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and Cause Different Types of Damage in Rat

Theo G. M. F. Gorgels? and Dirk van Norren*^

Purpose. To assess the influence of on retinal light damage in rat with funduscopy and histology and to determine a detailed action spectrum. Methods. Adult Long Evans rats were anesthetized, and small patches of retina were exposed to narrow-band in the range of 320 to 600 nm using a arc and Maxwellian view conditions. After 3 days, the retina was examined with funduscopy and prepared for light microscopy. Results. The dose that produced a change just visible in fundo was determined for each wavelength. This threshold dose for funduscopic damage increased monotonically from 0.35 J/cm2 at 320 nm to 1600 J/cm2 at 550 nm. At 600 nm, exposure of more than 3000J/cnr did not cause funduscopic damage. Morphologic changes in exposed to threshold doses at from 320 to 440 nm were similar and consisted of pyknosis of photorecep- tors. Retinas exposed to threshold doses of 470 to 550 nm had different morphologic appear- ances. Retinal epithelial cells were swollen, and their had lost the characteris- tic apical distribution. Some pyknosis was found in photoreceptors. Conclusions. Damage sensitivity in rat increases enormously from visible to ultraviolet - lengths. Compelling evidence is presented that two morphologically distinct types of damage occur in the rat retina, depending on the wavelength. Because two types also have been described in monkey, a remarkable similarity seems to exist across species. Invest Ophthahnol VisSci. 1995; 36:851-863.

In 1966, Noell and coworkers1 reported that the rat ity and histologic site of damage. Studies on mon- retina can be damaged by light even when levels are keys2'3 and, recently, also on rats4S showed an increase well below the threshold for thermal damage. They in damage sensitivity for shorter wavelengths (with a postulated that the damage mechanism involves nox- maximum in the ultraviolet wavelengths). Damage was ious chemical reactions initiated by absorption sometimes found to manifest itself in photoreceptors, in a in the retina. whereas in other studies it was localized in RPE, Miiller In these first experiments on photochemical dam- cells, or mitochondria throughout the retinal lay- 1 3 6 9 age to the retina, damage was found in photoreceptors ers. - - " and the retinal pigment epithelium (RPE). The action These diverse observations suggest that multiple spectrum of damage resembled the rhodopsin absorp- types of retinal light damage exist, as review articles tion curve, which suggested that rhodopsin is the dam- have indicated.6'7''0>11 However, the characteristics and aging chromophore. Since then, many studies have classification of these types are not well established, been conducted on retinal light damage. Their out- mainly because the various studies differed substan- comes revealed a large variety in both spectral sensitiv- tially in experimental procedures, which makes it dif- ficult to deduce the critical factors that determine damage type. Wavelength is most likely one of these From the *F. C. Danders Institute of Ophthalmology, Ulreckt Academic Hospital, Utrecht, and the f National Aerospace Medical Center, Soeslerberg, The Netherlands. factors because photochemical reactions depend on Supported by a grant from the Dr. F. P. Fischer Foundation. absorption spectra of their .1'2 In the Submitted for publication April 12, 1994; revisedjune 27, 1994; accepted November 15, 1994. present study, we aimed at carefully exploring the fac- Proprietary interest category: N. tor wavelength in setting the threshold for retinal light Reprint requests: Theo G. M. F. Gorgels, F. C. Danders Institute of Ophthalmology, Utrecht Academic Hospital, P.O. Box 85500, 3508 GA, Utrecht, The Netherlands. damage. A detailed action spectrum was determined

Investigative Ophtha nology & Visual Science, April 1995, Vol. 36, No. 5 Copyright © Associa on for Research in Vision and Ophthalmology 851

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P1 R'

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transmission (%) FIGURE l. . A set of neutral-density filters separated by bands was placed at a plane conjugate to the retina (R'). Thus, four patches of retina were simultane- ously irradiated at different levels. P' is a plane conjugate to the pupil. P = pupil plane; R = retinal plane.

with narrow-band irradiations. Because rat ocular me- rat eye. Two partly overlapping neutral-density filters dia transmit a large portion of ultraviolet-A radia- were placed in the light path in a plane conjugate to tion,13 the wavelength range was set at 320 to 600 nm, the retina. They divided the path into four beams with which encompasses all peak wavelengths of previous intensities of approximately 100%, 54%, 33%, and studies. As an experimental procedure, we chose to 17%. This filter set thus provided four simultaneous irradiate small patches of retina of anesthetized rats; irradiation levels in one exposure. Light reflected at advantages of this procedure are exact dosimetry and the fundus was directed by a mirror and lenses to the adjacent, unexposed, control retina. Funduscopy and observer's eye. In this way, the irradiated retinal field light microscopy were used for damage assessment, could be viewed. because these techniques can be performed relatively The output of this configuration was calibrated easily while they simultaneously allow for good com- every second experiment with a radiometer. The beam parison with data in the literature. had a maximum fall in irradiance of 25% toward the edges. Retinal irradiance was calculated from mea- sured irradiance according to the formula given by METHODS 5 Calkins et al,' with refraction index = 1.337 and dis- Animals tance from pupil to retina = 5.25 mm.16 The spectral transmission of rat ocular media was calculated from Male rats of the pigmented Long Evans strain were 17 13 obtained (Harlan CPB, Zeist, The Netherlands) when transmission data of rat and . they were 30 days of age. They were subsequently kept losses in the other ocular media were neglected. The in a 12-hour light/12-hour dark cycle under 10 to 90 calculated spectral transmission is shown in Table 1. lux illumination. The animals used in experiments Table 1 also lists the bandwidth of the irradiations were 60 to 144 days old. Treatment of the animals according to the specifications of the menochromator. conformed to the ARVO Statement for the Use of The spectral characteristics of the irradiations were Animals in Ophthalmic and Vision Research. checked with a 512- array spectroradiometer. In addition, exposures were carried out at 488 Optics nm using an ion (model 162A, Spectra- Optics were essentially the same as described by de , Mountain View, CA) as the light source. The Lint et al.14 Briefly, a 450 W Xenon arc provided the optical configuration included a lens (f = 10) that radiation, which passed through a monochromator diverged the laser light beam, but for the rest, the (MM 12, Carl Zeiss, Oberkochen, Germany) and a optical setup was similar to the one described above. lens to the rat eye, irradiating 18° X 13° of It provided irradiations with a similar field width. superior retina (Fig. 1). Maxwellian view conditions Irradiation were ensured because the quartz lens focused the Rats were sedated with ether and anesthetized by intra- monochromator's exit slit in the pupil plane of the peritoneal injection of pentobarbital (50 mg/kg body

