Controlled cortical impact TBI model

Our custom-designed controlled cortical impact (CCI) injury device consists of a microprocessor-controlled pneumatic impactor with a 3.5 mm diameter tip. Mice were anesthetized with isoflurane evaporated in a gas mixture containing 70% N₂O and 30% O₂ and administered through a nose mask (induction at 4% and maintenance at 2%). Depth of anesthesia was assessed by monitoring respiration rate and pedal withdrawal reflexes. Mice were placed on a heated pad, and core body temperature was maintained at 37°C. The head was mounted in a stereotaxic frame, and the surgical site was clipped and cleaned with Nolvasan and ethanol scrubs. A 10-mm midline incision was made over the skull, the skin and fascia were reflected, and a 4-mm craniotomy was made on the central aspect of the left parietal bone. The impounder tip of the injury device was then extended to its full stroke distance (44 mm), positioned to the surface of the exposed dura, and reset to impact the cortical surface. Moderate-level CCI was induced using an impactor velocity of 6 m/s and deformation depth of 2 mm as previously described (Fox et al., 1998; Loane et al., 2009). After injury, the incision was closed with interrupted 6-0 silk sutures, anesthesia was terminated, and the animal was placed into a heated cage to maintain normal core temperature for 45 minutes post-injury. The CCI model replicates several important secondary injury mechanisms after TBI, including apoptosis, edema, and secondary neuroinflammation (Byrnes et al., 2012; Piao et al., 2012), but does not produce acute intracranial hypertension. Sham animals underwent the same procedure as TBI mice except for the impact.

Sham (n=6) and CCI (n=10) of WT and NOX2^−/− mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) at 1 day post-injury and transcardially perfused with ice-cold 0.9% saline (100ml). Ipsilateral cortical tissue was rapidly dissected and snap-frozen on liquid nitrogen for RNA extraction.

Sham (n=6) and CCI (n=6) of WT and NOX2^−/− mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) at 3 day post-injury and transcardially perfused with ice-cold 0.9% saline (100ml). Ipsilateral cortical tissue was rapidly dissected and snap-frozen on liquid nitrogen for Western blotting.

Sham (n=9) and CCI (n=9) of WT and NOX2^−/− mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) at 3 days post-injury and transcardially perfused with ice-cold 0.9% saline (100ml). Ipsilateral cortical tissue was rapidly dissected and processed for CD11b positive selection and flow cytometry analysis. Ipsilateral cortical tissue from 3 mice were pooled for CD11b positive selection, and flow cytometry data represent 3 independent experiments.

Sham (n=6) and CCI (n=6) of WT and NOX2^−/− mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) at 1, 3, 7 and 28 days post-injury and transcardially perfused with ice-cold 0.9% saline (100ml), followed by 300 ml of 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde overnight, and cryoprotected in 30% sucrose for histological analysis.

Sham and CCI WT and NOX2^−/− mice were used for behavioral studies (sham WT, n=6; sham NOX2^−/− n=6; CCI WT, n=20; CCI NOX2^−/− n=14). At 21 days post injury mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) and transcardially perfused with ice-cold 0.9% saline (100ml), followed by 300 ml of 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde overnight, and cryoprotected in 30% sucrose and were processed for stereological based lesion volume and neuronal cell count analysis.

A CCR2 antagonist, RS102895, was administered to WT and NOX2^−/− mice starting at 36 hours prior to sham-injury (n=9) or CCI (n=9). Mice were treated with 2mg/kg RS102895 or equal volume vehicle (saline) by oral gavage every 12 hours (Matsubara et al., 2015) up until 24 hours post-injury. Mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) and transcardially perfused with ice-cold 0.9% saline (100ml), and ipsilateral cortical tissue was rapidly dissected and processed for flow cytometry or snap-frozen on liquid nitrogen for RNA extraction.

A selective NOX2 inhibitor, gp91ds-tat (5mg/kg), or equal concentration of ds-tat scrambled peptide (AnaSpec Inc. Fremont, CA) was delivered by intraperitoneal injection (I.P.) to WT CCI mice (n=8/group) starting at 1 day post-injury, with repeated I.P. administrations at 2 and 3 days post-injury. gp91ds-tat dosing was based on reported therapeutic effects in mice (Abais et al., 2013). ds-tat scrambled- and gp91ds-tat-treated CCI mice underwent motor function testing (beam walk) on 1, 3, 5, and 7 days post-injury, and cognitive function testing (Y-maze) at 6 days post-injury. Mice were anesthetized (100 mg/kg sodium pentobarbital, I.P.) at 7 days post-injury and transcardially perfused with ice-cold 0.9% saline (100ml), followed by 300 ml of 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde overnight, and cryoprotected in 30% sucrose for histological analysis. A non-treated sham control group of WT mice (n=6) were used to establish baseline motor and cognitive function performance.