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Fluorophotometric assessment of blood-retinal barrier function after white light exposure in the rabbit eye
Exp. Eye Res. (1990) 50, 297-304
Fluorophotometric After
Assessment of Blood-Retinal Barrier White Light Exposure in the Rabbit Eye
R.A.BORSJEa*, aDepartment The Netherlands,
G.F.J.M.VRENSEN*, of Ophthalmology, and b Department
(Received
J.A.vAN
BEST”?
AND
Function
J.A.OOSTERHUIS@
University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, of Morphology, The Netherlands Ophthalmic Research institute, Amsterdam. The Netherlands
19 May 7989 and accepted
in revised form 6 September
1989)
Fluorophotometry was performed in 14 rabbits after exposure of one eye to white light with an energy insuflicient to cause visible phototoxic retinal damage as determined by ophthalmoscopy and fundus photography. Fluorescence measurements in the vitreous were performed before and 1 hr after i.v. injection of fluorescein. Ratios between the fluorescein concentrations in the exposed and in the non-exposed fellow eye were calculated after correction for the autofluorescence. The average ratio directly after light exposure had significantly increased (P = 0005) as compared to pre-exposure values and was maximal one day after exposure (P < 0.005). Four days after exposure the ratios had returned to pre-exposure values (P > 0.05). A significant linear correlation between age and the ratios directly after exposure was found (r = - 06 7 : P < 00 1). Signs of phototoxic retinal damage were not found on ophthalmoscopy and fundus photography, nor on light and electron microscopic examination of the retinal pigment epithelium. neuro-retina or retinal capillaries 1 and 4 days after light exposure. A fluorophotometric assessment of the blood-retinal barrier (BRB) function after white light exposure appeared to be a more sensitive parameter of light-induced damage than morphological examination since light exposures at retinal irradiance levels below the threshold for ultrastructural damage resulted in a temporary BRB dysfunction that could be detected by tluorophotometry but not by the other methods. Key words: phototoxicity ; white light: fluorophotometry : electron microscopy : blood-retinal barrier: retinal pigment epithelium ; rabbit.
1. Introduction
2. Materials and Methods
In the past decades numerous studies have reported the harmful effects of light on the mammalian retina (Lanum, 1978; Mainster, Ham and Delori, 1983; Marshall, 1984; Kremers and Van Norren, 1988). Ophthalmoscopy, fundus photography, fluorescein angiography, electroretinography, light and electron microscopy have been used to evaluate light damage to the neuro-retina and the retinal pigment epithelium (RPE). Vitreous fluorophotometry proved to be a very sensitive method for assessing blood-retinal barrier (BRB) function after photocoagulation (Johnson, McNaught and Foulds, 1977; Noth, Vygantes and Cunha-Vaz, 1978; Zweig et al., 1981; Wallow, 1984; Tsukahara et al., 198 7) and has also been used
Animals
in
severe
phototoxic
retinopathy
(Bellhorn
and
Korte, 1983). The present fluorophotometric study was performed in rabbits after exposure to low-intensity white light levels and aimed to detect the earliest dysfunction of the BRB before damage was ophthalmoscopically visible. Light and electron microscopy of the retinas, and especially of the RPE and retinal capillaries as the structural counterparts of the BRB, were performed. * For reprint requests. t For correspondence. 00144835/90/030297+08
Pigmented rabbits with clear ocular media (strain Chinchilla greys, weight 1.7-2.9 kg, age 12.5-20.0 weeks) were used. Fourteen rabbits were exposed to light, three served as controls. Methods Light source and radiometry. The left eyes of 14 rabbits were exposed to light from a tungsten-halogen air-cooled projector bulb (24 V; 250 W) according to a method described by Calkins and Hochheimer (1980). Three infrared filters (KG 1, 2 mm, Schott Jena) were used to reduce infrared radiation to a negligible level. The radiance of the image source was measured with an EG&G Model 450 radiometer/ photometer (EG&G Electra-optics, USA) fitted with a multiprobe detector type 5 50-2. Retinal irradiance levels were calculated from the radiance value according to Calkins and Hochheimer (1980), using an index of refraction of 1.33 (Hughes, 1972) and a transmission factor of the ocular media of 09 (Wiesinger et al., 1956; Sliney, 1984). The distance from retina to image source focal point, in our case the focal length of the rabbit’s eye (see below), was 12.0 mm. This value was calculated from the average retinal spot size measured
$03.00/O
in three enucleated
rabbit
0 1990 Academic Press Limited
298
eyes positioned in the experimental set-up. The area of illumination lying centrally on the visual streak (Prince and McConnell, 1964) and including the radiating medullary nerve fibers was 8.8 mm in diameter, corresponding to an angIe of illumination of 40 degrees and a focal eyelength of 12-O mm. In our experiments a retinal irradiance level of about 100 mW crnm2with an exposure time of 90 min was aimed, as this level was considered to be below the threshold for visible retinal damage but sufficiently high to cause phototoxic damage of the rabbit retina. This value was calculated from interpolation of threshold data for minimal detectable damage in rabbits (Lawwill, 1973 ; McKechnie and Johnson 1977; Rinkhoff et al., 1986: Hoppeler et al., 1988). Light exposure. The light beam was focused by a coated Nikon + 20 D ophthalmoscopy lens at a 5-cm distance from this lens, During light exposure this focal spot (diameter 2 mm), as image source, was positioned in the center of the pupillary plane of the rabbit’s eye. During exposure an eyelid speculum was used and the cornea was continuously moistened by buffered saline. The eyelids of the fellow eye were occluded by adhesive tape. Body temperature was monitored by a rectal thermistor (Ellab, A-ISC, Denmark) and controlled within 1°C by means of thermal isolation. Fluorophotometry. Fluorophotometry was performed with the Fluorotron Master (Coherent Radiation Inc., Palo Alto, U.S.A.) 2-7 days prior to light exposure and 0.5-1.5 hr, 24 hr and 4 days after exposure. A plastic cylinder was mounted to the top of the adaptor to provide a fixed distance between lens and eye. Each fluorophotometric examination started with three prescans of both eyes before iv. administration of fluorescein to determine autofluorescence. Subsequently five scans were performed of each eye 5466 min after i.v. injection of fluorescein (0.125 ml per kg body weight of a solution containing 200 mg ml-l). Left and right eyes were measured alternately. The average fluorescence in the vitreous (F,,, expressed in ng equivalent ml-l) was computed along the optical axis from 18% of the distance between retina and anterior chamber fluorescence peaks (at about 5 mm from the retina) up to the point halfway between these peaks (at about the middle of the eye) (see Fig. 2). The mean fluorescein concentration value in the area of measurement, C,,, was obtained by subtracting the mean F,,-value of three prescans from that of the five scans performed after fluorescein administration. Effects of the light exposure on the BRB function were quantified by the ratio between C,, for the exposed (left) and the non-exposed (right) eye. Changes in the fluorophotometric ratios after light exposure were statistically tested using the Wilcoxon’s paired signed-rank test.
