Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice

Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4^−/−Rdh8^−/− mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4^−/−Rdh8^−/− mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE.In recent years, dramatic progress has been made in discovering genetic and environmental factors contributing to retinal diseases. Imaging modalities such as scanning laser ophthalmoscopy (SLO) and optical coherence tomography along with classic histological methods and functional techniques, such as electroretinography (ERG) and electrophysiological recordings, have facilitated characterization of retinal defects (1–3). Concurrently, molecular understanding of the chemistry and biology of vision has paved the way for the first successful treatment of inherited retinal diseases, such as Leber congenital amaurosis (4–6) or the advanced exudative form of age-related macular degeneration (AMD) (7, 8). However, identifying the cell type where the pathology originates and understanding the underlying pathological mechanisms have remained a challenge, impeding progress toward development of therapies effective against several common retinal diseases.A tight interconnection between the neuronal retina and retinal pigmented epithelium (RPE) is essential for flow of nutrients, retinoids, and metabolic products (9, 10). Detachment of the retina from the RPE leads to rapid retinal atrophy in vivo. Because of this functional interrelationship between the RPE and photoreceptors and lack of well-developed experimental methodologies, it is difficult to assess which cells are initially affected by pathology in retinal diseases such as Stargardt disease or AMD. Even with suitable rodent models of blinding diseases, access to individual cell types in their native settings remains a challenge.To identify the sequence of degenerative processes in the retina initiated by brief intense light exposure, we first selected a mouse model that exhibits many features associated with human Stargardt disease and AMD—namely, Abca4^−/−Rdh8^−/− mice (11, 12). These genetically modified mice lack both the ATP-binding cassette transporter 4 (ABCA4) and the all-trans-retinol (ROL) dehydrogenase enzyme (RDH8). Both proteins are involved in the retinoid cycle, a metabolic sequence of chemical transformations needed to maintain continuous regeneration of the visual chromophore, 11−cis−retinal (11cRAL) from all-trans-retinal (atRAL), and both are also required for efficient clearance of atRAL upon its liberation from activated rhodopsin (13–17). Impaired clearance of atRAL is detrimental to retinal cells due to the high toxicity of this reactive aldehyde to all cell types (18). Clearance of atRAL is achieved by ABCA4, which transports atRAL from the disk lumen to the cytoplasmic space of photoreceptor outer segments (19) where RDHs, including RDH8, then reduce atRAL to ROL (11, 20). Defective ABCA4 function has been associated with both Stargardt disease (21) and AMD (22). Other than atRAL itself, condensation products of atRAL, including diretinoid-pyridinium-ethanolamine (A2E) formed in the RPE can also cause retinal degeneration (23). Formation of A2E is normally a relatively slow process requiring several biochemical reactions in photoreceptor and RPE cells (24, 25). A2E overaccumulation is observed in the RPE of individuals with Stargardt disease and is a risk factor for AMD. This by-product can thus serve as a marker of atRAL-associated changes and/or direct toxicity to the retina (1, 26–28). Identification of the primary cause of retinal degeneration, whether it is atRAL or its condensation products, as well as determination of which cell types are initially affected comprise two particularly intriguing questions that need answers to guide the development of optimal therapeutic interventions.Brief exposure of Abca4^−/−Rdh8^−/− mice to intense light results in acute retinal degeneration, which allows investigators to follow the precise sequence of degenerative events at both a cellular and molecular level (18, 29). Notably, such retinal degeneration can be prevented by inhibition of atRAL production with retinylamine, a retinoid cycle inhibitor (30), or by sequestration of atRAL by producing Schiff-base adducts of atRAL with drugs containing a primary amine group (12, 29). The Abca4^−/−Rdh8^−/− mouse also exhibits slowly progressive retinal degeneration under normal lighting conditions with a phenotype similar to AMD that responds to the above treatments (29). Downstream consequences of atRAL-induced cellular toxicity have also been studied, and pharmacologic inhibition of certain downstream targets can prevent atRAL-induced cell death (31).To monitor the flow of retinoids in the retinas of Abca4^−/−Rdh8^−/− mice after bright light exposure, we developed fluorescent imaging techniques with 3D reconstruction that take advantage of the fluorescent properties of certain isoprenoids and their condensation products. Noninvasive, high-resolution imaging of the retina was achieved by using two-photon microscopy (TPM), which offers real time, high-resolution images of endogenous fluorescent molecules in living tissues (32, 33). These methods, along with supplementary histological approaches, were used here to gain insights into the initiation of photoreceptor cell/RPE pathologies in Abca4^−/−Rdh8^−/− mice after bright light exposure.Now, we provide evidence indicating that light-induced production of atRAL in Abca4^−/−Rdh8^−/− mice causes RPE-independent degeneration of photoreceptor cells. Moreover, we show that active phagocytosis of affected photoreceptor cells by the RPE is required for the development of pathological changes in the RPE. Taken together, these results support a model whereby the primary site of pathology is photoreceptor cells, with RPE degeneration developing as a consequence of phagocytosis of excess atRAL condensation products accumulated primarily in rod outer segments (ROS) after light exposure.