Dark Adaptation in Locally Detached Retina

Toshiro Mori,* David R. Pepperberg,t and Michael F. Marmor*

Nonrhegmatogenous retinal detachments were formed in the eyes of Dutch rabbits by subretinal injection of Hanks' balanced salt solution. The electroretinogram (ERG) was recorded locally from the acutely detached retina, and simultaneously from the surrounding attached retina (vitreal ERG [YERG]), before and after exposure to diffuse intense irradiation. Light adaptation elevated b-wave threshold for both the local ERG (LERG) and VERG by about 3 log units; thresholds for both responses recovered fully within 60-90 min after the irradiation. The normal time course of dark adaptation of the LERG suggests the occurrence of substantial rhodopsin regeneration in the rod photoreceptors of nonrhegmatogenously detached retina. These results differ from reports that visual pigment regeneration is slow in central serous chorioretinopathy, possibly because our detachments were studied within hours of formation, whereas some photoreceptor degeneration may be present in older clinical detachments. Invest Ophthalmol Vis Sci 31:1259-1263,1990

Under most conditions in vivo, the regeneration of ical experience that rhegmatogenous detachments rhodopsin in bleached rod photoreceptors requires typically cause an immediate loss of visual function, the delivery, to the rods, of 1 \-cis retinoid formed in measured psychophysically or electrophysiologically. the retinal pigment epithelium (RPE).1"6 Support for Even small serous detachments, such as found in pa- this view comes from studies of the isolated retina tients with central serous chorioretinopathy (CSC), preparation, in which the photoreceptors are re- are characterized by abnormally slow regeneration of moved from this RPE source of 1 l-cis retinoid. For visual pigment.13"15 However, in patients with CSC, example, after exposure to intense light, isolated ret- the focal electroretinogram (ERG) frequently re- inas ordinarily regenerate little if any rhodopsin and mains subnormal after resolution of the detachment, do not exhibit the component of electrophysiologic ie, after reassociation of the retina with the RPE.1617 recovery ("photochemical" dark adaptation7) that Thus, the retinal dysfunction in CSC may reflect de- depends on rhodopsin regeneration.89 However, the generative changes in the retina or RPE rather than, external application of l\-cis retinoid to bleached, or in addition to, the interruption of retinoid de- isolated retinas induces both the formation of rho- livery. dopsin and a substantial recovery of rod sensi- We recently have developed procedures that allow tivity.10"12 recording of the ERG from locally detached retina These findings raise a question relevant to the con- within the living rabbit eye as well as comparison of dition of retinal detachment in the living eye: To this response with the ERG generated by surrounding what extent does separation of the retina and RPE areas of attached retina.18 We report the use of this impair the delivery of 1 l-cis retinoid to the rods and technique to investigate the recovery of ERG sensitiv- thus retard or abolish photochemical dark adaptation ity in locally detached retina after strong light adapta- in the detached region of retina? We know from clin- tion.19 Since our detachments were made immediately before the electrophysiologic experiments, we were able to study the effects of detachment in the From the *Department of Ophthalmology, Stanford University absence of the deteriorative changes that can develop School of Medicine, Stanford, California, and the tDepartment of Ophthalmology, University of Illinois, College of Medicine, Chi- in the retina or RPE after detachment. cago, Illinois. TM is currently affiliated with the Department of Ophthalmol- Materials and Methods ogy, Iwate Medical University, Morioka, Japan. Supported by National Institutes of Health-National Eye Insti- Experiments were performed on Dutch rabbits and tute research grants EY-01678 and EY-05831 (MFM) and adhered to the ARVO Resolution on the Use of Ani- EY-05494 and EY-01792 (DRP). DRP is a Robert E. McCormick mals in Research. We studied seven animals, which Scholar of Research to Prevent Blindness, Inc. were anesthetized with ketamine (30 mg/kg IV) and Submitted for publication: May 10, 1989; accepted November 3, urethan (1.4 g/kg IP); additional doses of ketamine 1989. Reprint requests: Michael F. Marmor, MD, Department of Oph- were given as needed. The eyes were dilated with 10% thalmology, Stanford University School of Medicine, Stanford, CA phenylephrine and 1% atropine. Local retinal de- 94305. tachments, 4-5 mm in diameter, were made as de-

