The onset of pigment epithelialproliferation after retinal detachment

Don H. Anderson, Walter H. Stern, Steven K. Fisher, Page A. Erickson, andGerard A. Borgula

The adult mammalian retinal pigment epithelium (RPE) is mitotically inactive, yet retains thecapacity to proliferate under certain conditions. To determine the onset of RPE proliferationafter retinal detachment, toe examined experimentally detached cat retinas of 12, 24, 48, and 72hr duration. An additional animal served as a nondetached, sham-operated, control. 3H-thymidine ivas injected into the vitreous chamber and the eyes were processed for lightmicroscopic autoradiography. Autoradiograms from both the control and the 12 hr detachmentshowed no evidence of labeled RPE nuclei; however, labeled nuclei were present at both 24 and48 hr after detachment. Labeled nuclei per millimeter of RPE at 24 hr were 55% of the 48 hrrate. Mitotic figures were noted only at 48 and 72 hr after detachment. No labeled RPE nucleiappeared in autoradiograms that bordered the detachment zone. Electron micrographs showedthat proliferating RPE cells assume several configurations, some of which have been reported inother species. The proliferative response of the RPE occurs much sooner than had previouslybeen thought. It appears to be a local effect that does not involve retinal regions beyond thedetachment boundaries, and it may have potentially adverse effects when the retina and RPEare reapposed after retinal reattachment surgery.

Key words: retinal detachment, pigment epithelium, proliferation, retina,autoradiography

Larly in development, the retinal pigmentepithelium (RPE) cells undergo a rapid phaseof mitosis that is completed prior to the dif-ferentiation of the photo receptors.1 Aftermaturation, the RPE cells are generally con-

From the Department of Biological Sciences, Universityof California, Santa Barbara; the Department of Oph-thalmology, School of Medicine, University of Cali-fornia, San Francisco; and the Veterans Administra-tion Medical Center, San Francisco, Calif.

This study was supported by National Eye Institute re-search grants EY-02082 and EY-00888, by a researchgrant from the U.S. Veterans Administration, and byN.E.I. Research Career Development Award (RO-00174 to S. K. F.).

Submitted for publication June 25, 1980.Reprint requests: Don H. Anderson, Department of Bio-

logical Sciences, University of California, Santa Bar-bara, Calif. 93106.

10

sidered to be mitotically inactive, as are neu-rons and a few other cell types in adultmammals. One exception, however, mayoccur in the adult rat retina, where the RPEcells apparently turnover at a very low rate.2*3

Several studies have shown that the adultmammalian RPE retains the capacity to pro-liferate under certain conditions, i.e., incases of tumors of the RPE and choroid,4'5 inresponse to injury from intense light expo-sure,6"8 after cryotherapy,9 or after retinaldetachment.10

In this report we show that the onset ofRPE proliferation in the cat begins about 24hr after the retina is surgically detached fromthe RPE and that the response is confined tothe zone of detachment. The proliferationmay continue even after retinal reattachment

Volume 21Number 1, Part 1 RPE proliferation after retinal detachment 11

surgery.11 Apparently, the separation of theretina from the RPE is sufficient to releasethe RPE cells from their postmitotic state andto initiate the sequence of events leading tocell division.

MethodsTo determine the onset of proliferation, mon-

ocular retinal detachments were made in adultcats. Four eyes were enucleated and fixed at 12,24, 48, and 72 hr after detachment. An eye from afifth animal, which underwent all of the surgicalprocedures except for the detachment surgery,was used as a sham-operated control. All theanimals were maintained on the same lightingschedule (12L:12D).

Surgery. Animals were anesthetized and extra-capsular cataract extraction was performed througha 180-degree corneal incision. The posterior cap-sule was excised, and that was followed by a partialopen-sky vitrectomy. The cornea was suturedclosed and the eye was allowed to heal for 4 weeks.In the second stage of the surgery, the posteriorvitreous cortex was removed with a vitreous suc-tion cutter, which was then used to place a smallretinal hole in the superior nasal retina. The suc-tion cutter was replaced with a curved needlethrough which isosmotic Ringer's solution was in-jected into the subretinal space with a mechanicaldrive syringe. This resulted in a bullous retinaldetachment that radiated outward from the su-perior nasal quadrant to below the optic disc. Onlydetached regions distant from the retinal holewere studied histologically.

