Methods  Albino rats were exposed to 12 hours of 3000-lux cyclic light for 1, 3, or 6 months. Fundus examination, fundus photography, fluorescein and indocyanine green angiography, and optical coherence tomography were performed prior to euthanization. Light-exposed animals were euthanized after 1, 3, or 6 months for histopathological evaluation. Retinas were examined for the presence of 4-hydroxy-2-nonenal– and nitrotyrosine-modified proteins by immunofluorescence staining.

Results  Long-term intense cyclic light exposure resulted in retinal degeneration with loss of the outer segments of photoreceptors and approximately two-thirds of the outer nuclear layer as well as development of subretinal pigment epithelium neovascularization after 1 month. Almost the entire outer nuclear layer was absent with the presence of CNV, which penetrated the Bruch membrane and extended into the outer retina after 3 months. Absence of the outer nuclear layer, multiple foci of CNV, retinal pigment epithelial fibrous metaplasia, and connective tissue bands containing blood vessels extending into the retina were observed after 6 months. All intense light–exposed animals showed an increased presence of 4-hydroxy-2-nonenal and nitrotyrosine staining. Optical coherence tomographic and angiographic studies confirmed retinal thinning and leakiness of the newly formed blood vessels.

Conclusions  Our results suggest that albino rats develop progressive stages of retinal degeneration and CNV after long-term intense cyclic light exposure, allowing the detailed study of the pathogenesis and treatment of age-related macular degeneration.

Clinical Relevance  The ability to study the progressive pathogenesis of age-related macular degeneration and CNV will provide detailed knowledge about the disease and aid in the development of target-specific therapy.

The pathogenesis of AMD is not fully understood, but exposure to light is reported in epidemiological studies to be a risk factor for AMD in humans.⁸ Light energy is essential for visual function, but in excessive amounts it produces photochemical damage to retinal neurosensory cells. In 1966, Noell et al⁹ published an early histological study of retinal damage by light in rats. They documented the loss of photoreceptors and the outer nuclear layer (ONL) in albino rats after exposing them to constant light for periods ranging from a few hours to a few days. Subsequent light and electron microscopical studies revealed the sequence of histological events leading to the disappearance of photoreceptors and complete RPE–Müller cell adhesion^10-11 as well as RPE neovascularization by retinal capillaries.^12-14

In 1988, Kremers and van Norren¹⁵ proposed 2 classes of photochemical damage of the retina depending on the level of irradiance of exposed white light. In class 1 with low irradiance, the initial damage is restricted to photoreceptors; in class 2 with high irradiance, the RPE is the site of first insult. The rate and extent of retinal degeneration were increased with an increase in the duration and intensity of light exposure.^{11, 16} The appearance of the albino rat retina remained normal after 1 hour of 2500-lux light exposure, but pyknotic changes in the ONL were noted when light exposure was increased to 4 hours.⁹ Additionally, O’Steen et al¹⁷ reported that the susceptibility to light damage in the rat increases markedly at ages 16 to 24 weeks compared with ages 3 to 4 weeks, with approximately 95% loss of photoreceptors.

The exact molecular events involved in light-induced retinal degeneration have not been fully determined. However, formation of reactive oxygen species and consequent oxidative damage to retinal cells is one of the proposed mechanisms.^8-9,18-20 It is also suggested that the formation of bleached products of rhodopsin or other visual pigments leads to the formation of phototoxic molecules that mediate retinal cytotoxicity.^21-22 Both the caspase-dependent and caspase-independent pathways are involved in light-induced apoptosis of photoreceptors.^23-24 In recent years, vision scientists have increasingly used light-induced retinal degeneration in rodents for pathogenetic and therapeutic studies following long-term light exposure for a month or longer. However, the advanced stages of intense cyclic light–induced retinal degeneration have not been previously evaluated.

