Six-week-old male Sprague-Dawley rats (220 ± 20 g) were purchased from Hubei Research Center of Laboratory Animals (Wuhan, China) (No.: 211002300044504).
A Han’s Acupoint Nerve Stimulator (HANS-200A, Nanjing Jisheng Medical Technology Co., Ltd., Nanjing, China) was applied to “Baihui” (GV20, located at the intersection of sagittal midline and the line linking two rat ears), bilateral “Shenshu” (BL23, on both sides of the 2nd lumbar vertebra, and the distance was 6 mm from the dorsmedian) and “Sanyinjiao” (SP6, the tip of the medial malleolus of the hind limb is straight up 10mm) acupoints (Fig. 1a and b). BL23 and SP6 acupoints on the same side were used together as pairs, and a shallow needle was prickled at 0.5 cm near Baihui point as an auxiliary electrode to form a pair with GV20 acupoint. The needles (diameter 0.25 mm and length 25 mm) (Beijing Zhongyan Taihe Medical Instrument Co., Ltd., Beijing, China) were inserted into the skin. Then, a dilatational wave at 2/100 Hz and 1 mA with electrical stimulation was used for 10 min, and kept the needles alone without electrical stimulation for 5 min. The process was continually repeated 4 times for 1 h in total as EA pretreatment.
Middle cerebral artery occlusion (MCAO) model
Briefly, rats received intraperitoneal anesthesia with 5% chloral hydrate (0.6 mL/100 g). A monofilament nylon suture with a rounded tip coated with silicone rubber (2032/2432A5, Beijing Cinontech Co., Ltd., Beijing, China) was inserted into the internal carotid artery 18-20 mm to block the blood flow. When ischemia was reached for 2 h, the middle cerebral artery was re-perfused by withdrawing the silicone rubber coated monofilament to induce the MCAO model of focal cerebral ischemia-reperfusion injury in rats.
72 Rats were randomly distributed to four groups (n = 18 for each group): middle cerebral artery occlusion model group (MCAO), sham-operation group with surgery but no MCAO (Sham), EA pretreated-MCAO group (EA + MCAO), and sham EA pretreated-MCAO group (N-EA + MCAO). For sham EA pretreatment (N-EA), the needles were inserted vertically into five acupoints only about 2-3 mm deep under the skin without electricity output, but procedures, electrode placements, and other treatment settings were the same as EA pretreatment. The sensations of “de qi” were not achieved in the sham EA pretreatment. As shown in Fig. 1C, rats were pretreated with EA stimulation at GV20, bilateral BL23 and SP6 acupoints for 1 h. Next, the rats were yield to unilateral right middle cerebral artery occlusion for 2 h, and then re-perfused for 6 h. Lastly, rats were sacrificed to collect the samples.
60 Rats were randomly divided into four groups (n = 15 for each group): MCAO, EA + MCAO, capsaicin administration before MCAO model (Capsaicin + MCAO), and capsaicin administration before EA pretreated-MCAO group (Capsaicin + EA + MCAO). According to previous publication , capsaicin (the specific TRPV1 agonists, 0.2 mg/kg via s.c.) (S1990, Selleckchem, Houston, TX, USA) in advance was applied 2 h before EA pretreatment (Fig. 7a). Then, EA stimulation at GV20, bilateral BL23 and SP6 acupoints for 1 h prior to MCAO model. Lastly, rats were sacrificed to collect the samples.
Measurement of infarct volume
After rats were sacrificed, the brains were collected and infarct volume was measured according to previous description . The brain tissue was sectioned into six coronal blocks with an approximate thickness of 2 mm. Then, the sections were stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) (T8877, Sigma-Aldrich, St. Louis, MO, USA) for 20 min and immersed in 4% paraformaldehyde overnight. Normal area presented red and infarct area kept white. The percentage of infarct volumes were calculated following the formula: Infarct volumes (%) = (Vc-VL) / Vc × 100%
Vc = volumes of normal gray matter in the control hemisphere
VL = volumes of normal gray matter in the s lesioned hemisphere
At the end of the experiment, brain tissues of rats were removed and fixed in 4% paraformaldehyde. Fixed tissues were embedded in paraffin and serially sectioned to a thickness of 5 μm. Then, the sections were stained with hematoxylin and eosin (H&E) and examined for histological analysis. A minimum of three sections per animal experimentation was examined, and five visual fields of each sample were randomly selected to observe nerve cell injury in a blinded manner.