R. A. BORSJE
ET AL.
Anesthetics During exposure the animals were anesthetised with a 1: 1 mixture of ketamine 10 % (Aescoket’“, Aesculaap, The Netherlands) and 2- (2,6-xylidine)5,6-dihydro-4H-1,4-thiazine 2% (Rompun%, Bayer. Spain) ( 1.5 ml hr-’ ; after 1 hr a single dose of 0.5 ml was given). Mydriasis and cycloplegia were achieved by repeated instillation of tropicamide 0.5% (Mydriatricumm) and cyclopentolate 1% (Cyclogyl”) in both eyes. During fluorophotometry the animals were sedated by 1 ml Hypnorm” (containing 10 mg fluanison and 0.315 mg fentanylcitrate, Janssen Pharmaceutics, The Netherlands). The pupils were dilated by tropicamide 0.5 % and phenylephrine 5% before ophthalmoscopy, fundus photography and fluorophotometry. Histology Histological examination has been carried out in six eyes 1 day and 4 days after light exposure and in three control eyes. Three rabbits were killed 4 days after light exposure several hours after the last fluorophotometric scans. Their exposed eyes were enucleated at this day for morphological examination since phototoxic retinal lesions may not become apparent until 2-3 days after exposure (Lawwill, 1973; Rinkhoff et al., 1986). The non-exposed fellow eyes of these rabbits served as controls. The control eyes of three other rabbits with an increased C,,-ratio 24 hr after exposure were also exposed 3 weeks later. These animals were killed 24 hr after the last exposure with an overdose of pentobarbital sodium (Nembutal@, Ceva, France) and the most recently exposed eyes were enucleated for histological examination. After enucleation the eyes were immersed in a 0.08 M cacodylate buffered solution containing 1% glutaraldehyde and 1.2 5 % paraformaldehyde at pH 7.3. Liibal incisions were made to facilitate penetration of the fixative. After fixation for several days the eyes were dissected and four samples were taken from the visual streak and two samples from the nasal and temporal part of the area of the radiating medullary nerve fibers. After thorough rinsing in cacodylate buffer the retinal samples were post-fixed with OsO,, dehydrated and embedded in Epon 812. Ultrathin sections were stained with lead citrate and uranyl acetate and inspected in a Philips EM 400 transmission electron microscope. Light microscopic examination was carried out on semithin (1 pm) sections of the same material after staining with toluidine blue. As the main objective of the present study was to correlate morphological changes and leakage of fluorescein in the posterior eye segment as assessed by fluorophotometry we mainly studied the RPE in the visual streak and the capillaries in the area of the radiating medullary nerve fibers.
BLOOD-RETINAL
BARRIER
AND
LIGHT
EXPOSURE
I
t
I
I
400
500
600
700
Wavelength
(nm)
FIG. 1. Relative spectral power curve of the image source as measured behind the focussing lens (percent of maximum power). TABLE I
Fluorophqtometric results. Mean ratio and standarddeviations betweenfluorescein leakagein exposedand control fellow eyes. Mean ratio Group Total (n = 14) I* (n = 6) IIt (n = 8)
2-7 days pre-exposure 1.00+0.12 0.99 kO.14 1.01f0.11
0.5-1.5 hr post-exposure 1.26kO.37 1.50+0.39 1.08f0.23
1 day post-exposure 1.52 f 0.50 1.58*o-60 1.48+ 0.44
4 days post-exposure 097&0.18 092kO.17 l.OOf0.19
* Rabbit age < 17 weeks. t Rabbit age 3 17 weeks.
Ophthalmoscopy and fundus photography were carried out on all animals prior to, 24 hr and 4 days after light exposure.
3. Results Light exposure
The calculated mean retinal illumination level was 102*6&2*5 mW cmd2. The relative spectral power distribution of the image source, measured with a spectrophotometer (Photo Research Spectrascan S.N. 2186, U.S.A.), is shown in Fig. 1. The curve shows a peak at 572 nm, an effective suppression of infrared radiation and a relatively small contribution of the
blue range of the spectrum. Fluorophotometry
The average ratios between the fluorescein concentrations in the exposed and the control fellow eye prior 21
to and at different ties after exposure are presented in Table I. The average ratio immediately after exposure had significantly increased as compared to preexposure values (P = O-005). The increase was maximal one day after exposure (P < O-005). Four days after exposure the ratios had returned to pre-exposure values. There were no significant differences between the ratios immediately and 1 day after exposure and between the pre-exposure and 4 days post-exposure values (both P > 0.05). An example of a fluorophotometric scan showing an increased leakage of fluorescein into the vitreous after exposure is shown in Fig. 2. As we had the impression that the variation in fluorophotometic results was probably age-related we arbitrarily split the group into rabbits under and over 17 weeks of age, The average ratios for rabbits aged 12.5-l 7 weeks (group I; n = 6) and those aged 17-20-S weeks (group II; n = 8) are presented separately in Table I. In group I a signiiicant increase of the average ratio was found directly after exposure EER SO
300
R. A. BORSJE
II
I
10
1
ET AL.
I
20
Optical axis (mm) FIG. 2. Exampleof two fluorophotometricscansperformedat about 60 min after i.v. injection of fluorescein,1 day after light exposure.Upper curve: exposedeye; lower curve: fellow control eye. Vertical continuous lines: R = retina/choroid; A = anterior chamber.Vertical broken lines: limits usedfor leakageevaluation (F,-value).
0 0
0 0
0
q
o--------------
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-------
;,- ,---C-------------------A--D 0
UJ &I
,
I
I
I
I
I
12
16
20
12
16
2c
Age (weeks) FIG. 3. Ratiosbetweenexposed(OS)and control eyes(OD)directly after (left panel)and one day (right panel)after exposure as a function of the rabbit’s age. The obliquestraight line was obtainedby a linear regressionprocedureto the data points (r = -0.67; P < 0.01). A significantcorrelationwasnot found oneday after exposure.The horizontal straight line represents the averageratio of the total population beforeexposure.Broken lines representk 1 S.D.
(P = 0.02) and 1 day after exposure (P = 0.02). In group II a slight non-significant increase was found directly after exposure (P > 0.05) but a significant increase after 1 day (P = 0.01). The ratios of both groups had returned to pre-exposure values after 4
days (P > 0.05). The ratios directly after light exposure and 1 day after exposure are presented as a function of the rabbit’s age in Fig. 3. A significant linear correlation was found between age and ratios directly after exposure (decrease in ratio : O-1/week: r = - 0.67 ; P < O-01), but no significant correlation was found after 1 day (P = 0.73).
