Morphological analysis was done at ×10 magnification on a Nikon Eclipse Ni-E microscope. Cell density was obtained by counting the number of IBA1+ cell bodies in the dentate gyrus and cornu ammonis and normalizing to total area, while coverage was measured by dividing the thresholded IBA1 signal by the total area. Microglia in the hilus of the dentate gyrus and the stratum lacunosum-moleculare of the cornu ammonis were classified into five morphological phenotypes as previously described [14 (link), 16 (link), 43 (link)].
After checking for homogeneity of variance and normality, coverage and density data were analyzed using a two-tailed two-way ANOVA, while the morphological subtypes were analyzed with a general linear multivariate model, all using SPSS 25 (IBM software) and graphed using ggplot2 (v3.3.3.9000) [44 ] in R. Data were considered statistically significant when p < 0.05.