Downloaded from iovs.arvojournals.org on 10/02/2021 Two Spectral Types of Retinal Light Damage in Rat 853 TABLE l. Specification of Exposure Conditions for Funduscopic Threshold Damage Retinal Mean Funduscopic Wavelength Number of Transmittance Banduridth Itradiance Exposure Duration Threshold Dose (nm) Experiments Ocular Media* (nm) (mW/cm2) (range in minutes) (J/cm2) ± SEM

320 7 0.06 6 0.13 22-70 0.35 ± 0.09 340 4 0.28 5 0.66 15-25 0.79 ± 0.21 360 3 0.41 4 0.96 15-24 1.15 ± 0.25 380 13 0.52 5 1.76 8-23 1.37 ± 0.52 400 5 0.58 12 6.3 8-19 4.93 ± 1.22 420 6 0.60 11 8.3 60-70 31.5 ± 5.62 440 2 0.68 15 22.6 50-60 74.7 ± 9.6 470 7 0.72 19 46 134-285 577 ± 227 500 6 0.77 24 98 125-330 1145 ± 332 550 2 0.82 30 96 260-300 1612 ± 158 600 1 0.86 40 182 300 Subthreshold

1 Calculated from data on spectral transmittance of rat cornea" and lens.13

weight). An intravenous canula was applied to the tail, allowed to recover in the dark for 2 hours and was which ensured a constant intravenous infusion of pen- returned to the cage. tobarbital (15 mg/kg body weight per hour) and sa- line (1 ml/hour) during the experiment. Pupils were Analysis dilated with a drop of cyclopentolate HC1 1% and Three days after irradiation, the rat was sedated with phenylephrine HC1 5%. Atropine sulfate (0.3 ml of a ether and was anesthetized with an intraperitoneal solution of 0.5 mg/ml) was injected subcutaneously. injection of pentobarbital. Pupils were dilated as de- The rat was wrapped in an electric blanket and placed scribed above. The retina was inspected funduscopi- on a holder. Body was maintained at cally by following the drawing of the retinal position 37.5°C to 38.5°C using a rectal thermometer coupled of the irradiation. to the electrical blanket. Eyelids were kept open with For histology, the rat was then transcardially per- tape. The cornea was moistened by a continu- fused with a brief rinse of phosphate-buffered saline, ous flow of saline (4 to 6 ml/hour). Initially, the rat followed by a fixative of 2% glutaraldehyde, 2% para- was positioned in the beam (Fig. 1) with the mono- formaldehyde in 0.1 M sodium cacodylate buffer, pH chromator set at 570 nm and at a low intensity (retinal 7.4, containing 2 mM CaCl2. The eye was resected and irradiance <1 mW/cm2). Because the retina shows was kept, after removal of cornea and lens, in the regional differences in susceptibility to light dam- fixative at 4°C. The next day, the eyecup was inspected age,1819 care was taken to place the irradiations in the with a stereomicroscope. The irradiated areas were same location. The rat was positioned perpendicular, localized; tissue segments containing them and adja- but slightly tilted, to the light beam to irradiate the cent, unexposed, control retina were excised. The tis- superior retina. Extreme eccentricity was avoided. Us- sue segments were rinsed in buffer and fixed in 1% ing retinal vessels as landmarks, a drawing was made OsO4, 1.5% K,Fe(CN)6 in buffer for 1 hour. The tis- of the retinal position of the irradiation. Irradiation sue segments were then stained with 0.5% aqueous was then started with Xenon arc and monochromator uranyl acetate for 1 hour, dehydrated in , and set at the desired wavelength and bandwidth. To check embedded in Epon. Semithin (l-/xm) sections were for eye movements, the retinal position of the irradi- cut. Sections were collected every 40 /xm over at least ated field was checked at least every 10 minutes and at 200 //m into the irradiated area to avoid misinterpreta- the end of the irradiation. Irradiations with ultraviolet tion due to inhomogeneity of the irradiation or to wavelengths caused of the retina, oblique sectioning. Damage spots measured approxi- which enabled verification of the field position. In mately 0.5 X 0.4 mm. Sections were stained widi tolu- most experiments, no or only minor changes in posi- idine and were examined by light microscopy. tion occurred during irradiation. If there were signifi- To formalize the description of damage histology, cant changes, the experiment was stopped. In total, several morphologic parameters were quantified. This 46 rats were used and received 62 irradiations (some was done for irradiations at 320, 330, 420, and 470 animals received two subsequent irradiations). After nm. Using a light equipped with video- irradiations at 320 and 340 nm, the cornea was overlay, measurements were made direcdy on die checked for damage with , but no monitor screen. A representative sample of diree sec- damage was observed. After irradiation, the rat was tions was evaluated for each experiment. Irradiations

