Experimental Retinal Detachment with a Sulfated Polysaccharide

Experimental retinal detachment witha sulfated polysaccharide

Wallace M. Landholm and Robert C. Watzke

Research in the pathophysiology of retinal detachments would be aided if an experimentalmodel of such a lesion could be achieved. In an attempt to produce such a model, it wasfelt essential to cause vitreal as well as retinal alterations. This role of vitreoretinal adhesionsand liquefaction of the vitreous as the two main anatomic factors in the etiology of theidiopathic type of retinal detachments has been stressed by Teng and Chi.1 Balazs2 notedthat in the rabbit the hyaluronic acid has its highest concentration in the boundary layernext to the retina and possibly acts as a barrier to diffusion. Harris3 noted that hyaluronicacid sulfate caused vitreous liquefaction. We, therefore, injected hyaluronic acid sulfate intothe vitreous of a living rabbit and noted that this caused many vitreous alterations leadingto retinal detachments comparable to the idiopathic type of retinal detachment found inhumans.

C,'hodin4 first reported on experimentalretinal detachments in 1875. Since then,various authors have attempted to producea model for experimental retinal detach-ments in animals which would closely re-semble idiopathic retinal detachments inhumans. Until the present time, such amodel for the production of retinal detach-ments has not been achieved. Since spon-taneous reattachment of the retina occursin the normal eye of a rabbit following aphysically induced retinal tear,5-6 it ap-pears that the retinal tear is not the onlyfactor in maintaining a detached retina,but that some other abnormality of thevitreous, retina, or choroid is necessary.

Teng and Chi1 have stressed the role of

From the Department of Ophthalmology, Collegeof Medicine, State University of Iowa, IowaCity, Iowa.

Supported by a Training Grant from the NationalInstitute of Neurological Diseases and Blindness(NB-5080).

vitreoretinal adhesions and liquefaction ofthe vitreous as the two main anatomic fac-tors in the etiology of the idiopathic typeof retinal detachment. The presence ofvitreous shrinkage has also been noted bymany authors.7"13 Balazs2 has described thevitreous as a meshwork of collagen fibersinterlaced with coiled hyaluronic acidmolecules. He also noted that in the rabbitthe hyaluronic acid has its highest concen-tration in the boundary layer next to theretina and possibly acts as a barrier to dif-fusion. A sulfated form of hyaluronic acidwhich does not occur normally in nature14

was noted by Harris3 to produce vitreousliquefaction. From the foregoing it wouldseem clear that if one were to produce aretinal detachment which is at all compar-able to that in humans, a method should beused which would produce vitreous lique-faction with as little trauma as possible tothe retina and underlying choroid.

With this aim, it was the purpose of ourstudy to inject hyaluronic acid sulfate into

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Retinal detachment with sulfated polysacclwride 43

the posterior vitreous of a living rabbitand to observe alterations in the vitreousand the occurrence of retinal detachment.Periodically, the animals were to be killedfor gross and histologic examinations.

Materials and methodsRabbits of approximately equal weights (about

2.5 kilograms) and of either sex were used. Allthe rabbits but two were pigmented becausefundus details are seen more clearly in pigmentedrabbits. All intraocular injections were performedunder aseptic conditions and under Nembutal andThorazine anesthesia. The pupils were maximallydilated with 10 per cent Neo-Synephrine and 2per cent homatropine. The eye was fixated by fixa-tion forceps at the insertion of the superior andhorizontal rectus muscle. The lids were retractedby a Williams eye speculum. Solutions of 2.5 percent, 3.15 per cent, and 5 per cent were freshlyprepared by dissolving 100 mg. of sterile, purifiedhyaluronic acid sulfate with pyrogen-free, steriledistilled water.15 A tuberculin syringe was utilizedto measure accurately the solutions of 2.5 per cent,3.15 per cent, and 5 per cent into 0.1 c.c. and0.2 c.c. volumes for the injection of hyaluronicacid sulfate in measured amounts of 2.5 mg.,5 mg., 7.5 mg., and 10 mg. A one-half inch No.30 gauge needle on the tuberculin syringe wasthen placed temporally to the superior rectus mus-cle and approximately 4 mm. posterior to thelimbus. The needle was then inserted throughthe sclera at the edge of the pars plana ciliaris.Because of the fact that the pars plana ciliaris inthe rabbit is a very narrow structure, it was re-alized that, although it was planned to insert theneedle in this area, occasionally it might enter theperiphery of the retina, and on several occasionsa perforation of the periphery of the retina wasnoted. These perforation sites in the periphery ofthe retina were observed to heal quickly withoutthe retina becoming detached at this site.

