All procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the Chongqing Medical University.
Animals and Experimental Design
Approval and consent for our study were obtained from our institution’s Animal Care and Ethics Committee. A total of 72 adult (8-10 weeks) Sprague–Dawley rats were provided by the experimental animal center of Chongqing medical university. Rats were housed in a temperature-controlled (24℃) room on a 12/12 dark/night time cycle in the laboratory animal center before the experiment. Rats (n=72) were randomly divided into 3 groups on each time point: (1) the sham group (n = 8): rats underwent a sham operation without the perforation of the anterior cerebral artery which induces SAH. (2) the SAH group (n = 8): SAH was induced and rats were sacrificed on 7d, 14d, 21d, and 28d after SAH induction. (3) the EA group (n =8): rats received 10 minutes consistent EA treatment and were sacrificed on 7d, 14d, 21d, and 28d after SAH induction.
Induction of SAH was performed as previously described before (Feiler et al. 2010). Briefly, rats were anesthetized with 3% isoflurane in 70/30% medical air/oxygen by a rodent ventilator. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were exposed. A 5-0 prolene filament (Ethicon, USA) was advanced into the anterior cerebral artery (ACA) via the ECA and ICA. After a subtle resistance was encountered, the filament was advanced 2 mm further to perforate the ACA. Subsequently, the filament was immediately withdrawn. In the sham operation group, the same procedure was performed with the exception of the perforation of the ACA. The body temperature was maintained at 37.5 ± 0.5° C during the operation. Since SAH induces a high mortality rate, mice that died within 24 h were excluded and relevant mice were supplemented.
For the EA group, EA treatment was performed at Baihui (GV20), Dazhui (DU14) and Zusanli (ST36) acupoints under anaesthesia by isoflurane. Three pairs of needles were connected with the output terminals of an EA apparatus (Model G6805-2A, China). Alternating strings of dense-sparse frequencies (30 Hz for 1.05 s and 4 Hz for 2.85 s, alternately) were used for EA. Electrical stimulation lasted for 10 min per day on each rat.
Performed by an investigator blinded to group assignment, a modified Garcia’s method was used to obtain neurological scores for 1d, 2d, 3d, 7d, and 28d in SAH, EA and control group (Parrar et al. 2002). Briefly, a 27-point scoring system was used to assess the neurological deficits of rat, including spontaneous activity, limb symmetry, climbing, balance, body proprioception, vibrissae sensation, and tactile sensation.
Morris Water Maze
As a test for spatial learning and memory, Morris water maze (MWM) includes cued learning procedure, spatial acquisition task, reference memory task, and working memory task according to the previous study (JEON et al. 2010). The MWM consisted of a circular pool 2 m in diameter and 0.75 m in height. It was filled with water to a depth of 0.4 m and kept at room temperature. Four equally spaced points were arbitrarily designated as north (N), south (S), east (E), and west (W) around the circumference of the pool. This established four quadrants (NW, NE, SE, and SW). A clear plexiglass platform (10 cm 9 10 cm) was submerged 4 cm below the water level in the middle of one of the eight equally spaced arbitrary lines (N, S, E, W, NW, NE, SE, and SW). Three large unique shapes were placed on three walls to function as distal cues around the pool. A camera mounted in the center of the ceiling above the pool tracked the rat. Behavior testing was performed between 10:00 and 18:00. All animals were housed at a constant temperature of 22°C, under a 12-h light/dark cycle, with free access to food and water.
Cerebral Blood Flow Monitoring
Cerebral blood flow (CBF) of rats in each group was monitored by PeriCam PSI System (Perimed AB, China). Briefly, an incision was made along the sagittal axis on the rat's scalp under anaesthesia by isoflurane. Then rats were placed on the stereolocator, and the skull was completely exposed to the CBF detector. The monitoring lasted for 1 minute for each rat. Then rats were executed and the brain tissue was harvested.
Transmission Electron Microscopy
Rats in each group were perfused with 4°C 150ml normal saline and 100ml 4% glutaraldehyde solution, and then brain samples was extracted. Each specimen was immediately fixed with 2.5% glutaraldehyde for 2 days, post-fixed in 2% osmic acid for 1 h, dehydrated in serial alcohol and propylene oxide, and embedded in araldite for morphometric analyses on semithin sections. Ultrathin sections mounted on copper grids were stained with uranyl acetate and lead citrate. Images were obtained using transmission electron microscopy (TEM) (Hitachi, Japan).
The white matter samples of rats in sham, SAH, and EA groups were homogenized in 10 volumes of ice-cold RIPA (Beyotime, China) added by protease inhibitor cocktail (Roche, Indianapolis, USA). Prepared protein extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed overnight at 4 ℃with the following primary antibodies: PDGFR (1:1000, Abcam, UK), GPR17 (1:1000, Abcam, UK), SOX10 (1:1000, Abcam, UK),APC(1:1000, Abcam, UK) GAPDH (1:1000, Abcam, UK), PLP (1:1000, Abcam, UK), MBP (1:1000, Abcam, UK), and ID2 (1:1000, Abcam, UK) followed by incubation with secondary antibodies conjugated with horseradish peroxidase(1:10000, Beyotime, China). The bands were revealed using an ECL western blotting kit (Beyotime, China), and photographed by a chemiluminescence imaging system (Bio-Rad, USA). The amount of protein in each band was quantified using Image Lab software (Bio-Rad, USA).
Immunofluorescence staining was performed on frozen sections of rats in sham, SAH, and EA groups. The sections were incubated with mouse anti-rat primary antibody including Olig2 (1:250, Millipore, USA), SMI32 (1:250, Biolegend, China ), APC (1:250, Calbiochem, USA ), and rabbit anti-rat primary antibody including MBP (1:250, Abcam, USA), PDGFR-α (1:250, Abcam, USA), SOX10 (1:100, Abcam, USA), Olig2(1:250, Abcam, USA), and Id2(1:50, Genetex, USA) at 4 ◦C overnight. The sections were then incubated with second antibody including dylight 488-conjugated goat anti-rabbit antibody, 594-conjugated goat anti-rabbit antibody, 488-conjugated goat anti-mouse antibody, and 594-conjugated goat anti-mouse antibody accordingly. DAPI was used for nuclear staining.
All data was expressed as the mean ± standard deviation (SD) and compared with repeated measures of analysis of variance (ANOVA). Bonferroni’s post hoc method was applied for comparison among groups. The difference of mortality rate was analyzed using chi-square test. All statistic values were calculated using SPSS 28.0 (SPSS, USA). Significance was assumed at P < 0.05.