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Light Damage in Detached Retina
Light Damage in Detached Retina
John D. Zilis, M.D., and Robert Machemer, M.D.
We i n j e c t e d h o m o l o g o u s fibroblasts o v e r t h e r e t i n a l v a s c u l a r w i n g i n five r a b b i t s t o i n d u c e a fractional retinal detachment. Eleven days later, a focal area of d e t a c h m e n t was exposed to 30 m i n u t e s of visible light by a n i n t r a o c u l a r fiberoptic probe. Histologic damage to the detached retina exposed to light was demon strated. T h e o u t e r retina was affected m o s t severely.
L I G H T D A M A G E IN A T T A C H E D R E T I N A h a s
been
demonstrated with various ophthalmic light sources, including the intraocular fiberoptic p r o b e . T h i s damage primarily involves the o u t e r r e t i n a a n d p i g m e n t e p i t h e l i u m , ^ * b u t it c a n a l s o affect t h e i n n e r r e t i n a i f e x p o s u r e is i n t e n s e a n d o f l o n g duration.* T h e e x a c t m e c h a nisms of such retinal damage and the role of the melanin-containing retinal pigment epitheli u m " r e m a i n u n k n o w n . We e v a l u a t e d l i g h t d a m a g e in e x p e r i m e n t a l l y d e t a c h e d r a b b i t r e t i n a that h a d b e e n s e p a r a t e d from t h e r e t i n a l pigment epithelium.
Material and Methods Five p i g m e n t e d r a b b i t s w e i g h i n g b e t w e e n 2 . 5 a n d 3 . 5 kg w e r e h o u s e d u n d e r 5 0 f o o t - c a n d l e (ft-c) i l l u m i n a t i o n on a 1 2 - h o u r , l i g h t - d a r k c y c l e . At l e a s t t w o w e e k s b e f o r e t h e e x p e r i m e n t s , the rabbits were given anesthesia with an intra muscular injection of k e t a m i n e h y d r o c h l o r i d e (30 m g / k g of body weight) and xylazine hydro-
Accepted for publication Sept. 12, 1990. From the Devers Eye Institute, Portland, Oregon (Dr. Zilis), and Department of Ophthalmology, Duke Univer sity, Durham, North Carolina (Dr. Machemer). This study was supported by National Eye Institute grant F32 EY06046-01, the Helena Rubinstein Foundation, New York, New York, and Research to Prevent Blindness, Inc. Reprint requests to Robert Machemer, M.D., Duke University Eye Center, Box 3802, Durham, NC 27710.
c h l o r i d e (5 m g / k g o f b o d y w e i g h t ) . P u p i l s w e r e d i l a t e d b y a 1:1 m i x t u r e o f 0 . 2 5 % t r o p i c a m i d e and 5 % phenylephrine hydrochloride. The pe r i p h e r a l r e t i n a w a s t r e a t e d for 3 6 0 d e g r e e s at a distance o f 4 . 5 m m from the c o r n e o s c l e r a l limbus with transscleral cryopexy, under direct v i s u a l o b s e r v a t i o n , in p r e p a r a t i o n for future sclerotomy sites. At l e a s t t w o w e e k s l a t e r , t h e v i t r e o u s c a v i t i e s were injected with 2 5 0 , 0 0 0 h o m o l o g o u s tissuec u l t u r e d fibroblasts. F i b r o b l a s t s w e r e o f a s i n g l e l i n e a n d h a d b e e n h a r v e s t e d from r a b b i t r u m p dermis and tissue cultured in D u l b e c c o s modi fied E a g l e m e d i u m w i t h 1 0 % f e t a l b o v i n e s e r um, antibiotics, and a n t i m y c o t i c s , a n d main t a i n e d at 3 7 C in a n i n c u b a t o r w i t h a n a t m o s p h e r e o f 5 % c a r b o n d i o x i d e in a i r . T h e s e w e r e h a r v e s t e d for i n j e c t i o n in a m a n n e r p r e v i ously described.'" At t h e t i m e o f i n j e c t i o n , t h e a n i m a l s w e r e again given anesthesia, and the pupils were dilated. Under direct visualization by the indi r e c t o p h t h a l m o s c o p e , t h e c e l l s , s u s p e n d e d in 0.1 m l o f p h o s p h a t e - b u f f e r e d s a l i n e , w e r e i n jected slowly through the previously cryotreated peripheral retina into the vitreous body over the vascular wing o f the retina. T h e ani m a l s w e r e p l a c e d on t h e i r s i d e s for o n e h o u r t o allow settling of cells to occur on the vascular ized retina. Indirect o p h t h a l m o s c o p y was per f o r m e d at D a y 1 a n d D a y 7 a f t e r i n j e c t i o n t o monitor progression of the proliferative re sponse and resulting fractional retinal detach ment. E l e v e n days after fibroblast i n j e c t i o n , t h e a n i mals were again given anesthesia, and the pu pils of the injected eyes were dilated. R e t r o bulbar injections were not given. Rectal temperatures were monitored, and warming b l a n k e t s w e r e u s e d to m a i n t a i n b o d y t e m p e r a t u r e as n e e d e d . S c l e r o t o m i e s w e r e m a d e 3 . 0 m m p o s t e r i o r to the corneoscleral limbus through previously cryotreated peripheral retina, and cannulas w e r e p l a c e d t o m a i n t a i n an o p e n i n g . T w o a d d i t i o n a l s c l e r o t o m i e s w e r e p l a c e d for i n f u s i o n and outflow. Midvitreous temperature was ob-
