Animal modeling of cystoid macular edema

SURVEY OF OPHTHALMOLOGY

VOLUME 28 * SUPPLEMENT.

MAY 1984

Animal Modeling of Cystoid Macular Edema MARK O.M. TSO, M.D.

The Georgiana Theobald Ophthalmic Pathology Laboratory, Illinois, &Ye and Ear Infirmary, Chicago, Illinois

Department

of Ophthalmology,

University

oj

models of cystoid macular edema (CME) were developed along four sets of presumed primary causative factors: 1) disruption of the blood-retinal barrier at the retinal vasculature and the retinal pigment epithelium; 2) ischemic tissue injury with cyst formation; 3) intraocular events frequently observed in patients with CME, such as inflammation, ocular hypotony, and vitreous traction; and 4) the probable additive effect on the macula by systemic diseases such as diabetes mellitus and systemic hypertension. While each experiment produced some aspects ofCME in the human, none gave the complete picture. It is concluded that CME is the final common pathway of a multifactorial syndrome. (Surv Ophthalmol 28(Suppl):512519, 1984)

Abstract. Primate

Key words. ischemia . systemic

n most scientific

I

l

studies,

hypothesis,

prepare

prove

theory,

this

However,

are presented

and

it difficult

to establish

for clinical

seen in human

hypothesis

factors

Frequently, comparable

the animals

of the clinical

on pathogenesis

blood-retinal including tients

syndrome,

such

a

CME

vitreous

that

effect

and

they

factors;

mellitus,

traction,

must

be

with

events and ocu-

so frequently

in pa-

considered

and 4) the probable

on the macula

as diabetes

tissue injury

of intraocular

that are observed

the causative

additive

only

allowing

manifestations

CME.

2) ischemic

3) a number

inflammation,

with

among

at-

manifestations. may manifest

barrier;

lar hypotony

to those

repeated

of the

of human

cyst formation;

and proceed

similar

aspects

l

I believe that there are at least four sets of primary causative factors in CME: 1) disruption of the

situations,

insult on animals,

manifestations

some

mellitus epithelium

Primary Causative Mechanisms

and complicat-

frequently

primary

patients.

On other occasions,

a thesis.

To examine

diabetes pigment

pathophysiology

clinicians

a model.

edema l l retinal

illustrate

a

investigators

pathologic

fail to produce

some aspects

establish

investigations,

mechanisms,

to inflict a comparable

propose

and associated

select a set of presumed

tempts

then

l cystoid macular ocular inflammation

to verify or dis-

with a set of ill-defined

observations

pathogenetic

looking

investigators

experiments

in many clinical

ed clinical making

blood-retinal barrier ocular hypotony l hypertension

of systemic

systemic

diseases

hypertension,

and others.

to be generated.

DISRUPTION BARRIER

Although a long list of experiments exist on animal modeling of cystoid macular edema 67.S12~1Q2i no ideal models have been estab(CME),

OF THE BLOOD-RETINAL

One of the characteristic

features

of CME

is ex-

lished. The clinical and pathologic findings and the associated conditions of this syndrome are so varied, controversial, and complicated, that even the selec-

travasation of fluorescein demonstrated on the retinal angiogram. A number of experiments resulting in chronic disruption of the blood-retinal barrier

tion of presumed cult. Furthermore,

have been carried

nantly those While model been

primary causative factors is diffibecause CME occurs predomi-

out to examine

the response

of the

retinal tissues. Because CME is most frequently seen after lens extraction, this operation was performed on seven eyes of four rhesus monkeys.2” Fluorescein angiograms of these animals failed to show the classic petaloid leakage pattern noted in CME patients.

at the macula, we have to limit our models to animals that have maculas, namely primates. we currently do not have an ideal animal of CME, a series of animal experiments have performed and will be summarized here to 512

