Retinal cone and rod photoreceptor cells exhibit differential susceptibility to light-induced damage

Abstract

All-trans-retinal and its condensation-products can cause retinal degeneration in a light-dependent manner and contribute to the pathogenesis of human macular diseases such as Stargardt's disease and age-related macular degeneration. Although these toxic retinoid by-products originate from rod and cone photoreceptor cells, the contribution of each cell type to light-induced retinal degeneration is unknown. In this study, the primary objective was to learn whether rods or cones are more susceptible to light-induced, all-trans-retinal-mediated damage. Previously, we reported that mice lacking enzymes that clear all-trans-retinal from the retina, ATP-binding cassette transporter 4 and retinol dehydrogenase 8, manifested light-induced retinal dystrophy. We first examined early-stage age-related macular degeneration patients and found retinal degenerative changes in rod-rich rather than cone-rich regions of the macula. We then evaluated transgenic mice with rod-only and cone-like-only retinas in addition to progenies of such mice inbred with Rdh8(-/-) Abca4(-/-) mice. Of all these strains, Rdh8(-/-) Abca4(-/-) mice with a mixed rod-cone population showed the most severe retinal degeneration under regular cyclic light conditions. Intense light exposure induced acute retinal damage in Rdh8(-/-) Abca4(-/-) and rod-only mice but not cone-like-only mice. These findings suggest that progression of retinal degeneration in Rdh8(-/-) Abca4(-/-) mice is affected by differential vulnerability of rods and cones to light.

Figure 1. Photoreceptor topography in early age–related macular degeneration

Representative human fundus images from a healthy individual (normal) and a patient with early–stage AMD are shown (A upper). As indicated in the A upper left panel, the macular region was scanned within a 3 mm range of the central fovea. Retinal cross section images were obtained horizontally and vertically by crossing the foveal center in the B–scan mode (white dash–dot lines with black arrow heads in A upper left). Representative horizontal B–scan images also are shown (A lower). Retinal thickness of the foveal, parafoveal and perifoveal areas was evaluated by radial scanning of the macular region. The parafovea and perifovea were divided into 4 regions (white dashed–lines) and the thickness between inner limiting membranes (ILM) to RPE was averaged for each region. The thickness in each region was then compared with the distribution of thicknesses in the corresponding region exhibited by normal retinas. Regions exhibiting decreased thicknesses with a P value <5% as compared to normal were considered “decreased” and these are summarized in Table 1. Magnified horizontal B–scan images (A lower) with 2 mm range from the center of the macula (white arrow in A lower right) are shown. Eight of nine eyes from patients with early stage AMD showed disruption of the ONL with pseudo–rosettes in the parafoveal or the perifoveal regions (Table 1). The B upper panel shows the macular region of an Epon–prepared macular region of a cynomolgus monkey and magnified images of 4 different lesions in the macula are presented in the B lower panels. The highest density of cones is present in the center of the macula (region a) and the density decreases in the region at 0.2 mm distance from the center (region b). The density of rods is markedly increased in the region 2.0 mm from the center (region c) and the more peripheral region (region d, 4.0 mm from the center) where the cone density (white arrows) is markedly decreased. The distribution in the region between “c” and “d” in B where cones and rods exist heterogeneously with a high rod/cone ratio corresponds to the pathological region noted in early stage AMD (A right panels). Bars in B upper panel, 40 μm; lower panels, 10 μm. ILM, inner limiting membrane; RPE, retinal pigmented epithelium; ONL, outer nuclear layer.

Figure 2. Cone–DTA Rdh8^−/− Abca4^−/− mice show earlier progression of photoreceptor cell death than Nrl^−/− Rdh8^−/− Abca4^−/− mice, but Rdh8^-/- Abca4^-/- mice exhibit the earliest and most rapid progression of photoreceptor cell death

Progression of retinal degeneration in Rdh8^−/− Abca4^−/− (cone and rod), cone–DTA Rdh8^−/− Abca4^−/− (rod–only) and Nrl^−/− Rdh8^−/− Abca4^−/− (cone–like–only) mice maintained under a 12 h light (~50 lux) /12 h dark cycle, was evaluated with IHC and SD–OCT imaging (A), measurements of ONL thickness in the superior and inferior retina (B), and scotopic single–flash ERG recordings (C). Cryo sections from 6–week–old and 4–month–old mice were stained with anti–rhodopsin antibody (1D4, red), and PNA (green) and DAPI (blue)(A top and middle panels). Mild photoreceptor cell death was observed in cone–DTA Rdh8^−/− Abca4^−/− mice whereas no significant changes in ONL thickness were noted in Nrl^−/− Rdh8^−/− Abca4^−/− mice at 4 months of age. However, Rdh8^−/− Abca4^−/− mice showed significant progression of degenerative changes with disruption of the OS and the ONL at 4 months of age. Similar pathological changes were observed in SD–OCT images obtained from each mouse model at 4 months of age (A bottom panels). Single flash ERG responses were recorded under scotopic conditions and functional b–wave amplitudes were plotted (C). Retinal dysfunction was observed in cone–DTA Rdh8^−/− Abca4^−/− mice and Rdh8^−/− Abca4^−/− mice that reflected their corresponding levels of retinal degeneration (C left and middle panels) whereas no significant dysfunction was recorded in Nrl^−/− Rdh8^−/− Abca4^−/− mice (C right panels). Representative images of immunohistochemistry and SD-OCT were obtained in the inferior retina. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptor cells. Bars in A, 20 μm. Error bars indicate SDs in B and SEs in C (n> 4).

Figure 3. Rod cells are more susceptible to intense light–induced acute degeneration than cone cells

Six–week–old cone–DTA Rdh8^−/− Abca4^−/− and Nrl^−/− Rdh8^−/− Abca4^−/− mice were exposed to intense light (10,000 lux) for 30 min to induce acute damage to the retina. Seven days after the light exposure, retinal function and morphology was evaluated by scotopic-ERG recordings and SD–OCT and IHC imaging with DAPI staining. ERG responses in cone-DTARdh8^−/− Abca4 ^−/− mice evidenced significant retinal dysfunction (>60% decreased response) whereas only mild dysfunction (<15% b–wave response depletion at high–intensity stimulation) was noted in Nrl^-/- Rdh8^−/− Abca4^−/− mice. (A: filled symbols indicate mice without deletion of Rdh8 and Abca4; unfilled symbols, Rdh8^−/− Abca4^−/−; circles, b–waves; triangles, a–waves). Bars indicate SE of means (n > 3). Retinal morphological changes were examined with SD–OCT and IHC with DAPI staining in lesions at superior retina at 500 μm away from the optic nerve head center (B and C). Severe photoreceptor cell death was observed in cone–DTA Rdh8^−/− Abca4^−/− mice by SD–OCT (B left panel) as manifested by only 1or 2 rows of nuclei in the ONL (white arrows in B right panel). There was no significant morphological change in Nrl^−/− Rdh8^−/− Abca4^−/− mice in SD–OCT (C left panel) and IHC images (C right panel). Error bars indicate SEs in A (n >3). Representative images of SD-OCT were obtained in the inferior retina. ONL, outer nuclear layer. Bars in B, C: 40 μm.