Long-term light-induced reactive oxygen species can damage the Bruch membrane and may stimulate CNV by fostering a proangiogenic environment in the retina.^{20, 25} Numerous studies using various retinal degeneration models reported RPE neovascularization, which arises from the inner retinal capillaries with minimal contribution from the choroid vasculature and may be influenced by the rate at which photoreceptors degenerate.^12-13 However, the accumulation of lipofuscin in RPE cells is, at least in part, also related to light exposure.²⁶ Lipofuscin deposition contributes to the formation of large confluent drusen and pigmentary changes in the RPE layer and is also thought to lead to the development of CNV.²⁷ Thus, the relative contributions of the neurosensory retina and RPE degeneration to the development of CNV in the aging eye clearly require further study.

Based on the published work of others and our own previous observations of light-induced retinal degeneration in rodents, we hypothesized that rodents with late-stage intense cyclic light–induced retinal degeneration develop CNV. To test this hypothesis, we studied the effects of cyclic light exposure with long duration and high intensity on the eyes of Wistar and Sprague-Dawley (SD) albino rats. Here we show that exposure to high-intensity cyclic light for 1 to 6 months resulted in rapid retinal degeneration that ultimately progressed to CNV with minimal contribution from retinal capillaries and RPE neovascularization. These changes were associated with increased oxidative stress as revealed by increased immunostaining for the presence of 4-hydroxy-2-nonenal (HNE)–modified proteins, a product of lipid peroxidation,²⁸ and nitrotyrosine-modified proteins, a product of peroxynitrate reaction.²⁹ Thus, long-term intense cyclic light exposure of albino animals may provide a suitable model to study the progressive pathogenesis of exudative AMD and be useful in the development of targeted treatments.

METHODS

All experiments were carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Animal Care and Use Committee of the University of Wisconsin School of Medicine and Public Health, Madison. We used a total of 12 albino female SD and 12 albino female Wistar rats (Harlan Sera-Lab, Indianapolis, Indiana). The rats were aged 12 weeks at the start of the study and were kept in our vivarium under normal cyclic light (12 hours on/off, 7 AM to 7 PM Central time) for a week prior to experimentation. The SD and Wistar rats were each divided into normal and intense cyclic light–exposed groups. Both control and treated groups were further divided into 3 subgroups each. The 3 treated subgroups of SD and Wistar rats were exposed to 12 hours of cyclic diffuse cool white 3000-lux light (36-W compact fluorescent tubes [DL 930]; Uplift Technologies Inc, Dartmouth, Nova Scotia, Canada) for 1, 3, or 6 months. The corresponding 3 control subgroups were exposed to normal cyclic light used in the animal housing facility (approximately 70 lux) for the same periods. The 12-hour light/dark cycle was used to avoid alterations in the normal physiology of the rats by disturbing the circadian rhythms set by the eye.

In the treatment groups, pairs of clear plastic cages with wire tops were kept side by side with 2 light sources, one on each side of the 2 cages (ie, 1 light source per cage and 2 cages per shelf). Before placing rats inside their respective cages and exposing them to light, light meter measurements (Thermo Fisher Scientific Inc, Pittsburgh, Pennsylvania) were taken at the bottom of each cage to ensure equal light energy exposure inside the entire cage. The distance of the light source from either side of the cage was adjusted to provide the desired reading. The control groups were kept in identical cages in a separate room under the normal cyclic vivarium lights. The light exposure of the control rats was measured in a manner identical to that of the treatment group. All rats were monitored for signs of abnormal behavior and appearance and were weighed every other week for the duration of experimentation.

OCULAR EVALUATIONS

At the end of 1, 3, or 6 months, dilated funduscopy, optical coherence tomography, and angiography were carried out under anesthesia (intraperitoneal injection of 35 mg/kg of ketamine hydrochloride and 5 mg/kg of xylazine hydrochloride). The pupils were dilated with tropicamide, 0.5%, and phenylephrine hydrochloride, 2.5%, eyedrops. The topical endoscopy fundus imaging system was used to document changes in the retina following a technique previously described.³⁰ Cirrus high-definition optical coherence tomography (Carl Zeiss Meditec, Jena, Germany) and the Heidelberg retinal angiography II system (Heidelberg Engineering, Inc, Heidelberg, Germany) were used to evaluate retinal edema and CNV using intraperitoneal injection of fluorescein sodium (60 mg/kg; Akorn Inc, Buffalo Grove, Illinois) and indocyanine green (6 mg/kg; Akorn Inc). The rat eyes were manually positioned in front of the optical coherence tomography and angiography camera to get the optimal view. The interpretation of optical coherence tomographic and angiographic images was performed by 2 masked readers (A.N. and S.W.).