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irradiation. Threshold spots, however, were occasion- ally smaller than the irradiated area, probably because of inhomogeneity of the beam. Figure 3 shows the action spectrum of threshold doses for funduscopic damage. Doses are corrected for spectral transmittance of the ocular media. At 550 nm, the highest threshold dose was found (1612 J/ cm2). At 600 nm, the maximal available dose of 3275 J/cm2 did not result in damage. For shorter wave- lengths, the threshold dose decreased monotonically to 4.9 J/cm2 at 400 nm. In the ultraviolet range, the dose further decreased to 0.35 J/cm2 at 320 nm. Thus, the difference between the highest, and the lowest threshold doses encompassed nearly four orders of magnitude. FIGURE 2. Fundus photograph taken 3 days after irradiation Specifications of exposure conditions for thresh- at 440 nm. Four retinal patches received 380J/cm2 (1), 190 old damage are given in Table 1. At 380 nm, damage J/cm2 (2), 120 J/cm2 (3), and 64 J/cm2 (4). Three of them was produced in exposures of 8 to 23 minutes. For showed a distinct change in fundus appearance and were the other wavelengths, using maximal output at the classified as suprathreshold damage (1 to 3), whereas the 2 small bandwidths chosen, longer exposures were nec- dose of 64 J/cm caused a just-visible decoloration classified essary to sustain damage. For 500 nm, we had to ex- as threshold damage (4). pose for nearly 5 hours whereas, as stated above, a 5- hour exposure at 600 nm failed to cause damage. Longer exposure was not feasible with the anesthesia that did not produce a homogeneously damaged used. area—for example, as result of overlying blood ves- sels—were not measured. The effect of irradiation light Microscopy on the thickness of retinal layers was estimated by Damage spots detected with funduscopy remained visi- comparing layer thickness in irradiated and in adja- ble as whitish spots in the resected eyecup. With the cent control retina. Photoreceptor loss was deter- aid of a stereomicroscope, excision of the irradiated mined by comparing counts of outer nuclear layer spots was thus reliably performed, allowing for correla- (ONL) nuclei in irradiated retina with counts in an tion of histology and funduscopy. The position of sub- equal area of control retina in the same section. In threshold spots was indicated by retinal vasculature the irradiated retina, estimates were made of the per- and adjacent damage spots. centage of pyknotic photoreceptors and of the per- Analyzing the retinas exposed to the various wave- centage of cells in the RPE that had no apical distribu- lengths, we noticed that wavelengths in the range of tion of melanin. In addition, a possible inflammatory 320 to 440 nm caused a different damage morphology reaction in the choroid was assessed by counting the than those in the 470 to 550 nm range (Fig. 4). These choroidal cells located at Bruch's membrane in irradi- types will be successively described in detail. ated and in control retina. Damage at 320 to 440 nm. Damage morphology pro- duced by exposures of wavelengths in the range of RESULTS 320 to 440 nm will be exemplified by the effects of irradiation at 380 nm because the experiments at this Funduscopy wavelength were most numerous. Three days after irradiation, funduscopy showed a Retina exposed to doses of approximately 0.5 J/ change in fundus of irradiated patches (Fig. 2). cm2 showed no changes compared with adjacent con- As described in the Methods section, four retinal trol retina. At doses of approximately 1 J/cm2, ap- patches were simultaneously irradiated with different proaching the funduscopic threshold dose (Table 1), intensities spanning a range of approximately factor 6. the first signs of damage were found (Fig. 5A). A few Distinctly visible, homogeneous - patches pyknotic photoreceptors were present in the ONL. In were classified as having suprathreshold damage. the inner segment (IS) and outer segment (OS) lay- Threshold damage was defined as a change just visible ers, some segments were densely stained. The other in fundo. Irradiated patches without fundus change retinal layers showed no deviations from control. were classified as subthreshold damage. The decolora- With further increases in dose, progressively more tion closely corresponded Lo size and position of the photoreceptors were affected, until at approximately

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1 -• _ 5 ° o - - o threshold o A sub-threshold 0.1 -• FIGURE 3. Action spectrum for threshold damage of the retina assessed by fun- duscopy. Doses (±SEM) are 0.01 1 1 1 1 h- 1 11 corrected for spectral trans- 300 350 400 450 500 550 600 mittance of ocular media. wavelength (nm)

2J/cm2 (1.5 times the funduscopic threshold) virtu- dramatically (Fig. 7A), indicating a remarkably steep ally all photoreceptors showed pyknosis (Figs. 4, 5C). threshold for photoreceptor damage. In spite of this The thickness of the IS and OS layers was reduced. general pyknosis, cell loss (Fig. 7B) remained modest Rods had retracted from the villi of the RPE. They (note that all observations are at three days after irradi- were densely stained and appeared larger in diameter ation). In addition, changes occurred in the thickness than did rods in the adjacent control retina (Fig. 5C). of the retinal layers (Figs. 7C to 7F). An increase was Debris and phagocytic cells were observed in the pho- found in the RPE and INL. A decrease occurred in toreceptor layers, mostly between the rods. In compar- the IS and OS layers and, to a lesser extent, also in the ison to adjacent control retina, RPE cells occasionally ONL. No other retinal layers deviated from control. were slightly swollen and contained more phago- The other wavelengths in the 320- to 440-nm somes. These cells appeared otherwise normal, with range caused damage morphology similar to 380 nm. normal melanin content and distribution. However, a slight deviation was found in 3 of 6 expo- At still higher doses (2.5 J/cm2), additional fea- sures at 420 nm. At doses of two to three times thresh- tures could be observed, such as a loss of photorecep- old dose, the RPE was damaged more severely than at tors in the outer part of the ONL, mitoses of phago- corresponding doses of 380 nm. All irradiated RPE cytic cells (Fig. 5E), and the presence of dark spots in cells had lost their apical distribution of melanin and the outer plexiform layer and the inner nuclear layer contained numerous dark inclusions. Figure 8 shows (INL). Retinal pigment epithelial cells were swollen. an extreme example of this damage, in which a con- In a few RPE cells, melanin granules had lost their centration of cells in the choroid at Bruch's mem- apical distribution and were found throughout the brane is observed. cell body. Damage at 470 to 550 nm. Exposure to wavelengths In one experiment, a dose of 10J/cm2 (approxi- in the range of 470 to 550 nm resulted in a different mately seven times the threshold dose) was given. This damage morphology (Fig. 4), as will be described here resulted in the loss of most photoreceptors. The ONL for 470 nm. Damage became apparent at doses of and the IS and OS layers predominantly contained approximately 500 J/cm2, which closely corresponds debris and phagocytic cells. In the INL, mitoses were to the funduscopic threshold dose. Retinal pigment frequently observed, presumably of Muller cells (Fig. epithelial cells in the irradiated area were swollen and 6). At this dose, the apical distribution of melanin was had large pale nuclei, with distinct nucleoli. Cigar- lost in most RPE cells. shaped melanin granules were found throughout the Several morphologic parameters were quantified. cell body and not in the villi. Most of these cells were In Fig. 7, the most distinct parameters are plotted loaded with dark inclusions. The other retinal layers as a function of dose, scaled in units of funduscopic showed no deviation from control, except for a few threshold. Just above the funduscopic threshold dose, pyknotic photoreceptors in the ONL and a few densely the percentage of pyknotic photoreceptors increased stained rods (Fig. 5B).