Transmission electron microscopy
Fresh brain tissues were taken from the ischemic cerebral hippocampus, cut into 1mm3 size cubes and fixed in 2.5% glutaraldehyde for 24 h. Next, the samples were fixed in 1% osmium tetroxide for 2 h and dehydrated in graded ethanol and embedded in araldite. Then, the sections were cut at 50 nm and stained with uranyl acetate and lead citrate. Finally, the ultrastructure of neurons and mitochondria were observed with Tecnai G2 20 200kV transmission electron microscope (FEI/Philips Electron Optics, Eindhoven, Netherlands).
For immunohistochemical analysis, the paraffin sections of brain tissues were deparaffinized, blocked with 5% BSA, and treated with rabbit anti-rat Bax (ab32503), Bcl-2 (ab194583) (Abcam, Cambridge, Massachusetts, USA), cleaved caspase-3 (#9661) (Cell Signaling Technology Inc., Beverly, Massachusetts, USA) and TRPV1 (PA5-77317) (Invitrogen, Carlsbad, CA, USA) at 37°C for 2 h, separately. The sections were incubated with second antibody (ab6720, Abcam) at 37°C for 30 min. Then, SABC complex was added and the slides were stained with DAB solution. Finally, the slides were observed with a microscope (Olympus BX40, Tokyo, Japan). Under ×400 magnification, the morphometric examination was performed in a blinded manner by two independent investigators. Cells with brown granule staining of the membrane/cytoplasm were considered positive. For each section, five visual fields were chosen at random and mean number of the positive cells were represented.
Rat hippocampus were collected and extracted using ice-cold RIPA lysis buffer (P0013B, Beyotime, Shang, China). Protein concentration was determined with a BCA Protein Assay Kit (23227, ThermoFisher Scientific, San Jose, CA, USA). Equivalent amounts of protein (25 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then, it was transferred onto PVDF membranes. The membranes were blocked with 5% skim milk for 2 h and followed by incubation overnight at 4°C with primary rabbit anti-rat antibodies against Bax (ab32503), Bcl-2 (ab194583), Histone H3 (ab176842) (Abcam), cleaved caspase-3 (#9661), IκBα (#4812), p-IκBα (#2859), NF-κB (p65) (#8242), p-NF-κB (p65) (#3033) (Cell Signaling Technology Inc.), GAPDH (BM3874) (Boster Biological Technology Co., Ltd, Wuhan, China), and TRPV1 (PA5-77317) (Invitrogen). Then, the membranes were held with HRP-conjugated goat anti-rabbit antibody (#7074) (Cell Signaling Technology Inc.). The protein intensity was detected by the ChemiDoc™ XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA). Sample loading was normalized by quantity of GAPDH or Histone H3 detected parallel.
For dual immunofluorescence labeling, the slides of brain tissues were rinsed in 0.1 M PBS, blocked for 1 h with 2% BSA and then incubated with the following primary antibodies at 4°C overnight: rabbit anti-TRPV1(PA5-77317) and anti-NeuN (PA5-78499) (Invitrogen). For labeling, the primary antibodies were detected with corresponding secondary antibodies: donkey anti-rabbit IgG conjugated with Alexa Fluor®488 (711-545-152) or Alexa Fluor®594 (711-585-152) (Jackson ImmunoResearch, West Grove, PA, USA). Lastly, the sections were stained with DAPI (AR1177) (Boster Biological Technology, Wuhan, China) and examined under a fluorescence microscope (Olympus IX73, Tokyo, Japan).
Results were presented as the mean ± SD. Data were analyzed with SPSS 19.0 software (SPSS Inc., Chicago, IL, USA). Differences between two groups were evaluated by the Mann-Whitney U test, and multiple comparisons were analyzed using the Kruskal-Wallis test. When result of the Kruskal-Wallis test was significant, a Dunn test was performed for post hoc analysis. A value of P < 0.05 was considered statistically significant.