Experiment 1. Role of endogenous AHR in oxidative stress and neuronal apoptosis after ICH in mice

In vivo AHR CRISPR Knock out (KO.) was used to explore the role of endogenous AHR in oxidative stress and neuronal apoptosis 48 h after ICH, including neurobehavior, pathological histology changes, and protein biomarkers of oxidative stress and neuronal apoptosis. Mice were randomly divided into four groups (n = 6–10/group): Sham, ICH, ICH+AHR CRISPR (KO.), and ICH+Control CRISPR. Neurobehavior tests (Modified Garcia test and Forelimb Placement test) were performed in each mouse (n = 10/group).

Immunoblots were performed in the ipsilateral hemisphere to assess the protein levels of AHR, oxidative stress markers (4-HNE and HO-1), and intrinsic apoptosis markers (ratio of Bcl-2/Bax). Immunohistochemistry staining of Cleaved Caspase-3 and Terminal deoxynucleotidyl transferase dUTP (TUNEL POD) was performed to evaluate neuronal viability and proapoptotic activity (n = 4/group). In addition, to confirm the knockout efficacy of in vivo AHR CRISPR KO., two more groups (n = 6/group) were added for Western blot: Naive (Control CRISPR) and Naive (AHR CRISPR KO.).