Fig. 6

Riluzole treatment reduces cranial radiation-induced glial activation. Radiation-induced activated microglia in the hippocampal granule cell layer (GCL) and dentate hilus (DH) were assessed using dual immunofluorescence staining, laser scanning confocal microscopy and 3D algorithm-based volumetric quantification for IBA1 (green) and CD68 (magenta, and DAPI, blue) dual-labeling. A–C Exposure to cranial irradiation (RT + Vehicle) significantly elevated the immunoreactive volume of activated microglia (CD68⁺-IBA1⁺ co-localization) in the hippocampus compared with either Control + Vehicle or Control + RZ groups. Irradiated mice receiving RZ (RT + RZ group) showed a significantly reduced volume of CD68⁺-IBA1⁺ co-labeling compared with the RT + Vehicle group. CD68 alone (B) and CD68-IBA1 merged C fluorescence channels are shown for each group. a1 Higher magnification surface reconstruction for the IBA1⁺ (green) positive microglia expressing CD68 puncta (magenta) is shown for the selected cell (white arrow, B, C) from the RT + Vehicle group. D–E Surface reconstruction of GFAP⁺ (green, DAPI, blue) hippocampal astrocytes shows significantly elevated GFAP immunoreactivity volume in the RT + Vehicle group compared to either Control + Vehicle or Control + RZ group. Astrocytes showed hypertrophy with thicker and longer stelae in the irradiated hippocampus (RT + Vehicle). RZ administration to the irradiated mice (RT + RZ) showed a significant reduction in astrogliosis compared to RT + Vehicle group. Volumetric data for CD68⁺-IBA1⁺ co-expression and GFAP immunoreactivity are presented as mean ± SEM (N = 6–8 mice per group). P values were derived from two-way ANOVA and Bonferroni's multiple comparisons test. Scale bars, 30 μm, (B), 5 μm, (a1), and 50 μm, (E)