Animals
All animal experiments were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Harbin Medical University and strictly complied with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. A total of 207 male Wistar rats (260-280 g) were purchased from Changchun Yisi Company, which were fed with adequate food and water in a 12/12 hours light/dark room with humidity control and a temperature of 25℃.
SAH model and study design
An SAH rat model was established by endovascular puncture as previously reported[17].Pentobarbital (40 mg/kg) was injected intraperitoneally to anesthetize the animals. The animals were placed in a supine position, and a median incision was made in the neck to expose the common carotid artery and its branches. A single-stranded nylon thread penetrated the external carotid artery and encountered resistance after entering the internal carotid artery, indicating that it had reached the distal bifurcation of the internal carotid artery. The thread was then advanced approximately 3 mm beyond the resistance point and immediately withdrawn, allowing reperfusion of the internal carotid artery. The same operation was performed on rats in the sham group, except the vessels were not punctured.
24 rats were used to detect c-abl expression at 24, 48 and 72 hours after SAH by western blot(n=6).To test the role of c-Abl in SAH, 48 rats were randomly divided into four groups as follows: the sham (n=12), SAH (n=12), SAH+scramble small interfering RNA (siRNA) (n=12), and SAH+c-Abl siRNA (n=12) groups. c-Abl siRNA and scramble siRNA (500 pmol/5 µL) were injected intracerebroventricularly 24 h before SAH. Neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), and levels of the c-Abl, p-Akt, p-GSK3β and cleaved caspase-3 (CC-3) proteins were measured by Western blotting. The neurological function score and SAH severity were also evaluated. The PI3K/Akt inhibitor LY294002 was used to explore the underlying mechanism of the role of c-Abl in SAH-induced apoptosis. A total of 36 rats were randomly divided into three groups as follows: the SAH+scramble siRNA+vehicle (n=12), SAH+c-Abl siRNA+vehicle (n=12), and SAH+c-Abl siRNA+LY294002 (n=12) groups. LY294002 (5 mmol/L, 0.5 ml/animal) in a physiological saline solution was injected into the femoral vein 1 h before SAH induction, and rats in the vehicle group were given the same amount of physiological saline. Western blotting was used to detect protein levels. TUNEL was used to test apoptosis. To investigate the role of LRP-1 in the mechanism by which c-Abl regulates Akt/GSK3β, 54 rats were randomly divided into the following groups: the sham, SAH+vehicle (or scramble siRNA) (n=6), SAH+RAP (or LRP-1 siRNA) (n=6), SAH+c-Abl siRNA+vehicle (or scramble siRNA) (n=6), and SAH+c-Abl siRNA+RAP (or LRP-1 siRNA) (n=6) groups. RAP (0.7 nmol/g body weight) in a physiological saline solution was injected into the femoral vein 1 h before SAH operation, and rats in the vehicle group were given the same amount of physiological saline. Western blotting was conducted to detect protein levels.
Intracerebroventricular injection
Intracerebroventricular injection was conducted as previously reported[18].The rats were anesthetized and placed in stereotactic devices. A 10-μL Hamilton syringe (Hamilton Company, Reno, NV, USA) was inserted into the left ventricle through a burr hole made at the following coordinates: 1.5 mm posterior and 1.0 mm lateral to the bregma and 3.2 mm below the horizontal plane. siRNA in solution was administered at a rate of 0.5 μL/min 24 h before SAH operation. The needle remained in place for 5 min and was then withdrawn slowly. The c-Abl mixed sequences of the sense strands were as follows:
1: 5’-CGGCAGCCUAAAUGAAGAUtt-3’,
2: 5’-CCUAUGGCAUGUCACCUUAtt-3’,
3: 5’-GGUUUAUGAGCUGCUGGAAtt-3’.
Scramble siRNA sequence:5’-UUCUCCGAACGUGUCACGUtt-3’.
LRP-1-specific siRNA mixed sequences were as follows:
1: 5’- UUCUAUUCUUAUCCUUUCC tt-3’,
2: 5’- UUACUUCUUGUCCUCGCCC tt-3’,
3: 5’- AAUCUUUGCAUGUCUUGCC tt-3’.
Scramble siRNA sequence:5’-ACUGCUUAGCGUCAAUUCGtt-3’.
