Animals
Adult male Sprague-Dawley rats aged 9–10 weeks and weighing 250–280 g were used for the experiments. The experimental animals were purchased from the Animal Experiment Center of Chongqing Medical University. All animal experiments were approved by the Animal Ethics Committee of Chongqing Medical University. The experimental processes strictly complied with international guidelines for animal ethics and welfare. Every effort was made to reduce the number of rats used in each experiment and to minimize their distress. The rats were fed in a specific pathogen-free room of the Animal Experiment Center of Chongqing Medical University. The room conditions were as follows: ambient temperature, 21℃; relative air humidity, approximately 60%; and light condition, a light/dark cycle consistent with the external circadian rhythm for every 12 h. Five rats were raised in each cage to allow free access to food and water.
Construction of the middle cerebral artery occlusion mode
The thread occlusion method was used to construct a right middle cerebral artery I/R injury model[41, 42]. Briefly, after the rats were anesthetized, the common carotid artery, internal carotid artery, and external carotid artery were exposed. After clamping the common carotid artery, the external carotid artery was verified and excised. A nylon filament suture coated with paraffin wax was inserted at the tip of the excised external carotid artery and run along the internal carotid artery until the head of the thread plug reached the middle cerebral artery. The nylon filament was removed after 2 h.
A total of 231 rats were used in this study; of these, 21 rats died during the experiment (mortality rate: 9.09%), and 10 rats were noneligible for the experiments. A 5-point Zea-Longa scoring system[43] was used to determine the inclusion of the rats in the study (rats that scored 1–3 points were included). The remaining 200 eligible rats were randomly assigned to 5 groups: Sham; I/R; I/R + EE; AAV-Scramble + I/R + EE; and AAV-shALK5 + I/R + EE[44].
EE treatment
The EE treatment was performed as follows. The I/R + EE group rats were placed in EE cages of 120 cm length × 60 cm width × 50 cm height. Each EE cage was equipped with a steel mesh cover, a ladder, a chain, pipes of different shapes, plastic tunnels, colored blocks, and a wheel to provide sensory motor stimulation. Music was played and light stimulation was provided for 2 h at a regular time each day. Social stimulation was also provided by placing 10–12 rats in a cage. The content of the cage was changed daily to promote novelty and exploration. Rats in the I/R group and sham surgery group were placed in standard cages of 44 cm length × 32 cm width × 20 cm height. Five rats were kept in each standard cage with no objects for sensory motor stimulation[28, 45].
2,3,5-Triphenyltetrazolium chloride staining
Staining with 2,3,5-triphenyltetrazolium chloride (TTC) was used to detect the cerebral infarct volume. As reported earlier, the infarct volume was calculated based on the area of the ischemic zone. After 14 days of model construction, the rats were euthanized under anesthesia. The brain was then quickly excised. Next, 2-mm-thick brain sections (n = 5 per group) were cut and stained with standard 2% TTC at 37 ℃ for 20 min; for uniform staining, the slides were overturned once every 5 min. The noninfarct areas were stained deep red with TTC, while the infarct areas appeared white. ImageJ software from NIH was used to perform numerical analysis of the pale infarct and noninfarct hemispheric areas. The infarct area of each slide was summed and expressed as a percentage of the volume of the noninfarct hemisphere[33, 46]. The results were determined using the following formula: infarction rate (%) = A°/Aʹ × 100, where Aʹ represents the volume of both hemispheres and A° represents the infarct volume.
Neurobehavioral assessment
The Modified Neurological Severity Score (mNSS) test and the adhesive-removal somatosensory test (ARST), the limb placing test were performed 3 d before and 1, 7, and 14 d after middle cerebral artery occlusion/reperfusion (MCAO/R)[31, 47]. The mNSS is a motor, sensory, reflex, and balance composite test. Neurological function was graded based on a score from 0 to 18 (normal score, 0; maximal deficit score, 18). In the severity scores of injuries, 1 point indicates the inability to perform the test or the lack of a tested reflex. The ARST was used to evaluate sensorimotor deficits. Two pieces of adhesive-coated papers were used as bilateral tactile stimuli and stuck to the distal-radial region of each forelimb. The limb placing test is used to evaluate the sensory motor integration, including 7 limb placement tasks. The task score is: 2 points: rats perform normally; 1 minute, delayed (2 seconds) and/or incomplete performance in rats; 0 points, the rats showed abnormal behavior. Both sides of the body are included. The time to remove each paper was recorded in 3 trials per day (maximum limit: 120 s). Individual trials were separated by at least 5 min. Before MCAO/R, the animals were trained for 3 d.