©AMERICAN JOURNAL OF OPHTHALMOLOGY 111:47-50, JANUARY, 1991
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tained before beginning infusion and moni tored during i n f u s i o n b y a t e m p e r a t u r e p r o b e . L a c t a t e d R i n g e r ' s infusion fluid w a s w a r m e d to 3 9 . 5 ± 0 . 5 C in a w a t e r b a t h that w a s e l e v a t e d 2 6 i n c h e s a b o v e the l e v e l o f t h e e y e . I n t r a o c u l a r p r e s s u r e was m a i n t a i n e d b e t w e e n 14 a n d 2 1 m m Hg, a n d i n t r a o c u l a r t e m p e r a t u r e w a s m a i n t a i n e d at 3 8 . 3 ± 0 . 3 C b y c o n s t a n t p e r f u s i o n o f lactated Ringer's solution. A g l a s s fiberoptic p r o b e a t t a c h e d to a l i g h t source with a 1 5 0 - W , 2 1 - V tungsten-halogen projector bulb had a measured light output of 4 0 . 0 ± 0 . 8 ft-c. To confirm c o n s t a n t l i g h t out put, t h e l i g h t m e a s u r e m e n t w a s p e r f o r m e d b e fore a n d after t h e e x p e r i m e n t b y a r a d i o m e t e r / photometer with integrating sphere. T h e s c l e r o t o m y c r e a t e d for i n t r o d u c t i o n o f the fiberoptic p r o b e w a s m a d e i n f e r i o r l y . T h e p r o b e was a d v a n c e d s l o w l y b y a m i c r o m a n i p u lator to a d i s t a n c e o f 1.0 m m from t h e d e t a c h e d r e t i n a , w h i c h c r e a t e d an a p p r o x i m a t e c i r c l e o f light m e a s u r i n g 2 . 0 m m in its w i d e s t d i a m e t e r as m e a s u r e d b y t h e o c u l a r m i c r o m e t e r of t h e surgical m i c r o s c o p e . T h e a r e a o f t h e s m o o t h l y d e t a c h e d r e t i n a e x p o s e d to l i g h t w a s c e n t e r e d 1.5 m m b e l o w t h e e d g e o f t h e m y e l i n a t e d n e r v e layer a n d 1 disk d i a m e t e r t e m p o r a l to t h e disk in a v a s c u l a r r e t i n a . T h e c a l c u l a t e d r e t i n a l irra diance was 0 . 3 4 W / c m ^ which correlated well w i t h p r e v i o u s in v i t r o m e a s u r e m e n t s m a d e b y a s i m i l a r e x p e r i m e n t a l set-up."* L i g h t e x p o s u r e w a s m a i n t a i n e d for 3 0 m i n u t e s . T h e p o s i t i o n o f the fiberoptic p r o b e w a s c h e c k e d f r e q u e n t l y during the procedure. After 3 0 m i n u t e s , the i n s t r u m e n t s a n d c a n n u las w e r e r e m o v e d a n d the s c l e r o t o m i e s w e r e closed with 7-0 Vicryl sutures. The conjuncti v a e w e r e c l o s e d , a n d 5 m g of g e n t a m i c i n sulfate w a s i n j e c t e d s u b c o n j u n c t i v a l l y . A t r o p i n e 1% o i n t m e n t w a s p l a c e d on t h e c o r n e a s . P o s t o p e r a t i v e l y , the a n i m a l s w e r e e x a m i n e d daily by t h e i n d i r e c t o p h t h a l m o s c o p e . O n t h e third p o s t o p e r a t i v e day, the a n i m a l s w e r e k i l l e d , a n d the e y e s w e r e i n j e c t e d w i t h 3 % g l u t a r a l d e h y d e in 0.1 m o l / l of c a c o d y l a t e buff er i m m e d i a t e l y b e f o r e e n u c l e a t i o n . A d d i t i o n a l glutaraldehyde was injected through 3 . 0 - m m radial i n c i s i o n s b i s e c t i n g the i n f e r i o r c o r n e o scleral limbus, and the eyes were refrigerated a n d fixed for 4 8 h o u r s . P o s t e r i o r eye c u p s w e r e p r e p a r e d , a n d 3 . 0 x 3 . 0 - m m s e c t i o n s o f r e t i n a c e n t e r e d in t h e a r e a of l i g h t e x p o s u r e w e r e r e m o v e d . A d j a c e n t 3 . 0 x 3 . 0 - m m a r e a s of d e t a c h e d r e t i n a w e r e a l s o s e c tioned and examined. T h e s p e c i m e n s w e r e p r o c e s s e d in 2 % buff
ered osmium tetroxide through graded alcohol a n d e m b e d d e d in e p o x y r e s i n p l a s t i c . S e c t i o n s w e r e s t a i n e d w i t h m e t h y l e n e b l u e a n d a z u r e II for l i g h t m i c r o s c o p y , a n d t h i n s e c t i o n s w e r e prepared and stained with uranyl acetate and l e a d c i t r a t e for t r a n s m i s s i o n e l e c t r o n m i c r o scopy.