ANIMAL

MODELING

513

OF CME

However, light and electron microscopy of horseradish peroxidase tracer studies showed leakage from both the retinal pigment epithelium (RPE) and retinal vessels (Fig. 1). Some of the RPE was decompensated, with horseradish peroxidase tracer infiltrated within the cell; in other cells, the tracer passed through the RPE into the subretinal space. Leakage from retinal vessels, particularly the large veins, was also observed. Tracer passed into the cytoplasm of endothelial cells, and pinocytotic vesicles were increased. Tracer extended along the basement membrane of the endothelial cells and pericytes into the extracellular space between the glial and neuronal cells of the retina. Animals that had vitreous loss during lens extraction tended to show retinal vascular leakage. In this experiment, we demonstrated that a negative fluorescein angiogram does not necessarily imply that the blood-retinal barrier is intact. Lens extraction could cause disruption of the blood-retinal barrier at the retinal vasculature and RPE, giving rise to macular edema. Yet no cystoid spaces were seen, either clinically or pathologically, even though some of these animals demonstrated swelling of the Miiller cells, particularly in the outer layers of the retina.” A month after surgery, the retinal edema gradually subsided and no cystoid degeneration occurred in the macula. Trabeculectomy performed on rhesus monkeys produced clinical and pathologic changes in the retina similar to but milder than those seen after lens extraction. Recently, Drs. Arno Puck, Gholam Peyman, Jose Cunha-Vaz, Antonio Travassos, and IR examined live animals after subtotal vitrectomy through the pars plana approach. One of five animals showed diffuse leakage of fluorescein in the perimacular region, but the classical petaloid pattern of human CME was not noted. The horseradish peroxidase tracer study in this monkey showed leakage of tracer through the blood-retinal barrier at the pigment epithelium and the retinal vasculature. Some of the vessels had increased pinocytosis. No cystoid spaces were noted in the retina histologically. Some of the axons in the outer plexiform layer showed vacuolation and degeneration. The Miiller cells in the outer layers of the retina were not swollen (Fig. 2). This experiment illustrated that vitrectomy may cause disruption of the blood-retinal barrier at the RPE and retinal capillaries, although swelling of Miiller cells is not necessarily seen in the disruption of the blood-retinal barrier. In recent years, CME has been ascribed to photic injury by the operating microscope used by surgeons for lens extraction. The eyes of ten rhesus monkeys were exposed to the light of the indirect

Fig. 1. The macula

of a rhesus monkey that had lens extraction with vitreous loss. Two weeks before enucleation, extensive horseradish peroxidase tracer leaks from the retinal vasculature (v), extending into the perivascular tissues (arrows). The tracer also infiltrates the retinal pigment epithelial cells (arrowheads) (toluidine blue, x 260).

ophthalmoscope for one to two hours.‘Z.‘“,‘R.2’ Within the first 24 hours, the blood-retinal barrier at the RPE was disrupted, as demonstrated by leakage of fluorescein. Within 612 months a retinal scar had formed. Diffuse leakage of fluorescein from the pigment epithelium persisted for 2-5 years (Fig. 3).2’ Pathologically, chronic leakage of horseradish peroxidase through the pigment epithelium into the subretinal space was seen, yet no cystoid spaces formed in the retina, even though some of the proliferated RPE developed a plaque (Fig. 4) associated with sub-RPE neovascularization. This experiment showed that even though chronic leakage of the RPE was present for a long period of time, no classic CME was formed. Therefore, we concluded that while the disruption of the bloodretinal barrier is an important aspect of CME, it is not necessarily the primary pathogenetic mechanism. This experiment further illustrated distinct clinical and pathologic differences between CME

Fig. 2. The outer plexiform layer of rhesus monkey that had subtotal vitrectomy and showed diffuse leakage of fluorescein in the macular region. Note the vacuolation of some of the axons (A), but no swelling of the Miiller cells (M) surrounding the axons ( X 6000).

Fig. 3. Fluorescein scar (arrowheads)

angiogram of rhesus monkey that had been I exposed to the light ofan ophthalmoscope showing a retinal in the arteriovenous phase (A). The s(:ar ( arrowheads) stains with fluorescein in the late venous phase

Fig. 4. Macula of a rhesus monkey that had been exposed to the light of an indirect ophthalmoscope. A placoid proliferation of the retinal pigment epithelium (R) has developed. Horseradish peroxidase tracer (arrows) passes into the subretinal space and outlines the photoreceptor elements. No cystoid macular degeneration is noted in the inner and outer layers of the retina. Bruch’s membrane is indicated by arrowheads (toluidine blue, X 260).

ANIMAL

MODELING

OF CME

Fig. 5. Thh macula of a rhesus monkey that had repeated intravenous injections of talc emboli. Cystoid spaces are noted in the outer plexiform layer, inner nuclear layer, and ganglion cell layer (arrowheads) (toluidine blue, x 300).

and photic leakage

maculopathy.

of fluorescein

duce the petaloid

photic

directly

of CME

maculopathy

the diffuse

failed

to pro-

seen on fluores-

Pathologically,

in the photoreceptor

generation CME.

from the RPE

pattern

cein angiography. changes

In the latter,

the degenerative

elements

and RPE

differed

from

the cystoid

of the more inner

layers

of the retina

It is therefore from photic

unlikely

that

CME

in dein

resulted

injury. showed that the extracellular

ISCHEMIC DAMAGE WITH CYST FORMATION Patients

with CME

in the macula cally.” Tissue an important versial

that

extracellular,’ of the disease lar.

