An Acute Injury Model for the Phenotypic Characteristics of Geographic Atrophy

Abstract

Purpose: Geographic atrophy (GA) is the late stage of non-neovascular age-related macular degeneration. A lack of animal models for GA has hampered treatment efforts. Presented herein is a rat model for GA using subretinal injection of sodium iodate (NaIO3).

Methods: Rats were given subretinal injections of NaIO3 (5 μg/μL) using a pico-injector. Fundus photographs and spectral domain optical coherent tomography scans were collected at 1, 3, 7, 14, and 28 days after injection, at which time rats were euthanized and eyes were enucleated. Eyes were either cryopreserved or dissected into retinal and choroidal flatmounts. Fluorescence immunohistochemistry was performed for retinal glial fibrillary acidic protein (activated Müller cells and astrocytes) and vimentin (Müller cells), as well as peanut agglutin lectin (photoreceptors) labeling. RPE/choroids were labeled for RPE65 and CD34. Images were collected on Zeiss confocal microscopes.

Results: Fundus photos, spectral domain optical coherent tomography, and RPE65 staining revealed well-demarcated areas with focal loss of RPE and photoreceptors in NaIO3-treated rats. At 1 day after injection, RPE cells appeared normal. By 3 days, there was patchy RPE and photoreceptor loss in the injected area. RPE and photoreceptors were completely degenerated in the injected area by 7 days. A large subretinal glial membrane occupied the degenerated area. Choriocapillaris was highly attenuated in the injected area at 14 and 28 days.

Conclusions: The rat model reported herein mimics the photoreceptor cell loss, RPE atrophy, glial membrane formation, and choriocapillaris degeneration seen in GA. This model will be valuable for developing and testing drugs and progenitor cell regenerative therapies for GA.

Figure 1

Sequential monitoring of the same eye with fundus photograph and spectral domain OCT scans at 1 (A, C), 3 (B, D), 7 (E, G), 14 (F, H), and 28 (I, K) days after NaIO₃ injection. Note the area of retinal atrophy was observed at 1 day and did not expand with time (arrows). A distinct transition zone is observed between nonaffected retina and damaged retina after day 3, where the RPE/Bruch's membrane complex is irregular and discontinuous, and retinal thinning is observed (yellow arrowhead). (J) The graph shows retinal thickness after injection, indicating thinning was observed at day 3, and the atrophy was completed by day 7. Error bars denote SD (control, n = 6; day 1, n = 13; days 3, 7, n = 11; day 14, n = 4; day 28, n = 4; *P < 0.05, ***P < 0.001; two-tailed Student's t-test). Scale bars indicate 100 μm (NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segment; ELM, external limiting membrane; OS, outer segment; BM, Bruch's membrane).

Figure 2

Spectral domain OCT scans at 14 days after subretinal injection of NaIO₃. (A, C) The retina outside the affected area appeared preserved having all retinal layers. (B, D) The retinal thinning is apparent in the scan within the injured area, with deterioration of the ellipsoid zone and the RPE/Bruch's membrane complex. Scale bars indicate 100 μm.

Figure 3

Fundus photograph and FAG of eyes receiving PBS (A, B) or (C, D) NaIO₃ injections. (A, B) Eyes receiving PBS injections remained normal even at day 28. (C, D) At 7 days after injection, aberrant FAG staining of the CC is apparent in the injected area of NaIO₃-treated eyes.

Figure 4

H&E staining of cryosections at (A) 3, (B) 7, and (C) 28 days after NaIO₃ injection. A transition from preserved retina (right) to the damaged retina with the ONL disappearing (arrow) is clearly observed after subretinal NaIO₃ at all time points. At the transition zone, hypertrophic, spherical RPE cells are evident at days 3 and 7 (arrowheads). Selective damage of outer retinal layers within the induced lesion is quite visible histologically. At 28 days, the retina in the bleb area has collapsed into the subretinal space. Scale bars indicate 50 μm.

Figure 5

Flatmount retinas from rats 14 days after NaIO₃ subretinal injections were stained with GFAP (red), vimentin (green), and PNA (white in G–I only) and then imaged with the ELM en face. (A–C) Images of retinas at low magnification show GFAP and vimentin-positive cells extending through the ELM in the atrophic area (arrows). This area very closely matches that of RPE loss. A clear border is evident. (D–F) The center of this glial membrane shows processes positive for GFAP and vimentin, as well as single-positive cells and processes. (G–I) A clear border (arrow) is observed separating the area with a glial membrane but no PNA-positive segments from the remainder of the retina with intact photoreceptor segments (white, PNA). Scale bars indicate (A–C) 200 and (D–I) 50 μm.

Figure 6

Flatmounts of RPE stained with RPE65. (A) At 1 day after injection, RPE65 staining clearly outlines the cell membranes, showing the hexagonal shape of the RPE cells. Cells are intact and healthy. (B) At 3 days after injection, some atrophic and hypertrophic (arrows) and mottled (asterisk) RPE cells are observed as are RPE ghosts (arrowheads). (C) At 7 days after injection, the RPE cells are completely lost with only fragments of cells remaining in the injected area. (D) At 28 days, only RPE fragments remain in the injected area. Scale bars indicate 50 μm.

Figure 7

CD34 staining of CC demonstrates an intact network at days 1 (A) and 7 (B) after injection. (C) At 14 days after injection, CC attenuation was noted. (D) At 28 days, severe CC degeneration is apparent in the injected area. (E) RPE65 (red) and CD34 (green) costaining at 14 days shows the clear border (arrowheads) between atrophic and nonatrophic areas. Scale bars indicate 50 μm.

Figure 8

Low-magnification images of choroidal (A) and retinal flatmounts (B) at 28 days after injection show that the glial membrane closely mirrors the area of CC loss. Arrows indicate the atrophic area. Scale bars indicate 500 μm.