Results Tractional retinal detachments developed w i t h i n s e v e n days o f t h e fibroblast i n j e c t i o n in all r a b b i t s . T h e d e t a c h m e n t w a s h i g h e s t in t h e a r e a o f t h e v a s c u l a r m e d u l l a r y w i n g from w h i c h a p r o l i f e r a t i v e fibroblast s t r a n d e x t e n d e d to t h e a r e a of t h e i n j e c t i o n site ( F i g . 1 ) . T h e a v a s c u l a r retina b e l o w the wing was elevated with a defined i n f e r i o r d e m a r c a t i o n l i n e s e p a r a t i n g t h e r e m a i n i n g a t t a c h e d r e t i n a from t h e g r a y white detached retina. Subretinal white precip i t a t e s w e r e s o m e t i m e s n o t e d in t h e d e p e n d e n t portion of the d e t a c h m e n t . Rabbits that developed rhegmatogenous, bullous detach ments were excluded. A total of five p i g m e n t e d r a b b i t s fulfilled t h e experimental criteria. Daily postoperative indi rect o p h t h a l m o s c o p i c examinations were per f o r m e d . O n t h e first p o s t o p e r a t i v e day, all e y e s d i s p l a y e d s i g n i f i c a n t p o s t e r i o r i n t r a o c u l a r in-
Fig. 1 (Zilis and Machemer). Tractional retinal detachment of vascular medullary wings in a rabbit. Note proliferative fibroblast strand superiorly.
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flammation that o b s c u r e d r e t i n a l d e t a i l . P r o gressive clearing, however, occurred by Day 3, which allowed observation of the area of light exposure. Focal retinal edema was not found, although slight retinal thinning was suspected in two a n i m a l s . H i s t o l o g i c a l l y , t h e e y e s of all five a n i m a l s h a d m u c h m o r e s e v e r e d a m a g e in t h e a r e a o f l i g h t e x p o s u r e t h a n in o t h e r a r e a s o f r e t i n a l d e t a c h m e n t . In t h e a r e a o f r e t i n a l d e t a c h m e n t n o t e x p o s e d to l i g h t , p a t c h y l o s s o f p h o t o r e c e p t o r o u t e r s e g m e n t s a n d o c c a s i o n a l m a c r o p h a g e in filtration w e r e n o t e d . G e n e r a l l y , c e l l u l a r o r g a n i z a t i o n o f r e t i n a l layers w a s w e l l m a i n t a i n e d (Fig. 2 ) . C o n v e r s e l y , t h e a r e a s of d e t a c h e d r e t i n a e x p o s e d to light d i s p l a y e d e x t r e m e d e s t r u c t i o n (Fig. 3 ) . T h e p h o t o r e c e p t o r e l e m e n t s w e r e v i r tually e l i m i n a t e d w i t h o n l y s m a l l d e g e n e r a t e d inner segments remaining. Macrophages were found a l o n g t h e o u t e r l i m i t i n g m e m b r a n e . T h e o u t e r n u c l e a r layer d e m o n s t r a t e d n u c l e a r d r o p
Fig. 2 (Zilis and Machemer). Photomicrograph of detached retina without light exposure ( x 4 0 0 ) .
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o u t a n d p y k n o s i s . T h e i n n e r r e t i n a l layers w e r e relatively well preserved, although small cystic spaces were observed.
Discussion R e t i n a l d a m a g e c a u s e d b y v i s i b l e l i g h t is k n o w n to o c c u r w h e n an i n t r a o c u l a r fiberoptic l i g h t s o u r c e is used.^'' In a t t a c h e d r e t i n a , h i s t o logic damage has been demonstrated primarily in t h e o u t e r r e t i n a a n d p i g m e n t epithelium.^* Noell and a s s o c i a t e s ' speculated that the dam a g e is c a u s e d b y p h o t o c h e m i c a l e v e n t s t h a t are s e n s i t i v e t o c h a n g e s in t e m p e r a t u r e . The melanin-containing retinal pigment epi thelium demonstrated damage when exposed t o v i s i b l e l i g h t , a l t h o u g h i t s r o l e in m e d i a t i n g
Fig. 3 (Zilis and Machemer). Photomicrograph of detached retina with 30 minutes of light exposure. Note massive destruction of outer retina and loss of photoreceptors ( x 4 0 0 ) .
