Supplementary Figure 3: Spatiotemporal analysis of microglial expansion and redistribution in the injured facial nucleus.
From: A new fate mapping system reveals context-dependent random or clonal expansion of microglia
(a) Coronal brain section of a Cx3cr1GFP/+ mouse 7d post facial nerve lesion depicts areas analyzed for kinetics of microglial proliferation in the facial nucleus degeneration model. Microglial cells were marked by GFP (green). Neurons were immunolabeled with NeuN (blue) to locate the facial nucleus in the pons. The facial nucleus, a 200 μm broad perinuclear zone, and the parvicellular reticular nucleus, alpha part (PCRtA) were isolated in equal dimensions on lesion and contralateral sides for quantification of GFP+ microglia using the Imaris software.
(b) Quantification of GFP+ microglial cells in contralateral (black) and injured (red) facial nuclei from onset (1 to 2d) to peak (7 and 14d) to resolution of injury (60d) in 8-14 weeks old female Cx3cr1GFP/+ mice.
(c) Representative confocal images of microglia (GFP, green) with DAPI nuclear counterstain (blue) in matched facial nuclei during the time course of injury.
(d) Kinetics of microglial proliferation indicated by Ki67 immunohistochemistry.
(e) Representative confocal images for d. Microglia took on less ramified and more amoeboid morphology at 2 d after the lesion. GFP+ microglia (green), Ki67 (magenta) and DAPI (blue).
(f) Kinetics of microglial activation indicated by MHC class II immunohistochemistry.
(g) Representative confocal images for f. Microglia were highly activated and assumed a rod-like or amoeboid morphology 7d following nerve transection. GFP+ microglia (green), MHC class II (magenta) and DAPI (blue).
Data are represented as mean ± SEM in b, d and f. n = 4 (1 d), 5 (2 d), 6 (7 d), 5 (14 d) and 7 (60 d) mice pooled from five experiments, 3-6 sections each. Two-way ANOVA, * P = 0.0358 in b, ** P = 0.0036 in b, 0.00110 in f, *** P < 0.001 in b, d and f. Scale bars, 50 μm in c, 20 μm in e and g.
