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, Muller 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, Muller 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.