Fig 2: Astrocyte-specific CA1 MFN2 and OPA1 expression after global cerebral ischemia in mice. (A) Representative images of fluorescent IHC labeling of hippocampal GFAP (red) and MFN2 or OPA1 expression (green) 7 days after global cerebral ischemia with and without IV anti-miR-200c post-treatment. Quantification of co-labeled GFAP/mitofusin-2 (MFN2, B) and GFAP/OPA1 fluorescence (C) in CA1. N = 5–8 animals per treatment group, mean + SEM, *p < 0.05. Scale bar, 25 μm.

Fig 3: Astrocyte-specific CA1 SIRT1 expression after global cerebral ischemia in mice. Low power (200×, A) and high power (600×, B) representative images of fluorescent IHC labeling of hippocampal GFAP (red) and SIRT1 expression (green) 7 days after global cerebral ischemia with and without IV anti-miR-200c post-treatment. Quantification of co-labeled GFAP/SIRT1 (C) in CA1. N = 5–8 animals per treatment group, mean + SEM, *p < 0.05. Scale bar, 25 μm.

Fig 4: Inflammation levels in brain tissues after clusterzyme treatment.a–c Western blotting for IL-1β, IL-6, and TNFα in the brain tissues 1, 3, and 7 days post TBI after treatment (n = 3 per group), respectively. d Western blotting quantitative analysis of inflammatory factors at different time points (n = 3 per group). All the samples were derived from the same experiment and blots were processed in parallel. Data are presented as mean ± SEM and compared with the Sham and TBI groups, analyzed by one-way ANOVA with two-sided LSD test (adjusted p values are shown). It can be seen that Au24Cd1 can rapidly and significantly reduce the upregulated inflammatory cytokines of IL-1β and IL-6 after brain injury, while Au24Cu1 has a better ability to reduce the expression of TNFα. e–g ELISA quantitative analysis of IL-1β, IL-6, and TNFα levels in brain tissues on days 1, 3, and 7 with or without clusterzymes treatment (n = 5 per group), respectively. Data are presented as mean ± SEM and compared with the Sham and TBI groups, analyzed by one-way ANOVA with two-sided LSD test (adjusted p values are shown). h Immunofluorescence co-staining of IL-1β and microglia (Iba-1), astrocytes (GFAP), or neurons (NeuN) in injured cortex 3 days post injury with or without clusterzyme treatment. Quantitative analysis of i the number of IL-1β+ expression in different positive cells and j the pixels density of Iba-1/NeuN/GFAP cells in the injured cortex with or without clusterzyme treatment (n = 3 per group). Data are presented as mean ± SEM and compared with the Sham and TBI groups, analyzed by one-way ANOVA with two-sided LSD test (adjusted p values are shown). Experiments were repeated independently a–c twice and h three times with similar results.

Fig 5: Cell-type and regional-specific Sirt1 expression after MCAO in aged male and female mice. (A) Levels of Sirt1 expression co-localized with neurons (NeuN+) and astrocytes (glial acidic fibrillary protein, GFAP+) in ipsilateral (MCAO ipsi) and contralateral (MCAO contra) brain 1d after MCAO in aged males (left) and females (right). (B) Representative IHC images of hippocampal cornu ammonis 1 stained for Sirt1 (green), NeuN (blue) and GFAP (red) in aged males (left panels) and females (right panels) 1d after sham surgery (top four panels) or MCAO (bottom four panels). N=3-4 animals per treatment group, each data point represents averaged results from 5 separate sample images per animal, mean+SEM, *p<0.05 versus sham, 1-way ANOVA. Scale bar = 25 µm.