HISTOLOGICAL EVALUATIONS

The rats were euthanized at 1, 3, or 6 months in a carbon dioxide chamber. The eyes were enucleated and fixed in 10% neutral-buffered formalin. Further histological processing consisted of dehydration through increased concentration of alcohol, clearing with xylene, and embedding in a horizontal axis in paraffin. Six serial pupillary–optic nerve sections and 4 non–pupillary–optic nerve anterior-posterior sections of 8-μm thickness were obtained from each eye. These sections were stained with hematoxylin-eosin, periodic acid–Schiff, or Masson trichrome stain. Comparative histopathological evaluation of the neurosensory retina, RPE, Bruch membrane, and choroid were performed in each eye by light microscopy. The retinal thickness was measured from the internal limiting membrane to the outer limiting membrane. Measurements were taken at 4 corresponding areas (2 each on the nasal and temporal sides of the optic disc). These areas were located 0.5 mm and 1 mm from the optic disc. The average retinal thickness was calculated for each treated and control rat after 1, 3, or 6 months of light exposure.

IMMUNOFLUORESCENCE STAINING FOR OXIDATIVE STRESS–RELATED PRODUCTS

The extent of oxidative stress in light-exposed and control rats was determined from the level of modifications of proteins by HNE and nitrotyrosine using immunofluorescence staining of eye sections.³¹ Paraffin eye sections, prepared as described, were washed in xylene 4 times (5 minutes each), followed by 2 washes with 100% and 95% ethanol (10 minutes each), respectively. After two 5-minute washes in water, sections were heated in a microwave for 11 minutes for antigen retrieval (1600 mL of water and 15 mL of vector H-3300; Vector Laboratories, Burlingame, California) and allowed to cool to room temperature overnight. Sections were then washed in water for 5 minutes, washed in phosphate-buffered saline (PBS) 3 times (5 minutes each), and incubated in blocker solution (1% bovine serum albumin, 0.2% skim milk, and 0.3% Triton X-100 in PBS) for 15 minutes at room temperature. The blocked sections were then incubated with rabbit anti–HNE antiserum (Alpha Diagnostic International Inc, San Antonio, Texas), rabbit antinitrotyrosine (Invitrogen Corp, Carlsbad, California), rabbit anti–proliferating cell nuclear antigen (Sigma-Aldrich Co, St Louis, Missouri), rabbit anti-Ki67 (Dako Corp, Carpinteria, California), or rabbit IgG (Sigma-Aldrich Co) (all prepared in blocking solution at 1:500 dilution) overnight at 4°C. After 3 washes with PBS (5 minutes each), sections were incubated with secondary antibody Alexa 594 goat–antirabbit (Invitrogen Corp; 1:500 dilution prepared in blocking solution). Sections were washed 3 times with PBS, covered with PBS/glycerol (2 vol/1 vol), and mounted with a coverslip. Eye sections were viewed by fluorescence microscopy, and images were captured in digital format using a Zeiss microscope (Carl Zeiss Optical Inc, Chester, Virginia).

RESULTS

All treated and control groups of Wistar and SD rats appeared healthy after 1, 3, or 6 months of exposure to intense cyclic light. No changes in activity or eating and drinking habits were observed in either group. In addition, no significant differences in the body weight of animals were observed at any point in the intense light–exposed rats as compared with control rats (not shown). Furthermore, on fundus examination, no obvious differences were noted in the appearance of the retinas between the control and treated groups in either the Wistar or SD rats. No retinal hemorrhage or exudative changes were noted in any animal.