The complete experimental procedure with the exception of turning on the light bulb followed by fluorophotometry directly after and 1 day after the sham-treatment was performed in three rabbits. These rabbits did not show any significant increase of their left/right permeability ratio in any of the two fluorophotometric measurements (P > 005). Two animals showed anterior chamber fluorescein concentrations exceeding 3000 ng equivalent ml-’ in the fluorophotometric session before light exposure for unknown reasons. These animals were excluded from the study since such concentrations were assumed to interfere with measurements in the vitreous due to
” IL
FIG. 4. Electron micrograph of a RPE cell one day after light exposure. (a) RPE cell with normal smooth endoplasmatic reticulum (sER), mitochondria apical microvilli (Mv) and junctional complex (jc, between arrows) (Nu = nucleus: BrM = Bruch’s membrane; Cap = choriocapillary : Mel = melanin Detail of the lateral cell membrane with a tight junction (tj) and a desmosome (Des), both ultrastructurally normal in appearance ( x 97600).
(Mit), basal foldings (bf), granule) ( x 12 800), (b)
302
R. A. BORSJE
ET AL.
FIG. 5. Electron micrograph of a capillary with a normal endothelial cell (End) and a pericyte (Per) from the area of the radiati ing mednllary nerve fibers, 1 day after light exposure. Arrows indicate intercellular junctions (Lum = lumen ; bl = biasal lamina) ( x 12 800).
light absorption in the anterior chamber and/or leakage of fluorescein from the anterior chamber into the vitreous.
Ophthalmoscopy, Fundus Photography and Histology
Signs of retinal damage on ophthalmoscopy and fundus photography were not found 1 and 4 days after exposure. Light and electron microscopic examination of the samples of light exposed and control retinas 1 and 4 days after exposure did not reveal any changes in the RPE. Basal membrane invaginations facing Bruch’s membrane, nuclei, mitochondria and other cellular organeljes were normal [Fig. 4(a)]. Special attention was given to the junctional complex of the lateral cell membrane, as damage to the tight junctions was considered to be the main cause of fluorophotometrically assessed fluorescein leakage, but their aspect was completely normal [Fig. 4(b)]. There was
no evidence of increased disc shedding, or changes in the slender apical cell processes. Although the photoreceptor outer segments were somewhat irregular with respect to their membranes degenerative effects were not obvious. The neuro-retina exhibited no pathological changes. The junctions at the level of the external limiting membrane did not show signs of disturbance. The choriocapillaries, filled with numerous red blood cells due to the immersion fixation, were normal. The retinal blood vessels and capillaries, in the rabbit restricted to the region of the radiating medullary nerve fibers (Prince and McConnell, 1964), exhibited no changes. The endothelial lining was completely intact in both control and treated animals, and disruption of the tight junctions between neighboring endothelial cells could not be discerned (Fig. 5). The number of pinocytotic vesicles was unchanged. The myelinated fibers in the area of radiating nerve fibers were normal.
medullary
BLOOD-RETINAL
BARRIER
AND
LIGHT
EXPOSURE
4. Discussion After white light exposure of rabbit eyes at levels below the threshold for phototoxic damage to the retina, a significant leakage of fluorescein into the vitreous documented a temporary dysfunction of the blood-retinal barrier (BRB). Examination of the retina by ophthalmoscopy, fundus photography and light and electron microscopy failed to show evidence of structural damage. Animals younger than 17 weeks showed a significant increase in leakage directly after exposure whereas animals older than 17 weeks did not: 24 hr after exposure both groups showed identical increased leakage. Fluorescein leakage did return to pre-exposure values 4 days after exposure. The leakage of fluorescein was not associated with abnormal ophthalmoscopic findings or electron microscopic disturbances of the RPE, neuro-retina or retinal capillaries. Thus, a functional disturbance of the BRB is not necessarily reflected in morphological changes of this barrier and fluorophotometric assessment of BRB function was shown to be a more sensitive parameter of light-induced damage than morphological examination. The retinal irradiance level was chosen from literature on account of interpolations of threshold data for minimal detectable damage in rabbits (Lawwill, 19 73 : McKechnie and Johnson, 19 7 7 ; Rinkhoff et al., 1986; Hoppeler et al., 1988). Hoppeler et al. (1988) however, reported ophthalmoscopic and histological changes in the RPE and outer segments of photoreceptors at much lower irradiance levels than those used in the present study. This can be explained by the fact that the light they used had a spectral distribution ranging from 400 to 550 nm, measured on retinal surface. It has been proven that the highenergy (400-500 nm) part of the visible light is more hazardous than the low-energy part (500-700 nm) (Ham et al., 1979; Young, 1988). Moreover, the spectral distribution of our source was measured in air and showed little power in this short wave range and this was further reduced by the decrease in transmission of the rabbit ocular media under 500 nm (Wiesinger et al., 1956). A puzzling aspect of the present study is the source of the significant increase of fluorescein leakage into the vitreous of the exposed eyes in the absence of ultrastructural damage of RPE cells and the tight junctions cementing the lateral borders of the RPE cells and the endothelial lining of the capillaries. Tso and Woodford (1983) found mildly edematous RPE cells with ultrastructurally normal intercellular junctions in the periphery of a lesion of photochemical retinopathy. Horseradish peroxidase (HRP) studies showed that, despite the intact tight junctions, HRP leaked into the subretinal space, proving that a normal ultrastructure of tight junctions is not indicative for functional integrity. It might well be that levels of light below the threshold for ultra-structural
303
damage induce molecular changes of junctional membranes of RPE cells and/or endothelial cells, enabling fluorescein and/or the fluorescein-albumin complex to pass these junctions. An alternative explanation might be a disturbance of the transcellular route of the RPE cells which are actively involved in regulating molecular transport between the choriocapillaris and the neuro-retina. A disturbance of the homeostatic mechanism including enzyme inactivation has been found after light exposure (Hansson, 1971; Marshall, 1984; Ham et al., 1984). In addition, normally a tracer as fluorescein injected intravitreally is transported rapidly into the choroid across the outer BRB by an active transport mechanism (Kitano, Hori and Nagataki, 1988). Decreased pumping activity results in increased fluorescein concentrations in the vitreous (Tsukahara et al., 1987; Kitano et al., 1988). A relative pump defect is not likely to contribute to increased fluorescein concentrations in the vitreous since these concentrations were measured 1 hr after fluorescein injection while the plasma- and vitreousconcentration gradient still exceeded the pumping mechanism. Which intra- and intercellular components are participating in the transport of fluorescein and to what extent they are affected by light remains to be studied using tracers and morphometric assessment of affected RPE cells. Several authors reported histological and functional disturbances after light exposure as determined by light and electron microscopy and fluorescein angiography without ophthalmoscopically visible retinal damage (Hochheimer, D’Anna and Calkins, 1979 ; Parver, Auker and Fine, 1983). Michels et al., (1987) reported the threshold for light damage to the retina, using angiographic and histologic damage thresholds, to be probably several orders of magnitude higher than those for functional damage. The present results re-emphasize this discrepancy between functional and morphological assessments of retinal light damage and strongly plead for a functional assessment of light damage of which fluorophotometry seems to be the most sensitive method. In our fluorophotometric study a reversible dysfunction of the BRB was found with complete recovery within 4 days, after exposure to light with an irradiance level below the threshold for ultrastructural damage. Morphological and functional recovery from light has been reported before for both thermally and photochemically induced light damage ; the degree of recovery seems to be dose-related (Kuwabara, 1970; Johnson et al., 1977 ; Lanum, 1978; McKechnie and Foulds, 1980; Wallow, 1984). Tso and Woodford (1983) reported fast partial recovery of histological damage but complete recovery of the outer BRB function as determined by HRP studies, at the periphery of the retinal lesion. However, in eyes with pre-existent retinal disease or in eyes chronically exposed to high environmental light levels,
304
R. A. BORSJE
recovery may be impaired resulting in enhancement of retinal disease (Mainster et al., 1983: Young, 1988). Especially in these eyes, acute and repetitive light exposures may lead to more severe functional damage and visible damage, or to accelerated senescence. This animal study emphasizes the need to reduce instrumental light exposure as much as possible.