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d a m a g e to t h e o v e r l y i n g r e t i n a r e m a i n s u n known. Ham and a s s o c i a t e s " originally sug g e s t e d that t h e site of injury in t h e r h e s u s m o n k e y may b e t h e m e l a n i n g r a n u l e s o f t h e m e l a n o s o m e s o f the r e t i n a l p i g m e n t e p i t h e l i u m . D u r i n g the first 4 8 h o u r s after l i g h t e x p o sure, d a m a g e a p p e a r e d c o n f i n e d to t h i s layer, whereas overlying photoreceptor outer seg ments remained intact. W o r k i n g w i t h t h e rat, h o w e v e r , R a p p a n d Williams* s h o w e d a l m o s t e q u a l r e t i n a l s e n s i t i v ity to light d a m a g e in b o t h p i g m e n t e d a n d albino animals. Hoeppler and associates' found essentially no difference w h e n m o r p h o l o g i c d a m a g e w a s c o m p a r e d in p i g m e n t e d a n d a l b i n o r a b b i t s , w h i c h l e d t h e m to p o s t u l a t e t h a t m e l a nin may n o t b e c r i t i c a l l y i n v o l v e d in l i g h t d a m age to t h e r e t i n a . We d e s i g n e d t h i s s t u d y to a s s e s s l i g h t d a m a g e in r e t i n a that w a s d e t a c h e d from t h e r e t i n a l p i g m e n t e p i t h e l i u m . To m a x i m i z e l i g h t d a m age, a relatively long exposure (30 minutes) of v i s i b l e light w a s p l a c e d on a s m a l l a r e a ( 2 . 0 - m m d i a m e t e r ) o f d e t a c h e d , a v a s c u l a r r a b b i t r e t i n a at b o d y t e m p e r a t u r e . T h i s r e s u l t e d in s e v e r e h i s t o l o g i c d a m a g e to t h e r e t i n a (Fig. 3 ) , w h i c h w a s much more profound than the usual outer reti nal d e g e n e r a t i o n e x p e c t e d in d e t a c h e d r e t i na.'^'^ I n d e e d , t h e d a m a g e w a s d r a m a t i c w h e n c o m p a r e d to a d j a c e n t a r e a s o f d e t a c h e d r e t i n a n o t e x p o s e d to t h e fiberoptic l i g h t (Fig. 2 ) . In t h e l i g h t - e x p o s e d d e t a c h e d r e t i n a , d a m a g e w a s m o s t a p p a r e n t in t h e o u t e r r e t i n a w i t h resulting destruction of photoreceptor inner and outer segments and their nuclei. T h e same p a t t e r n o f d a m a g e w a s n o t e d in l i g h t e x p o s u r e to a t t a c h e d retina.'*' In a t t a c h e d r e t i n a , t h e o u t e r r e t i n a is e x t r e m e l y a c t i v e , m e t a b o l i c a l l y , a n d w e l l o x y g e n a t e d from t h e u n d e r l y i n g c h o roid. B e c a u s e o f t h i s , o n e p r o p o s e d m e c h a n i s m o f l i g h t d a m a g e to t h e r e t i n a i n v o l v e s p h o t o o x i dative a n d p h o t o d y n a m i c r e a c t i o n s i n v o l v i n g the retinal pigment epithelium with generation of d e s t r u c t i v e o x y g e n - d e r i v e d free r a d i c a l s . ' " Because we found a similar pattern of damage in the o u t e r d e t a c h e d r e t i n a t h a t w a s d e v o i d o f m e l a n i n a n d p r e s u m e d to b e i s c h e m i c , a d d i t i o n a l u n d e f i n e d m e c h a n i s m s or m e d i a t o r s m a y play a r o l e . W e a l s o c o n c l u d e from o u r e x p e r i m e n t s that light d a m a g e in d e t a c h e d r e t i n a m a y o c c u r w i t h light s o u r c e s c o m m o n l y u s e d b y clinicians, particularly during lengthy vitreo retinal surgical procedures.
References 1. Calkins, J. L., Hochheimer, B. P., and D'Anna, S. Α.: Potential hazards from specific ophthalmic devices. Vision Res. 20:1039, 1 9 8 0 . 2. Fuller, D., Machemer, R., and Knighton, R. W.: Retinal damage produced by the intraocular fiber optic light. Am. J. Ophthalmol. 85:519, 1 9 7 8 . 3. Noell, W., Walker, V. S., Kang, B. S., and Berg man, S.: Retinal damage by light in rats. Invest. Ophthalmol. Vis. Sei. 5:450, 1966. 4. Rinkoff, ] . , Machemer, R., Hida, T., and Chan dler, D.: Temperature-dependent light damage to the retina. Am. J. Ophthalmol. 102:452, 1986. 5. T'so, M. O. M., Fine, B. S., and Zimmerman, L. E.: Photic maculopathy produced by the indirect ophthalmoscope. I. Clinical and histopathologic study. Am. J. Ophthalmol. 73:686, 1972. 6. Lawwill, T.: Three major pathologic processes caused by light in the primate retina. A search for mechanisms. Trans. Am. Ophthalmol. Soc. 80:516, 1982. 7. Ham, W. T., Ruffolo, J. J . , and Mueller, H. Α.: The nature of retinal radiation damage. Dependence on wave length, power level and exposure time. Vision Res. 20:1105, 1980. 8. Rapp, L. M., and Williams, T. P.: The role of ocular pigmentation in protecting against retinal light damage. Vision Res. 20:1127, 1 9 8 0 . 9. Hoeppler, T., Hendrickson, P., Dietrich, C , and Reme, C : Morphology and time course of defined photochemical lesions in the rabbit retina. Curr. Eye Res. 7:849, 1988. 10. Chandler, D. B., Quansah, F. Α., Hida, T., and Machemer, R.: A refined experimental model for proliferative vitreoretinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 224:84, 1986. 11. Ham, W. T., Ruffolo, J. J . , Mueller, H . A . , Clarke, A. M., and Moon, M. E.: Histologic analysis of photochemical lesions produced in the rhesus retina by short wavelength light. Invest. Ophthal mol. Vis. Sei. 17:1029, 1 9 7 8 . 12. Machemer, R.: Experimental retinal detach ment in the owl monkey. II. Histology of retina and pigment epithelium. Am. J. Ophthalmol. 6 6 : 3 9 6 , 1968. 13. Kroll, A. J . , and Machemer, R.: Experimental retinal detachment in the owl monkey. III. Electron microscopy of retina and pigment epithelium. Am. J. Ophthalmol. 66:410, 1 9 6 8 . 14. Ham, W. T., Mueller, H. Α., Ruffolo, J. J . , Millen, J. E., Cleary, S. F., Guerry, R. K., and Guerry, D.: Basic mechanisms underlying the production of photochemical lesions in mammalian retina. Curr. Eye Res. 3:165, 1 9 8 4 .
