Transient middle cerebral artery occlusion (tMCAO) procedure

Mice at 10–12 weeks underwent a procedure to induce focal cerebral ischemia using silicone rubber-coated 7–0 nylon monofilaments (701956PK5Re; Doccol, Sharon, MA, USA) after TAM induction. Briefly, the mice were anesthetized with chloral hydrate (30 mg/kg), and analgesic gel (lidocaine) was applied evenly on the incision to relieve pain during the experiment. Then, the left common carotid artery, internal carotid artery, and external carotid artery were gently separated from the surrounding tissues. Occlusion was accomplished by slowly inserting monofilaments to block blood flow of the middle cerebral artery. After 60 min of occlusion, the monofilament was withdrawn to allow reperfusion. Throughout the surgery, body temperature was maintained at 37.0 ± 0.5 °C using an electric blanket. For disruption of the expression of ULK1 in the mouse brain, lentiviruses expressing the negative control or ULK1 RNAi were injected into the cortex using a stereotaxic instrument at day 7 before surgery, as our previous study reported [15]. The sham-operated mice were subjected to the same surgical procedures but without the artery occlusion procedures. Regional cerebral blood flow (rCBF) was monitored using laser speckle flowmetry (PeriCam PSI, Stockholm, Sweden) at different time points, including baseline, 30 min after ischemia, and 2 min after reperfusion. The value was expressed as a percentage of the value at baseline. All procedures were performed by the same operator (B.H.) throughout the study to minimize the systemic variation. A series of behavioral tests, including modified neurological severity score (mNSS), corner-turning test, foot-fault test, and rotarod test, were blindly performed to assess neurological deficits, as previously reported [16, 17]. The infarct volume was defined by 2,3,5-tripenyltetrazolium chloride (TTC) staining (Sigma) and quantified by using Image-Pro-Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Except for the data analyst, the researchers for neurobehavioral assessment were kept blind to the different genotypes of mice, outcome measurements, and trial results.