Acknowledgments The authors are indebted to Professor D. van Non-en, Ph.D. and J. J. M. Kremers, Ph.D., T.N.O. Institute for Perception, The Netherlands, for performing the measurement of the spectral power distribution of the image source, to D. F. Schaling, M.D., and T. Riimer for assistance in fluorophotometric measurements and to Mrs W. J. Baartse for typing the manuscript.
References Bellhorn, R. W. and Korte. G. E. (1983). Permeability of abnormal Blood-Retinal Barrier in rat phototoxic retinopathy : a clinicopathologic correlation study using fluorescent markers. Invest. OphthaJmoJ. Vis. Sci. 24, 972-5
Calkins,J. L. and Hochheimer,B. F. (1980). Retinal light exposurefrom ophthalmoscopes, slit lamps,and overhead surgical lamps.An analysisof potential hazards. Invest. OphthaJmoJ. Vis. Sci. 19, 1009-15. Ham, W. T., Jr., Mueller, H. A., Ruffolo,J. J,, Jr. and Clarke, A. M. (1979). Sensitivity of the retina to radiation damage as a function of wavelength. Photochem. Photobiol.29, 735-43. Ham, W. T.. Jr., Mueller, H. A., Ruffolo, J. J.. Millen, J. E., Cleary, S.F., Guerry, R. K. and Guerry III, D. (1984). Basicmechanismsunderlying the production of photochemical lesionsin the mammalianretina. Curr. Eye Res. 3, 165-74. Hansson, H. (1971). A hi&chemical study of cellular reactions in rat retina transiently damagedby visible light. Exp. Eye Res. 12, 2704. Hochheimer,B. F., D’Anna, S.A. and Calkins,J. L. (1979). Retinal damage from light. Am. I. Ophthalmol.88, 103944. Hoppeler,Th., Hendrickson,Ph., Dietrich, C. and Rem&Ch. (1988). Morphology and time-courseof delinedphotochemicallesionsin the rabbit retina. Curr. Eye Res. 7, 849-60.
Hughes,A. (19 72). A schematiceye for the rabbit. Vision Res.12. 123-37. Johnson,N. F., McNaught, E. I. and Foulds,W. S. (1977). Effectof photocoagulationon the barrier function of the pigmentepithelium.II. A study by electronmicroscopy. Trans.Ophthalmol.Sot. U.K. 97, 640-51. Kitano, S., Hori, S. and Nagataki, S. (1988). Transport of fluorescein in the rabbit eye after treatment with sodiumiodate. Exp. Eye Res. 46, 863-70. Kremers,J. J. M. and Van Norren, D. (1988). Two classes of
ET AL.
photochemical damage of the retina. Lasers Light Ophthalmol.2, 41-52. Kuwabara, T. (19 70). Retinal recovery from exposureto light. Am. J. Ophthalmol.70, 187-98. Lanum, J. (1978). The damaging effects of light on the retina. Empirical findings, theoretical and practical implications.Surv. Ophthalmol.22, 22149. Lawwill, T. (1973). Effectsof prolongedexposureof rabbit retina to low-intensity light. Invest.Ophthalmol.Vis. Sci. 12,45-51. Mainster, M. A., Ham, W. T. Jr. and Delori, F. C. (1983). Potential retinal hazards. Instrument and environmental light sources.Ophthalmology 90, 92 7-3 1. Marshall, J. (1984). Light damage and the practice of ophthalmology. In Intra-ocular Lens ImpZantation (MS Rosen,E., Arnott, E. and Haining, W.). Pp. 182-207. Mosebey-YearbookLtd : London. McKechnie,N. M. and Johnson,N. F. (1977). Light damage to the retina. Albrecht von Graefe’s Arch. Klin. Exp. Ophthalmol.203, 283-92. McKechnie,N. M. and Foulds,W. S.(1980). Recoveryof the rabbit retina after light damage(Preliminary observations). Albrecht von Graefe’s Arch. KJin. Exp. OphthaJmoJ. 212, 271-283. Michels,M.. Dawson.W. W., Feldman,R. B. andJarolem,K. ( 1987). Infrared. An unseenand unnecessaryhazard in ophthalmic devices.Ophthalmology 94. 143-8. Noth, J. M., Vygantas, C. and Cunha-Vaz, J. G. F. (1978). Vitreous fluorophotometry evaluation of xenon photocoagulation.Invest. OphthaJmoJ. Vis. Sci. 17, 1206-g. Parver, L. M., Auker. C. R. and Fine, B. S. (1983). Observationson monkeyeyesexposedto light from an operating microscope.Ophthalmology 90, 964-72. Prince, J. H. and McConnell,D. G. (1984). Retina and optic nerve. In The Rabbit in Eye Research (Ed. Prince, J. H.). Pp. 385415. CharlesC. Thomas: Springfield. Rinkhoff. J., Machemer. R., Hida, T. and Chandler, D. (1986). Temperature-dependentlight damageto the retina. Am. 1. Ophthalmol. 102, 452-62. Sliney. D. H. (1984). Quantifying retinal irradiancelevelsin light damageexperiments.Curr. Eye Res. 3. 175-9. Tso, M. 0. M. and Woodford, B. J. (1983). Effect of photic injury on the retinal tissues.Ophthalmology 90,952-63. Tsukahara.Y., Ogura, Y., Miura. M. and Kondo, T. (1987). Etrect of argon laser photocoagulation on the permeability of the Blood-RetinalBarrier. Ophthalmologica 94, 27-33. Wallow, I. H. (1984). Repair of the pigment epithelial barrier following photocoagulation.Arch. OphthaJmoJ. 102, 126-35. Weisinger,H., Schmidt, F. H., Williams, R. C., Tiller, C. 0.. Ruffin. R. S.. Guerry III. D. and Ham, W. T.. Jr. (1956). The transmissionof light through the ocular mediaof the rabbit. Am. 1. Ophthalmol. 42, 907-10. Young, R. W. (1988). Solar radiation and age-related macular degeneration.Surv. OphthaJmoJ. 32, 252-69. Zweig, K., Cunha-Vaz, J., Peyman, G., Stein, M. and Raichand. M. (1981). Effect of argon laser photocoagulationon fluoresceintransport acrossthe bloodretinal barrier. Exp. Eye Res. 32, 323-9.
