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. 2018 Mar 21;4(3):eaap8492. doi: 10.1126/sciadv.aap8492

Fig. 4. Repopulating microglia recapitulate endogenous microglial functions of dynamic process motility and response to injury signals.

Fig. 4

Ten-week-old CX3CR1+/GFP transgenic mice were administered PLX5622 for 1 week to induce depletion of retinal microglia and then allowed to undergo full repopulation for 60 days. Age-matched CX3CR1+/GFP mice maintained on a standard diet served as controls. (A) Ex vivo live-cell time-lapse imaging was performed in retinal explants to monitor process dynamics of repopulated microglia in PLX5622-fed mice versus endogenous microglia in control mice. Repopulated microglia relative to endogenous microglia demonstrated similar process motility at baseline and increased process motility and process elaboration in response to ATP stimulation (1 mM bath application). Scale bar, 30 μm. Quantitative analysis demonstrated that process motility measures, in terms of rates of process extension and process retraction, at baseline and in response to ATP stimulation were statistically similar between endogenous microglia (white bars) and repopulated microglia (red bars) (P values from Mann-Whitney test; control group = 21 cells from 9 recordings, repopulation group = 23 cells from 11 recordings, 4 animals in each group). (B and C) Response of endogenous versus repopulated cells in an in vivo model of light-induced injury. Wild-type mice were subjected to transient depletion (7 days of PLX5622 administration) and allowed to undergo full repopulation for 30 days and then subjected to light-induced photoreceptor injury. Nondepleted age-matched wild-type mice served as controls. Histological analysis (B) showed that repopulated Iba1+ cells, such as endogenous microglia, responded to photoreceptor injury by migration into the outer retina and the subretinal space and by up-regulation of CD68 expression (insets show the presence of subretinal Iba1+ and CD68+ cells in both groups). Scale bar, 40 μm. (C) Levels of post-injury inflammatory cytokines [interleukin (IL)-1β, IL-10, C-C motif chemokine ligand 3 (CCL3), CCL5, and tumor necrosis factor–α (TNFα)] were similarly elevated in retinas containing endogenous microglia versus repopulated cells (P > 0.05 for all comparisons; Mann-Whitney test, n = 5 retina from individual animals of mixed sex per group).