John D. Zilis, M.D., and Robert Machemer, M.D.
We i n j e c t e d h o m o l o g o u s fibroblasts o v e r t h e r e t i n a l v a s c u l a r w i n g i n five r a b b i t s t o i n d u c e a fractional retinal detachment. Eleven days later, a focal area of d e t a c h m e n t was exposed to 30 m i n u t e s of visible light by a n i n t r a o c u l a r fiberoptic probe. Histologic damage to the detached retina exposed to light was demon strated. T h e o u t e r retina was affected m o s t severely.
L I G H T D A M A G E IN A T T A C H E D R E T I N A h a s
been
demonstrated with various ophthalmic light sources, including the intraocular fiberoptic p r o b e . T h i s damage primarily involves the o u t e r r e t i n a a n d p i g m e n t e p i t h e l i u m , ^ * b u t it c a n a l s o affect t h e i n n e r r e t i n a i f e x p o s u r e is i n t e n s e a n d o f l o n g duration.* T h e e x a c t m e c h a nisms of such retinal damage and the role of the melanin-containing retinal pigment epitheli u m " r e m a i n u n k n o w n . We e v a l u a t e d l i g h t d a m a g e in e x p e r i m e n t a l l y d e t a c h e d r a b b i t r e t i n a that h a d b e e n s e p a r a t e d from t h e r e t i n a l pigment epithelium.
Material and Methods Five p i g m e n t e d r a b b i t s w e i g h i n g b e t w e e n 2 . 5 a n d 3 . 5 kg w e r e h o u s e d u n d e r 5 0 f o o t - c a n d l e (ft-c) i l l u m i n a t i o n on a 1 2 - h o u r , l i g h t - d a r k c y c l e . At l e a s t t w o w e e k s b e f o r e t h e e x p e r i m e n t s , the rabbits were given anesthesia with an intra muscular injection of k e t a m i n e h y d r o c h l o r i d e (30 m g / k g of body weight) and xylazine hydro-
Accepted for publication Sept. 12, 1990. From the Devers Eye Institute, Portland, Oregon (Dr. Zilis), and Department of Ophthalmology, Duke Univer sity, Durham, North Carolina (Dr. Machemer). This study was supported by National Eye Institute grant F32 EY06046-01, the Helena Rubinstein Foundation, New York, New York, and Research to Prevent Blindness, Inc. Reprint requests to Robert Machemer, M.D., Duke University Eye Center, Box 3802, Durham, NC 27710.
c h l o r i d e (5 m g / k g o f b o d y w e i g h t ) . P u p i l s w e r e d i l a t e d b y a 1:1 m i x t u r e o f 0 . 2 5 % t r o p i c a m i d e and 5 % phenylephrine hydrochloride. The pe r i p h e r a l r e t i n a w a s t r e a t e d for 3 6 0 d e g r e e s at a distance o f 4 . 5 m m from the c o r n e o s c l e r a l limbus with transscleral cryopexy, under direct v i s u a l o b s e r v a t i o n , in p r e p a r a t i o n for future sclerotomy sites. At l e a s t t w o w e e k s l a t e r , t h e v i t r e o u s c a v i t i e s were injected with 2 5 0 , 0 0 0 h o m o l o g o u s tissuec u l t u r e d fibroblasts. F i b r o b l a s t s w e r e o f a s i n g l e l i n e a n d h a d b e e n h a r v e s t e d from r a b b i t r u m p dermis and tissue cultured in D u l b e c c o s modi fied E a g l e m e d i u m w i t h 1 0 % f e t a l b o v i n e s e r um, antibiotics, and a n t i m y c o t i c s , a n d main t a i n e d at 3 7 C in a n i n c u b a t o r w i t h a n a t m o s p h e r e o f 5 % c a r b o n d i o x i d e in a i r . T h e s e w e r e h a r v e s t e d for i n j e c t i o n in a m a n n e r p r e v i ously described.'" At t h e t i m e o f i n j e c t i o n , t h e a n i m a l s w e r e again given anesthesia, and the pupils were dilated. Under direct visualization by the indi r e c t o p h t h a l m o s c o p e , t h e c e l l s , s u s p e n d e d in 0.1 m l o f p h o s p h a t e - b u f f e r e d s a l i n e , w e r e i n jected slowly through the previously cryotreated peripheral retina into the vitreous body over the vascular wing o f the retina. T h e ani m a l s w e r e p l a c e d on t h e i r s i d e s for o n e h o u r t o allow settling of cells to occur on the vascular ized retina. Indirect o p h t h a l m o s c o p y was per f o r m e d at D a y 1 a n d D a y 7 a f t e r i n j e c t i o n t o monitor progression of the proliferative re sponse and resulting fractional retinal detach ment. E l e v e n days after fibroblast i n j e c t i o n , t h e a n i mals were again given anesthesia, and the pu pils of the injected eyes were dilated. R e t r o bulbar injections were not given. Rectal temperatures were monitored, and warming b l a n k e t s w e r e u s e d to m a i n t a i n b o d y t e m p e r a t u r e as n e e d e d . S c l e r o t o m i e s w e r e m a d e 3 . 0 m m p o s t e r i o r to the corneoscleral limbus through previously cryotreated peripheral retina, and cannulas w e r e p l a c e d t o m a i n t a i n an o p e n i n g . T w o a d d i t i o n a l s c l e r o t o m i e s w e r e p l a c e d for i n f u s i o n and outflow. Midvitreous temperature was ob-