Fixation. All the eyecups were fixed by immer-sion in 1% glutaraldehyde, 1% paraformaldehydein phosphate buffer (pH 7.1) for approximately 12hr. After aldehyde fixation, the eye cups werewashed in phosphate buffer (plus sucrose at 45mg/ml), post-fixed in 2% barbital (Veronal)acetate-buffered osmium tetroxide, dehydratedin a graded ethanol-H2O series, and embedded inAraldite 6005.

Autoradiography. A solution of 200 /xCi of 3H-thymidine (50 mCi/mmol specific activity) in 0.2ml of distilled H2O was injected into the vitreouschamber of the control eye and into the eyes thathad had detachment surgery 12, 24, or 48 hr pre-viously. All injections were made through the parsplana with a 26-gauge needle. A preplaced suturesurrounding the injection site was closed imme-diately after withdrawal of the needle in order toensure that all the injected fluid remained withinthe eye. All the eyes were injected during the

middle part of the light cycle in order to control fordiurnal fluctuations in the cell cycle. After an in-cubation interval of 3 hr, the eyes were enucle-ated, the anterior one third of the globe was dis-sected away, and the eyecup was immersed infixative. Light microscopic autoradiograms wereprepared from 1 /uin tissue sections that weredipped in a 1:1 solution of Kodak NTB-2 and dis-tilled H2O maintained at 43° C. The slides werethen placed in light-tight boxes and exposed from5 to 10 days at 4° C. Finally, the slides were devel-oped for 2 min in full-strength D-19 (at 20° C),washed, fixed, and stained with methylene blue-azure II or basic fuchsin.

Samples for autoradiography were taken fromthe following regions: (1) normal retina from thesham-operated control eye; (2) an area of detach-ment (5 mm in length) from each of the 12, 24, and48 hr eyes; (3) ora serrata close to the zone ofdetachment from the 12 and 48 hr eyes; (4) ciliaryepithelium close to the zone of detachment fromthe 24 and 48 hr eyes; and (5) an area of attachedretina adjacent to the detachment zone (a transi-tion zone) from the 48 hr eye.

The number of labeled RPE nuclei per millime-ter of RPE was determined for the control, 12.24, and 48 hr animals (1 and 2 above) in the follow-ing manner. A minimum of 13 autoradiograms,each containing ten 1 /u,m tissue sections, wereprepared for each animal. One section was ran-domly selected from each of the 13 slides forcounting purposes. Because each of the tissueblocks was 5 mm long, a minimum of 65 mm ofRPE was examined and counted in each of theeyes.

ResultsLight microscopic examination of 1 /am

sections from the control eye showed that thesurgical procedures preceding the detach-ment had no effect on retinal-RPE mor-phology. The outer segments of both rodsand cones showed their usual cylindrical or-ganization, and the relationship between theouter segments and the apical RPE appearednormal (Fig. 1, a). Phagosomes could beidentified within the RPE cell bodies andwithin the apical RPE processes, indicatingthat the phagocytic role of the RPE cells re-mained unaffected. No labeled RPE cell nu-clei were ever observed in autoradiogramsfrom the control eye, and only a few scatteredsilver grains were present over the inner

12 Anderson et at.Invest. Ophthalmol. Vis. Sci,

July 1981

Fig. 1, a and b. a, Light microscopic autoradiogram from the control retina. The relationshipbetween the photoreceptor outer segments (OS) and the retinal pigment epithelium (RPE) isnormal. The RPE nuclei (N) are not labeled. A few scattered silver grains appear over thephotoreceptor inner segments (IS). (x840.) b, Autoradiogram from the 12 hr detachment.There is no evidence of labeled RPE cell nuclei (arrow) in this animal. (x450.)

segments of the photoreceptors (Fig. 1, a). Asexpected, there was no labeling of photore-ceptor nuclei or of tapetal cell nuclei.

In the 12 hr detachment some importantmorphological changes were detected in theRPE cells; however, no evidence of labeledRPE nuclei or mitosis was found (Fig. 1, b).Phagosomes that were clearly identifiablewithin the RPE cell bodies of the control eyewere absent in the RPE cells of the 12 hrdetachment. Furthermore, the apical pro-cesses that normally ensheath the outer seg-ments were gone and only a fringelike apicalborder could be detected in the light micro-scope. A few labeled cells, presumably invad-ing white blood cells, were found in the ret-

ina close to the outer margin of the innernuclear layer, thereby indicating that the la-beled compound had indeed been availablefor incorporation.