Neurological scoring
The modified Garcia scale was used to blindly assess neurological deficits in animals 24 h after SAH as previously reported[19].The assessment system consisted of six subtests to evaluate the following: spontaneous activity, spontaneous movement of the 4 limbs, forepaw outstretching, climbing, body proprioception and the response to whisker stimulation. Each subtest was scored from 0-3 or 1-3, and the total score (from 3 to 18) reflected neurological function (table 1). The lower a score was, the worse the neurological function was.
SAH grade assessment
SAH severity was quantified with an SAH grading scale based on the amount of bleeding as previously described[20].The basal cistern of the brain was photographed and divided into six predetermined segments (Fig 1B), and each segment was scored from 0 to 3, Grade 0:no subarachnoid blood; Grade 1: minimal subarachnoid blood; Grade 2: moderate blood clot with recognizable arteries; Grade3: blood clot obliterating all arteries. The total SAH grade was equal to the sum of all segment scores (maximum SAH grade=18). Rats in which the SAH grade was <8 and those for which SAH coexisted with subdural or epidural hemorrhage were excluded.
Western blot analysis
Western blotting was conducted as previously reported[18,21]. Briefly, the cerebral cortex from the left hemorrhagic site was collected as a sample 24 h after SAH. After anesthesia with pentobarbital(40 mg/kg), the rats were decapitated and then perfused with glacial PBS transcardially. The soft tissue and skull were separated to obtain the brain tissue. Fresh brain tissue was carefully removed from the basal cortex. The brain tissues were immediately frozen in liquid nitrogen and stored at 80 °C until used (all operations were performed on ice). Tissues were weighed and homogenized. RIPA lysis buffer and protease inhibitor (PMSF, NaF, etc.) were added to the homogenates, which were lysed on ice for 40-60 min and shaken once every 5-8 min. The samples were centrifuged to collect the supernatants. The protein concentration was detected with a detergent-compatible protein assay kit (Bio-Rad, Hercules, CA, USA). Samples containing the same amount of the target protein (30 μg) were separated by SDS-PAGE, and the proteins were then transferred to a PVDF membrane. The membrane was blocked with 5% nonfat dry milk for 2 h and incubated overnight at 4°C in the presence of the following primary antibodies: anti β-actin (Abcam, cat# ab6276, 1:1000), anti p-Akt (Abcam CST, cat# ab81283, 1:1000), anti p-GSK3β (Cell Signaling Technology, cat# 8566, 1:2000), anti-CC3 (Cell Signaling Technology, cat# 9661, 1:1000), anti-c-Abl (Abcam, cat# ab47315, 1:1000) and anti LRP-1 (Abcam, cat# ab92544, 1:1000).After washing with TBST buffer, the membrane was incubated with appropriate secondary antibodies (1:2000) at room temperature for 1 h. An ECL Plus chemiluminescence kit (Amersham Bioscience, Arlington Heights, IL) was used to display the protein bands according to the manufacturer's instructions. ImageJ software (NIH) was used to quantify band densities.
TUNEL staining and NeuN double immunofluorescence labeling
After anesthesia, the rats were perfused transcardially with 4℃ PBS followed by 4% paraformaldehyde. Then, the brain tissue was taken out immediately and fixed in 4% paraformaldehyde overnight. Sucrose solution was dehydrated in gradient, after rinsed, the brain tissue was quickly frozen on the machine. Finally, the continuous coronal frozen section was made. TUNEL and staining for NeuN, a neuronal marker, were used together to detect neuronal apoptosis. Briefly, frozen sections were rewarmed at room temperature for 20 min, blocked with 5% sheep serum(Equitech-Bio,SS-0100) for 1 h and then incubated with anti-NeuN primary antibody (Cell Signaling Technology, cat# 12943, 1:200 dilution). A TUNEL kit (Roche, cat# 11684795910) was used to label apoptotic cells after the use of anti-NeuN secondary antibody (Alexa Fluor® Plus 594-conjugated). The sections were incubated with 50 μL of TUNEL reaction mixture (enzyme solution:labeling solution = 1:9), incubated at 37°C in the dark for 60 min, and washed 3 times with PBS for 5 min each, then the sections were sealed and observed by fluorescence microscopy (Olympus, Tokyo, Japan). TUNEL-positive cells in five different fields were counted. The results are expressed as cells/mm2, and the apoptotic ratio was calculated as the number of apoptotic cells/the total cell number × 100%.
Statistical analysis
All data are expressed as the mean ± SD. Statistical significance was analyzed using one-way ANOVA followed by Tukey’s multiple comparison test. A probability value of p < 0.05 indicated statistical significance. SPSS 13.0 software for Windows was used to perform all statistical analyses.