After a recovery period of approximately 2 h, mice were placed in a plastic chamber containing 8% oxygen and 92% nitrogen for 25 min. The chamber was a standard mouse cage without bedding (26 × 18 × 2.5 cm), and was partially submerged

in a 37°C water bath to maintain normothermia during hypoxia. Fresh gas flowed continuously into the chamber throughout hypoxia through vents in the chamber lid. Following hypoxia mice were kept in a warm cage to maintain normothermia and had free access to soft food and water. Sham animals underwent surgical dissection of the right carotid artery, without carotid ligation or hypoxia. Stroke size To assess Inhibitors,research,lifescience,medical stroke size and location in our hands, we first performed TTC staining 24 h after hypoxic–ischemic stroke. To assess stroke size in surviving mice and determine whether stroke variability

was due to differences in surgical technique, two surgeons performed hypoxic–ischemic strokes in Inhibitors,research,lifescience,medical a larger group of 42 mice. Experimental design Mice were shipped from The Jackson Laboratories Inhibitors,research,lifescience,medical and acclimated to our mouse room for at least 1 week. They were then handled multiple times and most often sat calmly on the experimenter’s hand when removed from their cage. Mice were trained on all behavior tests prior to surgery, then tested at the intervals shown (Fig. 1). All behavior tests were performed in the light cycle and at the same time of day on each day of testing. All apparatuses were disinfected (with Simple Green (Huntington Beach, California) unless otherwise noted) between trials, and all tests and analyses Inhibitors,research,lifescience,medical performed by an evaluator blinded to experimental groupings. Figure 1 Experimental design. Mice were trained and tested as shown, Inhibitors,research,lifescience,medical and sacrificed for stroke volumes 6 weeks after stroke. Rotarod Motor coordination was tested using the rotarod apparatus on days 2, 9, 16, 23, 30, and 37 post surgery. Animals were placed on a ROTOR-ROD™ (San Diego Instrument, San Diego, CA; lane dimensions 11.4

cm wide per animal; rod diameter 3.2 cm) that accelerated from 5 to 10 rpm over 300 sec, and the latency to fall was automatically recorded. Roxadustat trials Levetiracetam were ended and recorded if mice clung to the rod without walking for two revolutions. Three trials were performed on each testing day, and animals received at minimum 30 min of rest between trials. Presurgery training consisted of 12 days of testing, and the average of the last three training days before surgery was considered baseline average. Animals that did not reach 250 sec by the end of presurgery training were excluded from the study. Ladder test The ladder test was adapted from the ladder rung walking task (Metz and Whishaw 2002; Farr et al. 2006). The apparatus consisted of two plexiglass walls (76.2 × 0.635 × 15.24 cm) spaced 3.175 cm apart, just wide enough for the animal to pass. Plexiglass rungs (10.16 cm long with a 0.