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FIGURE 5. Dose-effect relationship of damage at 320 to 440 nm (left) and 470 to 550 nm (right). (A, B) funduscopic threshold dose. (C, D) 2.5 dmes threshold. (E, F ,G) 3.5 times threshold. Adjacent unexposed retina is shown (A, D, FIGURE 4. Difference in damage morphology of irradiation right; B, C, E, left). Damage morphology of 320 to 440 nm: at 320 nm (A) and 470 nm (B). Dose: 2.5 times the fun- (A) Dark and pyknosis of a few photoreceptors. (C) duscopic threshold dose. The micrographs (magnification, Dark staining and pyknosis of all photoreceptors; RPE is X100) show irradiated retina in the central part, flanked by slightly swollen. (E) Retinal pigment epithelial cells show unexposed, control retina. (A) Outer nuclear layer cells are additional swelling and increased number of dark inclu- pyknotic and rods are densely stained. (B) Retinal pigment sions. Note of the macrophage close to the RPE. epithelial cells show severe swelling, accumulation of dark Damage morphology of 470 to 550 nm (B) Retinal pigment inclusions and loss of apical distribution of melanin. A few epithelial cells are swollen and contain numerous dark inclu- pyknotic photoreceptors and densely stained rods are pres- sions. Melanin granules are scattered over the cell body. A ent. Rods and outer nuclear layer cell columns appear bent, few photoreceptors are densely stained. (D) Same features away from the center of the irradiation. as in panel B, but more prominent. Note that rods and ONL cell columns are bent to the left, i.e., away from the center

2 of the irradiation. Melanin granules are present in the mac- At a higher dose (1000 J/cm , twice the thresh- rophage close to RPE. (F, G) Photoreceptor layers contain old) , the changes in the pigment epithelium remained much debris among normal-appearing photoreceptors. Mi- the most prominent feature (Figs. 4, 5D). In the cho- totic figures are present in RPE, ONL, and INL. Numerous roid, often an accumulation of cells at Bruch's mem- dark spots are observed in the INL. RPE = retinal pigment brane was noticed. A few photoreceptors were af- epithelium; ONL = outer nuclear layer; INL = inner nu- fected, showing pyknosis in the ONL and dark staining clear layer. Representative micrographs were taken of expo- of inner and outer segments. Debris and phagocytic sures at 380 nm (A, E), 320 nm (C), 550 nm

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470 nm, whereas in retina exposed to 380 nm and in control retina, only occasionally a cell was found in this position. At 470 to 550 nm, longer exposures were used than at 320 to 440 nm (Table 1). We performed con- trol experiments to check whether exposure duration instead of wavelength caused the difference in damage morphology. The first control experiments were 30- I to 60-minute exposures at 488 nm from an Argon ion laser. The laser provided retinal irradiances of 500 mW/cm2, five times higher than with the Xenon arc. Thus, similar exposure durations could be used as /. in the experiments at short wavelengths. These short exposures at 488 nm caused a similar morphology as FIGURES. Exposure to 380 nm at seven times the threshold the long exposures at 470 tp 550 nm (Fig. 9). As an dose caused abundant mitotic figures in inner nuclear layer. additional control, prolonged exposures of 2 to 3 In the outer nuclear layer, most photoreceptors are lost hours at 380 nm were conducted. Damage morphol- (compare with the border of the irradiation on the left). ogy was similar to that in experiments with short expo- Magnification, X345. sures at 380 nm. These control experiments also served to check photoreceptor layers (Fig. 5F, G). Cell loss was evident whether exposure duration influenced threshold in the ONL. Most of the photoreceptors, however, had dose. In the exposures of 2 to 3 hours at 380 nm, the a normal appearance. In the RPE, occasionally a cell threshold dose for funduscopic damage was 1.0 ± 0.3 was missing or a double layer was observed. Mitosis of J/cma (n = 3), which does not deviate from the thresh- RPE cells, phagocytes, and, presumably, Miiller cells in old dose of 1.3 ± 0.5 J/cm2, found in exposures of the INL was regularly observed. In the outer plexiform approximately 15 minutes. The short laser exposures layer and the INL, dark spots were discerned. at 488 nm caused threshold damage at 1100 ± 200 J/ Quantification of damage parameters highlights cm2 (n = 3), which compares well to the threshold the differences between the two damage types (Fig. dose of 1145 J/cm2 at 500 nm with a bandwidth of 24 7). As distinct from 380-nm damage, RPE swelling nm, determined in exposures of more than 3 hours. (Fig. 7E) and changes in melanin distribution (Fig. Thus, reciprocity of exposure duration and irradiance 7H) were evident at threshold. Furthermore, photore- appears to hold in this range. ceptor pyknosis was low near the funduscopic thresh- Relation Between Funduscopy and Light Microscopy. old dose and remained at a low level with increasing Figure 7 shows that a lesion visible with the technique doses (Fig. 7A). Measurement of ONL cell loss was of funduscopy always correlated with distinct histo- complicated by the aforementioned indication that logic changes in either photoreceptors (at 380 nm) ONL cell columns might have been pushed to the or RPE (at 470 nm). Below the funduscopic threshold, side, beyond the borders of the irradiation. We de- pyknotic photoreceptors were occasionally observed. cided to expand the area over which ONL cells were No deviations from control retina were evident at less counted to encompass displaced ONL cells. By this than half the threshold dose. procedure, the real cell loss was diluted because unir- radiated cells were included in the counting. Yet, this conservative estimate reveals a distinct ONL cell loss DISCUSSION (Fig. 7B), surprising in view of the relatively low level Examining the action spectrum of retinal light dam- of pyknosis. In addition, irradiation tended to increase age in the rat, we report two major findings. First, thickness of the IS and OS layers, most notably at there are two different damage types. Second, damage moderate doses (Fig. 7D). An increase in INL thick- sensitivity increases dramatically for shorter wave- ness and a slight decrease in ONL thickness was found lengths. (Figs. 7C, 7F). The other retina layers showed no devi- Threshold exposures at wavelengths in the range ation from control. The number of cells in the choroid of 320 to 440 nm produced a different retinal damage at Bruch's membrane was determined as a measure than exposures at 470 to 550 nm. This has not been of choroidal reaction to the irradiation (Fig. 7G). Irra- reported before in rat. Therefore, we carefully diation at 470 nm caused a concentration of cells at checked which experimental variable was responsible Bruch's membrane: On average, 10 cells/section were for this difference. Aside from wavelength, the two located at Bruch's membrane in retina exposed to groups of exposures also differed in retinal irradiance