Fluorophotometric After
Assessment of Blood-Retinal Barrier White Light Exposure in the Rabbit Eye
R.A.BORSJEa*, aDepartment The Netherlands,
G.F.J.M.VRENSEN*, of Ophthalmology, and b Department
(Received
J.A.vAN
BEST”?
AND
Function
J.A.OOSTERHUIS@
University Hospital, Rijnsburgerweg 10, 2333 AA Leiden, of Morphology, The Netherlands Ophthalmic Research institute, Amsterdam. The Netherlands
19 May 7989 and accepted
in revised form 6 September
1989)
Fluorophotometry was performed in 14 rabbits after exposure of one eye to white light with an energy insuflicient to cause visible phototoxic retinal damage as determined by ophthalmoscopy and fundus photography. Fluorescence measurements in the vitreous were performed before and 1 hr after i.v. injection of fluorescein. Ratios between the fluorescein concentrations in the exposed and in the non-exposed fellow eye were calculated after correction for the autofluorescence. The average ratio directly after light exposure had significantly increased (P = 0005) as compared to pre-exposure values and was maximal one day after exposure (P < 0.005). Four days after exposure the ratios had returned to pre-exposure values (P > 0.05). A significant linear correlation between age and the ratios directly after exposure was found (r = - 06 7 : P < 00 1). Signs of phototoxic retinal damage were not found on ophthalmoscopy and fundus photography, nor on light and electron microscopic examination of the retinal pigment epithelium. neuro-retina or retinal capillaries 1 and 4 days after light exposure. A fluorophotometric assessment of the blood-retinal barrier (BRB) function after white light exposure appeared to be a more sensitive parameter of light-induced damage than morphological examination since light exposures at retinal irradiance levels below the threshold for ultrastructural damage resulted in a temporary BRB dysfunction that could be detected by tluorophotometry but not by the other methods. Key words: phototoxicity ; white light: fluorophotometry : electron microscopy : blood-retinal barrier: retinal pigment epithelium ; rabbit.
1. Introduction
2. Materials and Methods
In the past decades numerous studies have reported the harmful effects of light on the mammalian retina (Lanum, 1978; Mainster, Ham and Delori, 1983; Marshall, 1984; Kremers and Van Norren, 1988). Ophthalmoscopy, fundus photography, fluorescein angiography, electroretinography, light and electron microscopy have been used to evaluate light damage to the neuro-retina and the retinal pigment epithelium (RPE). Vitreous fluorophotometry proved to be a very sensitive method for assessing blood-retinal barrier (BRB) function after photocoagulation (Johnson, McNaught and Foulds, 1977; Noth, Vygantes and Cunha-Vaz, 1978; Zweig et al., 1981; Wallow, 1984; Tsukahara et al., 198 7) and has also been used
Animals
in
severe
phototoxic
retinopathy
(Bellhorn
and
Korte, 1983). The present fluorophotometric study was performed in rabbits after exposure to low-intensity white light levels and aimed to detect the earliest dysfunction of the BRB before damage was ophthalmoscopically visible. Light and electron microscopy of the retinas, and especially of the RPE and retinal capillaries as the structural counterparts of the BRB, were performed. * For reprint requests. t For correspondence. 00144835/90/030297+08
Pigmented rabbits with clear ocular media (strain Chinchilla greys, weight 1.7-2.9 kg, age 12.5-20.0 weeks) were used. Fourteen rabbits were exposed to light, three served as controls. Methods Light source and radiometry. The left eyes of 14 rabbits were exposed to light from a tungsten-halogen air-cooled projector bulb (24 V; 250 W) according to a method described by Calkins and Hochheimer (1980). Three infrared filters (KG 1, 2 mm, Schott Jena) were used to reduce infrared radiation to a negligible level. The radiance of the image source was measured with an EG&G Model 450 radiometer/ photometer (EG&G Electra-optics, USA) fitted with a multiprobe detector type 5 50-2. Retinal irradiance levels were calculated from the radiance value according to Calkins and Hochheimer (1980), using an index of refraction of 1.33 (Hughes, 1972) and a transmission factor of the ocular media of 09 (Wiesinger et al., 1956; Sliney, 1984). The distance from retina to image source focal point, in our case the focal length of the rabbit’s eye (see below), was 12.0 mm. This value was calculated from the average retinal spot size measured
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eyes positioned in the experimental set-up. The area of illumination lying centrally on the visual streak (Prince and McConnell, 1964) and including the radiating medullary nerve fibers was 8.8 mm in diameter, corresponding to an angIe of illumination of 40 degrees and a focal eyelength of 12-O mm. In our experiments a retinal irradiance level of about 100 mW crnm2with an exposure time of 90 min was aimed, as this level was considered to be below the threshold for visible retinal damage but sufficiently high to cause phototoxic damage of the rabbit retina. This value was calculated from interpolation of threshold data for minimal detectable damage in rabbits (Lawwill, 1973 ; McKechnie and Johnson 1977; Rinkhoff et al., 1986: Hoppeler et al., 1988). Light exposure. The light beam was focused by a coated Nikon + 20 D ophthalmoscopy lens at a 5-cm distance from this lens, During light exposure this focal spot (diameter 2 mm), as image source, was positioned in the center of the pupillary plane of the rabbit’s eye. During exposure an eyelid speculum was used and the cornea was continuously moistened by buffered saline. The eyelids of the fellow eye were occluded by adhesive tape. Body temperature was monitored by a rectal thermistor (Ellab, A-ISC, Denmark) and controlled within 1°C by means of thermal isolation. Fluorophotometry. Fluorophotometry was performed with the Fluorotron Master (Coherent Radiation Inc., Palo Alto, U.S.A.) 2-7 days prior to light exposure and 0.5-1.5 hr, 24 hr and 4 days after exposure. A plastic cylinder was mounted to the top of the adaptor to provide a fixed distance between lens and eye. Each fluorophotometric examination started with three prescans of both eyes before iv. administration of fluorescein to determine autofluorescence. Subsequently five scans were performed of each eye 5466 min after i.v. injection of fluorescein (0.125 ml per kg body weight of a solution containing 200 mg ml-l). Left and right eyes were measured alternately. The average fluorescence in the vitreous (F,,, expressed in ng equivalent ml-l) was computed along the optical axis from 18% of the distance between retina and anterior chamber fluorescence peaks (at about 5 mm from the retina) up to the point halfway between these peaks (at about the middle of the eye) (see Fig. 2). The mean fluorescein concentration value in the area of measurement, C,,, was obtained by subtracting the mean F,,-value of three prescans from that of the five scans performed after fluorescein administration. Effects of the light exposure on the BRB function were quantified by the ratio between C,, for the exposed (left) and the non-exposed (right) eye. Changes in the fluorophotometric ratios after light exposure were statistically tested using the Wilcoxon’s paired signed-rank test.