©AMERICAN JOURNAL OF OPHTHALMOLOGY 111:47-50, JANUARY, 1991
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tained before beginning infusion and moni tored during i n f u s i o n b y a t e m p e r a t u r e p r o b e . L a c t a t e d R i n g e r ' s infusion fluid w a s w a r m e d to 3 9 . 5 ± 0 . 5 C in a w a t e r b a t h that w a s e l e v a t e d 2 6 i n c h e s a b o v e the l e v e l o f t h e e y e . I n t r a o c u l a r p r e s s u r e was m a i n t a i n e d b e t w e e n 14 a n d 2 1 m m Hg, a n d i n t r a o c u l a r t e m p e r a t u r e w a s m a i n t a i n e d at 3 8 . 3 ± 0 . 3 C b y c o n s t a n t p e r f u s i o n o f lactated Ringer's solution. A g l a s s fiberoptic p r o b e a t t a c h e d to a l i g h t source with a 1 5 0 - W , 2 1 - V tungsten-halogen projector bulb had a measured light output of 4 0 . 0 ± 0 . 8 ft-c. To confirm c o n s t a n t l i g h t out put, t h e l i g h t m e a s u r e m e n t w a s p e r f o r m e d b e fore a n d after t h e e x p e r i m e n t b y a r a d i o m e t e r / photometer with integrating sphere. T h e s c l e r o t o m y c r e a t e d for i n t r o d u c t i o n o f the fiberoptic p r o b e w a s m a d e i n f e r i o r l y . T h e p r o b e was a d v a n c e d s l o w l y b y a m i c r o m a n i p u lator to a d i s t a n c e o f 1.0 m m from t h e d e t a c h e d r e t i n a , w h i c h c r e a t e d an a p p r o x i m a t e c i r c l e o f light m e a s u r i n g 2 . 0 m m in its w i d e s t d i a m e t e r as m e a s u r e d b y t h e o c u l a r m i c r o m e t e r of t h e surgical m i c r o s c o p e . T h e a r e a o f t h e s m o o t h l y d e t a c h e d r e t i n a e x p o s e d to l i g h t w a s c e n t e r e d 1.5 m m b e l o w t h e e d g e o f t h e m y e l i n a t e d n e r v e layer a n d 1 disk d i a m e t e r t e m p o r a l to t h e disk in a v a s c u l a r r e t i n a . T h e c a l c u l a t e d r e t i n a l irra diance was 0 . 3 4 W / c m ^ which correlated well w i t h p r e v i o u s in v i t r o m e a s u r e m e n t s m a d e b y a s i m i l a r e x p e r i m e n t a l set-up."* L i g h t e x p o s u r e w a s m a i n t a i n e d for 3 0 m i n u t e s . T h e p o s i t i o n o f the fiberoptic p r o b e w a s c h e c k e d f r e q u e n t l y during the procedure. After 3 0 m i n u t e s , the i n s t r u m e n t s a n d c a n n u las w e r e r e m o v e d a n d the s c l e r o t o m i e s w e r e closed with 7-0 Vicryl sutures. The conjuncti v a e w e r e c l o s e d , a n d 5 m g of g e n t a m i c i n sulfate w a s i n j e c t e d s u b c o n j u n c t i v a l l y . A t r o p i n e 1% o i n t m e n t w a s p l a c e d on t h e c o r n e a s . P o s t o p e r a t i v e l y , the a n i m a l s w e r e e x a m i n e d daily by t h e i n d i r e c t o p h t h a l m o s c o p e . O n t h e third p o s t o p e r a t i v e day, the a n i m a l s w e r e k i l l e d , a n d the e y e s w e r e i n j e c t e d w i t h 3 % g l u t a r a l d e h y d e in 0.1 m o l / l of c a c o d y l a t e buff er i m m e d i a t e l y b e f o r e e n u c l e a t i o n . A d d i t i o n a l glutaraldehyde was injected through 3 . 0 - m m radial i n c i s i o n s b i s e c t i n g the i n f e r i o r c o r n e o scleral limbus, and the eyes were refrigerated a n d fixed for 4 8 h o u r s . P o s t e r i o r eye c u p s w e r e p r e p a r e d , a n d 3 . 0 x 3 . 0 - m m s e c t i o n s o f r e t i n a c e n t e r e d in t h e a r e a of l i g h t e x p o s u r e w e r e r e m o v e d . A d j a c e n t 3 . 0 x 3 . 0 - m m a r e a s of d e t a c h e d r e t i n a w e r e a l s o s e c tioned and examined. T h e s p e c i m e n s w e r e p r o c e s s e d in 2 % buff
ered osmium tetroxide through graded alcohol a n d e m b e d d e d in e p o x y r e s i n p l a s t i c . S e c t i o n s w e r e s t a i n e d w i t h m e t h y l e n e b l u e a n d a z u r e II for l i g h t m i c r o s c o p y , a n d t h i n s e c t i o n s w e r e prepared and stained with uranyl acetate and l e a d c i t r a t e for t r a n s m i s s i o n e l e c t r o n m i c r o scopy.