Through the dilated pupil the operator con-tinuously monitored the course of the needle withthe binocular indirect ophthalmoscope. The pointof the needle was directed toward the visual streakand when it reached the posterior vitreous, thematerial was injected. The fellow eye was usedas a control by injecting an equal volume of sterilewater in a like manner into the posterior vitreous.Immediately following the intraocular injections,the momentary increase in intraocular pressurecaused the cornea to become slightly steamy, butthis cleared in seconds.

A standard Schiotz tonometer was used to ob-tain some estimate of the intraocular pressure atvarious intervals. Measurements were taken priorto injection, one hour after injection, and threeweeks following injection.

Stereo fundus photographs were taken in Ko-dachrome and color Polaroid with the Zeiss fun-dus camera.10 The rabbits were sacrificed atperiodic intervals and the eyes were removed forgross and microscopic examination. All results arebased on the examination of such enucleated eyes.

Results

1. Alterations in the vitreous.A. Vitreous liquefaction. In 100 per cent

of the eyes injected with hyaluronic acidsulfate, liquefaction of the vitreous oc-curred in varying amounts. In all injectedeyes, liquefaction of the vitreous was ap-parent when such eyes were examined onboth gross and microscopic section. Suchalteration was characterized by the pres-ence of large intravitreal, fluid-filled cavi-ties surrounded by condensed vitreal bandsand membranes. The vitreous maintainedsome normal attachments to the retina, butsome pathologic vitreoretinal adhesionswere present. Liquefaction of the vitreoustook place as early as two days after injec-tion and was quite evident in the eyesexamined three months or more followinginjection. No attempt to measure theamount of liquefaction was made becauseof the heterogenous nature of the vitre-ous,17 but on gross section this changecould easily be observed. In the controlgroup vitreous liquefaction was not seenand the vitreous remained in its gel form(Figs. 1 to 4).

No vitreal or retinal changes were ob-served clinically in the enucleated controleyes, other than at the site of the injection.This rapidly healed without resulting in aretinal detachment by either ophthalmo-scopic or histologic examination.

B. Vitreous contraction. In addition tothe vitreous liquefaction as noted by Har-ris,3 we also noted in the majority of eyesother vitreous alterations including a defi-nite contraction of the vitreous. This changewas fairly characteristic; the vitreous wascontracted into a funnel-shaped, somewhatopaque mass with its apex at the opticnerve and with occasional vitreoretinal ad-hesions to the posterior pole (Figs. 1, 3,and 4). The base of this contracted vitre-


44 Landholm and Watzke Inocstigatioe OphthalmologyFebruary 1965

ous appeared to be firmly attached to theposterior lens capsule with many of itsattachments to the pars plana ciliaris stillintact. In vivo, even with the biomicro-scope, this contracted vitreous frequentlywas difficult to distinguish from a centralposterior lens opacity. However, in mostinstances one could visualize the funduswith the indirect ophthalmoscope through

the periphery of the lens, where the con-tracted vitreous was quite thin.

C. Vitreous strands. In several eyes vitre-ous strands were noted to extend from thecentral mass of contracted vitreous to theretina at a point which apparently con-tained a vitreoretinal adhesion (Figs. 1,3, and 4).

2. Retina. An even more interesting al-

\

Fig. 1. Eighteen clays after injection. There is vitreous liquefaction and contraction with anearly retinal detachment. S, sclera; Rd, retinal detachment; VI, vitreous liquefaction; a, vitreo-retinal adhesion; Vc, vitreous contraction; v, visual streak; L, lens; Jr} iris. (*9.)

Fig. 2. Control fellow eye of Fig. 1. The vitreous is clear and intact with no liquefaction orcontraction. A small artifactitious retinal detachment occurred during photography. S, sclera;R, retina; V, vitreous; L, lens; lr, iris; C, cornea. (x9.)


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Fig. 3. Five days after injection. There is vitreous liquefaction and contraction. S, sclera; R,retina; VI, vitreous liquefaction; a, vitreoretinal adhesion; Vc, vitreous contraction; L, lens;It, iris. (x7.)

Fig. 4. One month after injection. There is vitreous liquefaction and massive contraction. Notea thin vitreous strand (see arrow) and its shadow below on the visual streak. IT, iris; L, lens;Vc, vitreous contraction, V*, vitreous strand; v, visual streak; R, retina; S, sclera. (xlO.)

teration was the development of a retinaldetachment which resembled in many waysthe serous retinal detachment of humans.This detachment was noted in 13 of 21eyes injected.

A series of fundus photographs was takento record the development of one of theretinal detachments in the eye of a livingrabbit. This was followed with gross andmicroscopic studies of the same eye. Thefollowing sequence of events occurred inthis rabbit: Approximately two and one-

half days following the injection of 5 nig.of a 2.5 per cent solution of hyaluronic acidsulfate into the posterior vitreous, a slighthaze was noted in the vitreous with the ap-pearance of a white patch on the retinabelow (Fig. 5).