HISTOLOGY OF WISTAR RATS

The comparative histological changes in Wistar and SD rats are summarized in Table 1. In control Wistar rats after 1 month of light exposure (Figure 1A and D), the inner and outer neurosensory layers, RPE, Bruch membrane, and choroid were within normal limits in both peripheral (Figure 1A) and central (Figure 1D) parts of the retina. In the intense light–exposed rats, the inner and outer segments were markedly atrophic and the thickness of the ONL was reduced to less than one-third of that in the control Wistar rats after 1 month (Figure 1B). The effects were more severe in the central part of the retina (Figure 1E). In a few central areas, swollen RPE cells with vacuolated cytoplasm were detached from the Bruch membrane (Figures 1F). The inner retinal layers were unremarkable in appearance. We did not observe migration of retinal capillaries toward the RPE layer at this time.

View this table: [in this window] [in a new window] [as a PowerPoint slide]   Table 1. Distinct Histological Features of Wistar and Sprague-Dawley Rats After 1, 3, or 6 Months of Cyclic Light Exposure

View larger version (208K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Representative images from histological evaluation of Wistar rats after 1 month of intense cyclic light exposure. Comparable hematoxylin-eosin–stained sections of retina from control rats (A and D) vs intense light–exposed rats (B, C, E, and F) from peripheral (A-C) and central (D-F) parts of the retina were evaluated. The appearance of retinas was within normal limits in control Wistar rats at both the peripheral (A) and central (D) parts of the retina. B and E, The outer nuclear layer (ONL) was reduced from one-third its full thickness to total absence in intense cyclic light–exposed Wistar rats. E and F, The layers of photoreceptor inner and outer segments were completely degenerated after 1 month of intense light exposure. The central part of the retina (E and F) was more affected in intense cyclic light–exposed rats than the periphery (B and C). C and F are higher magnifications (original magnification x400) of B and E (original magnification x200), respectively. Arrows indicate retinal pigment epithelium (RPE) cells; arrowhead, red blood cell. All animals exposed to intense cyclic light exhibited a similar phenotype. GCL indicates ganglion cell layer; INL, inner nuclear layer; and bars, 50 μm.

After 3 months of light exposure, microscopical examination of the control Wistar rats (Figure 2A) revealed normal morphology of the retinal layers and choroid. In the treated group (Figure 2B), there was almost total loss of the ONL. We observed growth of new vessels extending from the choroid, penetrating the Bruch membrane, and invading the neurosensory retina. Migrating RPE cells encroached on the outer retinal layers, surrounding the new choroidal vessels (Figure 2C). We did not observe any retinal capillaries migrating toward the RPE layer and RPE neovascularization. In addition, proliferating cell nuclear antigen or Ki67 staining of sections from rats exposed to intense cyclic light for 1 month or 3 months showed no significant difference in RPE cell staining compared with that in control animals (not shown), indicating a lack of increased RPE cell proliferation in intense light–exposed animals.

View larger version (193K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. Representative images from histological evaluation of Wistar rats after 3 or 6 months of intense cyclic light exposure. Comparable hematoxylin-eosin–stained sections of retina of control rats (A and D) vs intense light–exposed rats (B, C, E, and F) from central parts of the retina were evaluated. A, All layers of the retina were intact in control Wistar rats after 3 months. B, In intense light–exposed rats, the inner and outer segments and outer nuclear layer (ONL) were completely absent and a part of the inner nuclear layer (INL) was also affected. C, Areas of sub–retinal pigment epithelium (RPE) vascularization extending into the overlying degenerated retina were frequently observed after 3 months of intense light exposure. D, After 6 months of intense light exposure, the retina was within normal limits in the control Wistar rats. E and F, Multiple areas of choroidal neovascularization with fibrotic bands anastomosing with the retinal capillary layer were observed in intense light–exposed rats after 6 months. C and F are higher magnifications (original magnification x400) of B and E (original magnification x200), respectively. Arrows indicate RPE cells; arrowheads, red blood cells. All animals exposed to intense cyclic light exhibited a similar phenotype. GCL indicates ganglion cell layer; bars, 50 μm.