R. A. BORSJE
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Anesthetics During exposure the animals were anesthetised with a 1: 1 mixture of ketamine 10 % (Aescoket’“, Aesculaap, The Netherlands) and 2- (2,6-xylidine)5,6-dihydro-4H-1,4-thiazine 2% (Rompun%, Bayer. Spain) ( 1.5 ml hr-’ ; after 1 hr a single dose of 0.5 ml was given). Mydriasis and cycloplegia were achieved by repeated instillation of tropicamide 0.5% (Mydriatricumm) and cyclopentolate 1% (Cyclogyl”) in both eyes. During fluorophotometry the animals were sedated by 1 ml Hypnorm” (containing 10 mg fluanison and 0.315 mg fentanylcitrate, Janssen Pharmaceutics, The Netherlands). The pupils were dilated by tropicamide 0.5 % and phenylephrine 5% before ophthalmoscopy, fundus photography and fluorophotometry. Histology Histological examination has been carried out in six eyes 1 day and 4 days after light exposure and in three control eyes. Three rabbits were killed 4 days after light exposure several hours after the last fluorophotometric scans. Their exposed eyes were enucleated at this day for morphological examination since phototoxic retinal lesions may not become apparent until 2-3 days after exposure (Lawwill, 1973; Rinkhoff et al., 1986). The non-exposed fellow eyes of these rabbits served as controls. The control eyes of three other rabbits with an increased C,,-ratio 24 hr after exposure were also exposed 3 weeks later. These animals were killed 24 hr after the last exposure with an overdose of pentobarbital sodium (Nembutal@, Ceva, France) and the most recently exposed eyes were enucleated for histological examination. After enucleation the eyes were immersed in a 0.08 M cacodylate buffered solution containing 1% glutaraldehyde and 1.2 5 % paraformaldehyde at pH 7.3. Liibal incisions were made to facilitate penetration of the fixative. After fixation for several days the eyes were dissected and four samples were taken from the visual streak and two samples from the nasal and temporal part of the area of the radiating medullary nerve fibers. After thorough rinsing in cacodylate buffer the retinal samples were post-fixed with OsO,, dehydrated and embedded in Epon 812. Ultrathin sections were stained with lead citrate and uranyl acetate and inspected in a Philips EM 400 transmission electron microscope. Light microscopic examination was carried out on semithin (1 pm) sections of the same material after staining with toluidine blue. As the main objective of the present study was to correlate morphological changes and leakage of fluorescein in the posterior eye segment as assessed by fluorophotometry we mainly studied the RPE in the visual streak and the capillaries in the area of the radiating medullary nerve fibers.
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t
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500
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FIG. 1. Relative spectral power curve of the image source as measured behind the focussing lens (percent of maximum power). TABLE I
Fluorophqtometric results. Mean ratio and standarddeviations betweenfluorescein leakagein exposedand control fellow eyes. Mean ratio Group Total (n = 14) I* (n = 6) IIt (n = 8)
2-7 days pre-exposure 1.00+0.12 0.99 kO.14 1.01f0.11
0.5-1.5 hr post-exposure 1.26kO.37 1.50+0.39 1.08f0.23
1 day post-exposure 1.52 f 0.50 1.58*o-60 1.48+ 0.44
4 days post-exposure 097&0.18 092kO.17 l.OOf0.19
* Rabbit age < 17 weeks. t Rabbit age 3 17 weeks.
Ophthalmoscopy and fundus photography were carried out on all animals prior to, 24 hr and 4 days after light exposure.
3. Results Light exposure
The calculated mean retinal illumination level was 102*6&2*5 mW cmd2. The relative spectral power distribution of the image source, measured with a spectrophotometer (Photo Research Spectrascan S.N. 2186, U.S.A.), is shown in Fig. 1. The curve shows a peak at 572 nm, an effective suppression of infrared radiation and a relatively small contribution of the
blue range of the spectrum. Fluorophotometry
The average ratios between the fluorescein concentrations in the exposed and the control fellow eye prior 21
to and at different ties after exposure are presented in Table I. The average ratio immediately after exposure had significantly increased as compared to preexposure values (P = O-005). The increase was maximal one day after exposure (P < O-005). Four days after exposure the ratios had returned to pre-exposure values. There were no significant differences between the ratios immediately and 1 day after exposure and between the pre-exposure and 4 days post-exposure values (both P > 0.05). An example of a fluorophotometric scan showing an increased leakage of fluorescein into the vitreous after exposure is shown in Fig. 2. As we had the impression that the variation in fluorophotometic results was probably age-related we arbitrarily split the group into rabbits under and over 17 weeks of age, The average ratios for rabbits aged 12.5-l 7 weeks (group I; n = 6) and those aged 17-20-S weeks (group II; n = 8) are presented separately in Table I. In group I a signiiicant increase of the average ratio was found directly after exposure EER SO
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Optical axis (mm) FIG. 2. Exampleof two fluorophotometricscansperformedat about 60 min after i.v. injection of fluorescein,1 day after light exposure.Upper curve: exposedeye; lower curve: fellow control eye. Vertical continuous lines: R = retina/choroid; A = anterior chamber.Vertical broken lines: limits usedfor leakageevaluation (F,-value).
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Age (weeks) FIG. 3. Ratiosbetweenexposed(OS)and control eyes(OD)directly after (left panel)and one day (right panel)after exposure as a function of the rabbit’s age. The obliquestraight line was obtainedby a linear regressionprocedureto the data points (r = -0.67; P < 0.01). A significantcorrelationwasnot found oneday after exposure.The horizontal straight line represents the averageratio of the total population beforeexposure.Broken lines representk 1 S.D.
(P = 0.02) and 1 day after exposure (P = 0.02). In group II a slight non-significant increase was found directly after exposure (P > 0.05) but a significant increase after 1 day (P = 0.01). The ratios of both groups had returned to pre-exposure values after 4
days (P > 0.05). The ratios directly after light exposure and 1 day after exposure are presented as a function of the rabbit’s age in Fig. 3. A significant linear correlation was found between age and ratios directly after exposure (decrease in ratio : O-1/week: r = - 0.67 ; P < O-01), but no significant correlation was found after 1 day (P = 0.73).