frequently

lose vision.

pathologic may

process. be initially

While

Cysts

it is contro-

intracellular

or

the cysts are so large at the late stage that I believe

they must be extracellu-

Drs. Jampol, Kaga, and I injected talc particles intravenously into rhesus monkeys twice a week for two to ten months.4,6.7 As collaterals developed in the lung, these small emboli passed into the systemic circulation and were choroidal circulations.

space contained

and that the cyst wall was lined by swollen

are observed clinically and histologidamage with cyst formation must be cysts

Pi,?. 6. A: Retina of a rhesus monkey that had retinal vein occlusion by photocoagulzition, showing cystoid edema (arrowheads) in the outer plexiform layer, inner nuclear layer, and nerve fiber layers (toluidine blue, X 260). B: Electron micrograph ofthe retina, as shown in Fig. 4. The glial cells (G) and the axons (A) in the vicinity of the cystoid spaces (S) are swollen. Fibrin (arrow) is also noted in the extracellular space ( X 5000).

thrown into the retinal and Microinfarcts were seen in

and glial cells (Fig. Animals

that

5).

have retinal

dary to photocoagulation toid spaces

in the outer

inner nuclear

fibrin

neuronal

vein occlusion

also have plexiform

layer in the acute

secon-

exhibited

layer

phase

MOM, Hayreh SS: unpublished data, tron microscopically, the extracellular

cys-

and in the (Fig.

6) (Tso

1983). Eleccysts were

filled with an exudate that was surrounded by swollen glial cells or swollen neuronal cells (Fig. 6). In this ischemic

retinopathy,

extracellular

associated with swelling of both neuronal cells. When the injury was severe enough, malemma ruptured and the intracellular

cysts

were

and glial the plasswelling

the retina in cystic spaces in the outer plexiform and inner nuclear layers, not unlike those observed in

became extracellular cysts. In CME, ischemia appears to be an important factor. On the other hand, many CME patients recov-

human

ered 20/20 vision

patients

with

CME.

Electron

microscopy

after

a remission

of CME.

How

516

Surv Ophthalmol

28 (Suppl)

May

1984

TSO Fig. 7. Macula of a rhesus monkey that had cyclocryotherapy and ocular hypotony. Horseradish peroxidase tracer infiltrates through the pigment epithelium into the outer layers of the retina. The tracer is picked up by photoreceptor cells and passed along the axons (arrows) to the cone pedicles and rod spher-

ules. The tracer is also taken up by Miiller cells (arrowheads), extending from the external limiting membrane to the internal limiting membrane of the retina (unstained sections, X 260).

can this be explained if the cysts are microinfarcts? Frisen’.” believes that only 44% of the neuronal channels in the retina are required for 20120 vision. Thus, it appears that even though more than half the retina may be infarcted, the patient may still regain 20120 vision when the active pathologic process has subsided. INTRAOCULAR CME

Fig. 8. Retinal pigment epithelium in a rhesus monkey that had cyclocryotherapy and ocular hypotony. Tracer material diffuses in the cytoplasm of a decompensated retinal pigment epithelial cell (E). The adjacent retinal pigment epithelial cell (E,) is free of tracer material. The tracer material accumulates in Bruch’s membrane (arrowheads) beneath E,. Bruch’s membrane adjacent to E is free of tracer, presumably having diffused into the decompensated cell ( X 12000).

EVENTS

ASSOCIATED

WITH

Many intraocular events have been noted so frequently in association with CME that it is thought they may play a role in the pathogenesis of this condition. To study some of these factors cryotherapy was applied to the ciliary body of rhesus monkeys. This produced an intraocular inflammation in the ciliary body and prolonged ocular hypotony for 30 to 60 days.lg Clinically the macula and optic disc were edematous, but no cystoid pattern was noted on fluorescein angiography. Histopathologic study showed extensive disruption of the blood-retinal barrier at the pigment epithelium and at the retinal Using horseradish peroxidase, we vasculature. could see that tracer materials leaked through the RPE into the subretinal space (Figs 7 and 8). Furthermore, leakage from the retinal vasculature was seen in association with increased pinocytotic vesicles and decompensation of the plasmalemma of endothelial cells, resulting in diffusion of tracer in the cytoplasm (Figs. 9 and 10). Many of these animals showed edema of the optic disc comparable to that noted in patients with CME after lens extraction (Fig. 11). However, none of these animals developed cystoid spaces in the retina histopathologitally. This experiment showed that inflammation and hypotony helped to produce disruption of the blood-retinal barrier and papilledema.