Administration of adeno-associated virus
To knock down ALK5 expression, we used an shRNA with the following sequence: GCCATAACCGCACTGTCATTC; vector: H12663, promoter: U6, reporter: EGFP, resistance marker: puromycin resistance gene. Vectors expressing ALK5-shRNA (4.44 × 1012 UT/mL) were supplied by OBIO Inc. (Shanghai, China). Vectors expressing GFP were used as the control (AAV-Scramble). Three weeks before MCAO/R, adeno-associated viruses (AAV) were stereotaxically injected into the ischemic cortex[48] at two sites on the right cortex as follows: A-P: 1.0 mm, M-L: -2.0 mm, D-V: -1.2 mm; A-P: -3.0 mm, M-L: -1.5 mm, D-V: -1.2 mm; 1 µL for each site. The injection rate was 200 nL/min, and at the end of the injection, the micro-injector was left in place for 5 min before withdrawal.
Western blotting assay
The rats were sacrificed by cervical dislocation, and the ischemic penumbra cortex tissues were rapidly removed on ice and preserved at -80℃. An appropriate amount of tissue was collected, and RIPA lysis buffer, a protease inhibitor, and a phosphatase inhibitor were added to the tissue; the tissue was then fragmented in a tissue grinder. The mixture was left to stand for 30 min and then centrifuged at 12,000 rpm for 20 min. The Beyotime BCA protein concentration determination kit was used to measure the protein concentration. The absorbance level was measured by a microplate reader, and the protein concentration was calculated and stored after balancing. For electrophoresis, Yarnase gel kits were used, with 6% and 12.5% electrophoretic gels. The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010, Millipore, USA) and blocked with 5% bovine serum albumin. The membranes were then incubated overnight at 4℃ with the following primary antibodies: anti-ALK5 (1:1000, AB235578, Abcam, USA), anti-p-Smad2/3 (1:1000, 8828, Cell Signaling Technology, USA), anti-Smad2,3 (1:1000, 8685, Cell Signaling Technology), anti-Gadd45β (1:1000, abs125760, Absin, China), anti-DCX (1:1000, 13925-3-AP, Proteintech) and anti-Nestin (1:1000, 13483-1-AP, Proteintech) and anti-GAPDH (1:4000, 10494-1-AP, Proteintech, China). GAPDH was used as a loading control. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:4000, SA00001-2, Proteintech) for 1 h at room temperature. Subsequently, the membranes were washed with TBST and visualized using an enhanced chemiluminescence reagent (P0018FS, Beyotime, China). Images were acquired with a Fusion FX5 analysis system (Vilber Lourmat, F-77601 Marne-la-Vallée Cedex 3, France) and quantified using Quantity One software (Bio-Rad Laboratories, USA).
Immunohistochemistry assay
Rats were first anesthetized and then subjected to thoracotomy. The brain was perfused with PBS and 4% paraformaldehyde from the left apex, dehydrated with a sucrose gradient, embedded in paraffin, and preserved after sectioning. The sections were dewaxed and then subjected to rehydration with a gradient alcohol series, followed by incubation with a peroxidase blocking agent for another 20 min[49]. Subsequently, the sections were blocked with 5% bovine serum albumin and incubated with the following primary antibodies at 4°C overnight: anti-ALK5 (1:50, ab235578, Abcam), anti-p-Smad2/3 (1:100,WL02305 ,Wanleibio), anti-Gadd45β (1:100, abs125760, Absin); anti-NF-H (1:50, 18934-1-AP, Proteintech). On the following day, the sections were incubated with goat anti-rabbit IgG antibodies or goat anti-mouse IgG antibodies (Zhongshan Golden Bridge Inc., China) for 30 min at 37°C. A positive result was revealed by staining with 3,3ʹ-diaminobenzidine (DAB) (Zhongshan Golden Bridge Inc.). Images were acquired with a LEICA DM600B automatic microscope (Leica Microsystems Heidelberg GmbH, Germany) and quantified using FIJI software. Protein expression levels were expressed as the mean optical density value (integrated optical density divided by the relevant area).