1259

Downloaded from iovs.arvojournals.org on 09/29/2021 1260 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / July 1990 Vol. 31

20 scribed previously. Briefly, a glass micropipette of I£R6 tip diameter 40-50 nm was advanced through a scleral incision near the limbus until it entered the subretinal space. Hanks' balanced salt solution was injected through the micropipette to form small, functionally nonrhegmatogenous detachments. Procedures used for recording the local ERG (LERG) were similar to those described.18 In brief, a double-barrelled micropipette filled with 3 M sodium chloride agar was advanced through the scleral incision so that the longer tip penetrated the detached retina through the small hole formed in creation of the detachment (Figure 1). The longer tip was posi- Fig. 2. Representative LERG and VERG responses from a dark- tioned within the subretinal space, while the shorter adapted eye to the unattenuated stimulus. Note the opposite polari- one remained in the vitreous. Signals from the mi- ties of the LERG and VERG responses. cropipettes were led through a silver-silver chloride bridge, alternating current (AC)-amplified and dis- played on a storage oscilloscope. The LERG was re- adapted eye. The LERG waveform is inverted, but corded between the tips of the subretinal and vitreal shows typical a- and b-waves. We have shown pre- electrodes. The mass, or vitreal, ERG (VERG) was viously that the LERG response arises only from de- recorded between the vitreal electrode and a reference tached retina and is free of contamination from sur- electrode on the sclera. The formation of retinal de- rounding, attached retina.18 The small size of the tachments and positioning of the recording electrodes LERG may in part reflect shunting of the transretinal were performed under dim red light. The animals current through the hole made in the formation of the were dark-adapted for an additional 30 min before detachment. Figure 3 shows the results of bleaching recording was begun. on the VERG. Before exposure to the bleaching light, ERG responses were recorded on the presentation a stimulus of —4.0 log units in intensity elicited a of 200-ms test flashes of white light from a 100-W criterion (40 nV) response. The 7-min bleach ele- lamp. The stimulating beam was controlled by an vated threshold for this criterion by 3 log units, as electronic shutter and attenuated with neutral density shown by the responses recorded § min after extinc- filters. The focused beam was led through a fiberoptic tion of the bleaching light. Over the next 90 min, guide that terminated 1 cm above the eye. The inten- threshold for the 40-/iV response returned to its origi- sity of the unattenuated light (log 1 = 0) was 8 X 103 nal level. lux at the cornea. The adapting irradiations, 7 min in Figure 4 shows LERG data recorded simulta- duration, used white light from a different source that neously with the VERG signals of Figure 3. Although produced an intensity of 6 X 105 lux at the cornea. the responses are of smaller amplitude than those of the VERG, a stimulus of log I = -4.0 was sufficient Results to elicit a detectable (20 ftV) response. Bleaching ele- Figure 2 shows representative LERG and VERG vated threshold for this criterion LERG response by responses recorded simultaneously from the dark- an extent similar to that observed for VERG threshold, 3 log units. At 90 min after bleaching, LERG responses were virtually identical to those recorded double-barrelled before the bleach. microelectrode Figure 5 shows the LERG and VERG thresholds from Figures 3 and 4. Figure 6 illustrates the averaged Ag-AgCI threshold data from all seven experiments of this electrode study. The thresholds for both the LERG and VERG were elevated to a similar degree (on average, about 2.6 log units) by the adapting irradiation, and thresholds for both responses declined with similar time retinal detachment course during the subsequent period of dark adaptation. In the three experiments in which thresholds Fig. 1. The recording procedure. The local ERG was recorded from the longer tip, within the retinal detachment. The ERG from were obtained at 90 min of dark adaptation, the final surrounding attached retina was recorded simultaneously from the thresholds for both the LERG and VERG were lower shorter vitreal tip. on average than those observed immediately before