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FIGURE 7. Quantification of histologic parameters in retina exposed to 380 nm (often squares) and 470 nm (black circles) as a function of dose, scaled in units of funduscopic threshold. (A) Percentage of pyknotic outer nuclear layer (ONL) cells in irradiated retina. (B) ONL cell number in exposed retina in percentage of control retina. (C, D, E, F) Thickness of retinal layers in exposed retina in percentage of control. (G) Concentration of choroidal cells at Bruch's membrane in irradiated retina, expressed as mean number of cells per section. (H) Percentage of irradiated retinal pigment epithelial cells without apical distribu- tion of melanin.

and duration. In retinal irradiance, there was a large short wavelengths is damage to the photoreceptors. overlap between exposures to 440 and 470 nm. At 440 At 470 to 550 nm, the most prominent feature is dam- nm, irradiances up to 60 mW/cm2, and at 470 nm age to the RPE, but some photoreceptor damage is irradiances down to 20 raW/cm2, were used and still also present. damage seem the difference in morphology was found. Regarding Thermal s highly unlikely.22 The 20 21 4 irradianc exposure duration, studies on monkeys ' and rats highest retinal e at threshold was 100 mW/ indicated that reciprocity of irradiance and duration cm2 at 550 nm (Table 1). This level does not lead to a holds in the range of irradiances and durations we significant rise in retinal temperature.23'2'1 In addition, used. In addition, we performed control experiments long exposures were needed for threshold damage, with different exposure durations at a single wave- damage was not visible immediately after exposure, length, which showed that duration had no influence and reciprocity between irradiance and exposure time on damage threshold dose, nor on morphology. We was found. Finally, 200 mW/cm2 at 600 nm did not conclude that wavelength (and neither irradiance nor cause damage at all. duration) is the factor responsible for the encoun- In studies on photochemical damage, two differ- tered morphologic differences. ent action spectra have been published. One action It appears then that there are two damage types, spectrum, first reported by Noel et al1 and later con- each with its own morphology and spectral sensitivity. firmed by Williams and Howell,23 peaks at approxi- The best way to extract the damage types in their pure mately 500 nm and resembles the rhodopsin absorp- form is to examine damage histology at doses close to tion spectrum. The other action spectrum shows an threshold. Following this rule, we found that, at 3 days increase in retinal sensitivity for shorter wavelengths. after exposure, the main feature of the type at the This action spectrum arose from studies on monkeys2'3 but has recently been described for the rat as well.4'5 The present findings agree well with the latter action spectrum. It is not known which factors determine the

FIGURE 8. Example of damage at 420 nm, deviating from the general damage morphology of 320 to 440 nm exposures FIGURE 9. Control experiment for exposure duration. Expo- {see Results). Photoreceptors as well as retinal pigment epi- sure of 30 minutes at 488 nm (Argon ion laser) produced thelium are damaged. In the choroid, cells are concentrated a morphology similar to long exposures at wavelengths in at Bruch's membrane and dark spots are present in the the range of 470 to 550 nm. Dose: funduscopic threshold. inner nuclear layer. For comparison, adjacent control retina Retinal pigment epithelial cells are swollen and contain dark is shown on the right. Dose was two times the threshold inclusions. Apical distribution of melanin granules is lost. dose. Magnification, X205. Photoreceptors are relatively spared. Magnification, X125.

