Animals
Adult male Sprague–Dawley rats (weighting 280-330g, n=185) obtained from Guizhou Laboratory Animal Engineering Technology Center (China) were used in this project. All animals were kept in a room with controlled humidity (60± 5%), constant temperature (25± 1°C), and remained in a 12 h light and dark cycle and with ad libitum access to food and water.
SAH model
The SAH model was performed in rats using a modified endovascular perforation model as previously described [25]. Induction of anesthesia in rats was achieved using 4% isoflurane, and was maintained using 2.5% isoflurane. After intubation, the mice were placed in the supine position and connected to the rodent ventilator to breathe medical air (70%) and oxygen (30%). The heart rate, respiration, skin color, and pedal reflex assessment were assessed every five minutes during the operation to confirm anesthesia status and prevent distress. After exposing the carotid artery and its bifurcation, a 4-0 sharp single nylon thread suture was inserted from the external carotid artery into the left internal carotid artery to the anterior and middle cerebral artery bifurcation. The nylon suture was withdrawn immediately, and isoflurane was reduced to 1.5%. After the operation, the endotracheal tube was removed and the animals were placed in the heating chamber (37.5℃) to recover. Animals in the sham group underwent the same procedure, but without arterial wall puncture.
SAH Grading
The degree of SAH was assessed according to the SAH grading scale system at 24 h after SAH as previously described [26]. The basal cistern was divided into six segments that were graded from 0 to 3 according to the amount of subarachnoid blood. The total score was calculated by adding all area scores (maximum SAH grade = 18). Rats with a score of 8 or less were excluded from the current study.
Experimental design
Four separate experiments were performed as shown in Fig. 2.
Experiment 1
To determine the time course of endogenous CXCL-12 and CXCR4 protein level expression in the sham group and each group after SAH. The rats were randomly divided into six groups (n=6/group): Sham, SAH-6 h, SAH-12 h, SAH-24 h, SAH-48 h, and SAH-72 h. Western blot analysis was performed to assess the protein levels of CXCL-12 and CXCR4 in the ipsilateral (left) hemisphere cerebral cortex. Additionally, the cellular localization of CXCR4 with calcium-binding adaptor molecule 1 (Iba-1) was evaluated using double immunofluorescence staining in the Sham and SAH-24 h group (n=2/group).
Experiment 2
To evaluate the neuroprotective effects of CXCL-12 on short-term neurological outcomes after SAH, rats were randomly assigned to five groups (n=6/group): Sham, SAH+vehicle (sterile distilled water), SAH+CXCL-12 (5 μg/kg), SAH+CXCL-12 (15 μg/kg), and SAH+CXCL-12 (45 μg/kg). CXCL-12 was administered intranasally (i.n.) at 1 h after SAH. The SAH grading score, neurobehavioral test (including modified Garcia test and beam balance test), and brain water content were assessed at 24 h after SAH in all groups. The best dose of CXCL-12 was selected based on the short-term neurological outcomes and brain water content results, which was also used for the following long-term outcome and mechanism experiments.
To explore the effects of CXCL-12 on microglia/macrophage activation and neutrophil infiltration at 24 h after SAH, rats were randomly assigned to three groups (n=4/group): Sham, SAH+vehicle (sterile distilled water), and SAH+CXCL-12 (optimal dose). Immunofluorescence staining of Iba-1 with CD68 and myeloperoxidase (MPO)-positive neutrophils was performed in the peri-hemorrhagic area at 24 h after SAH. Quantitative analysis of CD68-positive Iba-1 and relative fluorescence density of MPO were assessed. Brain samples of these three groups were shared with experiment 4.
Experiment 3
To evaluate the effects of CXCL-12 on long-term neurobehavioral outcomes after SAH, rats were randomly assigned to three groups (n=10/group): Sham, SAH+vehicle (sterile distilled water), and SAH+CXCL-12 (optimal dose). The Rotarod test was performed on days 7, 14, and 21 after SAH. Morris water maze was performed on days 23–27 after SAH.
Experiment 4
To explore the underlying mechanism of the CXCR4/PI3K/Akt signaling pathway-mediated anti-inflammatory effects after SAH, the selective CXCR4 inhibitor, AMD3100, and PI3K inhibitor, LY294002, were administered intraperitoneally (i.p.) at 1 h before SAH. Rats were randomly assigned to seven groups (n=10/group): Sham, SAH+Vehicle (sterile distilled water, i.n.), SAH+CXCL-12, SAH+CXCL-12+AMD3100, SAH+CXCL-12+PBS (Vehicle of AMD3100), SAH+CXCL-12+LY294002, and SAH+CXCL-12+DMSO (Vehicle of LY294002, i.p.). The ipsilateral (left) hemisphere of each group was collected for western blot analysis (n=6/group) and immunofluorescence staining (n=4/group) after neurological performances and SAH grades were evaluated at 24 h after SAH.
Drug administration
CXCL-12 or vehicle was given via intranasal administration at 1 h after SAH as previously described [27]. Animals were placed in the supine position and were administered 1.5% isoflurane anesthesia. A total volume of 20 μL of vehicle (sterile distilled water) or CXCL-12 (MedChem Express, NJ, USA) at three different doses (5 μg/kg, 15 μg/kg, and 45 μg/kg), with 5 μL administered every 5 minutes, alternating between the right and left nares. AMD3100 was diluted in PBS, LY294002 was diluted in 10% dimethyl sulfide (DMSO), and both were administered intraperitoneally (i.p.) at 1 h before SAH.
Assessment short-term neurological performance
The short-term neurobehavioral outcomes were assessed blindly using the 18 point modified Garcia scoring system and the 4 point beam balance test at 24h after SAH as previously described [28]. Higher scores indicated better neurological function.