Immunofluorescence assay
Rat tissue was obtained as mentioned above. The tissue was then embedded in an OCT compound and sectioned using a frozen microtome. The frozen sections were air-dried at room temperature, washed with PBS, permeabilized with 0.4% Triton X-100, and subjected to antigen retrieval with sodium citrate. After washing with PBS, the sections were blocked with normal goat serum for 1 h. The sections were then incubated overnight at 4°C with the following primary antibodies: anti-DCX (1:50, 13925-3-AP, Proteintech), anti-NeuN (1:200, MilliporeSigma, MAB377), anti-GFAP (1:100, BM0055, Boster, China), anti-Nestin (1:50, 13483-1-AP, Proteintech). On the following day, the sections were washed with PBS and incubated with a mixture of goat anti-rabbit IgG-CFL 488 (1:100, sc-362262, Santa Cruz Biotechnology, USA) and goat anti-mouse IgG-CFL 555 (1:200, sc-362267,Santa Cruz Biotechnology, USA) at 37°C for 1 h in the dark. The sections were counterstained with DAPI for nuclei staining. Images were acquired with a confocal laser scanning microscope (A1R, Nikon, Tokyo, Japan).
Golgi-Cox staining
Golgi-Cox staining was performed in accordance with the instructions of the Hito Golgi-Cox Optimstain™ kit (Hitobiotec Corp., USA). The brain tissue was rapidly removed and placed in a mixture of solution 1 and solution 2 from the kit for 14 days in the dark. The tissue was then soaked in solution 3 from the kit for 3 days. The tissue was then embedded in an OCT compound, and 100-µm-thick sections were prepared. The sections were dried in the dark and stained with solutions 4 and 5 from the kit in accordance with the manufacturer’s protocol. Subsequently, the sections were dehydrated with a gradient alcohol series, permeated with xylene, and then sealed. A total of 20–25 neurons were measured for each group. The length and distribution of dendrites were evaluated by Sholl analysis as follows: 2D reconstructions of the entire dendritic tree were generated; a series of concentric circles with 10-µm intervals were centered on the soma; and the number of intersections was used to estimate the total length and distribution of dendrites.
BDA-based neural pathway tracing
The NeuroTrace™ BDA-10,000 Neuronal Tracer Kit (N7167, Invitrogen, USA) was used for this analysis. In accordance with the instructions of the kit, after 4 weeks of I/R induction in rats, 15 µL of 10% BDA solution was injected into the sensorimotor cortex contralateral to the side of the lesion at the following 5 points: 1.0 mm rostral to bregma, 1.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 4.0 mm caudal to bregma, 1.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 1.0 mm rostral to bregma, 5.0 mm lateral to the midline, and 1.5 mm ventral to the dura; 4.0 mm caudal to bregma, 5.0 mm lateral to the midline, and 1.5 mm ventral to the dura; and 1.5 mm caudal to bregma, 2.5 mm lateral to the midline, and 1.5 mm ventral to the dura. Two weeks later, the brains were perfused, and continuous 50-µm-thick sections were prepared using a vibrating microtome. The sections were washed with PBS, incubated for 20 min in 3% hydrogen peroxide solution, and washed with 0.5% Triton X-100 solution for another 30 min. The sections were further incubated overnight with the avidin-biotin-peroxidase complex (Invitrogen, USA) at room temperature and DAB (Invitrogen, USA) for 5 min. The fibers projecting to the right red nucleus were examined by counting all midline-crossing BDA-positive fibers. The number of midline-crossing BDA-positive fibers was divided by the total number of corticospinal tract fibers. The method of counting positive fibers crossing the midline of the corpus callosum was similar to that for the red nucleus. BDA-positive fibers were counted using FIJI software.
Statistical analysis
The results are presented as mean ± standard deviation[50]. The data were collected by a researcher blinded to the study details. GraphPad Prism 9.5 (GraphPad, San Diego, CA, USA) was used for constructing the graphs. IBM SPSS Statistics 25.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis. The differences between the groups were analyzed by one-way or two-way analysis of variance followed by Tukey's post hoc multiple comparison tests. A P-value of < 0.05 indicated a significant difference.