The complete experimental procedure with the exception of turning on the light bulb followed by fluorophotometry directly after and 1 day after the sham-treatment was performed in three rabbits. These rabbits did not show any significant increase of their left/right permeability ratio in any of the two fluorophotometric measurements (P > 005). Two animals showed anterior chamber fluorescein concentrations exceeding 3000 ng equivalent ml-’ in the fluorophotometric session before light exposure for unknown reasons. These animals were excluded from the study since such concentrations were assumed to interfere with measurements in the vitreous due to
” IL
FIG. 4. Electron micrograph of a RPE cell one day after light exposure. (a) RPE cell with normal smooth endoplasmatic reticulum (sER), mitochondria apical microvilli (Mv) and junctional complex (jc, between arrows) (Nu = nucleus: BrM = Bruch’s membrane; Cap = choriocapillary : Mel = melanin Detail of the lateral cell membrane with a tight junction (tj) and a desmosome (Des), both ultrastructurally normal in appearance ( x 97600).
(Mit), basal foldings (bf), granule) ( x 12 800), (b)
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FIG. 5. Electron micrograph of a capillary with a normal endothelial cell (End) and a pericyte (Per) from the area of the radiati ing mednllary nerve fibers, 1 day after light exposure. Arrows indicate intercellular junctions (Lum = lumen ; bl = biasal lamina) ( x 12 800).
light absorption in the anterior chamber and/or leakage of fluorescein from the anterior chamber into the vitreous.
Ophthalmoscopy, Fundus Photography and Histology
Signs of retinal damage on ophthalmoscopy and fundus photography were not found 1 and 4 days after exposure. Light and electron microscopic examination of the samples of light exposed and control retinas 1 and 4 days after exposure did not reveal any changes in the RPE. Basal membrane invaginations facing Bruch’s membrane, nuclei, mitochondria and other cellular organeljes were normal [Fig. 4(a)]. Special attention was given to the junctional complex of the lateral cell membrane, as damage to the tight junctions was considered to be the main cause of fluorophotometrically assessed fluorescein leakage, but their aspect was completely normal [Fig. 4(b)]. There was
no evidence of increased disc shedding, or changes in the slender apical cell processes. Although the photoreceptor outer segments were somewhat irregular with respect to their membranes degenerative effects were not obvious. The neuro-retina exhibited no pathological changes. The junctions at the level of the external limiting membrane did not show signs of disturbance. The choriocapillaries, filled with numerous red blood cells due to the immersion fixation, were normal. The retinal blood vessels and capillaries, in the rabbit restricted to the region of the radiating medullary nerve fibers (Prince and McConnell, 1964), exhibited no changes. The endothelial lining was completely intact in both control and treated animals, and disruption of the tight junctions between neighboring endothelial cells could not be discerned (Fig. 5). The number of pinocytotic vesicles was unchanged. The myelinated fibers in the area of radiating nerve fibers were normal.
medullary
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4. Discussion After white light exposure of rabbit eyes at levels below the threshold for phototoxic damage to the retina, a significant leakage of fluorescein into the vitreous documented a temporary dysfunction of the blood-retinal barrier (BRB). Examination of the retina by ophthalmoscopy, fundus photography and light and electron microscopy failed to show evidence of structural damage. Animals younger than 17 weeks showed a significant increase in leakage directly after exposure whereas animals older than 17 weeks did not: 24 hr after exposure both groups showed identical increased leakage. Fluorescein leakage did return to pre-exposure values 4 days after exposure. The leakage of fluorescein was not associated with abnormal ophthalmoscopic findings or electron microscopic disturbances of the RPE, neuro-retina or retinal capillaries. Thus, a functional disturbance of the BRB is not necessarily reflected in morphological changes of this barrier and fluorophotometric assessment of BRB function was shown to be a more sensitive parameter of light-induced damage than morphological examination. The retinal irradiance level was chosen from literature on account of interpolations of threshold data for minimal detectable damage in rabbits (Lawwill, 19 73 : McKechnie and Johnson, 19 7 7 ; Rinkhoff et al., 1986; Hoppeler et al., 1988). Hoppeler et al. (1988) however, reported ophthalmoscopic and histological changes in the RPE and outer segments of photoreceptors at much lower irradiance levels than those used in the present study. This can be explained by the fact that the light they used had a spectral distribution ranging from 400 to 550 nm, measured on retinal surface. It has been proven that the highenergy (400-500 nm) part of the visible light is more hazardous than the low-energy part (500-700 nm) (Ham et al., 1979; Young, 1988). Moreover, the spectral distribution of our source was measured in air and showed little power in this short wave range and this was further reduced by the decrease in transmission of the rabbit ocular media under 500 nm (Wiesinger et al., 1956). A puzzling aspect of the present study is the source of the significant increase of fluorescein leakage into the vitreous of the exposed eyes in the absence of ultrastructural damage of RPE cells and the tight junctions cementing the lateral borders of the RPE cells and the endothelial lining of the capillaries. Tso and Woodford (1983) found mildly edematous RPE cells with ultrastructurally normal intercellular junctions in the periphery of a lesion of photochemical retinopathy. Horseradish peroxidase (HRP) studies showed that, despite the intact tight junctions, HRP leaked into the subretinal space, proving that a normal ultrastructure of tight junctions is not indicative for functional integrity. It might well be that levels of light below the threshold for ultra-structural
303
damage induce molecular changes of junctional membranes of RPE cells and/or endothelial cells, enabling fluorescein and/or the fluorescein-albumin complex to pass these junctions. An alternative explanation might be a disturbance of the transcellular route of the RPE cells which are actively involved in regulating molecular transport between the choriocapillaris and the neuro-retina. A disturbance of the homeostatic mechanism including enzyme inactivation has been found after light exposure (Hansson, 1971; Marshall, 1984; Ham et al., 1984). In addition, normally a tracer as fluorescein injected intravitreally is transported rapidly into the choroid across the outer BRB by an active transport mechanism (Kitano, Hori and Nagataki, 1988). Decreased pumping activity results in increased fluorescein concentrations in the vitreous (Tsukahara et al., 1987; Kitano et al., 1988). A relative pump defect is not likely to contribute to increased fluorescein concentrations in the vitreous since these concentrations were measured 1 hr after fluorescein injection while the plasma- and vitreousconcentration gradient still exceeded the pumping mechanism. Which intra- and intercellular components are participating in the transport of fluorescein and to what extent they are affected by light remains to be studied using tracers and morphometric assessment of affected RPE cells. Several authors reported histological and functional disturbances after light exposure as determined by light and electron microscopy and fluorescein angiography without ophthalmoscopically visible retinal damage (Hochheimer, D’Anna and Calkins, 1979 ; Parver, Auker and Fine, 1983). Michels et al., (1987) reported the threshold for light damage to the retina, using angiographic and histologic damage thresholds, to be probably several orders of magnitude higher than those for functional damage. The present results re-emphasize this discrepancy between functional and morphological assessments of retinal light damage and strongly plead for a functional assessment of light damage of which fluorophotometry seems to be the most sensitive method. In our fluorophotometric study a reversible dysfunction of the BRB was found with complete recovery within 4 days, after exposure to light with an irradiance level below the threshold for ultrastructural damage. Morphological and functional recovery from light has been reported before for both thermally and photochemically induced light damage ; the degree of recovery seems to be dose-related (Kuwabara, 1970; Johnson et al., 1977 ; Lanum, 1978; McKechnie and Foulds, 1980; Wallow, 1984). Tso and Woodford (1983) reported fast partial recovery of histological damage but complete recovery of the outer BRB function as determined by HRP studies, at the periphery of the retinal lesion. However, in eyes with pre-existent retinal disease or in eyes chronically exposed to high environmental light levels,
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recovery may be impaired resulting in enhancement of retinal disease (Mainster et al., 1983: Young, 1988). Especially in these eyes, acute and repetitive light exposures may lead to more severe functional damage and visible damage, or to accelerated senescence. This animal study emphasizes the need to reduce instrumental light exposure as much as possible.
