PCNA Staining of Retinal Cryosections to Assess Microglial/Macrophage Proliferation.

Detection of the protein PCNA (proliferating cell nuclear antigen) is used to identify cells in the S phase of the cell cycle to indicate cellular proliferation. Here we describe our method to detect PCNA expression by microglia and macrophages in retinal cryosections. We have used this procedure with zebrafish tissue, but this procedure could be applied to cryosections from any organism. Retinal cryosections are subjected to a heat-mediated antigen retrieval step in Citrate Buffer, then immunostained with antibodies to label PCNA and microglia/macrophages, and counterstained for cell nuclei. After fluorescent microscopy, the number of total and PCNA+ microglia/macrophages can be quantified and normalized to compare across samples and groups.

Microglia in the developing retina couple phagocytosis with the progression of apoptosis via P2RY12 signaling.

BACKGROUND: Microglia colonize the developing vertebrate central nervous system coincident with the detection of developmental apoptosis. Our understanding of apoptosis in intact tissue in relation to microglial clearance of dying cells is largely based on fixed samples, which is limiting given that microglia are highly motile and mobile phagocytes. Here, we used a system of microglial depletion and in vivo real-time imaging in zebrafish to directly address microglial phagocytosis of apoptotic cells during normal retinal development, the relative timing of phagocytosis in relation to apoptotic progression, and the contribution of P2RY12 signaling to this process. RESULTS: The depletion of microglia resulted in accumulation of numerous apoptotic cells in the retina. Real-time imaging revealed precise timing of microglial engulfment with the progression of apoptosis, and dynamic movement and displacement of engulfed apoptotic cells. Inhibition of P2RY12 signaling delayed microglial clearance of apoptotic cells. CONCLUSIONS: Microglial engulfment of dying cells is coincident with apoptotic progression and requires P2RY12 signaling, indicating that microglial P2RY12 signaling is shared between development and injury response. Our work provides important in vivo insight into the dynamics of apoptotic cell clearance in the developing vertebrate retina and provides a basis to understand microglial phagocytic behavior in health and disease.

Dynamic changes in microglial and macrophage characteristics during degeneration and regeneration of the zebrafish retina.

BACKGROUND: In contrast to mammals, zebrafish have the capacity to regenerate retinal neurons following a variety of injuries. Two types of glial cells, Müller glia (MG) and microglia, are known to exist in the zebrafish retina. Recent work has shown that MG give rise to regenerated retinal neurons, but the role of resident microglia, and the innate immune system more generally, during retinal regeneration is not well defined. Specifically, characteristics of the immune system and microglia following substantial neuron death and a successful regenerative response have not been documented. METHODS: The neurotoxin ouabain was used to induce a substantial retinal lesion of the inner retina in zebrafish. This lesion results in a regenerative response that largely restores retinal architecture, neuronal morphologies, and connectivities, as well as recovery of visual function. We analyzed cryosections from damaged eyes following immunofluorescence and H&E staining to characterize the initial immune response to the lesion. Whole retinas were analyzed by confocal microscopy to characterize microglia morphology and distribution. Statistical analysis was performed using a two-tailed Student's t test comparing damaged to control samples. RESULTS: We find evidence of early leukocyte infiltration to the retina in response to ouabain injection followed by a period of immune cell proliferation that likely includes both resident microglia and substantial numbers of proliferating, extra-retinally derived macrophages, leading to rapid accumulation upon retinal damage. Following immune cell proliferation, Müller glia re-enter the cell cycle. In retinas that have regenerated the layers lost to the initial injury (histologically regenerated), microglia retain morphological features of activation, suggesting ongoing functions that are likely essential to restoration of retinal function. CONCLUSIONS: Collectively, these results indicate that microglia and the immune system are dynamic during a successful regenerative response in the retina. This study provides an important framework to probe inflammation in the initiation of, and functional roles of microglia during retinal regeneration.