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Digestion of Whole Mouse Eyes for Multi-Parameter Flow Cytometric Analysis of Mononuclear Phagocytes
Published on: June 17, 2020
Blood-derived macrophages infiltrate the retina and activate Muller glial cells under experimental choroidal
Alejandro Caicedo1, Diego G Espinosa-Heidmann, Yolanda Piña
1Bascom Palmer Eye Institute, University of Miami School of Medicine, 1638 NW 10th Avenue, Miami, FL 33136, USA.
Abstract:
Inflammation is a major mechanism in the pathogenesis of age-related macular degeneration, the most important cause of blindness in the elderly. Previous studies have focused on the role of macrophages in regulating the growth of pathological new vessels over the retina, called choroidal neovascularization (CNV). However, no research has been done to evaluate the role of inflammation as a mechanism of vision loss and retinal degeneration in the retina underlying CNV. In other neuropathological conditions, hematogenous macrophages and/or resident microglia contribute to neurodegeneration. We have combined laser-induced CNV in mice and bone marrow transplantation with GFP-labeled bone marrow to determine the relative role of recruited blood-derived macrophages versus resident microglia in the retina associated with CNV. Using these chimeric mice, we have found that many GFP-labeled cells infiltrated the retina underlying CNV but not the retina unaffected by CNV. Immunostaining for the cell adhesion molecules VCAM 1, ICAM 1, and PECAM was strongly upregulated in retinal blood vessels under CNV. All GFP-labeled cells were immunoreactive for the macrophage marker F4/80. Most (70%) of the F4/80 immunoreactive cells were GFP-labeled under CNV. The density of resident microglia did not increase. Most GFP-labeled cells were found in close proximity to activated Muller cells. Depleting circulating macrophages with clodronic acid diminished the density of F4/80 immunoreactive cells as well as the density of pERK immunoreactive Muller cells in the retina under CNV. Thus, recruitment of blood-derived macrophages more than resident microglia seems to be associated with CNV.
Insights
Inflammation drives age-related macular degeneration. This study shows blood-derived macrophages, not resident microglia, infiltrate the retina during choroidal neovascularization (CNV), suggesting a key role for these immune cells in vision loss.
Area of Science:
- Ophthalmology
- Immunology
- Neuroscience
Background:
- Age-related macular degeneration (AMD) is a leading cause of blindness in the elderly, with inflammation playing a key role in its pathogenesis.
- While the role of macrophages in choroidal neovascularization (CNV) is known, their specific contribution to retinal degeneration underlying CNV remains unexplored.
- Neuroinflammation involving macrophages and microglia is implicated in other neuropathological conditions.
Purpose of the Study:
- To investigate the distinct roles of recruited blood-derived macrophages versus resident microglia in the retina during laser-induced choroidal neovascularization (CNV) in a mouse model.
- To elucidate the inflammatory mechanisms contributing to vision loss and retinal degeneration associated with CNV.
Main Methods:
- Utilized laser-induced CNV in mice, employing bone marrow transplantation with GFP-labeled cells to track macrophage origins.
- Analyzed chimeric mice for GFP-labeled cell infiltration into the retina under CNV.
- Assessed upregulation of cell adhesion molecules (VCAM-1, ICAM-1, PECAM) and macrophage marker (F4/80) in CNV retinas.
- Quantified resident microglia density and proximity of infiltrating cells to Muller cells.
- Depleted circulating macrophages using clodronic acid to evaluate their impact on retinal cells.
Main Results:
- Significant infiltration of GFP-labeled, blood-derived cells (macrophages) into the retina underlying CNV, unlike in unaffected retinas.
- Strong upregulation of VCAM-1, ICAM-1, and PECAM in retinal blood vessels associated with CNV.
- Majority (70%) of F4/80+ cells under CNV were GFP-labeled, indicating blood origin; resident microglia density did not increase.
- Infiltrating macrophages were found near activated Muller cells.
- Depletion of circulating macrophages reduced F4/80+ cells and activated Muller cells in CNV retinas.
Conclusions:
- Recruitment of blood-derived macrophages, rather than resident microglia, is significantly associated with choroidal neovascularization (CNV).
- These infiltrating macrophages appear to interact with Muller cells, potentially contributing to retinal pathology in CNV.
- Findings highlight the critical role of peripheral macrophage recruitment in the inflammatory processes underlying AMD-related vision loss.
