Middle Cerebral Artery Occlusion

Male mice, 10 to 12 weeks old (25 – 30g), were anesthetized with 2% isoflurane in 70% air and balanced O₂ using a face mask. Middle cerebral artery occlusion (MCAO) was induced by the insertion of a silicone-coated 6-0 monofilament (Doccol Corp, Redlands, CA) into the MCA internal carotid artery for 1 hr followed by reperfusion, as described^{17 (link)}. Rectal temperature was maintained at 37±0.5 °C with a heating pad (Harvard Apparatus, Holliston, MA). Heart rate, oxygen saturation, and respiratory rate were monitored continuously (STARR Life Sciences Corp, Allison Park, PA). Animals with no observable deficits immediately after ischemia, those that died within 48 hrs, and those with subarachnoid hemorrhage at the time of death were excluded from analysis. Brains were sectioned coronally after 48 hrs into 4 slices and stained in 2% 2,3,5-triphenyletrazolium chloride (TTC). Infarct size was analyzed, normalized blindly to the non-ischemic hemisphere and expressed as a percentage and corrected for edema using the NIH Image program (Image J 1.37v). Neurological deficit scores were evaluated at 48 hrs according to a neurological grading score,^{18 (link)} from 0 ( no observable neurological deficit) to 4 (unable to walk spontaneously and a depressed level of consciousness). The evaluator was blinded to experimental treatments. In separate animals, regional cerebral blood flow (rCBF) was monitored through a microtip fiber optic probe (diameter 0.5 mm) connected through a Master Probe to a laser Doppler computerized main unit (PerFlux 5000, Perimed AB, Stockholm, Sweden). A small incision was made at the coordinate 1 mm caudal to the bregma and 3.3 mm lateral to the midline in the ischemic hemisphere to expose the skull, and the laser Doppler probe was attached to the exposed skull. CBF was measured 10 min before ischemia onset, during (30 min after stroke onset) ischemia and 5 min after reperfusion. All data were normalized to the values of CBF measured before stroke in wild type animals and expressed as relative ratios.

We planned to enroll a maximum of 900 participants, 18 to 82 years of age, at 58 centers in the United States, Canada, Australia, and Europe. Eligibility criteria included receipt of intravenous t-PA within 3 hours after symptom onset and a moderate-to-severe neurologic deficit (defined as an NIHSS score ≥10 or, after approval of amendment 3, a score of 8 to 9 with CT angiographic evidence of an occlusion of the first segment of the middle cerebral artery [M1], internal carotid artery, or basilar artery at institutions where CT angiographic imaging at baseline was the standard of care for patients with acute stroke). Written informed consent was obtained from the patient or a legal representative before enrollment. Detailed inclusion and exclusion criteria are provided in Table 1 in the Supplementary Appendix.