Figure 9.

HO₂-EVs decrease cell survival and increase cell death in the hippocampus. (A) Merged representative immunofluorescence staining for Ki67 (pink, white arrow) and DAPI nuclear staining (blue) in the hippocampus of control EV–injected, LO₂-EV–injected, and HO₂-EV–injected mice. (B) Quantification of the cell proliferative index (Ki67⁺ nuclei/total nuclei × 100) showed a highly significant decrease in the HO₂-EV–injected group compared with the control group and LO₂ group, while the LO₂-EV–injected group also had a small but significant reduction compared with the control EV–injected group. (C) TUNEL assay (teal, white arrow) and DAPI nuclear stain (blue) were used to identify dead cell nuclei. (D) Quantification of cell death index (TUNEL⁺ nuclei/total nuclei × 100) revealed that it was increased in the hippocampus of HO₂-EV–injected mice compared with LO₂-EV–injected mice and control EV–injected mice. n = 5 per group. (E) Double immunofluorescent staining with GSDMD (green) and MLKL (red) showed they were colocalized in some cluster cells near the subgranular zone in HO₂-EV–injected mice. (F) Zoomed magnification in the white box. (G–L) qRT-PCR detected increased expression of casp3 (G) and casp8 (H) (apoptosis related) and Tnf (I), Ripk1 (J), Ripk3 (K), and Mlkl (L) (necroptosis related) in the hippocampus of HO₂-EV–injected mice compared with control EV–injected and LO₂-EV–injected mice. n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. MLKL = mixed-lineage kinase domain-like. Objective magnification, 20×. Scale bars, 50 μm.