Animal preparation and controlled cortical impact (CCI)
Adult male Sprague-Dawley rats (weighing 300–320 g) were used in the present study. The rats were housed individually under controlled conditions (temperature, 22±1°C; humidity, 60%) with a 12‐hr light/dark cycle. food and water were available ad libitum. All protocols were approved by the Capital Medical University Institutional Animal Care and Use Committee. Controlled Cortical Impact (CCI) was performed as previous described . Briefly, all Rats were anesthetized by isoflurane inhalation and the head was fixed in a stereotaxic frame. A 6mm craniotomy was made in the middle of the right parietal bone, leaving the underlying dura intact. Then rats were then subjected to impact with parameters we have described before (moderate TIB model: velocity 2.8 m/s; compression time 85 ms; and depth 2 mm; diameter of impactor tip 5 mm). Sham operated rats underwent the same procedure without percussion.
Experimental groups and CORT treatments
All 168 rats (n=24 per group) were grouped by CCI and treatments into: 1) sham control group (Sham); 2) CCI + dimethyl sulfoxide (DMSO, with a final concentration <1%) group (CCI); 3) CCI + CORT (0.3, 3, 30mg/kg, sigma) group (CCI+CORT1; CCI+CORT2; CCI+CORT3), 4) CCI + Spironolactone (50mg/kg, ab141289, Abcam) + CORT (0.3mg/kg) group (CORT1+SPIRO); and 4) CCI + Mifepristone (50mg/kg, ab141289, Abcam) + CORT group (30mg/kg) (CORT3+RU486). Drugs were administered intraperitoneally for 3 days after CCI. To effectively block MR and GR, SPIRO and RU486 (twice a day) were given for 2 days before CCI and 3 days after CCI. All drug dosages were chosen based on pilot experiments from our laboratory and our previous study .
Morris water maze (MWM)
MWM test was used to test the spatial memory ability in this study. Rats (n=24 per group) were trained using MWM before injury according to the protocol described before . Each rat received 4 trials per day for 5 consecutive days (8-4 days before CCI) to find the platform submerged below the water (20±2°C, with nontoxic black ink). The pool (150 cm in diameter) was divided into four equal quadrants (northwest, northeast, southwest, southeast). Each trial (with 5 min interval) was started from different positions (north, east, southeast, and northwest) and lasted at most 120 s. if the animal reached the platform within 120 s, the time was recorded as the latency time. If it failed to find the platform, the trial was terminated and the animal was placed on the platform for 15 s, the latency time was recorded as 120 s. To assess the spatial memory, probe trial was conducted 1 day before and 3 days after CCI. The hidden platform was removed, and the rats started at a northeast position. The percentage of time spent in the goal quadrant during the 30-s (total) swimming period was recorded.
TUNEL assay and H&E staining
The rats (n=8 per group) were sacrificed by decapitation on day 3 after CCI, and then 5 μm-thick coronal paraffin sections at the level of the hippocampus were made according to the Paxinos atlas of the rat brain . To assess the survival apoptosis of neurons in the hippocampus, H&E staining and TUNEL assay (Roche, Germany) were carried out as we previously described . Briefly, after rehydration, tissue sections were incubated with proteinase K working solution (20 mg/ml in 10 mM Tris-HCl buffer, pH 7.5–8.0) for 10 min at 37°C. They were rinsed twice with 0.01 M PBS (pH 7.4) and subsequently incubated at 37°C with the TUNEL reaction mixture for 1 h. Finally, color development was conducted for 10 min using 3, 3-diaminobenzidine (DAB) substrate. A MIDI FL (3D Histech, Hungary) system was used to obtain the digital images of the brain sections. A digital image analysis system (3D Histech, Hungary) was used to count the number of neurons and apoptotic cells in the ipsilateral hippocampus (three sections) as previously described . Data are presented as the number of hippocampal neurons per mm and the total number of TUNEL-positive cells. All analysis was performed in a blinded fashion.
Immunofluorescence staining for cleaved-caspase 3 were performed as we previously
described . Briefly, the brain sections were incubated in 3% hydrogen peroxide for 0.5 h and then blocked with normal bovine serum for 0.5 h. The brain sections were washed three times and incubated with rabbit polyclonal anti-cleaved caspase-3 (1:400, CST, #9664), overnight at 4°C. The slices were then washed with PBS and incubated with Alexa Fluor 647-conjugated donkey anti-rabbit IgG at room temperature for 2 h. Finally, the samples were counterstained with DAPI (Sigma-Aldrich, St. Louis, MO) for 10 min. Digital images of the whole brain sections were obtained by a MIDI FL (3D Histech, Hungary) system.
The rats (n=16 per group) were sacrificed by decapitation on day 3 after CCI and the right hippocampus was removed at 4°C. Total (n=8 per group) and nuclear protein (n=8 per group) extraction were performed as previously described . Equal amounts of proteins (30 μg) were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride membranes. Blots were incubated in 5% nonfat milk or BSA for 2 hr and then reacted overnight at 4°C with primary antibody P-Akt (ser 473, 1:5000; ab81283), P-Ert (1:1000, CST, #9010), P-CREB (ser133, 1:5000; ab32096), P53 (1:1000, ab131442), Bax (1:1000, ab32503), P-Bad (S136, 1:1000, ab28824), cleaved caspase-3 (1:1000, #9664, CST), Bcl-2 (1:1000; ab196495), MR (1:400, ab2774), GR (1/200 dilution; ab3578), β-Actin (1:5000; ab179467, Abcam, UK), and Histone H3 (1:400; Millipore Co.), followed by incubation with secondary antibodies for 2 hr at room temperature. Blots were visualized using chemiluminescence (Bio Spectrum 500 Imaging System; UVP Co., Upland, CA, USA) and ImageJ software determined the intensity of each band. The percent expression compared with that of the sham controls was calculated for each sample.
Co-immunoprecipitation was performed to assess protein-protein interaction among Bcl-2, Bad, and Bax. According to previously described , an aliquot of 500 μg total protein was first pre-treated with either rabbit polyclonal anti-Bcl-2 (1:1000; ab196495, Abcam, UK) or rabbit polyclonal anti-Bad (1 µg/ml, ab90435, Abcam). A total of 20 μL Protein A/G agarose (Sigma) was added to each sample, and the mixture was incubated overnight at 4 °C, and then centrifuged for 1 min at 12,000g. To remove non-specifically bound proteins, the precipitates were rinsed with NP-40 buffer four times. Agarose-bound immunocomplexes then were released by resuspension in a loading buffer containing denaturing solution. IgG was used as a negative control for precipitation.
All data are depicted as the mean ±SDs. Data were analyzed using SPSS 22.0 (IBM Corporation, USA). The percentage of time spent in the goal quadrant, cell counts, and western blot were statistically analyzed using one-way analysis of variance (ANOVA). Repeated ANOVA was used to analyze the Escape latency. A P-value < 0.05 was considered statistically significant.