Volume 14Number 9

Reports 707

RPE (Fig. 4, B). With loss of the basal infoldingsdesmosome-like sites between basal infoldings arenot seen. However, the hemidesmosome-like struc-tures continue to connect the basal plasma mem-brane with the markedly infolded basal laminawhich follows the contours of the basal cell sur-face (Fig. 4, B, inset).

Discussion. Many fine ultrastructural studies ofthe retinal pigment epithelium in various specieshave been reported among which are those byCohen,H Dowling and Gibbons,1 and Feeney,Grieshaber, and Hogan,r> but, to our knowledge,the cellular connections described here have notpreviously been reported.

The hemidesmosome-like attachment sites ob-served at the basal surface of the RPE are dif-ferent from the classical hemidesmosomes whichconnect the basal surface of the corneal epitheliumwith its underlying basement membrane. The at-tachments of the RPE are smaller in size, haveless extensive cytoplasmic filaments, and lack anintracytoplasmic density. The attachment sites ofthe RPE have a condensation of small cytoplasmicfibrils, a slight increase in density of the cyto-plasmic membrane, and a few fine fibrils extend-ing across the electron-lucent space into thebasement lamina of the RPE. These structures aresimilar to those on the inner limiting lamina atthe vitreoretinal juncture as described by Foos.(!

This is not unexpected considering that the innerlimiting lamina and basement lamina of the RPEline the basal surfaces of cells that embryologi-cally were two layers of apically apposed neuro-ectodermal cells. Considering the common origin,one might expect to see the same types of attach-ment sites in the epithelial layers of the iris andciliary body.

The desmosome-like attachment sites connect-ing apposed membranes forming the basal in-foldings of the RPE may possibly join the plasmamembranes of an individual cell rather than beingintercellular connections, as is usually the casewith desmosomes. Alternatively, there may be ex-tensive, long-range interdigitation of processesfrom neighboring cells which give the appearanceof attachment sites within a single cell.

There is regional variation of the basal infold-ings in that they are less marked within a fewcells of both the optic nerve head and ora ser-rata. In the rat, cat, and dog, it has been observedthat there may be focal attenuation of the basalinfoldings as a normal anatomic variation of theRPE, in contrast to the findings in normal humaneyes.2

Desmosomes are generally considered to beimportant in adding structural stability to cell-cellcontacts between epithelia.7 A similar functionis presumed for hemidesmosomes which connectepithelia to their basal lamina. The attachmentsites observed at the basal surface of the RPE

might possibly aid in maintaining cohesion be-tween the RPE and Bruch's membrane of thechoroid. It would be interesting to see if theseattachment sites are disrupted under conditionswhich result in a separation of the retinochoroidaljuncture.

From the Department of Ophthalmology, Monte-fiore Hospital and Medical Center/Albert EinsteinCollege of Medicine, 111 E. 210 St., Bronx,N. Y. 10467. This study supported in part byUnited States Public Health Service Grant No.EY00613-4 and No. EY01103A; Fight for Sight,Inc., New York, N. Y.; Research to Prevent Blind-ness, Inc., New York, N. Y.; and The Seeing Eye,Inc., Morristown, N. J. Submitted for publicationSept. 16, 1974. "Dr. Miki is an InternationalResearch Scholar of Research to Prevent Blindness,Inc.

Key words: retinal pigment epithelium, Bruch'smembrane, basal infoldings, choroid, attachmentsites, basement lamina.

REFERENCES1. Karnovsky, M. J.: A formaldehyde-glutaralde-

hyde fixative of high osmolarity for use inelectron microscopy, J. Cell Biol. 27: 137a,1965.

2. Hogan, M. J., Alvarado, J. A., and Weddell,J. E.: Retina, in: Histology of the Human Eye,Philadelphia, 1971, W. B. Saunders Company,p. 393.

3. Cohen, A. I.: A possible cytological basis forthe "R" membrane in the vertebrate eye, Na-ture 205: 1222, 1965.

4. Dowling, J. E., and Gibbons, I. R.: The finestructure of the pigment epithelium in thealbino rat, J. Cell Biol. 14: 459, 1962.

5. Feeney, L., Grieshaber, J., and Hogan, M. J.:Studies on human ocular pigment, in: TheStructure of the Eye. Rohen, J. W., editor.Stuttgart, 1965, Schattauer-Verlag, p. 535.

