2.2. Middle cerebral artery occlusion and bilateral ovariectomy

We induced focal transient cerebral ischemia by a 60-min right middle cerebral artery occlusion (MCAo) under isoflurane anesthesia followed by reperfusion for either 3, 15, or 30 days. We selected the three-day outcome because the infarct matures completely and shows minimal variation in its volume by that time. The 15- and 30-day time points represent sub-acute and chronic recovery as discussed previously (Verma et al., 2014, 2016). Briefly, we performed a midline ventral neck incision and unilateral right MCAo by advancing a 6.0 silicone rubber-coated monofilament (Doccol Corporation, CA) 10–11 mm from the internal carotid artery bifurcation via an external carotid artery stump. We monitored rectal temperatures with a temperature control system (Fine Science Tools, Canada), maintaining the temperature at ~37 °C during surgery with an automatic heating pad. We used laser doppler flowmetry (DRT 4/Moor Instruments Ltd, Devon, UK) to measure cerebral blood flow and to confirm occlusion (reduction to 15% of baseline cerebral blood flow) and reperfusion. All animals were fed with wet mash for one week after surgery to ensure adequate nutrition for chronic endpoints, as animals have rearing deficits after stroke. In sham mice, we performed identical surgeries except the suture was not advanced into the internal carotid artery. For the gonadal hormone-mediated outcome study, we ovariectomized (ovxed) female mice two weeks prior to stroke surgery. Details of the ovariectomy procedure are provided in the Supplementary file.