Rat cerebral ischemia model

Animal experimental procedures were conducted according to the guidelines established and approved by Animal Care Committee at Tohoku University. Animal arrive guidelines were followed in the preparation of the manuscript. Adult male rats (3 months old, 260–290 g) were subjected to tFCI by intraluminal MCA blockade with a nylon suture as described previously [30]. The rats were anesthetized with 1.5 % isoflurane in 30 % oxygen and 70 % nitrous oxide using a face mask. The rectal temperature was controlled at 37 °C. Blood gas was analyzed with a pH/blood gas analyzer (Chiron Diagnostics Ltd). After a midline skin incision, the left external carotid artery was exposed and its branches were electrocoagulated. A 22.0 mm 3–0 surgical monofilament nylon suture, blunted at the end, was introduced into the left internal carotid artery through the external carotid artery stump. After 90 min of middle cerebral artery occlusion, blood flow was restored by the withdrawal of the nylon suture. For probe (bare FMNP or SR-FLIVO-FMNP, n = 4 each) administration, the left jugular vein was exposed between the sternocleidomastoid muscle and anterior perusal muscle. 100 μg of SR-FLIVO were diluted in 50 μl of DMSO followed by addition of 200 μl of PBS buffer. The volume ratio of DMSO/PBS buffer was kept 1:5. The left jugular vein was cannulated with a 23–gauge needle and 600 μl of SR-FLIVO-FMNP or bare FMNP/PBS (1.0 mg/ml) solution was injected and kept in blood circulation for 60 min before MRI signal recording.