6 0 suture

Animal studies were reported in accordance with Animal Research: Reporting in Vivo Experiments: ARRIVE guidelines. The procedure for using laboratory animals was approved by the Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University, Shanghai, China. The surgery of MCAO was performed as described previously^{59 (link)}. Adult mice (10–12 weeks, n = 30 per group, male: female = 1:1) were anesthetized with 1.5–2% isoflurane and 30%/70% oxygen/nitrous oxide. Body temperature was maintained at 37 °C using a heating pad. In brief, the common carotid artery, internal carotid artery and external carotid artery were separated. A 6-0 suture (Covidien, Mansfield, MA) coated with silicon (Heraeus Kulzer, Germany) was inserted from the external carotid artery, followed by the internal carotid artery, and gently stopped at the opening of middle cerebral artery. The success of occlusion was determined by monitoring the decrease in surface cerebral blood flow (CBF) to 10% of baseline CBF using a laser Doppler flowmetry (Moor Instruments, Devon, UK). Reperfusion was performed by withdrawing the suture 90 min after MCAO.

Two weeks after virus injection, mice underwent a 60-minute tMCAO surgery. The protocol of tMCAO surgery was described in the previous study [18 (link)]. Briefly, the middle cerebral artery (MCA) was occluded by a 6–0 suture (Covidien, Mansfield, MA) with a silicone-coated round tip. During the surgery, mice body temperature was maintained at 37 ± 0.5°C using a heating pad (RWD Life Science) with a feedback control. The blood flow in MCA was measured by Laser Doppler monitor (Moor instruments, Devon, U.K.) before MCAO, at the beginning of occlusion and 1 minute after reperfusion. Animals whose MCA blood flow did not decrease to 10%~20% of baseline during occlusion or did not recover to 40%~60% of baseline after reperfusion were excluded from this study.

The surgical procedure for transient middle cerebral artery occlusion (tMCAO) has been previously described [15 (link)]. Briefly, animals were anesthetized with 1.5% isoflurane in a 30% O²/70% NO mixture under spontaneous breathing conditions on a heating pad (RWD Life Science, Shenzhen, China). A 6-0 suture (Covidien, Mansfield, MA, USA) coated with silicon was inserted through the external carotid artery (ECA) with an advancement of 0.9–0.95 mm from the internal carotid artery (ICA) until reaching the intersection of the middle cerebral artery (MCA). After 90 min of ischemia, the suture was withdrawn to allow reperfusion. A laser Doppler Monitor (Moor Lab, Moor Instruments, Devon, UK) was used to monitor the blood flow in the MCA territory before surgery, immediately after occlusion, and upon reperfusion. Successful occlusion of the MCA was defined as a decline in the regional blood flow of the ipsilateral hemisphere to 20% of the baseline. A recovery of cerebral blood flow to more than 70% of the baseline was regarded as a successful reperfusion. The mortality rate of mice that underwent tMCAO was 7.6%. Animals were randomly assigned into different groups. Both the surgeon and the behavioral analyst were blinded to treatment assignments.