Results Tractional retinal detachments developed w i t h i n s e v e n days o f t h e fibroblast i n j e c t i o n in all r a b b i t s . T h e d e t a c h m e n t w a s h i g h e s t in t h e a r e a o f t h e v a s c u l a r m e d u l l a r y w i n g from w h i c h a p r o l i f e r a t i v e fibroblast s t r a n d e x t e n d e d to t h e a r e a of t h e i n j e c t i o n site ( F i g . 1 ) . T h e a v a s c u l a r retina b e l o w the wing was elevated with a defined i n f e r i o r d e m a r c a t i o n l i n e s e p a r a t i n g t h e r e m a i n i n g a t t a c h e d r e t i n a from t h e g r a y white detached retina. Subretinal white precip i t a t e s w e r e s o m e t i m e s n o t e d in t h e d e p e n d e n t portion of the d e t a c h m e n t . Rabbits that developed rhegmatogenous, bullous detach ments were excluded. A total of five p i g m e n t e d r a b b i t s fulfilled t h e experimental criteria. Daily postoperative indi rect o p h t h a l m o s c o p i c examinations were per f o r m e d . O n t h e first p o s t o p e r a t i v e day, all e y e s d i s p l a y e d s i g n i f i c a n t p o s t e r i o r i n t r a o c u l a r in-
Fig. 1 (Zilis and Machemer). Tractional retinal detachment of vascular medullary wings in a rabbit. Note proliferative fibroblast strand superiorly.
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flammation that o b s c u r e d r e t i n a l d e t a i l . P r o gressive clearing, however, occurred by Day 3, which allowed observation of the area of light exposure. Focal retinal edema was not found, although slight retinal thinning was suspected in two a n i m a l s . H i s t o l o g i c a l l y , t h e e y e s of all five a n i m a l s h a d m u c h m o r e s e v e r e d a m a g e in t h e a r e a o f l i g h t e x p o s u r e t h a n in o t h e r a r e a s o f r e t i n a l d e t a c h m e n t . In t h e a r e a o f r e t i n a l d e t a c h m e n t n o t e x p o s e d to l i g h t , p a t c h y l o s s o f p h o t o r e c e p t o r o u t e r s e g m e n t s a n d o c c a s i o n a l m a c r o p h a g e in filtration w e r e n o t e d . G e n e r a l l y , c e l l u l a r o r g a n i z a t i o n o f r e t i n a l layers w a s w e l l m a i n t a i n e d (Fig. 2 ) . C o n v e r s e l y , t h e a r e a s of d e t a c h e d r e t i n a e x p o s e d to light d i s p l a y e d e x t r e m e d e s t r u c t i o n (Fig. 3 ) . T h e p h o t o r e c e p t o r e l e m e n t s w e r e v i r tually e l i m i n a t e d w i t h o n l y s m a l l d e g e n e r a t e d inner segments remaining. Macrophages were found a l o n g t h e o u t e r l i m i t i n g m e m b r a n e . T h e o u t e r n u c l e a r layer d e m o n s t r a t e d n u c l e a r d r o p
Fig. 2 (Zilis and Machemer). Photomicrograph of detached retina without light exposure ( x 4 0 0 ) .
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o u t a n d p y k n o s i s . T h e i n n e r r e t i n a l layers w e r e relatively well preserved, although small cystic spaces were observed.
Discussion R e t i n a l d a m a g e c a u s e d b y v i s i b l e l i g h t is k n o w n to o c c u r w h e n an i n t r a o c u l a r fiberoptic l i g h t s o u r c e is used.^'' In a t t a c h e d r e t i n a , h i s t o logic damage has been demonstrated primarily in t h e o u t e r r e t i n a a n d p i g m e n t epithelium.^* Noell and a s s o c i a t e s ' speculated that the dam a g e is c a u s e d b y p h o t o c h e m i c a l e v e n t s t h a t are s e n s i t i v e t o c h a n g e s in t e m p e r a t u r e . The melanin-containing retinal pigment epi thelium demonstrated damage when exposed t o v i s i b l e l i g h t , a l t h o u g h i t s r o l e in m e d i a t i n g
Fig. 3 (Zilis and Machemer). Photomicrograph of detached retina with 30 minutes of light exposure. Note massive destruction of outer retina and loss of photoreceptors ( x 4 0 0 ) .