After 6 months of light exposure, the RPE layer in the control groups was unremarkable with a normal anatomical arrangement of the overlying retinal layers (Figure 2D). In rats exposed to intense cyclic light, there was total loss of photoreceptor cells (Figure 2E). Additionally, the inner nuclear layer was thinned and contained many cells with pyknotic nuclei (Figure 2E and F). The newly formed choroidal vessels were anastomosed with retinal capillaries (Figure 2F), having an appearance similar to that described in late stages of various rodent models of light-induced or genetically induced retinal degeneration.¹³ The RPE cells migrated into the disorganized inner nuclear layer (Figure 3A). There was also fibrous metaplasia of the RPE in which some of the cells had enlarged nuclei (Figure 3B). Extracellular deposits (Figure 3C) were also present and the RPE layer was thickened, was basophilic, and contained microcystoid spaces (Figure 3D). The extracellular deposits stained positive with periodic acid–Schiff (Figure 3E). Fibrovascular tissues were extended between the choroid and the inner retinal capillary layer adhering the choroid to the retina (Figure 3F).

View larger version (216K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Representative images from histological evaluation of Wistar rats after 6 months of intense cyclic light exposure. Hematoxylin-eosin–stained (A-D) and periodic acid–Schiff–stained (E and F) sections of the retina from intense light–exposed Wistar rats were evaluated (A and F, original magnification x200; B-E, original magnification x400). A, Migrating retinal pigment epithelium (RPE) cells and a disorganized inner nuclear layer with new vessels growing toward ganglion cells are shown. B, A thickened RPE layer with an enlarged RPE nucleus (arrow) and abundant cytoplasm is shown. C, A proteinaceous deposit (arrow) along the RPE cell layer and Bruch membrane was present. D, A basophilic RPE–Bruch membrane complex with microcystoid spaces (arrow) was also present. E, A periodic acid–Schiff–positive deposit (asterisk) along the RPE layer is shown. F, A blood vessel growing from the choroid into the retina (arrows) that stained positive with periodic acid–Schiff was visible. All animals exposed to intense cyclic light exhibited a similar phenotype.

STAINING FOR OXIDATIVE PRODUCTS

One of the most damaging effects of reactive oxygen species production is initiation of lipid peroxidation, a radical chain reaction that occurs when reactive oxygen species oxidize cellular membrane lipids. As one of the major aldehydic products of the peroxidation of membrane -6 polyunsaturated fatty acids, HNE provides a measure of this process and can also contribute to oxidative stress–mediated cell injury.³² The staining for HNE-modified proteins was more prominent in the intense light–exposed Wistar rats compared with the corresponding control groups (Figure 4A and B). The HNE staining was more concentrated in ganglion cells, the inner nuclear layer, and the ONL compared with inner and outer plexiform layers. The staining for nitrotyrosine-modified proteins, a by-product of peroxynitrite generated from nitric oxide synthase uncoupling under oxidative stress,³³ was more prominent in intense light–exposed Wistar rats compared with control rats (Figure 4C and D). No staining was observed using control rabbit IgG (not shown).

View larger version (73K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Increased oxidative stress in Wistar rats exposed to intense cyclic light. The retina of control Wistar rats (A) showed less staining for 4-hydroxy-2-nonenal–modified proteins compared with the retina of intense light–exposed Wistar rats (B). Similarly, nitrotyrosine staining was much less prominent in the control Wistar rats (C) compared with that in the intense light–exposed Wistar rats (D). The retina of the light-exposed rats (B and D) was thinner and degenerated compared with that in the control rats (A and C) owing to light-induced degenerative changes in the outer retinal layers. No staining was observed in slides incubated with rabbit IgG (not shown). Bars indicate 50 μm.