Assessment long-term neurological performance
Long-term neurobehavioral effects were assessed using the rotarod test to evaluate sensorimotor coordination and balance on days 7, 14, and 21 after SAH, and the Morris water maze was used to evaluate spatial learning capacity and memory ability on day 23–27 after SAH as previously described [29]. For the rotarod test, the animals were placed on the rotarod at the starting rate of 5 revolutions per minute (RPM) or 10 RPM, followed by gradual acceleration of 2 RPM every 5 seconds. The duration that the rats were able to stay on the accelerating rotating cylinder was recorded and used for statistical analysis. In regard to the Morris water maze test, animals were taken to the platform on the first day of cueing test. For the spatial learning test in the following days, the animals were placed in a set of semi-random starting positions, and were tasked to find the submerged platform within the 60-second time limit. The probe test was performed with the actual platform removed on day 27 after SAH. Swimming distance and trace, escape latency, and probe quadrant duration were recorded by the Computer Tracking System (San Diego Instruments Inc., CA, USA).
Brain water content
Brain edema was assessed by measuring brain water content using the wet-dry method as previously described [6]. The rats were euthanized at 24 h after SAH, and the brains were quickly removed and separated into four parts (right hemisphere, left hemisphere, cerebellum, and brain stem). Afterwards, each part of the brain was weighed immediately to obtain the wet weight, and then placed into an oven for 72 h at 100℃. The dried brain was weighed again. The percentage of brain water content was calculated as follows: (wet weight − dry weight) /wet weight × 100%.
Immunofluorescence staining
The rats were deeply anesthetized (5% isoflurane), and were euthanized via trans-cardiac perfusion with 100-150 mL of pre-cooled PBS (4 °C) and 100 mL of 10% formalin. Whole brains were rapidly collected and fixed in 10% formalin (4 °C, 24 h), followed by dehydration with 30% sucrose (4 °C, 72 h). Brain samples were embedded in OCT (Scigen Scientific Gardena, CA, USA), and then frozen at − 80 °C. The brains were sliced into 10 μm thick coronal brain sections using a cryostat (CM3050S, Leica Microsystems, Bannockburn, Germany), and then mounted onto normal Poly-L-Lysine coated slides. The slices were washed with 0.01 M of PBS three times for 5-10 min, and then incubated in 0.3% Triton X-100 in 0.01 M of PBS for 10 min at room temperature. After being blocked with 5% donkey serum in 0.01 M of PBS for 2 h at room temperature, the sections were incubated overnight at 4 °C with the following primary antibodies: anti-Iba-1 (1:200, Abcam), anti-CXCR4 (1:200, Abcam), anti-CD68 (1:200, Santa Cruz Biotechnology), rabbit anti-IL-1β (1:200, Abcam), and mouse anti-MPO (1:200, Santa Cruz Biotechnology). Next, the slices were incubated with fluorescence-conjugated secondary antibodies (1:500, Jackson ImmunoResearch) for 1 h at room temperature. The slides were visualized and photographed using a fluorescence microscope (DMi8, Leica Microsystems). The number of CD68-positive microglia cells were identified and counted in three different fields from the left basal cortex of five random coronal sections of each rat. The positive cells were quantified under microscopic field of 200x magnification, and data were expressed as cells/field. To assess neuroinflammation levels, six randomly selected tubules were examined to count IL-1β- and MPO-positive cells under the microscopic field at 400x and 200x magnification. The fluorescence intensity was quantified by ImageJ software (ImageJ 1.5, NIH, USA).
Western blot analysis
At 24 h after SAH, rats were deeply anesthetized (5% isoflurane) and transcardially perfused with chilled PBS, followed by decapitation. The brain sections were separated into ipsilateral and contralateral hemispheres. The ipsilateral hemisphere brain tissues were snap frozen in liquid nitrogen and stored in −80 °C freezer for storage until used. Brain samples were homogenized in RIPA lysis buffer (Santa Cruz Biotechnology) with protease inhibitor for 15 min and then centrifuged at 14,000 g (4 °C, 30 min). The supernatant was collected, and protein concentration was measured by detergent compatible assay (DC Protein Assay, Bio-Rad Laboratories). Equal amounts of protein were loaded onto the 10% SDS-PAGE gel for electrophoresis and then transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat blocking grade milk (Bio-Rad) for 2 h (37 °C), and incubated overnight at 4 °C with the following primary antibodies: anti-CXCR4 (1:1000, Abcam), anti-CXCL-12 (1:1000, Abcam), anti-p-Akt (1:1000, Cell Signaling Technology), anti-Akt (1:1000, Cell Signaling Technology), anti-PI3K (1:1000, Thermo Fisher Scientific), anti-IL-1β (1:1000, Abcam), anti-IL-6 (1:1000, Abcam), anti-TNF-α (1:1000, Abcam), and anti-β-actin (1:5000, Santa Cruz Biotechnology). The membranes were incubated with the appropriate peroxidase-conjugated secondary antibodies (1:5000, Santa Cruz Biotechnology) for 1h at 37°C. The bands were then visualized with the ECL Plus chemiluminescence reagent kit (Amersham Bioscience, Pittsburgh, PA) and quantified with the ImageJ software (ImageJ 1.5, NIH, USA).
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7 (Graph Pad Software, San Diego, CA, USA). All data were presented as mean ± SD. One-way ANOVA followed by Tukey’s post-hoc test was used for comparison among multiple groups. Two-way ANOVA was used to analyze the long-term neurobehavioral results. P < 0.05 was considered statistically significant.