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prevalence of either type of action spectrum. Kremers phology. We can think of two possible explanations and van Norren" suggested that presence or absence for the discrepancy with the present findings. First, of rhodopsin is crucial. When retinal irradiance is low the action spectrum of our damage type at short wave- and rhodopsin is present, the rhodopsin action spec- length is remarkably steep, especially in the range of trum occurs. When retinal irradiance is high and rho- 400 to 470 nm. Consequently, a small fraction of wave- dopsin is bleached, the short wavelength action spec- lengths of approximately 440 nm present in the green trum emerges. In the present study, relatively high light exposure may well determine the damage mor- retinal irradiances were used (Table 1). For example, phology at threshold instead of the wavelengths of Noell et al1 used a retinal irradiance of 0.01 mW/cm2 approximately 500 nm. The observation of Rapp and of green light, which reduced visual pigment to 35% Smith5 that threshold doses damaged only the photo- of dark-adapted control as measured biochemically. receptors corroborates this explanation. Second, From data on the relation between light intensity and strain differences in susceptibility for light damage bleaching in the rat,26'27 we calculate that our retinal have been demonstrated.30'31 In addition, the intracel- irradiances bleached rhodopsin completely within 1 lular distribution of melanin appears to be a sensitive minute. Only at 340 nm, 4% to 10% remained un- indicator for the RPE damage by 470 to 550 nm (Fig. bleached after 1 minute, and at 320 nm, it took 5 7). This feature was conspicuous in the pigmented minutes to reduce rhodopsin to 5%to 12% of the dark- Long Evans rat we used but does not appear in the adapted value. albinotic retina. Peculiar in this context is Rapp and 82 Comparison of our histologic data with other stud- Tolman's earlier study on Long Evans rats, in which ies is complicated by the great variation in experimen- they report different damage morphologies in long tal procedures. Some general remarks appear appro- wave ultraviolet radiation and in green light (1 day priate in this respect. First, having established that after exposure). Later, these findings were retracted because the threshold factor had not been carefully there are two spectral types of damage, exposures to 5 broad-band irradiations become difficult to interpret, controlled for. particularly when no details on the spectral output are Strong support for the existence of two spectral given. Second, the postexposure time point of analysis types of light damage can be found in studies with is relevant for damage histology because repair in narrow-band irradiations in the monkey and the squir- some layers might occur or secondary damage might rel. In the monkey, Ham et al9 report that after 2 days, spread to different layers.3'28'29 Third, it is extremely 441 nm radiation caused RPE edema and agglutina- important to analyze damage at the lowest possible tion of melanin granules with only minor damage to (threshold) dose because, at higher doses, both dam- photoreceptors. Lawwill6 evaluated exposures to vari- age types may be present and secondary effects may ous laser lines from 457.9 to 590 nm. He reported be greater.5 At high doses, we found damage to photo- that in light microscopy, the most striking changes receptors, RPE, and INL as well at all wavelengths. were localized in the RPE, although ultrastructural Several aspects of our experimental setup ensured a analysis revealed damage to mitochondria in all retinal sensitive analysis of damage at low doses. For example, layers. On the other hand, irradiation at 325 nm33 and a postexposure interval of 3 days was used because a 350 nm3 primarily damaged photoreceptors. Re- previous study in rat14 showed that fundus changes peated exposures to long wave ultraviolet radiation, were then maximally visible. By using the unique fea- however, primarily affected the RPE.34 In the squirrel, ture of four simultaneous irradiance levels in each Collier and Zigman also found two damage types, com- experiment, retina irradiated with four different paring damage histology of exposures to 366 nm and doses, including doses below funduscopic threshold, 440 nm (at 1 day after exposure). At threshold, ultra- and an unexposed control retina could be compared. radiation damaged only photoreceptors,35 Fourth, as stated above, there is a dichotomy in the whereas light of 440 nm damaged photoreceptors and literature on light damage with regard to spectral sen- RPE.36 Longer wavelengths were not tested. Possibly, sitivity. This dichotomy, probably related to exposure light of 440 nm produced in the squirrel a combina- conditions and particularly to irradiance levels, may tion of both damage types. also occur in damage morphology. We, therefore, first Concerning the morphology of light damage, we compared our histologic data to studies that used ex- conclude that the similarities across species are more posure conditions similar to ours. striking than the differences. A similar conclusion was High retinal irradiances were used in a recent reached earlier for the action spectrum.4 Similarity in study by Rapp and Smith.5 They exposed albino rats to experimental conditions is crucial for a valid compari- broad-band irradiations (70 nm bandwidth) centered son across species. around either 355 or 500 nm. Their conclusion was The question remains unresolved whether the that both irradiations caused identical damage mor- present data are also valid for other exposure condi-