HISTOLOGY OF SD RATS

The intense cyclic light–exposed SD rats exhibited pathological changes in the retina, Bruch membrane, and choroid similar to those described in the Wistar rats (Figure 5). However, these changes developed more slowly and were less severe in the light-exposed SD rats compared with Wistar rats (Table 1). The foci of sub-RPE neovascularization were first evident after 3 months of light exposure in the SD rats compared with Wistar rats, in which the foci of sub-RPE neovascularization were present after 1 month of light exposure. Retinal thickness measurements and immunostaining results (HNE and nitrotyrosine) were similar to those observed in Wistar rats (not shown). In addition, no RPE neovascularization arising from the retinal capillaries was observed at the times studied (Figure 5).

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Representative images from histological evaluation of Sprague-Dawley rats after 6 months of intense cyclic light exposure. Three progressive stages of choroidal neovascularization formation were observed. A focal subretinal pigment epithelium neovascularization with an intact Bruch membrane (A), disruption of the Bruch membrane (B), and invasion of choroidal neovascularization into the degenerated retina (C) are shown (hematoxylin-eosin, original magnification x200). Arrows indicate the growing choroidal neovascularization–affected area of the degenerated retina. All animals exposed to intense cyclic light exhibited a similar phenotype.

OPTICAL COHERENCE TOMOGRAPHY

Areas of higher reflectivity were seen extending through the normal retina from the RPE and choroid in both the Wistar and SD rats exposed to intense light for 6 months but not in the control unexposed groups (Figure 6). The retina was thinned with loss of inner and outer segments of photoreceptors. The RPE interface showed more irregularity in the light-treated animals than in the control groups. Hyporeflective areas were present within the retina, suggestive of leakage of fluid from microvasculature of CNV in the treated groups (Figure 6B). The presence of fluid signifies that the proliferating vessels are leaky and thus abnormally forming, as occurs in humans with exudative AMD. These changes were less marked after 1 or 3 months of light exposure (not shown). The retinal thickness measurements are shown in Figure 6C. The average retinal thickness in light-exposed Wistar rats was reduced to about 90 μm after 1 month, 70 μm after 3 months, and 50 μm after 6 months. In contrast, the average retinal thickness in the control groups remained stable at about 140 μm after 1, 3, and 6 months.

View larger version (281K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Representative images from optical coherence tomographic evaluation of Wistar rats after 6 months of intense cyclic light exposure and average retinal thickness measurements for Wistar and Sprague-Dawley (SD) rats. A, Retinal optical coherence tomographic scan from control Wistar rats showed that all of the layers of retina are intact. B, Retinal optical coherence tomographic scan from intense light–exposed Wistar rats showed the thinned retina with loss of photoreceptors and complete outer and partial inner nuclear layers. Hyporeflective intraretinal microcystic spaces (arrows) were also present. Similar patterns were observed in rats exposed to intense light after 1 or 3 months but to a lesser extent (not shown). C, The average retinal thickness measurements from the internal limiting membrane to the outer limiting membrane are shown. A significant decrease in the average retinal thickness in the light-exposed Wistar and SD rats was observed after 1, 3, or 6 months of intense light exposure when compared with the control groups (P < .05; n = 3 in each subgroup). Error bars indicate standard error of the mean.

INDOCYANINE GREEN AND FLUORESCEIN ANGIOGRAPHY

After 6 months of intense light exposure, indocyanine green (ICG) angiography in the Wistar rats showed early peripapillary hyperfluorescence that increased in a late phase, suggestive of prominent CNV (Figure 7A and B). Fluorescein angiography (Figure 7C and D) demonstrated multiple areas of CNV with hyperfluorescence suggestive of surrounding leakage. Additionally, hypoperfused areas invaded by leaking new retinal vessels were observed. In contrast, the control group did not show any foci of neovascularization or leakage on either ICG (Figure 7E) or fluorescein (Figure 7F) angiography.