Downloaded from iovs.arvojournals.org on 09/29/2021 No. 7 ADAPTATION IN LOCAL DETACHMENT / Mori er ol 1261

Dark- Adapted Recovery Stimulous Intensity (Log) 90 min -4.6 Fig. 3. Example of VERG 30 min recovery after light adaptation. The threshold for a 40-MV criterion response was raised 3 log units by bleaching, but recovered to its original level within 90 min.

100 ^V | 50 msec

light adaptation. This slight pre-bleach desensitiza- hibits complete recovery of ERG b-wave sensitivity, tion may represent a residual effect of the light with a normal time course. Since recovery of the needed for the surgical procedures. VERG and LERG exhibited similar properties, one might argue that the LERG is contaminated by elec- Discussion tric currents generated in surrounding, attached ret- The current results show that, after intense illumi- ina. However, in our previous studies using these nation, locally detached retina in the rabbit eye ex- methods,18 we found no evidence of such contamina-

Dark- Recovery Adapted

Stimulous Bleaching Intensity 90 min (Log) Fig. 4. LERG recovery after light adaptation in the -4.6 - same eye as shown in 30 min Figure 3. The LERG responses were recorded simultaneously with the VERG responses. Threshold for a 20-MV response of the LERG was raised 3 log units by bleaching and recovered fully within 90 min after the O min bleach. I 50 msec

Downloaded from iovs.arvojournals.org on 09/29/2021 1262 INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / July 1990 Vol. 01

ment regeneration in the isolated retina prepara- 8 12 BLEACHING tion " ). OLERG The apparent delivery of 1 l-cis retinoid with even o- • a small serous detachment is remarkable, considering o • VITREAL ERG 1_J that the greatest distance over which retinoid diffuses -1 .0- • between RPE and the proximal portion of the outer O I segments is in the range of 50 jam. Local elevation of the retina by even 1 mm would be expected to in- UJ -2.0- 9 crease the volume of the subretinal compartment by irr several orders of magnitude and thus severely dilute h -3 .0- 9 the concentration of retinoid and its hypothesized o W carrier, interphotoreceptor retinoid-binding protein (IRBP).3031 Mechanisms that might preserve efficient -4 .0- 9 9 delivery under such conditions include the inter- membranous diffusion of retinoid to the local detachment from surrounding, normally positioned retina. For example, retinoid can move easily among 0 10 20 30 40 50 60 70 80 90 membranous structures32"34 and through the retina TIME (minutes) itself.3536 There may, of course, be some height of Fig. 5. Graph of the data from Figures 3 and 4 to show the detachment and some associated volume of the sub- recovery of LERG and VERG b-wave thresholds after bleaching. retinal compartment, sufficient to interrupt rhodopsin regeneration. It would be of relevance to examine the recovery of the b-wave using higher detachments, tion. For example, no LERG response is recorded to determine if recovery becomes abnormal at a when the area of retinal detachment has previously "threshold" height. been photocoagulated. Furthermore, the injection of Our data, which imply normal pigment regenera- aspartic acid beneath a local detachment eliminates tion kinetics within a localized detachment, suggest a the b-wave component of the LERG but preserves situation different from CSC, in which regeneration is the VERG b-wave. abnormally slow.13"15 It is possible that this may re- Under our experimental conditions, the average late to species differences, or to differences in height values of log threshold for the LERG continued to between clinical detachments and ours. However, the decline for up to 90 min after the adapting irradiation. This time scale resembles that required for rhodopsin regeneration in the rabbit eye after major bleaching,21 and is comparable to the duration of BLEACHING photochemical, or regeneration-dependent dark ad- 0- oLERG 7 22 23 aptation in the rat and human eye. ' ' Thus, we J • VITREAL ERG believe that the adapting irradiation used in the cur- -1.0- o (n=7)T rent experiments caused substantial bleaching. A cal- I culation based on the photic sensitivity of our ERG

