Ethic oversight
Animal experimental procedures were performed under the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of People’s Republic of China. All experiments were conducted in accordance with animal care guidelines approved by the Animal Ethics Committee of the Army Military Medical University. The animal study proposal was approved by the Institutional Animal Care and Use Committee (IACUC) of the Southwest Hospital of the Army Military Medical University, with the permit number: IACUC2012-0012.
Study design and subjects
All experiments were performed in the central laboratories in Southwest Hospital except for the ex vivo DTI acquisition and analyses, which were performed in the laboratory of department of Molecular Imaging in West China Hospital.
Adult female Sprague-Dawley (SD) rats (aged approximately three-months-old, 200-250g) were obtained from the Army Military Medical University. Animals were housed in a controlled pathogen-free environment at a temperature of 23~25 °C, 70% humidity, 12 h light–dark cycles, free access to food and water.
The sample size was calculated according to the previous study [20]. Except for 2 rats died from anesthesia accident, the rest 42 rats were randomized into 3 groups: control (n=6), moderate group(n=18), and severe group(n=18). Six rats from each group were tested for Basso, Beattie, and Bresnaha (BBB) score and MEPs test at different timepoints to evaluate the graded SCI injury in rats, and then animals were sacrificed on day 14 for ex vivo DTI and histology examination to provide imaging evidences. The rest animals from the moderate and severe group were sacrificed on day 3/ 7(n=6/group at each timepoint) for ex vivo DTI and histology examination. The euthanasia was performed with an intraperitoneal injection of 1% sodium pentobarbital overdoses (100 mg/kg body weight).
SCI Model
SCI was induced according to the previous method [21, 22]. Briefly, rats were anesthetized with 1% sodium pentobarbital (40mg/kg body weight, i. p.), and fixed in a prone position to perform a dorsal laminectomy at the T7-9vertebrae. A 50-g aneurysm clip was used to laterally compress the T8 spinal cord with no spacer for 30 seconds to establish the severe injury model, while adding a 0.4-mm thickness spacer to establish the moderate injury model. The control group received identical laminectomy without crush injury. The skin incision was then closed. SCI rats were isolated to a separate cage in animal house. Antibiotics (Cefazolin, 50 mg/kg body weight, i. p.) were given daily to treat/prevent bladder infection immediately after the surgery for 7 continuous days. Bladders were pressed twice daily until the rats recovered to normal spontaneous micturition.
Behavioral assessment of motor function
All animals were assessed in an open field walking according to the BBB locomotor scale [23], the day before inducing injury, and on day 1/3/7/14 after operation. Hindlimb function was assessed according to the 0–21 BBB scoring, where 0 is flaccid paralysis and 21 is normal gait.
Electrophysiological assessments of hindlimb motor function
MEPs were induced according to the previous method [24, 25]. Briefly, rats were anesthetized with 1% pentobarbital sodium (20mg/kg i.p.). The stimulation needle electrode (DSN1620, Medtronic, USA), acting as the anode, was inserted subcutaneously at the base of the nose with the tip point touching the scalp. The needle electrode acting as the cathode was placed subcutaneously at the midpoint of the two ears. The recording electrode was inserted into the tibialis anterior (TA) muscle. A ground electrode was placed subcutaneously at the base of tail. A single pulse of electrical stimulation (10 mA, 0.1 ms, 1 Hz) to excite the brain was delivered via stimulator (Keypoint, Medtronic, USA). The electrical stimulation was repeated five times at the intervals of 15 s in each rat. The base-to-peak amplitude of the trace after single stimulation was recorded for each type of evoked potential.
Histology
Rats were sacrificed at 3days (3d), 7 days (7d) and 14 days (14d) post SCI. The spinal cords were harvested, fixed, dehydrated, embedded, and sliced into 20μm successively. After blocked with 5% bovine serum albumin (BSA) for 1.5 h at room temperature, the slices were incubated with the primary antibodies of goat anti-myelin basic protein (anti-MBP; 1:250; Santa Cruz; Cat: sc-13914) overnight at 4◦C, and were probed subsequently with AlexaFluor-488-conjugated secondary antibodies against goat (1:200; Invitrogen) antibodies for 2 h at 37 °C. The nuclei were counterstained with 4′ -6-diamidino-2-phenylindole (DAPI; Santa Cruz Biotechnology). Sections were imaged under a Zeiss confocal microscope (Zeiss, LSM780).
Ex vivo DTI acquisition
Magnetic resonance imaging was performed with a 7.0 Tesla Bruker Biospec 70/30 USR preclinical scaner, using a volume coil with a 23-mm inner diameter and 44-mm outer diameter for both transmission and reception. The spinal cord specimens were loaded into 2mL syringes filed with Fomblin (Solvay, Brussels, Belgium) [26] and stabilized by a parallel plastic rod (6-mm diameter). RARE T2-weighted scans were acquired to locate the epicenter of the injury. 15 thick slices arranged around the epicenter were acquired for diffusion tensor imaging (sequence parameters: spin echo DTI-EPI, b=1319s/mm2, δ = 4ms, Δ = 18ms, 30 diffusion sampling directions, 16 averages, TE/TR = 32/2750ms, 100μm in-plane resolution, acquisition time = 3h6min40s).
Paravision version 5.0 (Bruker BioSpin) was used for B0, fractional anisotropy (FA) and MD map analysis. The cross-sectional areas of the spinal cord were defined by manually outlining the white matter (WM) areas on transverse B0 and FA scans for each time point. Of the 15 diffusion axial images acquired, the 11 axial slices centered on the lesion core were used for subsequent. Regions of interest (ROIs), which chose the total WM were manually outlined on the transverse images.
The DTI parameters including λ1, λ2, λ3, Mean Diffusivity (MD), and FA were used with the Paravision version 5.0 (Bruker BioSpin, Karlsruhe, Germany) diffusion tensor calculation module according to the previous study.[26] The axial diffusivity (AD) is a measure of the diffusivity along the principal axis of the diffusion tensor (AD = λ1). The radial diffusivity (RD) was calculated using the two minor diffusion axes (RD = (λ2 + λ3) / 2). The FA was calculated using the equation: FA = √1/2(√((λ1 − λ2)2 + (λ1 − λ3)2 + (λ2 − λ3)2) / √(λ12 + λ22 + λ32)).
Fibertracks were visualised and analysed using TrackVis (v. 0.6.1) by drawing ROIs on epicenter mask in ex vivo b0 maps for ROI filers. Then count the number of track greater than 3mm.
Statistical analysis
All DTI parameters were presented as mean and standard error of the mean (SEM). BBB score, MEPs amplitude and number of tracks were presented as mean and standard deviation (SD). BBB score, MEPs and DTI parameters data were analyzed using one-way analysis of variance (ANOVA), and group differences were ascertained using Tukey’s post hoc comparisons when appropriate. Comparisons between 2 groups were analyzed using 2-tailed Student t tests. DTI parameters (including FA, AD and RD) of the epicenteral spinal cord were analyzed relative to BBB and MEPs via Pearson’s correlation. All statistical analyses were performed via the SPSS 23.0 software. The significance level for all tests were set at p <0.05.