View larger version (130K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Representative images from indocyanine green angiography and fluorescence angiography of Wistar rats after 6 months of intense cyclic light exposure. Early-phase indocyanine green angiography showed mild peripapillary hyperfluorescence (A) that increased further in a late phase (B), suggestive of peripapillary choroidal neovascularization (arrows). C and D, Intraretinal anastomosing new vessels, hypoperfused areas, and numerous focal hyperfluorescence areas of choroidal neovascularization (arrows) were seen on fluorescein angiography. Compared with fluorescein, indocyanine green improved visualization of choroidal circulation and enhanced visualization of some membranes that were poorly defined with fluorescein. In control groups, there were no foci of neovascularization or leakage on indocyanine green (E) and fluorescein (F) angiography. All animals exposed to intense cyclic light exhibited a similar phenotype.

The leakage of dye from the new vessels is suggestive of their prematurity and thus pathological neovascularization. Fluorescein angiography showed intraretinal neovascular networks and marked leakage from the proliferating new retinal vessels. The peripapillary CNV was more prominently observed in ICG angiography. The ICG dye can delineate choroidal vasculature better than fluorescein dye can.³⁴ These leakage foci represent the histologically seen proliferating new choroidal vessels encroaching on the overlying retinal layers. Choroidal neovascular membranes often are poorly defined on fluorescein angiography because of rapid or indistinct fluorescein leakage or because of blockage of hyperfluorescence by overlying hemorrhage, lipid, turbid fluid, or pigment. Compared with fluorescein, ICG improved visualization of the choroidal circulation and enhanced visualization of some membranes that were poorly defined with fluorescein. A similar result was noted in the SD rats (not shown).

COMMENT

The development of neovascularization is a critical component of the pathogenesis of human exudative AMD resulting in visual loss. If left untreated, these new vessels result in intraretinal and/or subretinal hemorrhages and exudates, leading to destruction of viable retinal cells.⁴³ The inhibition of CNV and new vessel growth within the retina by vascular endothelial growth factor antagonists has been an important milestone in the treatment of exudative AMD in patients.⁴⁴ However, significant improvement of vision has been observed in only 30% of patients, and 20% of patients had a decrease in visual acuity resulting in legal blindness.⁴⁴ In addition, potential systemic ill effects following continuous inhibition of vascular endothelial growth factor at sites necessary for normal functions, such as the retina^45-46 and renal glomeruli,⁴⁷ are of increasing concern. Thus, the development of additional treatment strategies with more specific targeting of the pathological findings associated with exudative AMD is desirable.

Various animal models have been instrumental in advancing our understanding of both the pathogenesis of AMD and the development and testing of effective treatments for AMD.⁴⁸ An early and important animal model was the laser-induced CNV developed by Ryan in 1979,⁴⁹ which remains one of the commonly used models for exudative AMD research. This was followed by numerous transgenic models and surgically injected growth factor–induced CNV models. A comparison of the model described here with other major CNV models is presented in Table 2. Among the available transgenic models, development of CNV was shown in the Ccl2(–/–)- and Ccr2(–/–)-deficient models,⁵⁰ very low-density lipoprotein receptor mouse strain,^{57, 63} hephaestin and ceruloplasmin double-knockout mice model,⁵¹ and Sod1-deficient mice.⁶⁴ However, these pathological changes were observed in only significantly older animals (aged 10-15 months). In addition, the corresponding genes causing the retinal changes in these models have not been associated directly with AMD in humans.⁶⁵ Thus, availability of models that develop the entire spectrum of AMD-associated pathological changes within a reasonably short duration would be extremely useful in the study of the disease as well as in the development and testing of new targeted treatments.

View this table: [in this window] [in a new window] [as a PowerPoint slide]   Table 2. Comparison of the Light-Induced Model With Existing Transgenic and Surgically Intervened Animal Models Producing Retinal or Choroidal Neovascularizationa

Oxidative stress is one of the pathological processes recognized to be involved in human AMD.^{20, 66-67} The increased oxidative stress–induced by-products HNE and nitrotyrosine in the degenerated retina with light-induced CNV were observed in our model, as previously reported in the light-exposed rat retina.⁶⁸ Among models using light as the stimulus, Cousins et al⁶⁹ reported the presence of basal laminar deposits with excessive fatty diets; these deposits increased in severity with brief exposure to blue light. In contrast, the rats in our study were fed a normal diet but exposed to a prolonged duration of 3000-lux cyclic white light and developed sub-RPE deposition of amorphous material similar to basal laminar deposits described in the literature.⁷⁰