In contrast to both the control and the 12hr detachments, heavily labeled RPE nucleiwere evident at both 24 and 48 hr after de-tachment, and mitotic figures were observedat both 48 and 72 hr after detachment (Fig. 1,d). At 24 hr, labeled RPE nuclei were foundoccasionally, and at 48 hr it was not unusualto find two or more adjacent RPE nuclei la-beled (Fig. 1, c). Labeled nuclei per millime-ter of RPE 24 hr after detachment were about55% of the 48 hr rate (Fig. 2). Neither thephotoreceptor nuclei nor the tapetal cell mi-

Volume 21Number 1, Part 1 RPE proliferation after retinal detachment 13

Fig. 1, c and d. c, Autoradiogram from the 48 hr detachment. Four adjacent RPE cell nuclei areintensely labeled, indicating that they were in the S-phase of the cell cycle for at least a portionof the 3H-thymidine incubation period. The tapetal cell nuclei (arrow), which lie immediatelyabove the RPE monolayer, remain unlabeled. (xl200.) d, Light micrograph from the 72 hrdetachment. In the center of the micrograph, a mitotic figure in anaphase appears within theRPE cell monolayer. The chromatids have migrated to opposite poles of the cell. The cell onthe right side of the micrograph appears to be in the final stages of cleavage prior to division(arrow). (X1650.)

clei showed any evidence of 3H-thymidineincorporation at these times.

We examined auto radiograms of the RPEthat bordered the area of detachment inorder to determine whether the proliferation

was strictly localized to the detachment zoneor was a more generalized response. Wefound no mitotic figures or labeled RPE cellsin these areas at 12, 24, or 48 hr after de-tachment. Furthermore, we found no evi-

14 Anderson et al. Invest. Ophthalmol. Vis. Set.July 1981

LJ 4Q.Ct

li.

oe 3EUJ

iHi i i i

C 12 24 48TIME AFTER RETINAL DETACHMENT (h)

Fig. 2. Number of labeled RPE nuclei/mm as afunction of the time (hr) since retinal detachmentsurgery. Each vertical bar represents the meannumber of RPE nuclei labeled during one 3 hrinterval after a single injection of 3H-thymidine.C, Control animal; error bars = ±1 S.D.

dence of mitosis in the ciliary epithelium ad-jacent to the detachment zone and, with oneexception, no ciliary epithelial cells were la-beled at the times sampled.

After 72 hr, small areas of RPE hyperplasiabegan to appear in the RPE of the detachedretina. In Fig. 3, the proliferating RPE cellsappear histologically as a second layer of cellsparallel to the original monolayer. Their nu-clear membranes are more scalloped thanthose of normal RPE cell nuclei, and theusual apical-basal surface polarity is not evi-dent. The cell surfaces are lined with manyshort, undifferentiated processes that some-times interdigitate with similar processesfrom adjacent cells. The cells are joined byadhering junctions to each other and to cellsin the original monolayer (Fig. 3, inset).There is no sign of any fibrous extracellularmatrix or basement membrane associatedwith the proliferating cells at this stage.

DiscussionRPE proliferation has been shown to occur

in several species, including humans, in re-sponse to a variety of different stimuli such asxenon arc6 and laser7 photocoagulation, andexposure to ophthalmoscopic light8 and as aresult of overlying choroidal tumors,5 cryo-therapy,9 or retinal detachment.10 The mor-

phological features of the proliferative re-sponse are similar in all these studies despitethe different conditions that elicit it. Theareas of proliferation assume one of sev-eral specific configurations. With time, theseareas may appear as placques of spindle-shaped cells. The normal polarity of the cellsis usually modified, and they are intercon-nected by adhering junctions. Their pig-mentation is altered to some degree, andthey are often associated with a fibrous inter-cellular matrix presumed to be secreted col-lagen. The RPE cells are capable of separat-ing from Bruch's membrane, and there issuggestive evidence that they become mi-grating phagocytes in the subretinal space.