Acknowledgments The authors are indebted to Professor D. van Non-en, Ph.D. and J. J. M. Kremers, Ph.D., T.N.O. Institute for Perception, The Netherlands, for performing the measurement of the spectral power distribution of the image source, to D. F. Schaling, M.D., and T. Riimer for assistance in fluorophotometric measurements and to Mrs W. J. Baartse for typing the manuscript.
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Calkins,J. L. and Hochheimer,B. F. (1980). Retinal light exposurefrom ophthalmoscopes, slit lamps,and overhead surgical lamps.An analysisof potential hazards. Invest. OphthaJmoJ. Vis. Sci. 19, 1009-15. Ham, W. T., Jr., Mueller, H. A., Ruffolo,J. J,, Jr. and Clarke, A. M. (1979). Sensitivity of the retina to radiation damage as a function of wavelength. Photochem. Photobiol.29, 735-43. Ham, W. T.. Jr., Mueller, H. A., Ruffolo, J. J.. Millen, J. E., Cleary, S.F., Guerry, R. K. and Guerry III, D. (1984). Basicmechanismsunderlying the production of photochemical lesionsin the mammalianretina. Curr. Eye Res. 3, 165-74. Hansson, H. (1971). A hi&chemical study of cellular reactions in rat retina transiently damagedby visible light. Exp. Eye Res. 12, 2704. Hochheimer,B. F., D’Anna, S.A. and Calkins,J. L. (1979). Retinal damage from light. Am. I. Ophthalmol.88, 103944. Hoppeler,Th., Hendrickson,Ph., Dietrich, C. and Rem&Ch. (1988). Morphology and time-courseof delinedphotochemicallesionsin the rabbit retina. Curr. Eye Res. 7, 849-60.
Hughes,A. (19 72). A schematiceye for the rabbit. Vision Res.12. 123-37. Johnson,N. F., McNaught, E. I. and Foulds,W. S. (1977). Effectof photocoagulationon the barrier function of the pigmentepithelium.II. A study by electronmicroscopy. Trans.Ophthalmol.Sot. U.K. 97, 640-51. Kitano, S., Hori, S. and Nagataki, S. (1988). Transport of fluorescein in the rabbit eye after treatment with sodiumiodate. Exp. Eye Res. 46, 863-70. Kremers,J. J. M. and Van Norren, D. (1988). Two classes of
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photochemical damage of the retina. Lasers Light Ophthalmol.2, 41-52. Kuwabara, T. (19 70). Retinal recovery from exposureto light. Am. J. Ophthalmol.70, 187-98. Lanum, J. (1978). The damaging effects of light on the retina. Empirical findings, theoretical and practical implications.Surv. Ophthalmol.22, 22149. Lawwill, T. (1973). Effectsof prolongedexposureof rabbit retina to low-intensity light. Invest.Ophthalmol.Vis. Sci. 12,45-51. Mainster, M. A., Ham, W. T. Jr. and Delori, F. C. (1983). Potential retinal hazards. Instrument and environmental light sources.Ophthalmology 90, 92 7-3 1. Marshall, J. (1984). Light damage and the practice of ophthalmology. In Intra-ocular Lens ImpZantation (MS Rosen,E., Arnott, E. and Haining, W.). Pp. 182-207. Mosebey-YearbookLtd : London. McKechnie,N. M. and Johnson,N. F. (1977). Light damage to the retina. Albrecht von Graefe’s Arch. Klin. Exp. Ophthalmol.203, 283-92. McKechnie,N. M. and Foulds,W. S.(1980). Recoveryof the rabbit retina after light damage(Preliminary observations). Albrecht von Graefe’s Arch. KJin. Exp. OphthaJmoJ. 212, 271-283. Michels,M.. Dawson.W. W., Feldman,R. B. andJarolem,K. ( 1987). Infrared. An unseenand unnecessaryhazard in ophthalmic devices.Ophthalmology 94. 143-8. Noth, J. M., Vygantas, C. and Cunha-Vaz, J. G. F. (1978). Vitreous fluorophotometry evaluation of xenon photocoagulation.Invest. OphthaJmoJ. Vis. Sci. 17, 1206-g. Parver, L. M., Auker. C. R. and Fine, B. S. (1983). Observationson monkeyeyesexposedto light from an operating microscope.Ophthalmology 90, 964-72. Prince, J. H. and McConnell,D. G. (1984). Retina and optic nerve. In The Rabbit in Eye Research (Ed. Prince, J. H.). Pp. 385415. CharlesC. Thomas: Springfield. Rinkhoff. J., Machemer. R., Hida, T. and Chandler, D. (1986). Temperature-dependentlight damageto the retina. Am. 1. Ophthalmol. 102, 452-62. Sliney. D. H. (1984). Quantifying retinal irradiancelevelsin light damageexperiments.Curr. Eye Res. 3. 175-9. Tso, M. 0. M. and Woodford, B. J. (1983). Effect of photic injury on the retinal tissues.Ophthalmology 90,952-63. Tsukahara.Y., Ogura, Y., Miura. M. and Kondo, T. (1987). Etrect of argon laser photocoagulation on the permeability of the Blood-RetinalBarrier. Ophthalmologica 94, 27-33. Wallow, I. H. (1984). Repair of the pigment epithelial barrier following photocoagulation.Arch. OphthaJmoJ. 102, 126-35. Weisinger,H., Schmidt, F. H., Williams, R. C., Tiller, C. 0.. Ruffin. R. S.. Guerry III. D. and Ham, W. T.. Jr. (1956). The transmissionof light through the ocular mediaof the rabbit. Am. 1. Ophthalmol. 42, 907-10. Young, R. W. (1988). Solar radiation and age-related macular degeneration.Surv. OphthaJmoJ. 32, 252-69. Zweig, K., Cunha-Vaz, J., Peyman, G., Stein, M. and Raichand. M. (1981). Effect of argon laser photocoagulationon fluoresceintransport acrossthe bloodretinal barrier. Exp. Eye Res. 32, 323-9.