Traumatic brain injury model and experimental procedure
All procedures were approved by the guidelines for the care and use of animals in China. The subjects were eighty male Sprague-Dawley rats obtained from the Animal Experimental Center of Tongji Medical College of Huazhong University of Science and Technology. Adult male Sprague-Dawley rats weighing about 260-300g were used in the experiment. Animals were kept under a 12-h light/dark cycle and allowed free access to food and water. Experimental TBI was performed using a modified weight-drop device previously developed in our laboratory[35-37]. Briefly, a cylindrical steel weight of 450 g was allowed to fall through the tube at a designated height of 2 m to impact the disk. The scalp was sterilized and sutured when their respiratory condition was regular, and the rats were randomized at the desired time points at 1, 3, 6, 12, 24, 48 and 72 h after TBI (n = 8 for each time point). In the sham group, rats underwent the surgical procedure without impact.
Tissue preparation and histopathological assessments
On the basis of previously described concerning the brain tissue preparation, the rats recovered and were observed for 1 - 72 hours, after which they were euthanized using a lethal dose of sodium pentobarbital given IP [35, 38, 39]. In short, the animals were perfused with heparinized saline to remove blood from the intravascular compartment, followed by perfusion-fixation with 4% paraformaldehyde. The brain was harvested and photographed to document surface hemorrhages. The BS was then divided equally by a sagittal cut to include the basal interpeduncular regions of the brain, pons, and pyramids. One part of the brain was processed and embedded in paraffin for histopathological examination. Tissue sections (4 µm thickness) were prepared for hematoxylin and eosin (HE), Luxol-fast blue (LFB), immunohistochemistry (IHC) and immunofluorescence analyses. Rat BS sections were stained with HE and LFB as described previously [40-43]. The other half was quickly dropped in liquid nitrogen for western blot analysis.
Immunohistochemistry
The procedure of IHC was based on the previous study [35, 44]. IHC analysis was performed on paraffin-embedded 4 µm BS sections. After de-paraffinization, the slides were washed three times with distilled water and incubated for 20 min in citrate buffer (pH 6.0) for antigen retrieval using microwave oven, and then blocked for endogenous peroxides with 3% hydrogen peroxide. After the citrate buffer cooled to room temperature and washed three times with phosphate buffered saline (PBS, pH 7.4). The sections were incubated with rabbit monoclonal anti-β-APP antibody (ab32136, 1:2000, Abcam, American), polyclonal rabbit anti-Aβ antibody (ab2539, 1:2000, Abcam, American) and monoclonal mice anti-NGB antibody (ab37258, 1:400, Abcam, American) overnight at 4℃. The following day, the sections were washed three times in PBS for 10 min each, followed by the incubation with ready-to-use SABC (rabbit IgG) kit (Google Biological Technology, Wuhan, China) for 1 h at room temperature Thereafter, the sections were washed three times in PBS for 5 min each. The immunostaining was visualized with 3, 3’-diaminobenzidine (DAB) for brown color development, and sections were counterstained with hematoxylin (Google Biological Technology, Wuhan, China). Final three PBS washes were performed for 5 min each. The sections were allowed to dry and dipped for 5 min each in 75–90–95–95–100–100 % ethanol solutions, followed by two treatments for 10 min each in xylene. Finally, the sections were cover slipped. For control experiments, primary antibodies were omitted. The slides were scanned and examined under Nikon Eclipse 90i microscope (Tokyo, Japan) system using 20 x objective. As for the quantitative analyses of immunohistochemistry, the number of Aβ and NGB immunopositivity staining were counted in the BS using ImageJ software. The image analysis was performed with the default parameter setting, and visual inspection of markup images was performed to confirm that the algorithm results were sufficiently accurate for the purpose of specific biomarker quantification. The positivity value was expressed as a ratio of positive pixels detected (brown staining) to total area. Analysis was performed at full 200 x magnification.
Western blot analysis
The protein expression of Aβ and NGB were examined by western blot analysis. Rats were killed, and the brain tissues were separated. The protein was extracted with RIPA Lysis Buffer (Beyotime, Haimen, China) according to the manufacturer’s instructions. Protein concentrations were quantified using a BCA Protein Assay Kit (Beyotime, Haimen, China). Equal amounts of protein samples (20μg) were subjected to 10% SDS-PAGE separation and transferred onto polyvinylidene difluoride membranes. The blots were blocked with 5% fat-free milk in TBS mixed with 0.1% Tween and then incubated overnight at 4 °C with specific antibodies against Aβ (1:1000, Abcam, Cambridge, UK, USA) and NGB (1:1000, Abcam, Cambridge, UK, USA). After three washes with TBST, the membranes were incubated with the corresponding secondary antibodies for 1h at room temperature. The protein bands were visualized using enhanced chemiluminescence kit (Thermo, USA) according to the manufacturer's instructions. β-actin was used as an internal control.
Immunofluorescence assay
We detected the distribution of Aβ and NGB using an immunofluorescence method. Rats were anesthetized and transcardially perfused with saline followed by 4% paraformaldehyde. Brains were removed, post-fixed and dehydrated in graded ethanol solutions. We blocked the nonspecific binding with donkey serum albumin following antigen retrieval. Then the sections were incubated in primary antibody overnight at 4 °C (Aβ 1:100 Abcam, Cambridge, UK, USA; NGB 1:100 Abcam, Cambridge, UK, USA). The following day, the sections were washed and incubated with fluorescently tagged secondary antibody. Negative controls were performed in the absence of primary antibody.
Statistical Analysis
All experiments were performed independently at least in triplicate. Statistical analysis was conducted by using GraphPad Prism 5.0. The values were the mean ± S.E.M. Statistical comparisons were analyzed using a one-way analysis of variance (ANOVA) followed by a Fisher post hoc test to correct for multiple comparisons. Values of P less than 0.05 were considered to indicate statistical significance.