6. Foos, R. Y.: Vitreoretinal juncture, topograph-ical variation, INVEST. OPHTHALMOL. 11: 801,1972.

7. Kelly, D. E.: Fine structure of desmosomes,hemidesmosomes, and an adepidermal globularlayer in developing newt epidermis, J. CellBiol. 28: 51, 1966.

Biopsy of human scleral-chorioretinal tis-sue. G H O L A M A. PEYMAN, GERALD A.

FISHMAN, DONALD R. SANDERS, DAVID J.

APPLE, AND JOSEPH K. VLCHEK.

The usefulness of a technique for scleral-clwrio-retinal biopsies was evaluated on a human eyedestined for enucleation due to a malignantmelanoma of the choroid. The operative procedurewas without complication. The biopsy specimenwas removed and carefully evaluated both by light

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708 Reports Investigative OphthalmologySeptember 1975

and electron microscopy. The underlying choroidand retinal tissues were successfully preserved bythis technique.

Successful scleral-chorioretinal biopsies havebeen performed on animal models at our institu-tion.1- •-' Because of initial encouraging results, weattempted a similar procedure on an eye that con-tained a malignant melanoma of the choroid andwas destined for enucleation. A modification ofthe experimental technique was used to removethe biopsy specimen. Methods of the biopsy pro-cedure, histologic sections of the specimens, aswell as potential clinical implications will be dis-cussed.

Case report. A 77-year-old white woman firstnoticed blurred vision in her left eye in August1974, four months prior to admission. She wasseen in October 1974 by an ophthalmologist whena diagnosis of malignant melanoma of the choroidwas made. The patient's general health was good.She was referred to our institution for furtherevaluation and therapy.

General physical examination was normal. Onocular examination, vision was correctable to 20/20in the right eye and 20/100 in the left eye. Ex-ternal and motility examinations were normal.Both pupils reacted normally to direct and con-sensual light stimuli. The biomicroscopic examina-tion of the anterior segment showed a normalcornea, anterior chamber depth, angle, and iris.Ocular pressures were normal and equal in botheyes. There were 2+ bilateral nuclear scleroticchanges while the vitreous was clear. A fundusexamination of the right eye was normal. In theleft eye there was a large black tumor mass ap-proximately 8 by 10 mm. just superotemporal andslightly inferotemporal to the left fovea extendingfrom 12 to 4 o'clock. A serous detachment of theretina was seen superiorly and inferotemporallycapping the periphery of the tumor from 12 to 5:30o'clock. A relative loss in visual field demonstratedon a Goldmann perimeter corresponded to theareas of tumor and serous detachment. B-scanultrasonography showed a large tissue echo masselevated in the posterior segment of the left eyewith a serous detachment. An electroretinogramshowed a scotopic b-wave recording which washalf the amplitude of the right eye. Fluoresceinangiography showed a late staining pattern of thelesion consistent with a melanoma. A P-32 testwas positive. Because of extensive subretinal fluidaccumulation and both the tumor size and itslocation, a local excision of the tumor was notadvised.'1 With appropriate patient consent, a localbiopsy of retinal and choroidal tissue precededenucleation of the patient's left eye.

Technique. Under general anesthesia, a 360 de-gree peritomy was performed. The rectus muscleswere isolated with 4-0 silk sutures. A Peymaneye basket (Fig. 1, A) was sutured to the sclera

in the superior nasal area (Fig. 1, B). By indirectophthalmoscopy the retina was attached withinthis area. The ring sutured to the sclera was 12mm. in diameter. An 8 mm. trephine (Fig. 1, C)was utilized to demarcate approximately half thethickness of the sclera. The sclera was then dis-sected (Fig. 1, D) using a No. 64 Beaver blade.After the removal of a partial-thickness disciformscleral button, the scleral tissue was stored in asaline solution containing 200 /tg per milliliter ofgentamicin. A 4 mm. skin trephine was then usedto demarcate a portion within the previous 8 mm.partial scleral dissection (Fig. 1, E). The tissuesurrounding the 4 mm. trephined area was thendiathermized (Fig. 1, F). Using a No. 52 Beaverblade, the sclera, choroid, and retina were pene-trated through the margin of the central 4 mm.button. The section was then completed with acurved corneal-scleral scissors (Fig. 1, G). Thespecimen was obtained without complication asthere was no hemorrhage from choroidal tissue.During the procedure a moderate amount ofvitreous loss occurred through the biopsy site. Theprotruding vitreous was excised with scissors andremoved. After removal of the biopsy specimen,the resected area was covered with the previouslytrephined 8 mm. partial thickness sclera using firstfour interrupted and then running 9-0 nylon sutures(Fig. 1, H). The intraocular pressure was thenreestablished by the injection of air through thegraft edge utilizing a 30-gauge cannula (Fig. 1, I).After removal of the eye basket, enucleation wasperformed using standard techniques.

