Suture

The tMCAO model was established to mimic unilateral focal cerebral ischemic stroke as described previously [40 (link)]. After being anesthetized, a midline neck incision was made under a dissecting microscope, and then the right common carotid artery and external carotid artery (ECA) were isolated. A suture (Doccol Corporation, MA, USA) was introduced into a wedge-shaped incision on the ECA and further inserted to obstruct the middle cerebral artery. After one hour of occlusion, the filament was withdrawn for reperfusion. Cerebral blood flow (CBF) was evaluated by a Doppler laser. Mice would be excluded from the study if their relative CBF failed to reduce to 20% of baseline or showed no neurologic deficits after anesthesia recovery. Sham-treated mice were subjected to the same procedure without tMCAO. During the surgery, the body temperature of mice was maintained at 37 ± 0.5 °C with a heating pad.

Mice were randomly allotted to five groups: sham, MCAO+solvent, MCAO+300 mg/kg γ-GC, MCAO+600 mg/kg γ-GC, and MCAO+900 mg/kg γ-GC. Except for mice in the sham group, all mice were subjected to MCAO to establish a transient focal cerebral ischemia model. In brief, mice were deeply anesthetized with an intraperitoneal injection of pentobarbital sodium. Next, after dissecting the right common carotid artery, external carotid artery, and internal carotid artery, a suture (Doccol Corporation, MA, USA) was inserted via the external carotid artery into the internal carotid artery until a steep drop in regional cerebral flow was observed. After 1 h, the suture was removed to establish reperfusion. Fifteen minutes from the onset of reperfusion, the mice were intragastrically administered γ-GC or saline. During the surgery, body temperature was maintained at 37 ± 0.5°C with a heating pad. Sham-operated mice were subjected to all procedures except for insertion of the suture.
To ensure the success of the MCAO model, LSCI was used to visualize cerebral blood flow. The surface of the mouse skull was exposed to a laser (Perimed Corporation, Stockholm, Sweden), and regions of interest were then selected to assess the speckle contrast over time. Images were captured to further analyze the changes in cerebral blood flow.

Two-month-old male C57/BL6J mice weighing about 25 g were purchased from the Animal Model Center of Nanjing University (Nanjing, Jiangsu, China). All procedures were approved by the Animal care and Use Committee at Nanjing University. In Middle Cerebral Artery Occlusion (MCAO) model, mice were anesthetized with pentobarbital sodium (45 mg/kg i.p.). A 6/0 sutures (Doccol Corporation, MA, United States) was inserted through internal carotid artery into the beginning of the middle cerebral artery (MCA) until the ipsilateral blood flow of MCA supply territory decreased to below 30% of baseline monitored by a laser Doppler flowmetry (Perimed Corporation, Stockholm, Sweden). After 60 min, the filament was withdrawn to allow blood reperfusion. Sham-operated groups underwent the same procedures expect for the insertion of the filament into the MCA. The mice were subjected to the dose of 5 mg/kg T16ainh-A01 and Caccinh-A01 or the same amount of saline by caudal vein injection within 15 min after the onset of reperfusion.