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tions. In the vast majority of light damage experi- elusive.45 This is partly because of the existence of ments, particularly those on rats, unanesthetized ani- multiple types of light damage. The identification and mals are exposed for several hours or days to very low morphologic characterization of two spectrally differ- levels of white30'37"40 or broad-band green light.1-41"44 ent types of damage in the rat may serve to clarify some The histology of this damage, originally described by issues and to direct further studies on its mechanism. Noell and coworkers for green light (490 to 580 nm), involves photoreceptors only or photoreceptors and Key Words RpE_7.10,41.43 The latt£r morphology is favored by dark light damage, retinal pigment epithelium, green light, ultra- rearing of the animals and elevated body temperature violet light, photoreceptors during exposure.7'9 These characteristics may not match the results we obtained with our exposure con- References ditions. Cyclic-light-reared rats were irradiated at nor- 1. Noell WK, Walker VS, Kang BS, Berman S. Retinal mal body temperature and, yet, the RPE was damaged damage by light in rats. Invest Ophthahnol. 1966; 5:450- by green light (500 nm). Therefore, there may be 473. factors besides wavelength and light history of the ani- 2. Ham WTJr, Mueller HA, Sliney DH. Retinal sensitivity mal that influence damage morphology. As stated, to damage from short wavelength light. . these may be related to exposure conditions, such as 1976;260:153-155. light intensity or level of bleaching of rhodopsin.11'19'45 3. Ham WTJr, Mueller HA, RuffoloJIJr, Guerry D III, Guerry RK. Action spectrum for retinal injury from Several molecules have been proposed as the near-ultraviolet radiation in the aphakic monkey. Am chromophore for initiating light damage. The damage J Ophthahnol. 1982;93:299-306. action spectrum provides, in principle, a clue to the 4. van Norren D, Schellekens P. Blue light hazard in rat. identity of the chromophore by comparison to the Vision Res. 1990;30:15l7-1520. absorption spectrum.12 Noell et al1 suggested rhodop- 5. Rapp LM, Smith SC. Morphologic comparisons be- sin, or its photointermediates, as the chromophore. tween rhodopsin-mediated and short-wavelength In our exposure conditions, rhodopsin is nearly fully classes of retinal light damage. Invest Ophthahnol Vis. bleached. For both damage types, blue wavelengths Sci. 1992; 33:3367-3377. were more damaging than green, which does not cor- 6. Lawwill T. Three major pathologic processes caused respond with the absorption characteristics of rhodop- by light in the primate retina: A search for mecha- nisms. Trans Am Ophthahnol Soc. 1932;80:517-579. sin. The absorption of its products711'46 shows 7. Noell WK. Possible mechanisms of photoreceptor resemblance to the action spectrum of the damage damage by light in mammalian . Vision Res. type, found at short wavelengths. Rhodopsin is 1980;20:1163-1171. bleached to all-^rans retinal (absorption peak at 387 8. Berler DK. Muller cell alterations from long-term am- nm), which in vivo is rapidly reduced to all-fran$-reti- bient fluorescent light exposure in monkeys: Light 49 nol 47.48 jne jatler pigment peaks at 330 nm, in line and electron microscopic, fluorescein and lipofuscin with the present findings. Two other candidate chro- study. Trans Am Ophthahnol Soc. 1990;87:515-576. mophores, melanin50 and cytochrome c oxidase, may 9. Ham WTJr, RuffoloJJ, Mueller HA, Clarke AM, Moon match the admittedly ill-defined action spectrum of ME. Histological analysis of photochemical lesions the damage type found at 470 to 550 nm. The absorp- produced in rhesus retina by short wavelength light. tion of melanin increases slowly with decreasing wave- Invest Ophthahnol Vis Sci. 1978; 17:1029-1035. length.50 The action spectrum of free- produc- 10. Mainster MA. Light and : A bio- physical and clinical perspective. Eye. 1987; 1:304-310. tion by melanin has a stronger wavelength depen- 51 11. Kremers JJM, van Norren D. Two classes of photo- dency, and in vitro studies have indicated that chemical damage of the retina. and Light in melanin may initiate noxious photochemical reac- Ophthalmology. 1988; 2:41-52. 52 tions. However, in vivo studies have failed to demon- 12. Coohill TP. Action spectra revisited. / Photochem Pho- strate a correlation between retinal melanin and light tobiolB. 1992; 13:95-100. damage.20'53"56 Cytochrome c oxidase has an absorp- 13. Gorgels TGMF, van Norren D. Spectral transmittance tion peak at approximately 420 nm.57 The action spec- of the rat lens. Vision Res. 1992;32:1509-1512. trum or RPE damage in culture of bovine RPE cells58 14. de Lint PJ, van Norren D, Toebosel AM. Effect of body closely resembles this absorption spectrum. Blue light temperature on threshold for retinal light damage. (404 nm) inhibited this in vivo in rat retina,59 Invest Ophthahnol Vis Sci. 1992; 33:2382-2387. and, recently, Putting et al60 found that the action 15. CalkinsJL, Hochheimer BF, D'Anna SA. Potential haz- ards from specific ophthalmic devices. Vision Res. spectrum of leakage of the blood-retina barrier in 1980;20:1039-1053. rabbit peaks at approximately 420 nm. 16. Hughes A. A schematic eye for the rat. Vision Res. In conclusion, 25 years after its discovery by 1979; 19:569-588. Noell,' many aspects of retinal light damage remain 17. Hemmingsen EA, Douglas EL. Ultraviolet radiation

