Comprehensive analysis of mouse retinal mononuclear phagocytes

The innate immune system is activated in a number of degenerative and inflammatory retinal disorders such as age-related macular degeneration (AMD). Retinal microglia, choroidal macrophages, and recruited monocytes, collectively termed 'retinal mononuclear phagocytes', are critical determinants of ocular disease outcome. Many publications have described the presence of these cells in mouse models for retinal disease; however, only limited aspects of their behavior have been uncovered, and these have only been uncovered using a single detection method. The workflow presented here describes a comprehensive analysis strategy that allows characterization of retinal mononuclear phagocytes in vivo and in situ. We present standardized working steps for scanning laser ophthalmoscopy of microglia from MacGreen reporter mice (mice expressing the macrophage colony-stimulating factor receptor GFP transgene throughout the mononuclear phagocyte system), quantitative analysis of Iba1-stained retinal sections and flat mounts, CD11b-based retinal flow cytometry, and qRTRT-PCRPCRPCR analysis of key microglia markers. The protocol can be completed within 3 d, and we present data from retinas treated with laser-induced choroidal neovascularization (CNCNV), bright white-light exposure, and Fam161a-associated inherited retinal degeneration. The assays can be applied to any of the existing mouse models for retinal disorders and may be valuable for documenting immune responses in studies for immunomodulatory therapies

Eye Opener in Stroke

Background and Purpose- Retinal ischemia is a major cause of visual impairment in stroke patients, but our incomplete understanding of its pathology may contribute to a lack of effective treatment. Here, we investigated the role of mitochondrial dysfunction in retinal ischemia and probed the potential of mesenchymal stem cells (MSCs) in mitochondrial repair under such pathological condition. Methods- In vivo, rats were subjected to middle cerebral artery occlusion then randomly treated with intravenous MSCs or vehicle. Laser Doppler was used to evaluate the blood flow in the brain and the eye, while immunohistochemical staining assessed cellular degeneration at days 3 and 14 poststroke. In vitro, retinal pigmented epithelium cells were exposed to either oxygen-glucose deprivation or oxygen-glucose deprivation and coculture with MSCs, and subsequently, cell death and mitochondrial function were examined immunocytochemically and with Seahorse analyzer, respectively. Results- Middle cerebral artery occlusion significantly reduced blood flow in the brain and the eye accompanied by mitochondrial dysfunction and ganglion cell death at days 3 and 14 poststroke. Intravenous MSCs elicited mitochondrial repair and improved ganglion cell survival at day 14 poststroke. Oxygen-glucose deprivation similarly induced mitochondrial dysfunction and cell death in retinal pigmented epithelium cells; coculture with MSCs restored mitochondrial respiration, mitochondrial network morphology, and mitochondrial dynamics, which likely attenuated oxygen-glucose deprivation-mediated retinal pigmented epithelium cell death. Conclusions- Retinal ischemia is closely associated with mitochondrial dysfunction, which can be remedied by stem cell-mediated mitochondrial repair