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. 2022 May 12;10:903436. doi: 10.3389/fcell.2022.903436

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Copyright © 2022 Tribble, Kastanaki, Uslular, Rutigliani, Enz and Williams.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Microglial morphological changes associated with pro-inflammatory phenotypes occur in all retinal microglia niches in retinal explants. Retinal explants were maintained for 1 day (D1) or 2 days (D2) ex vivo and compared to D0 controls (eyes fixed immediately after enucleation). IBA1 labeled microglia were imaged and individually reconstructed using Imaris software from the three retinal microglia niches (A) nerve fiber layer/ganglion cell layer (NFL/GCL) (B) inner plexiform layer/inner nuclear layer (IPL/INL), and (C) outer plexiform layer (OPL). (D) Across all microglial niches, at D1 there was a significant reduction in the number of microglia processes, total process length, field area, and normalized volume, demonstrating that microglia became less complex and more voluminous, as occurs with a shift towards pro-inflammatory phenotypes. The degree of change was no greater in any one layer, suggesting a global retinal inflammatory environment. There was no significant difference in microglial density within niches demonstrating that there is no active proliferation, migration, or apoptosis of microglia by D1. By D2, microglia in the NFL/GCL had significantly increased number of microglia processes, total process length, field area, and normalized volume, demonstrating an outgrowth of processes reminiscent of microglia in cell culture. This was similar in the IPL/INL, but not in the OPL. Iba1 = microglia specific marker, n = 4 retinas for all conditions, scale bars = 50 μm, * = p < 0.05, ** = p < 0.01, *** = p < 0.001, NS = non-significant.