After fixation in a 10 per cent formaldehydesolution, the eye was sectioned vertically. The graftsite was grossly evident and free of hemorrhage.The area surrounding the biopsy site was attached.These gross observations were reconfirmed byhistologic preparation (Fig. 2). The biopsy seg-ment was immediately fixed in a neutral bufferedsolution of 1 per cent formaldehyde/1 per centglutaraldehyde. After overnight fixation, all tissuewas washed in Sorenson's phosphate buffer, pH7.35, postfixed with Dalton's chrome osmium fortwo hours, again washed in phosphate buffer, de-hydrated in graded ethanols and propylene oxide,and embedded in Araldite. Thick sections weremade along the diameter of the tissue with aSorvall MT2-B ultramicrotome and stained withMallory's stain for five seconds. Appropriate areaswere thin-sectioned and stained with uranyl ace-tate and lead citrate, and subsequently examinedwith a JEM 100B electron microscope.

Results. On light microscopy the tumor wasfound to be a typical spindle B melanoma on thechoroid. Observations of the retinal biopsy siterevealed that the retina was attached (Fig. 3). Ahigh-powered view of the light microscopy sec-tion demonstrated normal retinal and choroidalstructures. The choroidal and retinal vessels weremoderately congested. Electron microscopy re-

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Reports 709

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Fig. 1. The scleral-chorioretinal biopsy procedure.

vealed normal structures including the photo-receptors, retinal pigment epithelium, and remain-ing retinal structure (Fig. 4).

Discussion. Our report emphasizes that a chorio-retinal biopsy, with this technique, can be donewith little complication. We did not experienceany retinal or choroidal hemorrhage during theremoval of the specimen. In the future, the initialtechnique will be modified by performing a vitrec-tomy in the area of the biopsy site to preventpossible postoperative adhesions to the excisedarea. Of equal importance, the technique demon-strates that retinal tissue can be removed withoutdestruction to the underlying retinal structures.This was clearly demonstrated by light and elec-tron microscopic examinations. The moderate con-gestion of both retinal and choroidal vessels waspresumably related to the diathermy applied to thesurrounding biopsy site. The development of thistechnique will enable the further investigation ofretinal diseases previously unavailable to both lightand electron microscopic examinations. One im-portant area of regard includes the category ofhereditary retinal dystrophies in which early tis-sue examinations are lacking. Additionally, thetechnique would be of value in patients with un-diagnosed fundus lesions. This would include thedifferential diagnosis of sympathetic ophthalmiafrom nonspecific uveitis. As with most new pro-cedures careful patient selection is mandatory.Presently, these diagnostic procedures are beingapplied to eyes that have a long history of legalblindness. Further modifications and other areasfor implementation are under investigation at our

"Vfc

Fig. 2. Section along diameter of the biopsy but-

Fig. 3. Enlargement of specimen shown in Fig. 2,emphasizing normal morphologic relationshipsamong all retinal layers and choroid ( C ) . ONLdenotes outer nuclear layer (x260).

' - 'A *

ia^><

Fig. 4. The outer retinal layers shown in Fig. 2,illustrating normal pigment epithelial cells ( P ) ,associated outer (OS) and inner (IS) photorecep-tnr segments, CC denotes choriocapillaris (x5,000).

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710 Reports Investigative OphthalmologySeptember 1975

institution. Further investigations and clinical trialsare necessary to appreciate the full diagnostic valueof this technique.

From the Department of Ophthalmology, Uni-versity of Illinois Eye and Ear Infirmary, Chicago.Research funded by Grants EY 1107-02 and EY24-16 from the National Institutes of Health, andin part by grants from the Illinois Lions Club andthe National Retinitis Pigmentosa Foundation. Sub-mitted for publication March 18, 1975. Reprintrequests: Dr. C. A. Peyman, University of IllinoisEye and Ear Infirmary, 1855 W. Taylor St., Chi-cago, 111. 60612.