Downloaded from iovs.arvojournals.org on 09/29/2021 No. 7 ADAPTATION IN LOCAL DETACHMENT / Mori er ol 1263

latter possibility seems unlikely, since CSC detach- lar electroretinographic responses in idiopathic central serous ments are typically of a size similar to those studied chorioretinopathy. Am J Ophthalmol 106:546, 1988. 17. Nagata M and Honda Y: Macular ERG in central serous reti- here. One factor clearly distinguishing our experi- nopathy. Jpn J Ophthalmol 15:9, 1971. mental conditions from CSC is the interval between 18. Mori T, Tsue T, and Marmor MF: Electrical responses from the onset of detachment and the physiologic record- detached retina inside the intact rabbit eye. Invest Ophthalmol ing. Photoreceptors are known to degenerate progres- Vis Sci 29:1040, 1988. sively after retina becomes detached1617-37 (although 19. Marmor MF, Mori T, and Pepperberg DR: The recovery of the retinal sensitivity after bleaching in small non-rhegmatogenous the clinical preservation of vision in CSC suggests retinal detachments. ARVO Abstracts. Invest Ophthalmol Vis that this degeneration may be slower in serous than in Sci29(Suppl):414, 1988. rhegmatogenous detachments). Our findings suggest 20. Marmor MF, Abdul-Rahim AS, and Cohen DS: The effect of that abnormalities of visual pigment regeneration metabolic inhibitors on retinal adhesion and subretinal fluid observed in CSC may derive in part from degenera- absorption. Invest Ophthalmol Vis Sci 19:893, 1980. tive changes in the retina or RPE rather than from the 21. Rushton WAH, Campbell FW, Hagins WA, and Brindley GS: The bleaching and regeneration of rhodopsin in the living eye acute interruption of retinoid movement between the of the albino rabbit and of man. Optica Acta 1:183, 1955. RPE and retina. 22. Dowling JE: Chemistry of visual adaptation in the rat. Nature 188:114, 1960. Key words: dark adaptation, retinal detachment, electroret- 23. Rushton WAH: Rhodopsin measurement and dark-adapta- inogram, visual pigment regeneration, central serous cho- tion in a subject deficient in cone vision. J Physiol 156:193, rioretinopathy 1961. 24. Ames A, III, Walseth TF, Heyman RA, Barad M, GraeffRM, References and Goldberg ND: Light-induced increases in cGMP metabolic flux correspond with electrical responses of photorecep- 1. KUhne W: Zur Photochemie der Netzhaut. Unters Physiol Inst tors. J Biol Chem 261:13034, 1986. Heidelberg 1:1, 1877. 25. Green DG and Powers MK: Mechanisms of light adaptation in 2. Wald G: Carotenoids and the visual cycle. J Gen Physiol rat retina. Vision Res 22:209, 1982. 19:351, 1935. 26. Bridges CDB: 1 \-cis vitamin A in dark-adapted rod outer seg- 3. Hubbard R and Dowling JE: Formation and utilization of ments is a probable source of prosthetic groups for rhodopsin 1 \-cis vitamin A by the eye tissues during light and dark adap- biosynthesis. Nature 259:247, 1976. tation. Nature 193:341, 1962. 27. Bridges CDB, Alvarez RA, Fong S-L, Gonzalez-Fernandez F, 4. Zimmerman WF: The distribution and proportions of vitamin Lam DMK, and Liou GI: Visual cycle in the mammalian eye: A compounds during the visual cycle in the rat. Vision Res Retinoid-binding proteins and the distribution of 1 l-cis retin- 14:795, 1974. oids. Vision Res 24:1581, 1984. 