On the eighth day following injection,the misty haze in the vitreous, which wasnot unlike the typical vitreous contraction,was still present. This haze extended to thepatch of pale retina below. This locale ap-peared to be migrating anteriorly as though


46 Landholm and Watzke Investigative OphthalmologyFebruary 1965

Fig. 5. Two days after injection. Fundus photoof retina below visual streak. There is a slightretinal haze with a white patch on the retina be-low. R, retina; Rp, pale retina. (x40.)

Fig. 7. Three weeks after injection. There is nowa hole in the retina, and about 30 per cent of theretina is now detached. Rd} retinal detachment;Rh, retinal hole. (x30.)

Fig. 6. Eight days after injection. There is nowa retinal detachment forming. A misty haze in thevitreous extends down to the pale area of retina.The pale retina appears to be migrating anteriorlyas though being pulled by a vitreous strand. R,retina; Rd, detached retina; Vh, vitreous haze;Rp. pale retina. (x30.)

it was being pulled by a vitreous strand(Fig. 6).

It was now noted that the retina sur-rounding the spot of pale retina was be-coming detached. Gradually, the locale ofwhite retina was becoming separated fromits neighboring detached retina. In Fig. 7a retinal hole is present in the area pre-

viously occupied by the pale retina, threeweeks following injection.

The area of detached retina surroundingthe hole gradually enlarged and stereofundus photographs were taken of theretinal detachment.113 The animal was killedon the thirty-first day after injection andthe area of retinal detachment examinedmicroscopically. Fig. 8 illustrates the retinalhole on a mounted section with vitreousstrands extending from the edge of the holetoward the pars plana ciliaris.

In Fig. 9 is illustrated a large strand ofcontracted vitreous, Vs, with attachedretinal tissue, O, from the area of the reti-nal hole; such retinal tissue has the samestaining characteristics as the detachedretina.

The use of the Rhinehart, Abul-Haj staindemonstrated the presence of acid muco-polysaccharide in the vitreous cavity ofboth injected and control eyes and alsoin the rod and cone layer of the retina.Treating mounted sections with hyaluroni-dase for one hour indicated the vitreal acidmucopolysaccharide to be hyaluronidasesensitive while that of the rod and conelayer was hyaluronidase resistant.

Since we also found that hyaluronicacid sulfate stains as an acid mucopoly-


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Retinal detachvient with sulfated polysaccliaride 47

saccharide with the Rhinehart, Abul-Hajstain and is also hyaluronidase sensitive,the presence of injected material could notbe demonstrated in. the vitreous body bythis technique. The staining intensity ofthe injected vitreous was slightly greaterthan the counterpart controls, but thismay have been caused by vitreous con-traction.

The gross specimen in Fig. 10 illustratesthe retinal hole and surrounding retinaldetachment one month after injection. In

Fig. 11 is illustrated the retinal hole withthe typical rolled edges and a cellular vitre-ous strand attached to one edge of theretinal break and partially covering it. InFig. 12 a higher magnification illustratesthe firm attachment of the cellular vitreousstrand to the rolled edge of the retinal hole.

The intraocular pressure as measured bythe Schiotz tonometer was utilized only fora gross estimation of the intraocular pres-sure. There was no significant differencein intraocular pressure between the pre-

Fig. 8. Mounted section showing retinal hole and detachment with vitreous strands attachedto the rolled edge of the hole. Vs, vitreous strands; Rh, retinal hole; Rd, retinal detachment.(Rhinehart, Abul-Haj stain.>s xG.)

Fig. 9. Contracted vitreous strand, Vs, with attached operculum, O, which has the same stain-ing characteristics as the detached retina, Rd. (Rhinehart, Abul-Haj stain.1S x50.)


48 Landholm and Watzke Investigative OphthalmologyFebruary 1965

injection pressure and the pressure threeweeks following intraocular injection. Asnoted previously by Harris,3 an occasionalcorneal and/or lens opacity developed. Incertain cases there was a spontaneous dis-location of the lens with fragmentation ofthe zonular fibers being noted on histologicexamination. With all these changes, therewas little or no inflammatory response. Anoccasional eye showed a slight beam butno cells were noted in the aqueous. Theeye was usually white but occasionallycircumcorneal injection was noted.

Fig. 10. Cross section showing retinal hole anddetachment. Vs, vitreous strand; Rh, retinal hole;R, retina; Rd, retinal detachment; S, sclera. (*10.)

With the use of special staining tech-niques for acid mucopolysaccharides, therelationship between the vitreal and retinalelements was more easily determined.18

Discussion

Previous experimental models of retinaldetachment have not duplicated success-fully the vitreal contraction, liquefaction,and formation of vitreoretinal adhesionsseen so frequently in humans. By the in-jection of hyaluronic acid sulfate into theposterior vitreous, liquefaction and con-traction of the vitreous produced vitreousstrands extending to the sites of vitreo-retinal adhesions. At these sites of vitrealtraction, a retinal hole and subsequent de-tachment were observed in 13 of 21 in-jected eyes. The detachments in all 13 eyeswere progressive with two becoming total,one as early as one month and one as lateas three months following injection. Nospontaneous reattachment was noted forthe three-month period of observation.More extensive studies are presently inprogress.

