Transient middle cerebral artery occlusion (tMCAO)

WT CD-1 mice (male, 8–10 weeks old) were subjected to transient MCAO for 60 min and reperfusion for 48 h according to a previous report with some modification⁸⁵. Briefly, mice were anesthetized with 1.5% isoflurane in oxygen. Mice breathed 100% oxygen with spontaneous breathing from the induction of anesthesia to 15 min after MCA reperfusion. Rectal temperature was monitored and kept at 37 ± 0.5 °C using a heating pad under mice during surgery until mice recover from anesthesia. Changes in cerebral blood flow (CBF) were monitored during surgery to confirm the occlusion of MCA using a laser Doppler flowmetry (Moor Instruments, Inc.) probe positioned at 2 mm posterior, 5 mm lateral from the Bregma. Common carotid artery (CCA) was occluded with a microclip and a 7-0 monofilament was inserted via the isolated external carotid artery (ECA) through an internal carotid artery to occlude MCA. MCA was reperfused by removing a monofilament at 60 min after starting MCAO. CCA was reperfused after ECA closure by removing a microclip at 15 min after MCA reperfusion. Saline or SS20 at 250 µmol/kg was administered IV at 15 min before MCA reperfusion. Taking circadian rhythm into account, all mice were subjected to surgery in the daytime (9:00 to 17:00). Mice received 1 mL of 5% dextrose-enriched lactated Ringer’s solution (ip) daily to avoid dehydration. After evaluating neurological functional score⁸⁴, brains were harvested to measure cerebral infarct volume using TTC staining at 48 h after MCAO.