7 0 monofilament suture

Male mice (8–12 weeks, 23–28 g) were anesthetized with isoflurane (3% for induction, 1.5% for maintenance) in mixed O₂ and N₂O. Focal cerebral ischemia was induced by introducing a 7–0 monofilament suture (Doccol Corporation) in the left common carotid artery, advanced to the origin of the middle cerebral artery for 1 h as described previously (19 (link), 49 (link)). Body temperature was measured with a rectal thermometer and maintained at 37.0 ± 0.5 °C by a temperature-controlled heating pad during the ischemic period. Only those mice with cerebral blood flow (CBF) reduced to less than 25% of baseline were considered to be successfully MCA occluded (MCAo). Reperfusion was performed by removing the suture after 1 h-occlusion. Animals that did not show reperfusion of CBF over 60% of baseline were excluded. Regional cerebral blood flow was measured using a laser speckle imager (Perimed PeriCam PSI HR) at 15 min before MCAo surgery, 15 min after occlusion, and 15 min after the onset of reperfusion. Two identical elliptical regions of interest were selected. The relative CBF was calculated as the ratio of the ipsilateral and contralateral hemispheres, which was then normalized to the mean value of pre-surgery CBF baseline level for each animal. Animals were allowed 24 h or 48 h reperfusion before being sacrificed according to the design of the experiment.

To investigate PTEN expression after MCAO, three month old male C57BL/6J (Jackson Lab) mice were used. MCAO was conducted following a similar protocol as described previously (Li et al., 2013 (link)). Briefly, mice were anesthetized by inhalation of isoflurane. The left MCA was occluded by a 7-0 monofilament suture (Doccol Corporation) introduced via internal carotid artery. After 90 minutes occlusion, the suture was withdrawn for reperfusion. Ischemia preconditioning was conducted by occluding MCA for 10 minutes following previous study (McLaughlin et al., 2003 (link)). Twenty four hours after preconditioning, mice were sacrificed; cortex and sub-cortex were dissected separately for Western blot analysis.