ANIMAL

MODELING

517

OF CME

Fig. 9. Retina in a monkey that had cyclocryotherapy and ocular hypoton?. Note marked leakage of tracer material from retina1 vessels into the perivascular space (arrowheads) ( X 4000). Inset shows leakage of tracer (arrows) around a large retinal vein (v) (unstained section, x 130).

rig. 10. Disruption of blood-retinal barrier at the retinal vasculature of an animal that had cyclocryotherspy and ocular hypotony. A: Note increase of pinocytotic activity with tracer material (arrowheads) in the cytoplasm of the endothelial cells (E) and pericytes (P) ( X 5000). L, lumen ofblood vessel. B: Tracer (arrows) infiltrates the cytoplasm of an endothelial cell (E), the basement membrane of the endothelial cell and pericyte (P), and the perivascular interstitial space (arrow) ( X 2500). L, lumen of blood vessel.

SYSTEMIC

DISEASES

ASSOCIATED

WITH

CME

Systemic diseases, such as diabetes mellitus, hypertension, and aging have been shown to be associated with CME. We have produced diabetes in rhesus monkeys either by pancreatectomy or intravenous streptozotocin injections. These animals were observed for a period of2-11 years. The pathologic study using the horseradish peroxidase tracer technique showed that they developed leakage at the pigment epithelium (Fig. 12). Small focal leakage from the retinal vessels was also seen.‘” However, we did not note the typical microangiopathy of

diabetic retinopathy and CME that is so commonly observed in diabetic patients with perimacular capillary closure. One cynomologus monkey developed diabetes mellitus naturally. Its medical history was difficult to trace, but it probably had been diabetic for at least two years. Lens extraction on this animal was performed. So far we have not observed the classic signs of CME. We continue to observe this and similarly treated animals for possible decompensation of the retinal vasculature. Furthermore, we have studied baboons and rhe-

518

Sure Ophthalmol

sus monkeys

with

systemic

28 (Suppl)

May

hypertension

TSO

1984 induced

by

the modified Goldblatt’s technique. Unfortunately, none of these animals has yet developed a classic fluorescein pattern of CME, even though their maculas exhibited generalized edema, shown by slitlamp examination and fluorescein angiography. Histopathologic examination disclosed leakage in the pigment epithelium and the retinal vessels, with

occlusive microangiopathy of the retina. We hope that some of these animals, if given time, will eventually develop CME after lens extraction.

Conclusion To examine experimental models ofCME, I have studied four sets of presumed primary factors: 1) chronic disruption of the blood-retinal barrier; 2)

Fig. 11. Papilledema in an animal that had cyclocryotherapy and ocular hypotony. A: Note the lateral displacement (arrowheads) of the peripapillary retina secondary to swelling of the optic nervehead (unstained section, X 90). B: Horseradish peroxidase tracer is seen around the central section, X 200). C: Tracer material leaks through the retinal artery (A) and vein (V) ( unstained intermediate tissue of Kuhnt (arrowheads) and extends into the subretinal space (unstained sections, X 30).

ANIMAL

MODELING

OF CME

519

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2.

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, ,+.

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6.

7.

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Fig. 12. hlacula

of tracer

of a diabetic monkey showing inliltration material into an isolated pigment epithelial cell

(arrow) and into the perivascular tissue or the retinal vasculature (arrowhead) (unstained sections, X 270). 14.

ischemic conditions that produce retinal tissue damage associated with cyst formation; 3) several intraocular events, such as inflammation and ocular hypotony; and 4) associated systemic diseases, such as diabetes, hypertension, and aging. However, none of these experimental models gave the complete picture of human CME, although each produced some aspects of it. These studies led me to conclude that CME is a multifactorial syndrome that combines a number of these factors to produce a classic picture ofCME in the human patient. This also explains the unpredictable occurrence of CME in our patients and the difficulties in tracking a single pathogenetic mechanism. We are now progressing into the second phase of our animal investigations. By combining the various sets of factors, we may produce a classic picture of CME in animals. Until then, we shall not have a comprehensive understanding of the pathogeneic mechanisms of CME, and preventive and therapeutic measures will be largely empirical.

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This work was supported in part by Public Health Service Grants EYO1903 and Core Grant IF30 EYO1792. Reprint requests should he addressed to Dr. Mark ‘I’so, Departmcnt of Ophthalmology, University of Illinois. Eye and Ear I&rmary. 1855 West Taylor Street, Chicago. IL 60612.