Key words: scleral-chorioretinal biopsy, retinaldystrophies, malignant melanoma, sympatheticophthalmia.

REFERENCES1. Peyman, G. A., Meisels, H. E., Batko, K., et al.:

Full-thickness eye wall biopsy. I. An experi-mental approach to the tissue diagnosis andstudy of choroidal and retinal lesions, INVEST.OPHTHALMOL. In press.

2. Peyman, C. A., Homer, P., Kasbeer, R., et al.:Full-thickness eye wall biopsy. II. In primates,INVEST. OPHTHALMOL. 14: 565, 1975.

3. Peyman, C. A., Diamond, J., and Apple, D. J.:Clinical pathological correlation of six pig-mented choroidaT neoplasms treated with fullthickness eye wall resection, Can. J. Ophthal-mol. In press. 1975.

The response of mouse ocular tissues tocontinuous near-UV light exposure. S.ZLGMAN, J. GROFF, T. YULO, AND T.VAUCHAN.

Continuous exposure of mice to near-ultraviolet(UV) light (black light) over a period of 19 weeksinduces adverse alterations in lens protein chemis-try, in lens epithelial cell differentiation, and inretinal photoreceptor structure at more than adoubled rate as for 12 hours a day of intermittentexposure. No histologic changes were found in thecornea. The results may indicate the presence ofrepair mechanisms in these ocular tissues for dam-age induced by radiant energy.

We have shown that intermittent exposure ofmice to black light for many weeks resulted indamage to their lenses and retinas. These includedsevere thinning of the outer segments and macro-phagic invasion between 10 and 17 weeks, andtotal destruction of the photoreceptors by 60 to 70weeks.1 Between 30 and 60 weeks, lens epithelialcells lost their ability to differentiate into fibercells and pyknotic nuclei permeated the cortex.Lens protein synthesis and growth were alsoinhibited, and a buildup of insoluble proteins to

levels much higher than those of aging controlswas found.2 From 60 weeks on, many lens corticalopacities were seen.

In this study, mice of the same strain wereexposed to black light 24 hours a day. By com-paring the times of appearance of the changes in-duced by continuous light to those induced byintermittent light, the dose dependency and re-pair potential of the ocular tissues damaged bynear-ultraviolet (UV) light can be assessed.

Materials and methods. Two hundred 8-weekold female A/J mice (Jackson Laboratories, BarHarbor, Me.) were divided into two groups andhoused, five per plastic cage. One week after theanimals were received, the experimental conditionswere imposed. Using heavy black plastic sheeting,the air conditioned mouse room was divided intotwo separate chambers, each additionally venti-lated by a fan. In one chamber, 40 W black lighttubes (General Electric, BLB) were fastened tothe top of the shelf 3 inches above the cages. Theaverage intensity of the lamps, as measured withan Ultraviolet Products, Inc. Long wave-UV lightmeter, was 450 /tW per square centimeter. Allcages received the same amount of UV-irradiationand were periodically rotated. Both chambers hada 12-hour on-off period of incandescent visiblelight at 50 foot candles. Animals were fed, housed,and cared for as described in Reference 1.

The mice were weighed at time zero, four,eight, thirteen, and nineteen weeks. Four groupsof six mice each were then selected at randomfrom the two chambers and were killed withether. Eyes were immediately removed after deathand dissected. Lenses were quickly examined,weighed, and frozen. Eight eyes from each groupwere removed and fixed in 4 per cent glutaralde-hyde for histologic studies. Eye sections of 7 fiwere stained with hematoxylin and eosin.

The lenses of each group were pooled and usedfor biochemical study as indicated in Reference 1.

Results. The only grossly observable differencesbetween the irradiated and control animals wereredness and irritation of the skin of the ears andtails, a situation which continued throughout theduration of the experiment. Erosion of the earsand tails after scab formation was common duringthe second half of the experiment, but no skintumors were seen.

No differences in body weights were observedbetween the irradiated and control animals duringthe 19 weeks of the experiment, but the averagecontrol mouse lens weight was greater from 4weeks on by 0.4 to 0.8 mg. (Fig. 1). The meanvalues are significantly different at a confidencelevel of one chance in 175.

No significant differences were observed in thetotal soluble protein increment between controland irradiated mouse lenses during the 19-week

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