Sixty 8-week-old healthy adult female Sprague-Dawley (SD) rats weighing about 200–250 g were purchased from the Guangdong Experimental Animal Center and were housed in ventilated polycarbonate rat cages under controlled temperature, humidity, and light. The room temperature was maintained at 23 ℃. The rats were acclimatized for 7 days before starting the experiment. Over the whole period, the rats were provided with enough food and water.
After acclimatization, the rats were pre-trained to balance and walk across a bar for 5 s. On the day of surgery, the rats were starved of food for 4 h and pre-assessed for their basal performance using the balance beam task. Rats with mobility issues, injuries, and behavioral disturbance during the 1–7 days pre-training period were removed. The rats were randomly divided into four groups; the sham group, sham + EE group, TBI group, and TBI + EE group. The weight-drop (WD) model and the controlled cortical impact (CCI) model are both commonly used animal models for mechanical force-induced TBI. The Feeney WD model is used to simulate concussion and brain contusion after TBI[11]. WD modeling in the present study was developed (Fig. 1). For the sham and sham + EE groups, the bone window was opened without tapping. Specifically, tapping was performed using the modified Feeney modeling method after incising the scalp. After the rats in all the groups could move spontaneously, those in the EE and TBI + EE groups were placed in an EE, whereas those in the TBI and sham groups continued recovery in standard cages. The EE was a three-tier wire cage (78 × 54 × 137 cm) with a ladder containing various toys and nesting materials (tissue paper). The items were rearranged daily to maintain freshness. Each time the cage was cleaned, the rats were moved to a different floor. Rats in the TBI and Sham groups were housed in standard laboratory rat cages (32 × 21 × 17 cm, 1 rat per cage) as shown in Fig. 1, for 19 d.
Acute neurological assessment
Acute neurological assessments were performed on all the rats immediately after anesthesia awakening by gently squeezing the rat's paw every 5 s and recording the time to respond. The roll-over reflex score was based on the time taken to switch from supine to prone position on three consecutive occasions, and the severity of nerve injury after modeling was determined using these neurological indices.
Immunofluorescence staining for neural cell proliferation
Neural cell proliferation was assessed using Immunofluorescence staining. The cells were stained using 5-Bromo-2-deoxyuridine (BrdU), a thymidine analog that binds cellular DNA during the S phase cell cycle. The rats were injected twice a day at 12 h intervals for 3 days intraperitoneally with 120 mg/kg of BrdU before the water maze experiment to monitor the proliferation of hippocampal dentate gyrus and neural cell proliferation during the water maze.
BrdU-positive cells were detected by injection of BrdU reagent in vivo and selected paraffin sections were read by immunofluorescence technique. Sections were dewaxed into the water according to the conventional method, and after dewaxing, sections were subjected to hyperthermic repair, then cooled at 23°C and subjected to a water bath at a temperature of 65°C for 2 h using 50% formamide, followed by 3 dips using 2× sodium citrate buffer. The sections were then treated with cold hydrochloric acid 0.1 mol/L for 10 min and incubated with hydrochloric acid 2 mol/L at 37°C for 30 min. A 10 min dip was performed using borate buffer 0.1 mol/L (pH 8.5) and 3 rinses with PBS 0.01 mol/L; sheep serum 10% blocking solution was blocked for 60 min; mouse anti-BrdU (1:100) was added dropwise for a 2-night incubation at 4°C. Then, Alexa Flur488-labeled goat anti-mouse IgG (1:00) was added dropwise for 2 h of incubation at 37°C under no light, followed by 5 rinses with PBS solution 0.01 mol/L and blocking with DAPI blocker. The number of positive cells was analyzed using the software Image-Pro plus 6.0.
Translated with www.DeepL.com/Translator (free version)Learning and Memory
The maze was placed in a room with visual cues that remained constant throughout the experiment. The maze consisted of a plastic pool (120 cm wide and 41 cm high) and a transparent Plexiglas holder platform (10 cm wide and 33 cm high) 15 cm from the maze wall. The pool water was 35 cm deep and at 26 °C. For the positioning navigation test, the platform was put 2 cm below the water surface. Ink was added into the water to make the platform invisible to the rats. All the rats were trained from day 14 by placing them in the water facing the wall at a fixed time each day. The time taken by the rats to find and climb the escape platform in the water was recorded. If the rat did not climb the escape platform after 120 s, the search time was recorded as 120 s. The rat would then be guided to the platform and stay there for 10 s to memorize the location. A spatial exploration experiment was conducted on day 19 to test the memory retention of the rat. Here, the platform was removed from the pool, and the rat was placed in the water at a random location. The movement trajectories of the rats over 120 s, the distance covered in the target quadrant, and the percentage of time spent in the target quadrant were recorded.
Tissue preparation
After 19 d, 5 brain tissues of rats in each group were extracted, fixed 24 h in 4% paraformaldehyde, rinsed with running water for 30 min, and dehydrated using alcohol. After tissue clearing, infiltration, and waxing, the sections were cut into thin slices. Other processes included gluing and baking, dewaxing, de-benzene and rehydration, and hematoxylin and eosin stainning (HE).
Western blot analysis
The remaining rats were sacrificed, and the cortical tissue of the brain injury foci was separated on ice. Thereafter, 0.1 g of contused brain tissue was put in a pre-cooled grinding rod, ground to homogeneity, splitting, and centrifuged at 4 ℃. The supernatant was collected for protein extraction and concentration and concentration measurement. The concentration of the separation gel was selected according to the molecular weight of the protein, and the voltage of the electrophoresis instrument was adjusted after setting the parameters, A constant voltage of 60 V for gel concentrate electrophoresis and 90 V for separation gel electrophoresis. The proteins were then extracted from the gels after electrophoresis and analyzed.
Statistical Analysis
The concentration of proteins on the protein strip plots was analyzed for grayscale values using ImageJ software, and the expression of the target protein was calculated Grayscale value analysis. Continuous normally distributed data were expressed as mean ± standard deviation (Mean ± SD). Data sets for comparing two groups were plotted using Origin 2017. The test data were expressed as mean±standard deviation (Mean±SD), with two decimal places retained, and data were plotted between groups using Prism 5.0. If p<0.05, the difference between groups is significant "*" indicates a significant difference between the experiment group compared with the control group.