrows). In the control (B) no such alterations
in transport were noted at the lamina scleralis. Some histologic evidence of vacuolization and
early gliosis is present in the treated optic nerve (A). There is also a reduction in grain density.
The site of ischemic alteration in axoplasmic
transport is identical to that seen with pressure
elevation.3 The effect becomes most apparent at
the terminus of the watershed of capillaries that
supply the optic nerve head, namely at the posterior extent of the lamina scleralis. The focal obstruction of axoplasmic transport occurred at the
posterior margin of the lamina scleralis, the terminus of the circulatory watershed. The extent of
change was elsewhere diffuse both autoradiographically and histologically. Morphologically, the
disc changes resembled acute ischemic optic neuropathy, with edema of the optic nerve and macula
and some blurring of the disc margins.
In longer duration studies, the neurons were
histologically replaced with gliotic tissue. There
was no evidence of glaucomatous cupping, either
by funduscopy or histologically, but a slight saucerization was present due to the gliotic replacement of lost neurons.
The phenomenon of cupping appears to require
pressure elevation associated with ischemia. As
vacuoles appear in the laminar and retrolaminar
areas in the face of pressure elevation, the disc is
capable of collapsing into the defect resulting from
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the tissue loss. In our study the pressure was lowered, rather than raised, by the surgical interruption, so that there was no significant pressure gradient across the lamina scleralis. There was no cupping. Pressure, as well as laminar ischemia with
vacuolization, appears necessary to effect the irreversible cupping process seen in chronic intraocular pressure elevation.
A three-hour interval was used between injection of the tritiated leucine and the surgical interruption of blood flow, since the observations of
Karlsson and Sjostrand7 in the rabbit had suggested that this time is sufficient for the completion of protein synthesis by the retina. It is apparent that more time is probably required in the
rhesus monkey, since a decrease in specific activity
of retinal labeled protein was observed in those
eyes in which the short posterior ciliary arteries
were interrupted.
This observation also implies that a reduction
in the blood flow required to sustain the retina
occurred during the period of protein synthesis.
Nutrition to the outer retina via the choriocapillaris
was undoubtedly altered. Some flow destined for
the retina was probably lost by shunting into the
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Volume 15
Number 6
2. Anderson, D. R., and Hendrickson, A.: Effect
of intraocular pressure on rapid axoplasmic
transport in monkey optic nerve, INVEST.
OPHTHALMOL. 13: 771, 1974.
3. Levy, N. S.: The effect of elevated intraocular pressure on slow axoplasmic transport in
the rhesus monkey. Doctoral Dissertation.
University of Chicago, 1975.
4. Armaly, M. F., and Araki, M.: Optic nerve
circulation and ocular pressure: Contribution
of central retinal artery and short posterior
ciliary arteries and the effect on oxygen tension, INVEST. OPHTHALMOL. 14: 475, 1975.
SKI
Fig. 3. This section (xlOO) of the optic nerve head
of animal No. 30 shows the maximal extent of
histologic change and cupping observed six weeks
after severing the short posterior ciliary arteries.
There is glial proliferation and replacement of
lost neurons. No retrodisplacement of the cup, nor
glaucomatous cupping is present. A small amount
of saucerization is noted.
severed short ciliary arteries. In addition, spasm of
the central retinal artery may have occurred as a
result of the surgery. Histologic evidence of intraretinal hemorrhages in two eyes supports this. All
probably contributed to the compromise in retinal
nutrition during the period of protein synthesis.
Our results contrast with those of Anderson and
Davis10 who found no evidence of any histologic
change in the optic nerve head of young squirrel
monkeys following surgical interruption of the
short posterior ciliary arteries. This may reflect
their selection of the young squirrel monkey for
study. Armaly and Araki4 have found a 79 per
cent reduction in blood flow to the retrolaminar
optic nerve of rhesus following ligation of the
short posterior ciliary arteries. A decrease in blood
flow of this magnitude would probably be sufficient over a period of time to explain the 80 per
cent reduction in axoplasmic transport and the
histologic changes observed.
The technical assistance of Cheryl Curington is
appreciatively acknowledged.
From the Veterans Administration Hospital
(MRIS 5221-02) and the Department of Ophthalmology, University of Florida, Gainesville, Fla.
This study was supported in part by Fight for
Sight, Inc. (G-507), New York, N. Y. Submitted
for publication Nov. 3, 1975. Reprint requests:
Dr. N. S. Levy, 4020 Newberry Rd., Gainesville,
Fla. 32607.
REFERENCES
1. Levy, N. S.: The effect of elevated intraocular pressure on slow axonal protein flow,
INVEST. OPHTHALMOL. 13: 691, 1974.
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5. Hayreh, S. S., and Baines, J. A.: Occlusion of
the posterior ciliary artery. Ill, Br. J. Ophthalmol. 56: 754, 1972.
6. Levy, N. S., and Adams, C. K.: Slow axonal
protein transport and visual function following retinal and optic nerve ischemia, INVEST.
OPHTHALMOL. 14: 91, 1975.
7. Karlsson, J. C , and Sjostrand, J.: Synthesis,
migration, and turnover of protein in retinal
ganglion cells, J. Neurochem. 18: 749, 1971.
8. Williard, M., Cowan, W. M., and Bagelos, P.
R.: The polypeptide composition of intraaxonally transported proteins. Evidence for
four transport velocities, Proc. Natl. Acad. Sci.
71: 2183, 1974.
9. Leone, J., and Ochs, S.: Reversibility of fast
axoplasmic transport following differing durations of anoxic block in vitro and in vivo. Program/Abstracts, Annual Meeting of the Society for Neuroscience, 3: 147, 1973.
10. Anderson, D. R., and Davis, E. B.: Retina
and optic nerve after posterior ciliary artery
occlusion: an experimental study in squirrel
monkeys, Arch. Ophthalmol. 92: 422, 1974.
Centrioles and cilia in the mesothelial cells
of the pericanalicular region. M. GARY
WlCKHAM AND DAVID M. WORTHEN.
An evaluation of 70 trabecular meshwork biopsies
obtained at the time of therapeutic surgery in
glaucomatous and cataractous eyes revealed that
the mesothelial cells in the iridocorneal angle had
a marked abundance of cilia and centrioles. The
distribution of cells showing cilia and/or centrioles
is positively correlated with the apparent aqtieaus
humor outflow pathway. The morphology and
arrangement of the cilia-centriole complexes in
the angle are highly variable and show many
forms not previously reported in a single tissue.
There were no obvious correlations between organelle abundance and the identifiable factors affecting the patients involved in this study.
Scherft and Daems1 presented a review of the
literature on the various types of cilia in vertebrates. Their data showed that most single or