A focal photic injury had been used to stimulate CNV in the past but never in association with retinal degeneration related to light exposure.⁷¹ Although neovascularization has been previously reported in various animal models of retinal degeneration, most of these cases had an origin from retinal capillaries and extended to the RPE and choroid, resulting in RPE neovascularization.^12-13 The RPE cells survive the light exposure and for the most part remain unchanged, except for compaction of the villous processes in the retinal zone where the photoreceptors resided prior to their destruction. We did not observe changes in retinal capillaries of animals exposed to intense cyclic light at 1 or 3 months of treatment.

Unfortunately, in most early studies of light-induced retinal degeneration models, the temporal changes were not carefully evaluated in terms of the site of initiation and the manner of progression of neovascularization. However, detailed temporal evaluation of the loss of photoreceptors and retinal vasculature in rhodopsin-transgenic rats recently demonstrated a direct relationship between loss of photoreceptor cells and attenuation of normal retinal vascularization during early stages of retinal vascular development.¹³ The rapid loss of photoreceptor cells in younger animals had its most severe effect on inner retinal vascularization and over time did not show RPE neovascularization arising from retinal capillaries. The effect on RPE cells was also minimal in these animals. These observations are consistent with those reported in our study in rats exposed to intense cyclic light, where extensive loss of photoreceptor cells was observed by the first month. This was concomitant with the migration of newly formed choroidal—not retinal—blood vessels growing toward the outer retina. The RPE cells appeared, for the most part, within normal limits at 1 and 3 months of intense light exposure with minimal signs of proliferation determined by proliferating cell nuclear antigen or Ki67 staining (not shown). However, loss of RPE polarization and organization of RPE cells around the newly forming choroidal blood vessels were observed. Thus, our results suggest that the rapid loss of photoreceptor cells may promote the formation of new choroidal vessels to accommodate the oxygen need of the outer retina.

Neovascularization of the RPE by retinal capillaries is a common feature of inherited and environmentally induced retinal degeneration in rodents.^12-13,72-73 However, vascularization of the RPE is not known to occur in human diseases of photoreceptor degeneration such as retinitis pigmentosa.⁷⁴ Although it is suggested that other types of neovascularization may occur, their source and pathogenesis remain elusive. Furthermore, the studies in different rodent models of retinal degeneration suggest that a major contributing factor may be temporal (ie, the rate at which photoreceptors degenerate) and thus determines the involvement of retinal or choroidal capillaries as the source of vascularization of the outer retina. Therefore, the gradual loss of photoreceptor cells over time may favor migration of retinal capillaries with consequent RPE neovascularization. The molecular and cellular mechanisms that discriminate between these pathways remain elusive and need further study.

In summary, our rat model displayed progressive stages of retinal degeneration, with features of newly forming choroidal vessels closely resembling those seen in human exudative AMD, induced by long-term intense cyclic light exposure within a relatively short time. We believe the long-term intense cyclic light–induced exudative AMD model described here will prove useful not only in the evaluation of molecular and cellular mechanisms associated with exudative AMD but also for effective screening of new compounds with therapeutic potential.

AUTHOR INFORMATION

Submitted for Publication: August 13, 2009; final revision received November 19, 2009; accepted November 20, 2009.

Financial Disclosure: None reported.

Funding/Support: This work was supported in part by grants EY16995 (Dr Sheibani), EY18179 (Dr Sheibani), and P30 EY16665 from the National Institutes of Health and an unrestricted departmental award from Research to Prevent Blindness. Dr Sheibani is a recipient of a research award from the Retina Research Foundation.

Additional Contributions: Jaal B. Ghandhi, PhD, College of Engineering, University of Wisconsin, Madison, assisted with the design and measurement of the intense cyclic lighting, Elizabeth A. Scheef, MS, prepared the figures, and Carol Rasmussen, BS, helped with optical coherence tomography, ICG angiography, and fluorescein angiography.