Supplementary Figure 9: Characterization of apoptotic cells in the adult brain.
From: Epigenetic regulation of brain region-specific microglia clearance activity
(a) 15% of the dying cells are of neuronal origin. Left: Immunolfluorescence images to assess overlap between Nissl+ neurons / GFAP+ astrocytes / Bergmann glia / OLIG2+ oligodendrocytes / CD11b+ macrophages/microglia (green), with cCASP3+ apoptotic cells (red) in the cerebellum of PLX-treated mice. Arrows point to cCASP3+ cells. Dotted circles indicate CD11b+ cells in the meninges. DAPI: blue. Scale: 100 μm. Right: Quantification of overlap of cCASP3+ cells with Nissl+ (mean=11.58, SEM=0.9391), NeuN (mean=12.78, SEM=1.470), GFAP+ (mean=0, SEM=0), OLIG2+ (mean=0, SEM=0), CD11b+ (mean=0, SEM=0), and GFP+ (mean=0, SEM=0) cells. p<0.0001, F=78.31; 3-5 cerebellum/striatum sections from n=3 wild-type mice. PLX-treated Cx3cr1GFP/+ mice were used to test for the existence of GFP+ apoptotic macrophages/microglia. ***p ≤ 0.001. (b) Apoptotic cells in the cerebellum are distributed within all layers. Top: Representative immunofluorescence images are shown from the Purkinje cell layer and the molecular layer of cerebellum of brain sections from 4mo PLX-treated mice. Nissl+ neurons: green; DAPI: blue. Scale: 10 μm. Arrows point to cCASP3+ apoptotic cells (red). Bottom: Bar graph with individual data points shows percent localization of cCASP3+ cells. Granule cell layer: mean=0.4950, SEM=0.07649, molecular layer: mean= 0.1868, SEM= 0.04645, white matter: mean= 0.2042, SEM= 0.06745, and Purkinje cell layer: mean=0.1140, SEM=0.02625; p=0.0072, F=8.516; 3-5 cerebellum / striatum sections from n=3 PLX-treated wild-type mice. (c) Apoptotic cells numbers in the cerebellum are comparable to neurogenic regions. Quantification of cCASP3+ apoptotic cells (cm-2) in neurogenic and non-neurogenic regions. olf: mean=101.3, SEM=18.20; st: mean= 3.333, SEM=1.667, cx: mean=4.938, SEM=4.938, dg: mean=500.0, SEM=19.24; cb: mean=110.0, SEM=25.17; p<0.0001, F=155.0. Ratio of cCASP3+ apoptotic cells per 100 microglia: olf: mean=0.73, SEM=0.1311; st: mean= 0.0196, SEM=0.0196, cx: mean= 0.0403, SEM= 0.0403, dg: mean= 3.387, SEM= 0.1304; cb: mean=2.450, SEM=0.2917; p<0.0001, F=95.10. Ratio of cCASP3+ apoptotic cells per percent area covered by microglia olf: mean=0.8832, SEM=0.235; st: mean=0.0337, SEM=0.01985, cx: mean=0.02604, SEM=0.02604, dg: mean=1.988, SEM=0.2696; cb: mean=7.201, SEM=0.6095; p<0.0001, F=64.29. 12 whole brain sections from n=3 mice. (d) Cell division rates in the cerebellum are not comparable to neurogenic regions. Quantification of density of Ki67+ cells (cm-2) undergoing cell division in neurogenic and non-neurogenic regions. olf: mean=486.6, SEM=41.30; st: mean= 55.32, SEM= 8.583, cx: mean= 64.76, SEM= 8.330, dg: mean= 2050, SEM= 132.3; cb: mean=125.0, SEM=8.660; p<0.0001, F=187.6. 16 whole brain sections from n=4 mice. Bar graphs with individual data points show mean ± SEM. One-way ANOVA with Tukey’s Multiple Comparison post-hoc analysis to compare all pairs of columns. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
