A total of 140 Sprague-Dawley (SD) rats weighing 250 g to 320 g were randomly divided into three groups: (1) normal group (N，n = 20); (2) model group:, which was divided into the following subgroups: model 0 day group (D0，n = 20), model 1 day group (D1，n = 20), model 7 day group (D7，n = 20), model 14 day group (D14，n = 20), model 21 day group (D21，n = 20); (3) intervention group (I，n = 20). All rats were provided by the Experimental Animal Center of Chongqing Medical University. All experimental procedures were performed in accordance with the Animal Care and Ethics Committee and were approved by the Ministry of Science and Technology of the People’s Republic of China. The rats were housed in a 26℃ room on a 12:12 dark/light cycle and were supplied with sufficient food and water.
Model groups: the CSCI model (Additional File 1) was constructed based on a previous method developed by our group [5, 6, 21]. The rats were fasted 24 hours prior to surgery, anesthetized by intraperitoneal injection of 3.5% chloral hydrate and subjected to continuous oxygen inhalation. The rats were then fixed prone on a surgical table. The anterior and the posterior articular processes were excised without damaging the spinal cord. A small rectangular stainless steel board (3mm×2mm) was placed in the center of the spinal cord surface at L1. A custom-made swallow tail-like stainless steel fixation device was positioned between T12 and L2. To establish a rat model with a compressed spinal cord, the center of the stainless steel fixation device was inserted using a small screw (2 mm in diameter and 2 mm long with a flat and smooth screw tip) slowly and vertically until the front end of the screw reached the small rectangular stainless steel board. The incision was then gradually sutured in layers. The rats were allowed to recover from anesthesia and housed as described previously. Thereafter, the screws were turned inside 1/4 circle every 4 days until occurrence of double lower limbs paralysis and incontinence. After successful model construction, spinal cord decompression was performed. At 0, 1, 7, 14 and 21 days after decompression, the rats from different groups were anesthetized. Physiological saline (250 mL, 4°C) and 4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS 500 mL, 4°C, pH 7.4) were perfused into the left ventricle and outflowed from right auricle. When the right auricle efflux became clear, spinal cord sections from L1 (1cm) were quickly harvested.
Normal group: the rats were not subjected to any experimental procedure.
Intervention group: after successful model construction and decompression, pEGFR inhibitor (0.6mg/kg•d, ab141839/PD153035) was injected intraperitoneally and the spinal cord was removed at day 14.
Neurological function assessment
Locomotor activities were examined based on the BBB rating scale in an open field, according to the indications of published articles by two independent observers in a double-blind manner[22, 23]. A total of 21 points from 0 (complete paralysis) to 21 (normal) were recorded after decompression of CSCI.
Luxol fast blue staining
The spinal cord sections were removed and fixed in 4% paraformaldehyde for 24 hours at 4°C. Sections from the center of the injury were selected along with those from 0.5cm to 0.7cm rostral and caudal to the injury site. The tissues were then dehydrated successively in 10%, 20% and 30% sucrose solutions, and embedded in optimal cutting temperature (OCT) compound. Transverse sections (10 µm in thickness) of the spinal cord were prepared on a frozen slicer. Spinal cord sections were stained with luxol fast blue at 60°C for 2h. The sections were then rinsed with 95% ethanol and differentiated in 0.05% lithium carbonate solution followed by 70% ethanol. Differentiation was stopped by distilled water until unmyelinated tissue turned white. Twelve random micrographs from the lateral funiculus were obtained under an Olympus microscope with an objective lens of 40×. Images of at least 10 randomly sections were captured. The total number of myelinated ﬁbers in the micrographs was further determined using the Image-J software (National Institutes of Health, USA) by a blinded investigator.
Double-labeling immunofluorescence assay
To detect co-expression of p-EGFR, pAkt1 and NG2+ (the marker of OPCs) in the spinal cord sections, we used the primary antibodies listed in Table 1 (see supplemental materials). Tissue sections were rewarmed, rinsed and incubated in 5% donkey serum (Jackson ImmunoResearch, Lancaster, PA, USA) for 1 h at 37°C in a humidified atmosphere to permeabilise the tissue and block non-specific protein-protein interactions. The tissues were then incubated with the primary antibody overnight at +4℃. The tissue sections were rinsed again with 0.01mol/L PBS. The secondary antibody (red) was cy3-conjugated goat anti-rabbit IgG (H+L) used at a 1/200 dilution for 1.5h at 37°C in a humidified atmosphere in the dark. Alexa Fluor 488 goat anti-mouse IgG (H+L) was used to label NG2 (green) at a 1/200 dilution for 1.5h at 37°C in a humidified atmosphere in the dark. A nuclear dye (4′,6-diamidino–2-phenylindole, 1:20; Bestbio Inc., China) was used to stain the cell nuclei (blue) for 5 min. The tissue sections were then washed and mounted in 50% glycerol dissolved in PBS. The samples were observed under a confocal microscope (Leica TCS SP2, Germany). All the digital images from lateral funiculus were captured in a double-blind manner from four random fields per section of the injured epicenter of the cross sections in the rats. The number of p-EGFR+-NG2 and pAkt1+-NG2 signals per field were counted for further analysis.
Tissues soaked at 4°C in a buffer containing 50 mmol/L ethylenediaminetetraacetic acid, 2μg/mL leupeptin, 2μg/mL pepstatin A, 2 mmol/L phenylmethylsulfonyl fluoride and 200 KIE/mL aprotininwere broken down mechanically using a blender. The homogenates were then centrifuged at 10,000×g for 20 min at 4°C. The supernatant was collected, and protein concentration was determined using a Bradford assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins of the sample were separated using 10% SDS-PAGE and then transferred to a polyvinylidenedifluoride membrane. The blotted membranes were incubated in 5% skim milk to block non-specific protein-protein interactions. For immunoblotting, the following primary antibodies were used: polyclonal rabbit anti-AKT1 antibody (1:1000; Abcam, Cambridge, UK, ab66138) and monoclonal rabbit anti-EGFR antibody (1:1000; Abcam, Cambridge, UK, ab52894). Alkaline phosphatase-conjugated anti-IgG antibody (1:10000, Santa Cruz Biotechnology) was used as the secondary antibody. Immunoreactive bands were visualized using a chemiluminescent substrate (Pierce Inc., Rockford, IL, USA). Western blot bands were quantified by a gel densitometry (Bio-Rad). The ratio of protein–to -actin was obtained for each sample, and each point was measured in triplicate.
Statistical analyses were performed using the SPSS Statistics 20.0 software（IBM, Inc., USA）. Number of Myelinated nerve fibers, and expression level of pEGFR and pAkt1 proteins for the specimens were expressed as means±SD. Differences between individual groups were initially compared using one-way ANOVA. The data were then analyzed with LSD multiple-comparison post hoc test. Differences between the intervention group and model group on day 14 were compared using independent samples t test. All of the reported P values were two-sided, and P < 0.05 was considered statistically significant.