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d a m a g e to t h e o v e r l y i n g r e t i n a r e m a i n s u n known. Ham and a s s o c i a t e s " originally sug g e s t e d that t h e site of injury in t h e r h e s u s m o n k e y may b e t h e m e l a n i n g r a n u l e s o f t h e m e l a n o s o m e s o f the r e t i n a l p i g m e n t e p i t h e l i u m . D u r i n g the first 4 8 h o u r s after l i g h t e x p o sure, d a m a g e a p p e a r e d c o n f i n e d to t h i s layer, whereas overlying photoreceptor outer seg ments remained intact. W o r k i n g w i t h t h e rat, h o w e v e r , R a p p a n d Williams* s h o w e d a l m o s t e q u a l r e t i n a l s e n s i t i v ity to light d a m a g e in b o t h p i g m e n t e d a n d albino animals. Hoeppler and associates' found essentially no difference w h e n m o r p h o l o g i c d a m a g e w a s c o m p a r e d in p i g m e n t e d a n d a l b i n o r a b b i t s , w h i c h l e d t h e m to p o s t u l a t e t h a t m e l a nin may n o t b e c r i t i c a l l y i n v o l v e d in l i g h t d a m age to t h e r e t i n a . We d e s i g n e d t h i s s t u d y to a s s e s s l i g h t d a m a g e in r e t i n a that w a s d e t a c h e d from t h e r e t i n a l p i g m e n t e p i t h e l i u m . To m a x i m i z e l i g h t d a m age, a relatively long exposure (30 minutes) of v i s i b l e light w a s p l a c e d on a s m a l l a r e a ( 2 . 0 - m m d i a m e t e r ) o f d e t a c h e d , a v a s c u l a r r a b b i t r e t i n a at b o d y t e m p e r a t u r e . T h i s r e s u l t e d in s e v e r e h i s t o l o g i c d a m a g e to t h e r e t i n a (Fig. 3 ) , w h i c h w a s much more profound than the usual outer reti nal d e g e n e r a t i o n e x p e c t e d in d e t a c h e d r e t i na.'^'^ I n d e e d , t h e d a m a g e w a s d r a m a t i c w h e n c o m p a r e d to a d j a c e n t a r e a s o f d e t a c h e d r e t i n a n o t e x p o s e d to t h e fiberoptic l i g h t (Fig. 2 ) . In t h e l i g h t - e x p o s e d d e t a c h e d r e t i n a , d a m a g e w a s m o s t a p p a r e n t in t h e o u t e r r e t i n a w i t h resulting destruction of photoreceptor inner and outer segments and their nuclei. T h e same p a t t e r n o f d a m a g e w a s n o t e d in l i g h t e x p o s u r e to a t t a c h e d retina.'*' In a t t a c h e d r e t i n a , t h e o u t e r r e t i n a is e x t r e m e l y a c t i v e , m e t a b o l i c a l l y , a n d w e l l o x y g e n a t e d from t h e u n d e r l y i n g c h o roid. B e c a u s e o f t h i s , o n e p r o p o s e d m e c h a n i s m o f l i g h t d a m a g e to t h e r e t i n a i n v o l v e s p h o t o o x i dative a n d p h o t o d y n a m i c r e a c t i o n s i n v o l v i n g the retinal pigment epithelium with generation of d e s t r u c t i v e o x y g e n - d e r i v e d free r a d i c a l s . ' " Because we found a similar pattern of damage in the o u t e r d e t a c h e d r e t i n a t h a t w a s d e v o i d o f m e l a n i n a n d p r e s u m e d to b e i s c h e m i c , a d d i t i o n a l u n d e f i n e d m e c h a n i s m s or m e d i a t o r s m a y play a r o l e . W e a l s o c o n c l u d e from o u r e x p e r i m e n t s that light d a m a g e in d e t a c h e d r e t i n a m a y o c c u r w i t h light s o u r c e s c o m m o n l y u s e d b y clinicians, particularly during lengthy vitreo retinal surgical procedures.
References 1. Calkins, J. L., Hochheimer, B. P., and D'Anna, S. Α.: Potential hazards from specific ophthalmic devices. Vision Res. 20:1039, 1 9 8 0 . 2. Fuller, D., Machemer, R., and Knighton, R. W.: Retinal damage produced by the intraocular fiber optic light. Am. J. Ophthalmol. 85:519, 1 9 7 8 . 3. Noell, W., Walker, V. S., Kang, B. S., and Berg man, S.: Retinal damage by light in rats. Invest. Ophthalmol. Vis. Sei. 5:450, 1966. 4. Rinkoff, ] . , Machemer, R., Hida, T., and Chan dler, D.: Temperature-dependent light damage to the retina. Am. J. Ophthalmol. 102:452, 1986. 5. T'so, M. O. M., Fine, B. S., and Zimmerman, L. E.: Photic maculopathy produced by the indirect ophthalmoscope. I. Clinical and histopathologic study. Am. J. Ophthalmol. 73:686, 1972. 6. Lawwill, T.: Three major pathologic processes caused by light in the primate retina. A search for mechanisms. Trans. Am. Ophthalmol. Soc. 80:516, 1982. 7. Ham, W. T., Ruffolo, J. J . , and Mueller, H. Α.: The nature of retinal radiation damage. Dependence on wave length, power level and exposure time. Vision Res. 20:1105, 1980. 8. Rapp, L. M., and Williams, T. P.: The role of ocular pigmentation in protecting against retinal light damage. Vision Res. 20:1127, 1 9 8 0 . 9. Hoeppler, T., Hendrickson, P., Dietrich, C , and Reme, C : Morphology and time course of defined photochemical lesions in the rabbit retina. Curr. Eye Res. 7:849, 1988. 10. Chandler, D. B., Quansah, F. Α., Hida, T., and Machemer, R.: A refined experimental model for proliferative vitreoretinopathy. Graefes Arch. Clin. Exp. Ophthalmol. 224:84, 1986. 11. Ham, W. T., Ruffolo, J. J . , Mueller, H . A . , Clarke, A. M., and Moon, M. E.: Histologic analysis of photochemical lesions produced in the rhesus retina by short wavelength light. Invest. Ophthal mol. Vis. Sei. 17:1029, 1 9 7 8 . 12. Machemer, R.: Experimental retinal detach ment in the owl monkey. II. Histology of retina and pigment epithelium. Am. J. Ophthalmol. 6 6 : 3 9 6 , 1968. 13. Kroll, A. J . , and Machemer, R.: Experimental retinal detachment in the owl monkey. III. Electron microscopy of retina and pigment epithelium. Am. J. Ophthalmol. 66:410, 1 9 6 8 . 14. Ham, W. T., Mueller, H. Α., Ruffolo, J. J . , Millen, J. E., Cleary, S. F., Guerry, R. K., and Guerry, D.: Basic mechanisms underlying the production of photochemical lesions in mammalian retina. Curr. Eye Res. 3:165, 1 9 8 4 .