Fig. 2.

Morphological correlates of microglial activation as revealed by Iba1 immunohistochemistry. (A) CA3 region of control (CTL) and slice cultures exposed to LPS (10 µg/mL), IFN-γ (100 ng/mL), or NMDA+KA (5 µM) for 48 h to induce excitotoxic death. (B) Design-based stereology in cryosections (25 µm) of individual slice cultures. The dentate gyrus is in yellow, and CA3-CA1 is in blue. (Inset) Interpolated 3D reconstruction of sampling contours with randomly sampled microglial cells (dots). Estimates refer to the total hippocampus. (C) Stereological estimation of Iba1-positive cells per slice culture with the optical fractionator probe. *P < 0.05 vs. control, ANOVA on ranks. n/N as in D. (D) Stereological estimation of total process length per cell with the spaceballs probe. *P < 0.05 vs. control, one-way ANOVA. For n/N cultures/preparations: CTL, 10/5; LPS, 13/5; IFN-γ, 9/4; NMDA+KA, 10/4. (E) Parameterization of 2D somatic projections based on maximum length (L) and projection area (A). The somatic shape index (L/A) increases in rod-shaped somata. Images, spectrum of microglial shapes. (F–H) Microglial somatic L, A, and L/A. *P < 0.05, rank-sum test. CTL, 114 cells/9/7; LPS, 102 cells/10/5. (I) Three-dimensional Sholl grid of concentric spheres describing microglial branch length and number as a function of distance from the geometric center of the cell somata (red cross). Branch intersections with the spherical grid quantify the number, whereas the branch length between consecutive spheres estimates the degree of convolution. (J and K) Sholl analysis of microglial processes. IS, intersections. CTL, 116 cells/9/7; LPS, 102 cells/10/5. (L) Fluorescent images of ramified and ameboid microglia. (M) Stereological quantification of ameboid cells with enlarged, round somata (cell bodies) showing no more than a single process and a few filopodia, with the optical fractionator probe. P > 0.05, rank-sum test. CTL, 11/11; LPS, 7/7.