TBI was induced in mice using a controlled cortical impact (CCI) device TBI-0310 (Precision Systems and Instrumentation, Fairfax, VA, USA), as described previously (43). Mice were anesthetized with 3% isoflurane in 67% N2O/30% O2 until they were nonresponsive to the tail-pinch test. Then, 1.5% isoflurane was used to maintain anesthesia. The diameter of the impactor was 3 mm. The machine was set at a velocity of 5.0 m/s, a depth of 2.0 mm, and a dwell time of 100 ms, which produced a moderate contusion in the right cortex and underlying hippocampus, with pronounced behavioral deficits but virtually no mortality, thereby mimicking moderate TBI in humans. Mice were maintained at 36° to 37°C throughout the procedure. At 24 hours after CCI, mice were sacrificed under deep anesthesia, and the brain tissues around the injury site were subsequently harvested for Western blotting.

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