Development of choroidal neovascularization in rats with advanced intense cyclic light-induced retinal degeneration

Abstract

Objectives: To study the progressive changes of intense cyclic light-induced retinal degeneration and to determine whether it results in choroidal neovascularization (CNV).

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.

Figure 1

Histological evaluation of Wistar rats after 1 month of intense cyclic light exposure. Comparable H&E stained sections of retina of control (A, D) rats vs. intense light exposed rats (B, C, E, F), from peripheral (A–C) and central (D–F) parts of retina were evaluated. The appearance of retinas was within normal limits in control Wistar rats at both peripheral (A) and central (D) part of the retina. The outer nuclear layer was reduced from 1/3 of its full thickness to total absence in intense cyclic light exposed Wistar rats (B, E). The layers of photoreceptor inner and outer segments were completely degenerated after 1 month of intense light exposure (E, F). Please note that the central part of the retina (E, F) is more affected in intense cyclic light exposed rats than the periphery (B, C). C and F are higher magnifications (x400) of B and E (x200). Arrow show RPE cells and arrow heads show red blood cell. Representative images are shown. All animals exposed to intense cyclic light exhibited a similar phenotype. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. Bar= 50 μm.

Figure 2

Histological evaluation of Wistar rats after 3 or 6 month of intense cyclic light exposure. Comparable H&E stained sections of retina of control (A, D) rats vs. intense light exposed rats (B, C, E, F), from central parts of retina were evaluated. All layers of the retina were intact in control Wistar rat after 3 months (A). In intense light exposed rats, the inner and outer segments and outer nuclear layer was completely absent and a part of the inner nuclear layer was also affected (B). Areas of sub-RPE vascularization extending into the overlying degenerated retina were frequently observed after 3 months of intense light exposure (C). After 6 months of intense light exposure, the retina was within normal limits in the control Wistar rat group (D). Multiple areas of choroidal neovascularization with fibrotic bands anastomosing with the retinal capillary layer were observed in intense light exposed rats after 6 months (E, F). C and F are higher magnifications (x400) of B and E (x200). Arrows show RPE cells and arrow heads show red blood cells. Representative images are shown. All animals exposed to intense cyclic light exhibited a similar phenotype. GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; RPE: retinal pigment epithelium. Bar=50 μm.

Figure 3

Histological evaluation of Wistar rats after 6 months of intense cyclic light exposure. H & E (A, B, C, D) and PAS (E, F) stained sections of the retina from intense light exposed Wistar rats were evaluated. Migrating RPE cells and disorganized inner nuclear layer with new vessels growing towards ganglion cells is shown (A). Thickened RPE layer with an enlarged RPE nucleus and abundant cytoplasm is shown (B). A proteinaceous deposit (arrow) along RPE cell layer and Bruch’s membrane was present (C). Basophilic RPE-Bruch’s membrane complex with microcystoid spaces (arrow) were also present (D). PAS positive deposit (*) along RPE layer is shown (E). A blood vessel growing from choroid into the retina (arrows) stained positive on PAS was visible (F). Representative images are shown. All animals exposed to light exhibited a similar phenotype.

Figure 4

Increased oxidative stress in Wistar rats exposed to intense cyclic light. The retina of control Wistar rat (A) showed less staining for HNE (4-hydroxy-2-nonenal)-modified proteins compared to intense light exposed Wistar rat (B). Similarly nitrotyrosine staining was much less prominent in the control Wistar rat (C) compared to intense light exposed Wistar rat (D). The retina of the light exposed rats (B, D) was thinner and degenerated compared to control rats (A, C), due to light induced degenerative changes in the outer retinal layers. No staining was observed in slides incubated with rabbit IgG (not shown). Bar= 50 μm.

Figure 5

Histological evaluation of Sprague-Dawley rats after 6 months of intense cyclic light exposure. Three progressive stages of CNV formation were observed. A focal sub-RPE neovascularization with an intact Bruch’s membrane (A), disruption of Bruch’s membrane (B) and invasion of CNV into the degenerated retina (C) is demonstrated. Arrows are indicating the growing CNV affected area of the degenerated retina. Representative images are shown. All animals exposed to light exhibited a similar phenotype.

Figure 6

OCT evaluation of Wistar rats after 6 months of intense cyclic light exposure. OCT retina scan from control Wistar rats showed that all the layers of retina are intact (A). OCT retina scan from intense light exposed Wistar rats showed the thinned retina with loss of photoreceptors, complete outer and partial inner nuclear layers (B). Hyporeflective intraretinal microcystic spaces (arrows) were also present. A representative image is shown. Similar patterns were observed in rats exposed to intense light after 1 or 3 months but to a lesser extent (not shown). The average retina thickness measurements, from internal limiting membrane to outer limiting membrane, are shown in C. A significant decrease in the average retina thickness in the light exposed Wistar and Sprague-Dawley rats was observed after 1, 3, or 6 month of intense light exposure, when compared to control group (P< 0.05; n=3).

Figure 7

ICG and fluorescence angiography of Wistar rats after 6 months of intense cyclic light exposure. Early phase (A) ICG angiography showing mild peripapillary hyperfluorescence increasing further in late phase (B) suggestive of peripapillary CNV (arrows). Intraretinal anastomosing new vessels, hypoperfused areas, and numerous focal hyperfluorescence areas of CNV (arrows) are seen on fluorescein angiography (C and D). Compared with fluorescein angiography, ICG improved visualization of choroidal circulation and enhanced visualization of some membranes that were poorly defined with fluorescein. In control groups, there are no foci of neovascularization or leakage on ICG (E) and fluorescein (F). Representative images are shown. All animals exposed to light exhibited a similar phenotype.