The Laboratory Animal Care and Use Committee of Shenzhen University approved all animal related experimental protocols. Male Sprague–Dawley rats (purchased from the Experimental Animal Center Southern Medical University, Guangzhou, Guangdong, China) weighing 300 g to 400 g were anesthetized with isoflurane (4% for induction, 1.75% for maintenance) in N2O:O2 (70%:30%) during surgical procedures and the body temperature was maintained through a heated pad. A 4–0 silicone-coated monofilament nylon suture was introduced into the right intra-carotid artery to occlude the opening of the MCA as we previously described (Liu et al., 2009). MCAO was lasted for 3 h, and the animals were then deeply anesthetized with isoflurane and euthanized by decapitation. Successful MCAO was confirmed by 2,3,5-triphenyltetrazolium chloride (TTC, Sigma-Aldrich, St. Louis, MI, USA) staining of the 2-mm-thick brain coronal section 6 mm away from the tip of the front lobe as we previously described (Liu et al., 2008).