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thresholds for corneal injury in antarctic and temper- aphakic and pseudophakic monkey eyes: A prelimi- ate-zone animals. Comp Biochem Physiol. 1970;32:593- nary report. Retina. 1990; 10:301 -314. 600. 35. Collier RJ, Waldron WR, Zigman S. Temporal se- 18. Rapp LM, Williams TP. A parametric study of retinal quence of changes to the gray squirrel retina after light damage in albino and pigmented rats. In: Wil- near-UV exposure. Invest Ophthalmol Vis Sci. 1989; liams TP, Baker BN, eds. The Effects of Constant Light 30:631-637. on Visual Processes. New York: Plenum Press; 1980:135- 36. Collier RJ, Zigman S. Comparison of retinal photo- 159. chemical lesions after exposure to near-UV or short- 19. Noell WK. There are different kinds of retinal light wavelength visible radiation. Prog Clin Biol Res. damage in the rat. In: Williams TP, Baker BN, eds. The 1989; 314:569-575. Effects of Constant Light on Visual Processes. New York: 37. Bush RA, Williams TP. The effect of unilateral optic Plenum Press; 1980:3-28. nerve section on retinal light damage in rats. Exp Eye 20. Ham WT Jr, Mueller HA, Rufollo JJ Jr, Clarke AM. Res. 1991;52:139-15. Sensitivity of the retina to radiation as a function of 38. Faktorovich EG, Steinberg RH, Yasumura D, Matthes wavelength. Pholochem Photobiol. 1979;29:735-743. MT, La Vail MM. Basic fibroblast growth factor and 21. KremersJJ, van Norren D. Retinal damage in macaque local injury protect photoreceptors from light damage after white light exposures lasting ten minutes to in the rat. JNeurosci. 1992; 12:3554-3567. twelve hours. Invest Ophthalmol Vis Sci. 1989; 30:1032- 39. Bush RA, Reme CE, Malnoe A. Light damage in the 1040. rat retina: The effect of dietary deprivation of N-3 22. MellerioJ. The interaction of light with biological tis- fatty acids on acute structural alterations. Exp Eye Res. sues and the potential for damage. In: Marshall J, ed. 1991:53:741-752. Vision and Visual Dysfunction. Vol 16. The Susceptible Vi- 40. Henton WW, Sykes SM. Changes in absolute threshold sual Apparatus. New York: Macmillan Press; 1991:30- with light-induced retinal damage. Physiol Behav. 53. 1983;31:179-185. 23. Friedman E, Kuwabara T. The retina pigment epithe- 41. Li ZL, Lam S, Tso MO. Desferrioxamine ameliorates lium: fV. The damaging effects of . Arch retinal photic injury in albino rats. Curr Eye Res. Ophthalmol. 1968; 80:265-279. 1991;10:133-144. 24. Clarke AM, Ceeraets WJ, Ham WT Jr. An equilibrium 42. Li J, Edward DP, Lam TT, Tso MOM. Amelioration thermal model for retinal injury from optical sources. of retinal photic injury by a combination of flunarizine ApplOpt. 1969;8:1051-1054. and dimethylthiourea. Exp Eye Res. 1993;56:71-78. 25. Williams TP, Howell WL. Action spectrum of retina 43. Organisciak DT, Jiang YL. Wang BM, Pickford M, light-damage in albino rats. Invest Ophthalmol Vis Sci. Blanks JC. Retinal light damage in rats exposed to 1983;24:285-287. intermittent light: Comparison with continuous light 26. Cone RA. Quantum relations of the rat electroretino- exposure. Invest Ophthalmol Vis Sci. 1989; 30:795-805. gram./Gen Physiol. 1963;46:1267-1286. 44. Organisciak DT, Darrow RM, Jiang YI, Marak GF, 27. Perlman I. Kinetics of bleaching and regeneration of Blanks JC. Protection by dimethylthiourea against reti- hodopsin in abnormal (RCS) and normal albino rats nal light damage in rats. Invest Ophthalmol Vis Sci. in vivo./Physiol. 1978;278:141-159. 1992;33:1599-1609. 28. Tso MOM, Woodford BJ. Effect of photic injury on 45. Organisciak DT, Winkler BS. Retinal light damage: the retinal tissues. Ophthalmology. 1983;90:952-963. Practical and theoretical considerations. Prog Retinal 29. Hoppeler T, Hendrickson P, Dietrich C, Reme C. Mor- Eye Res. 1994; 13:1-29. phology and time-course of defined photochemical 46. Delmelle M. An investigation of retinal as a source of lesions in the rabbit retina. Curt Eye Res. 1988; 7:849- singlet . Pholochem Photobiol. 1978;27:731-734. 860. 47. Bok D. Processing and transport of retinoids by the 30. LaVail MM, Gorrin GM, Repaci MA, Yasumura D. retinal pigment epithelium. Eye. 1990;4:326-332. Light-induced retinal degeneration in albino mice 48. Bongiomo A, Tesoriere L, Livrea MA, Pandolfo I. Dis- and rats: Strain and species differences. Proc Clin Biol tribution of vitamin A compounds in bovine eyes after Res. 1987; 247:439-454. bleaching adaptation. Vision Res. 1991;31:1099-1106. 31. BorgesJM, Edward DP, Tso MO. A comparative study 49. Lerman S. Radiant Energy and the Eye. New York: Mac- of photic injury in four inbred strains of albino rats. millan; 1980. CurrEyeRes. 1990;9:799-803. 50. Sarna T. Properties and function of the ocular mela- 32. Rapp LM, Tolman BL, Dhindsa HS. Separate mecha- nin: A photobiophysical view. / Photochem Photobiol B. nisms for retinal damage by ultraviolet-A and mid- 1992; 12:215-258. visible light. Invest Ophthalmol Vis Sci. 1990;31:1186- 51. Sarna T, Sealy RC. Free radicals from cumclanins: 1190. Quantum yields and wavelength dependence. Arch 33. Schmidt RE, Zuclich JA. Retinal lesions due to ultravi- Biochem Biophys. 1984;232:574-578. olet laser exposure. Invest Ophthalmol Vis Sci. 52. Glickman RD, Lam K-W. Oxidation of ascorbic acid 198O;19:1166-1175. as an indicator of photooxidative stress in the eye. 34. Li ZL, Tso MO, Jampol LM, Miller SA, Waxier M. Photochem Photobiol. 1992;55:191-196. Retinal injury induced by near-ultraviolet radiation in 53. La Vail MM, Gorrin GM. Protection from light dam-

Downloaded from iovs.arvojournals.org on 10/02/2021 Two Spectral Types of Retinal Light Damage in Rat 863

age by ocular pigmentation: Analysis using experi- 57. Yoshikawa S, Caughey WS. Heart cytochrome c-6xi- mental chimeras and translocation mice. Exp Eye Res. dase: An study of effects of oxidation state on 1987;44:877-889. binding./flio/ Chem. 1982;257:412- 54. Rapp LM, Smith SC. Evidence against melanin as the 420. mediator of retinal phototoxicity by short-wavelength 58. Pautler EL, Morita M, Beezley D. Hemoprotein(s) me- diate blue light damage in the retinal pigment epithe- light. Exp Eye Res. 1992;54:55-62. lium. Photochem Photobiol 1990;51:599-605. 55. Rapp LM, Williams TP. The role of ocular pigmenta- 59. Chen E, Soderberg PG, Lindstrom B. Cytochrome oxi- tion in protecting against retinal light damage. Vision dase activity in rat retina after exposure to 404 nm Res. 1980;20:1127-1131. blue light. CurrEyeRes. 1992;11:825-831. 56. Putting BJ, van BestJA, Vrensen GFJM, OosterhuisJA. 60. Putting BJ, van BestJA, Zweypfennig RCVJ, Vrensen Blue light induced dysfunction of the blood-retinal GFJM, OosterhuisJA. Spectral sensitivity of the blood- barrier at the pigment epithelium in albino versus retinal barrier for blue light in the 400-500 nm range. pigmented rabbits. Exp Eye Res. 1994;58:31-40. Qraefe'sArch Clin Exp Ofihthalmol. 1994;231:600-60(5.

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