Fig 2: Double immunofluorescence staining of KDM6B with calcium-binding adaptor molecule 1 (Iba-1), glial fibrillary acidic protein (GFAP), and neuronal nuclei (NeuN) at 24 h after GMH. Top panel indicates the location of staining (small black box). Scale bar = 50 µm. n = 2 for each group

Fig 3: GPR40 CRISPR, PAK4 CRISPR, or KDM6B CRISPR downregulated the expression of GPR40, p-PAK4, or KDM6B after GMH. The representative bands and densitometric quantification of GPR40, p-PAK4, and KDM6B revealed the efficacy of CRISPR knockout in naive and GMH rats, n = 5 per group. Vehicle: 10% dimethyl sulfide. Data was represented as mean ± SD. *p < 0.05 vs. naive group + scr CRISPR group; &p < 0.05 vs. GMH + scr CRISPR group; one-way ANOVA, Tukey’s post hoc test; scr CRISPR, scrambled CRISPR; KO CRISPR, knockout CRISPR

Fig 4: GPR40 K.O CRISPR abolished the beneficial effects of GW9508 at 24 h after GMH. a, b The representative bands and quantification of GPR40, PAK4, p-PAK4, CREB, p-CREB, KDM6B, IL-1ß, TNF-a, CD206, and IL-10. Data was represented as mean ± SD (n = 6 per group). One-way ANOVA was used followed by Tukey’s HSD post hoc test and Holm-Bonferroni correction. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. Sham group; @p < 0.05, @@p < 0.01 vs. GMH + vehicle group; &p < 0.05 and &&p < 0.01 vs. GMH + GW9508, #p < 0.05 vs. GMH + GW9508 + scramble CRISPR group. Vehicle, 10% dimethyl sulfide

Fig 5: Temporal expression of GPR40, p-PAK4, PAK4, KDM6B, CD16, and CD206 in the brain after GMH. a The representative bands. b–d GPR40, p-PAK4, and KDM6B expression levels decreased significantly from 12 h to 24 h, reaching the lowest point at 24 h after GMH. e CD16 was increased and peaked rapidly at 3 h after GMH, but gradually tended to decrease afterwards. f The expressions of CD206 were upregulated as early as 3 h and gradually increased till 7 days after GMH. **p < 0.01 vs sham. Data are represented as mean ± SD, n = 6 for each group