5. Bridges CDB: Vitamin A and the role of the pigment epithe- 28. Lai Y-L, Tsin ATC, Lam K-W, and Garcia JJ: Distribution of lium during bleaching and regeneration of rhodopsin in the retinoids in different compartments of the posterior segment of frog eye. Exp Eye Res 22:435, 1976. the rabbit eye. Brain Res Bull 15:143, 1985. 6. Bernstein PS, Law WC, and Rando RR: Isomerization of all- 29. Catt M, Ernst W, and Kemp CM: The products of photore- trans retinoids to 1 \-cis retinoids in vitro. Proc Natl Acad Sci versing rhodopsin bleaching by microsecond flashesi n the iso- USA 84:1849, 1987. lated vertebrate retina. Vis Res 23:971, 1983. 7. Dowling JE: Neural and photochemical mechanisms of visual 30. Chader GJ, Wiggert B, Lai Y-L, Lee L, and Fletcher RT: In- adaptation in the rat. J Gen Physiol 46:1287, 1963. terphotoreceptor retinol-binding protein: A possible role in 8. Weinstein GW, Hobson RR, and Dowling JE: Light and dark retinoid transport to the retina. Progr Retinal Res 2:163, 1983. adaptation in the isolated rat retina. Nature 215:134, 1967. 31. Okajima T-IL, Pepperberg DR, Ripps H, Wiggert B, and 9. Pepperberg DR, Brown PK, Lurie M, and Dowling JE: Visual Chader GJ: Interphotoreceptor retinoid-binding protein: Role pigment and photoreceptor sensitivity in the isolated skate retin delivery of retinol to the pigment epithelium. Exp Eye Res ina. J Gen Physiol 71:369, 1978. 49:629, 1989. 10. Pepperberg DR, Lurie M, Brown PK, and Dowling JE: Visual 32. Rando RR and Bangerter FW: The rapid intermembraneous adaptation: Effects of externally applied retinal on the light- transfer of retinoids. Biochem Biophys ResCommun 104:430, adapted, isolated skate retina. Science 191:394, 1976. 1982. 11. Pepperberg DR and Masland RH: Retinal-induced sensitiza- 33. Ho M-TP, Massey JB, Pownall HJ, Anderson RE, and Holly- tion of light-adapted rabbit photoreceptors. Brain Res 151:194, field JG: Mechanisms of vitamin A movement between rod 1978. outer segments, interphotoreceptor retinoid-binding protein, 12. Perlman JI, Nodes BR, and Pepperberg DR: Utilization of and liposomes. J Biol Chem 264:928, 1989. retinoids in the bullfrog retina. J Gen Physiol 80:885, 1982. 34. Fex G and Johannesson G: Studies of the spontaneous transfer 13. Alpern M and Krantz DH: Visual pigment kinetics in abnor- of retinol from the retinol:retinol-binding protein complex to malities of the uvea-retinal epithelium interface in man. Invest unilamellar liposomes. Biochim Biophys Acta 901:255, 1987. Ophthalmol Vis Sci 20:183, 1981. 35. Marmor MF and Martin LJ: Visual pigment regeneration: Oc- 14. van Meel GJ, Smith VC, Pokorny J, and van Norren D: Foveal currence in frog retina upside down upon the pigment epithe- densitometry in central serous choroidopathy. Am J Ophthal- lium. Experientia 34:384, 1978. mol 98:359, 1984. 36. Marmor MF and Martin LJ: 100 years of the visual cycle. Surv 15. Chuang EL, Sharp DM, Fitzke FW, Kemp CM, Holden AL, Ophthalmol 22:279, 1979. and Bird AC: Retinal dysfunction in central serous retinopa- 37. Machemer R: Experimental retinal detachment in the owl thy. Eye 1:120, 1987. monkey: II. Histology of retina and pigment epithelium. Am J 16. Miyake Y, Shiroyama N, Ota I, and Horiguchi M: Local macu- Ophthalmol 66:396, 1968.

Downloaded from iovs.arvojournals.org on 09/29/2021