Experimental groups
Mice were randomly assigned to receive either a sham or mild CCI surgery (TBI). 30 minutes following impact, a 200μL intravenous tail vein injection of 750nM C-176 (Enamine, Cat. No. EN300-6503757) diluted in phosphate-buffered saline (PBS) (Gibco, Cat. No. 18912-014) or a PBS vehicle was administered. Blinding of both the operator and data analysis was conducted by concealing the identity of the solution administered to mice post-TBI. Magnetic resonance imaging (MRI) and DigiGait™ analysis performed measuring lesion size and behavioural outcome respectively at 24h after TBI, with biochemical analysis performed at 2 and 24h after TBI. In this study the identity of each animal with respect to treatment was blinded to researchers who conducted the experiments and analysis.

Controlled cortical impact

The controlled cortical impact (CCI) procedure performed in this study was based on standard protocols as previously described and reported by our group . This model of TBI was selected as it is well documented to model secondary-injury inflammatory processes in addition to its high degree of reproducibility (Osier & Dixon 2016). Mice were anesthetised with ketamine (100mg/kg, Parnell)/xylazine ( 10mg/kg, Sigma, Cat. No. X-1251) via intra-peritoneal injection. A sagittal incision was made to expose the skull before a 2mm diameter craniotomy was performed using a handheld electrical drill (Dremel, 10.8V) at 2.5mm lateral the midline and 1.5mm posterior to the bregma to expose the right parietal cortex. The mice were restrained in a stereotaxic frame device for the injury to be delivered using a 2mm flat computer-controlled impactor (LinMot-Talk 1100). An impact 1.5mm deep was applied to the exposed cortex at a velocity of 1m/s and a dwell time of 100ms. The skull flap previously removed during the craniometry was replaced over the exposed cortex and the sagittal incision was sutured by hand using wax coated braided silk (Covidien, Cat. No. GS-832). 30 mins following successful CCI, mice were administered either C-176 or PBS intravenously. Post-surgery, all mice were administered buprenorphine (0.6mg/kg, Lyppard Australia Pty Ltd) intraperitonially and placed on a heat mat to recover from anaesthesia and their condition was monitored hourly over 6 hours and the subsequent morning. Sham mice underwent identical anaesthesia, sagittal incision and craniometry as TBI mice, omitting the impact by the computer-controlled impactor. The mice were housed for 2- or 24-hours following surgery and euthanised via cervical dislocation before subsequent analysis.

MRI acquisition

MRI scans were performed for this study using a hybrid Agilent 9.4 Tesla small animal MRI scanner using Bruker imaging hardware and software (Monash Biomedical Imaging) to quantify the progression of tissue damage. Mice were anesthetized with ∼3% isoflurane in medical-grade oxygen. Anesthesia was maintained throughout scanning with 0.25 to 1.5% isoflurane through a nosecone placed over the animal’s snout and anesthetized animals were laid supine on a purpose-built small-animal holder and their heads fixed into position with ear and bite bars and respiration rate was continuously monitored. A Bruker mouse heart surface receiver coil was placed under the animals’ head and the cradle was inserted into a Bruker 86mm transmitter coil fixed inside an Agilent SGRAD 120/HD/S gradient set for imaging. The MRI protocol consisted of a three-plane localizer sequence followed by multiecho T2 and diffusion-weighted sequences. The total scanning time was kept to <1 h per animal. Multiecho T2 -weighted images were acquired using a rapid acquisition, relaxation enhanced (RARE) sequence with RARE factor = 2; repetition time = 2500 ms; effective echo time (TEeff) = 10, 30, 50, 70, 90, and 110 ms; field-of-view = 1.6 Å∼ 1.6 cm2; matrix = 192 Å ∼192; and 24 slices with thickness = 0.5 mm. Volumetric analysis was carried out on T2-weighted images using MRIcroGL software, available from www.nitrc.org/projects/mricrogl .

Gait analysis

The gait dynamics of the mice pre-and post-surgery were recorded non-invasively using a DigiGaitTM apparatus (v11.5, Mouse SpecificsTM). The apparatus consists of a transparent conveyor belt over a recording device to capture the gait dynamics of the mice from underneath the belt. The belt was set to operate at a fixed speed of 15cm/s at an incline of 0 degrees. Light fur and tails were coloured with black ink to reduce noise in recording. Mice were given one ‘test run’ prior to recording. The gait dynamics of the mice were recorded in triplicate over a running period of 4 seconds for each recording. Gait indices were quantified using the DigiGaitTM Imaging System. The duration of each gait interval, which includes the duration of the stride, stance, swing, braking and propulsion phase (seconds) was used for gait analysis (Table 1).