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).