The exact mechanism by which the in-jected hyaluronic acid sulfate causeschanges in the ocular structures is, ofcourse, unknown. Such changes may wellbe due to a fundamental alteration andpossibly a replacement of the polysac-charide of the ground substance of ocular

Rh

Fig. 11. Hole, Rh., in detached retina, Rd, with vitreous strand, Vs, attached to one edge ofthe retinal break and partially covering it. (Rhinehart, Abul-Haj stain.^ x50.)


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Retinal deMchment with sulfated -polysaccliaride 49

/ - •

Rd

Rh

Fig. 12. Higher power view of rolled edge of retinal hole with the attached cellular vitreousmembrane. Rd, retinal detachment; Rh, retinal hole; Vs, vitreous strand. (Rhinehart, Abul-Haj

xl50.)

tissues. It is also possible that there wasa direct toxic effect on the retina and vit-reous at the site where the solution wasinjected. The effect of other sulfated andnonsulfated mucopolysaccharides on theretina and vitreous will have to be de-termined by further research. However,the most striking result of this experimentalprocedure was the production of vitreousliquefaction, contraction, vitreoretinal ad-hesions, and a progressive retinal detach-ment. Assessment of the role of hyaluronicacid sulfate in the pathogenesis of humanretinal detachments will have to await fur-ther study as no etiologic significance canbe drawn from these results. However, theexperimental models so created should beuseful for further research in this field.

We would like to acknowledge G. D. Searle &Co. for the hyaluronic acid sulfate, the talents ofLee Allen for the preparation of the stereoscopicfundus photographs, and Myra McClenahan, whoprepared the microscopic sections.

REFERENCES1. Teng, C. C , and Chi, H. H.: Vitreous

changes and the mechanism of retinal detach-ment, Am. J. Ophth. 44: 335, 1957.

2. Balazs, B.: Cone, ophth. XVIII, Belg. acta2: 1296, 1960.

3. Harris, J. E.: Structural changes in rabbit

eyes induced by hyaluronic acid sulfate, Am.J. Ophth. 43: 293, 1957.

4. Chodin, A.: Recherches sur la chute ducorps vitre, Rev. de sc. med. (Paris) 5: 704,1875.

5. Duke-Elder, W. S.: Textbook of ophthalmol-ogy, vol. 3, St. Louis, 1947, The C. V. MosbyCompany, p. 2888.

6. Castroviejo, R.: Experimental detachment ofthe retina, Am. J. Ophth. 17: 1112, 1934.

7. von Sallmann, L., and Rieger, H.: "Qber hin-tere Claskorperabhebung bei Ablatio retinae,Arch. f. Ophth. 133: 75, 1934.

8. Lindner, K.: Zur klinik des Glaskorpers, Arch,f. Ophth. 137: 158, 170, 1937.

9. Amsler, M.: Detachment of vitreous, Arch.Ophth. 4: 433, 1930.

10. Vogt, A.: Handbook and atlas of the slit-lampmicroscopy of the living eye: Iris, vitreousbody, conjunctiva (English translation), ed.2, vol. 3, Zurich, 1941, Schweizer Druck iindVerlagshaus, pp. 949, 973.

11. Pischel, D. K.: Detachment of the vitreousas seen by slit-lamp examination, Am. J.Ophth. 36: 1497, 1953.

12. Amiga, H.: Detachment of retina (translatedby Castroviejo), New York, 1936, Wester-mann.

13. Schepens, C. L.: Importance of the vitreousbody in retina surgery, St. Louis, 1960, TheC. V. Mosby Company, pp. 15-127.

14. Hogan, M. J., and Zimmerman, L. E.:Ophthalmic pathology, Philadelphia, 1962,W. B. Saunders Company, p. 75.

15. Tolman, E. L., and Harris, J. E.: Ocularchanges induced by polysaccharides, Am. J.Ophth. 47: 428, 1959.


50 Landhohn and Watzke Investigative OphthalmologyFebruary 1965

16. Allen, L.: Ocular fundus photography, Am.J. Ophth. 57: 13, 1964.

17. Mayer, L. L.: The vitreous in experimentalretinal detachment, Arch. Ophth. 7: 884,1932.

18. Rhinehart, J. F., and Abul-Haj, S. K.: Animproved method for histologic demonstra-tion of acid mucopolysaccharides in tissues,Arch. Path. 52: 189, 1951.


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Experimental Retinal Detachment with a Sulfated Polysaccharide