2.3. Fluid percussion injury

We employed our standard lateral fluid percussion injury (FPI) protocol as described earlier [30]. Briefly, 3% isoflurane (1.0 mL/min in 100% oxygen) was provided in a chamber (VetEquip Inc., CA, USA), and then maintained with 2–2.5% isoflurane via nose cone while rats were in a stereotaxic frame. Body temperature was controlled (37–38 °C) by a heating pad. Under aseptic surgical conditions, a midline skin incision was made to expose the skull. Using a high-speed drill (Dremel, WI, USA), craniotomy (3.0 mm diameter) was made 3.0 mm posterior to bregma and 6.0 mm lateral (left) of midline to expose the intact dura. A hollow plastic injury cap was placed over the craniotomy, secured with dental acrylic cement and was later filled with 0.9% saline. When the dental cement hardened, the anesthesia was discontinued and the rat was attached to the FPI device via the head cap. At the first response of hind-limb withdrawal to a paw pinch, rats received a moderate fluid percussion pulse (2.7 atm). Upon resumption of spontaneous breathing the head cap was removed and the skin was sutured. Neomycin was applied on the suture and the rats were placed in a heated recovery chamber to be fully ambulatory before being returned to their cages. The sham animals were prepared using the identical surgically procedure but without the fluid pulse.