Fig. 4

Fig. 4

Neuronal injury caused by radiation-induced microglial proinflammatory responses. a Representative fluorescence micrographs of intracellular ROS detection via DCFH-DA staining in BV2 microglia at 6 hpi with 5, 10, and 15 Gy. b Quantification of ROS fluorescence intensity normalized to that of the control (n = 3 fields; one-way ANOVA). c Representative fluorescence micrographs of mtROS detection via MitoSOX staining in BV2 microglia at 6 hpi with 5, 10, and 15 Gy. d Quantification of MitoSOX fluorescence intensity normalized to that of the control (n = 3 fields; one-way ANOVA). e Immunofluorescence images of iNOS in BV2 cells at 24 hpi. f Quantification of iNOS fluorescence intensity relative to that of the control (n = 3 fields; one-way ANOVA). g Quantification of the NO concentration in the supernatant of BV2 cells (n = 6; one-way ANOVA). h–j Quantitative reverse transcription PCR analysis of the levels of the proinflammatory cytokines TNF-α (h), IL-6 (i), and IFN-γ (j) in irradiated BV2 cells (normalized to β-actin via the 2^−ΔΔCt method; n = 4 biological replicates; one-way ANOVA). k NeuN immunofluorescence micrographs of HT22 hippocampal neurons after 24 h of exposure to conditioned media from irradiated BV2 microglia. l Quantification of surviving neurons (NeuN⁺ cells/mm²), normalized to neurons treated with control conditioned media (n = 3; one-way ANOVA). All the statistical data are presented as the means ± SEMs. Scale bars: 20 μm (a, c, e, and k)