This morphological profile is also found incat retinas detached for longer than a fewdays.11' 12 Proliferating RPE cells appear his-tologically as localized mounds of cells joinedby adhering junctions (Fig. 3, inset) to eachother and to the original layer, as mul-tilayered sheets of cells parallel to the origi-nal monolayer (Figs. 3), or as folds of cellsthat protrude into the subretinal space. Inaddition, electron microscopic observationsshow that some RPE cells in the detachedretina separate from Bruch's membrane andfloat freely in the subretinal space. Thesecells tend to be pigmented in areas of pig-mented retina and nonpigmented in tapetalregions, thereby indicating their probableRPE origin. The cells appear to migrate to-ward the tips of the degenerating outer seg-ments, where they phagocytize outer seg-ment debris.

Only rough estimates of when the pro-liferative response first begins have beenmade in previous investigations. Proliferatingcells were first observed as early as 2 weeksin light-damaged retinas6 and between 1 and8 weeks in experimentally detached retinas.10

The issue is a significant one because of theinvolvement of proliferating RPE cells inmassive periretinal proliferation13* 14 and be-cause of the potentially adverse effects ofRPE proliferation with respect to retinal reat-tachment surgery. If the experimentally de-tached cat retina accurately reflects the timecourse of the response in other mammalianretinas, then the onset of the proliferative re-

1 r'

Volume 21Number 1, Pert 1 RP£ proliferation after retinal detachment 15

Fig. 3. Area of proliferated RPE cells 72 hr after experimental detachment in the cat. A secondlayer (L2) of RPE cells parallels the original monolayerf LI) in the upper portion of the figure.The new layer of RPE cells has not retained the apical-basal polarity characteristic of normalRPE cells. BM, Bruch's membrane. Inset, New cells are linked by adhering junctions. (X9600;inset x 150,000.)

sponse occurs much sooner than had previ-ously been reported.

In the cat retina and in all other vertebrateretinas examined thus far, processes from theapical RPE surface envelop or ensheath theouter portion of both rod and cone outersegments.15 This close intercellular associa-tion between the ensheathing processes andthe outer segments provides the structuralframework for the exchange of ions and mole-cules between the outer segments and theRPE cells,16' l7The loss or disruption of thisnormal metabolic exchange through detach-ment may act as the stimulus for the dedif-ferentiation of RPE cells and the resultingproliferative response. This type of prolifera-tion is not without precedent in other tissues.

Other in vivo examples of stimulus-depen-dent DNA synthesis and cell division in mi-totically quiescent cell populations occur inmammalian liver, kidney, uterine and lensepithelium, mammary gland, and severalother tissues.18 In all these examples, theproliferative response occurs after a singlestimulus application and becomes evidentonly after an interval of 12 to 72 hr. In someof these systems, the response involves asignificant fraction of the cell population.Whether or not this is true for the RPE re-mains an intriguing but unanswered question.

There are several potential pathologicalconsequences of reapposing the retina toproliferating RPE cells. Our results pointtoward two related conclusions that could ad-

16 Anderson et at. Invest. Ophthalmol. Vis. Sci.July 1981

versely affect the re-establishment of the nor-mal photoreceptor-RPE interface and there-fore the return of vision after retinal reat-tachment surgery. First, in the earlier stagesafter reattachment surgery, the RPE maycontinue to proliferate. We have identifiedRPE mitotic figures in cat retinas that havebeen detached for 2 weeks and reattached for1 month.n What is not yet certain is whetherthis represents the residual response to de-tachment or whether the proliferation con-tinues uninterrupted despite reapposition ofthe photoreceptors to the apical RPE. Sec-ond, the presence of multiple layers of RPEcells may interfere with the re-ensheathmentof regenerating outer segments by apicalRPE processes and therefore with the phago-cytosis of shed-disc packets and with thetransfer of metabolites from the choroidalvasculature to the retina. It remains to beseen whether the daughter cells adjacent tothe original RPE monolayer are able to fulfilltheir specialized role in the outer segmentrenewal process after retinal reattachment.

Many of the major characteristics of RPEproliferation in the detached cat retina havealso been identified in cases of human reti-nal detachment.19 Consequently, the experi-mentally detached retina can be used as amodel system in providing quantitative esti-mates of the rate and time course of RPEproliferation, in determining the effective-ness of various pharmacological agents in in-hibiting proliferation, and in assessing theviability of the restructured RPE-photore-ceptor relationship after retinal reattachmentsurgery.

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