Chloral hydrate

Chloral hydrate was obtained from Abbott Laboratories (North Chicago, IL, USA). FITC-dextran (3–5 kDa), dimethyl sulfoxide (DMSO), Triton X-100 and paraformaldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). Alizarin carmine was obtained from Chroma (Stuttgart, Germany). Texas Red-X phalloidin and the rabbit polyclonal antibody against ZO-1 were purchased from Zymed-Invitrogen (Carlsbad, CA, USA). Mouse monoclonal antibodies against Na,K- ATPase were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and the rabbit monoclonal antibody against Ki67 was purchased from Abcam (Cambridge, MA, USA). Alexa Fluor 488-conjugated donkey anti-rabbit IgG and Alexa Fluor 594-conjugated donkey anti-mouse IgG were purchased from Invitrogen-Gibco (Carlsbad, CA, USA). Hoechst 33342 and BSA were obtained from Vector Laboratories (Burlingame, CA, USA).

EA was applied to the acupuncture points Baihui (GV20) and Dazhui (GV14) with a pair of bipolar stimulation electrodes after placing the rats under intraperitoneal injection of 10% chloral hydrate (Abbott, Illinois, USA). Stainless acupuncture needles of 0.3 mm in diameter (HuaTuo, Suzhou Medical Appliance Factory) were applied to both Baihui (GV20) and Dazhui (GV14) acupoints in each rat (10 mm EA penetration depth, sparse-dense wave with a frequency of 2/15 Hz and a current intensity of 1~3 mA) using an electrical needle stimulator (WQ1002K, Electro-Acupuncture Equipment Company, China). For the EA pretreatment group, the animals underwent acupuncture once per day for a duration of 30 min for 5 consecutive days and then received MCAO. For the EA treatment group, after 2 h of MCAO, rats received EA stimulation for 30 min. Moreover, the stimulation parameters were the same as EA pretreatment.