Metronidazole

To inhibit the growth of commensal bacteria, rats were treated with a cocktail of antibiotics (ABX) containing ampicillin (1 g/L), vancomycin (500 mg/L), neomycin (1 g/L), and metronidazole (1 g/L) (Meilun Biotechnology, Dalian, China) in drinking water for 4 weeks. Bottles with antibiotic‐containing water were inverted every day, and the antibiotic solution changed every 1–2 days, together with the cage bedding. The ABX selection is based on previous reports showing that they effectively deplete gut bacteria and may be used to model a germ‐free condition (23 (link), 24 (link)). Four weeks after treatment, HT was induced by 50% glucose (6 ml/Kg, IP) 15 min MCAO. ABX was continued until the rats were euthanized, 5 days after surgery.

To eradicate commensal bacteria, filter-sterilized drinking water was supplemented with ampicillin (0.5 mg/ml, Meilunbio), gentamicin (0.5 mg/ml, Meilunbio), metronidazole (0.5 mg/ml, Meilunbio), neomycin (0.5 mg/ml, Meilunbio), and vancomycin (0.25 mg/ml, Meilunbio) for 1 week. Antibiotics were purchased from Dalian Meilun Biotechnology Corporation Ltd. (Dalian, China). Before fecal microbiota transplantation, the native gut microbiota in one group of C57 female mice (n = 10 biologically independent animals per group) was deleted by administering drinking water containing a cocktail of antibiotics for 1 week. Fecal samples of ∼200 mg were then collected from arbutin (0.4 mg/ml)-fed mice and resuspended in 2.0 ml normal saline. Fecal samples were mixed and centrifuged at 1,000 × g, and the microbiota supernatants were transplanted into the microbiota-depleted mice by gavaging with 0.2 ml per mice for 1 week. After transplantation, two groups of mice were administrated with a standard diet and regular water.