Radiation-induced mitochondrial DNA leakage activates microglial cGAS-STING neuroinflammation. a Representative TEM images of mitochondria in control and 15 Gy-irradiated BV2 cells. Higher-magnification images revealed crista fragmentation (black arrowheads) and matrix vacuolization in irradiated mitochondria, in contrast to the intact crista architecture in the control (red arrowheads). b Live-cell mitochondrial DNA visualization (MitoTracker: PK Mito Deep Red, MtDNA: SYBR; yellow arrows: cristae-localized mtDNA; white arrows: cytosolic mtDNA). c Quantification of mtDNA leakage frequency (n = 6 cells/group; Student’s t test). d–g Mitochondrial morphology analysis: Branch length (d), number of branches (e), mean area (f), and mean perimeter (g) (n = 6 mitochondria; Student’s t test). h, i Representative confocal images and quantification of relative fluorescence intensity of BV2 cells co-stained with MitoSOX Red (mtROS, red) and MitoTracker Green (mitochondria, green) at 6 h post-irradiation. BV2 cells were pretreated with NAC (10 mM) or MitoTEMPO (5 μM) for 1 h. j Cytosolic mRNA levels of the mtDNA-encoded genes ND1 and Cox1 (normalized to 18S rRNA). k p-STING immunofluorescence images of the control, irradiated (15 Gy), and irradiated + H151 (STING inhibitor) groups (nuclei: DAPI; STING: Alexa Fluor 488). l Western blot analysis of cGAS-STING pathway proteins (cGAS, p-STING, p-NF-κB, p-IRF3), inflammatory and neuronal markers (iNOS, NeuN), and GAPDH as a loading control. m Schematic diagram illustrating the mechanism by which radiation-induced mtDNA leakage triggers cGAS-STING-mediated neuroinflammatory cascades. Image was created in BioRender. Shang, Y. (2025) https://BioRender.com/mo dthph (agreement number: LW28JKG73J). All the statistical data are presented as the means ± SEMs. Scale bars: 2 μm (a), 5 μm (b, Whole mitochondria), 1 μm (b, insets), and 20 μm (h, k)
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