reagent
Reagents purchased from commercial sources include: Pentobarbital sodium (Yipin Pharmaceutical Co., Ltd., Hebei, China). TTC staining reagent, 4% paraformaldehyde, diaminobenzidine, and hematoxylin (Solarbio Science & Technology Co., Beijing, China). Fluorescein isothiocyanate (FITC)-conjugated and tetramethylrhodamine (TRITC)-conjugated secondary antibodies (Invitrogen, CA, USA). Tunicamycin (TM) (MCE, Shanghai, China); goat serum (Thermo Scientific, Rockford, IL, USA); primary antibodies specific for p-PERK, p-IRE1, ATF6, and p-JNK (Affinity Biosciences, Jiangsu Ministry, China). Primary antibodies specific for LC3, CHOP, cleaved caspase-3, and cleaved caspase-9 (Proteintech, Wuhan, China). Primary antibody specific for KDEL peptide (Santa Cruz Biotechnology, Shanghai, China). Primary antibody specific for FAM134B (Cell Signaling Technology, Danvers, MA, USA). and secondary antibodies (Bioss Antibodies, Beijing, China).
animal
Male Sprague-Dawley (SD) rats (8 weeks old, weight 300 ± 20 g) were purchased from Yisi Experimental Animal Technology Co. (Changchun, China). Rats were housed in a dedicated specific pathogen free (SPF) laboratory under 12-h light/12-h dark cycle conditions with free access to food and water. All animal experiments described in this study were conducted in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Changchun University of Traditional Chinese Medicine (Changchun, China IACUC 2021092, May 8, 2021) before the start of the experiments. Approved.
experiment
Forty rats were randomly assigned to four groups (10 animals/group): control (CON) group, MCAO group, MCAO + acupuncture (ACU) group, and MCAO + TM + acupuncture (TM + ACU). ) group. Three weeks before her MCAO surgery, TM (ER stress agonist) was stereotaxically injected into the brains of rats in the TM+ACU group. Three weeks later, TM-injected rats underwent MCAO surgery with or without acupuncture. The experiment is shown schematically in Figure 5A.
MCAO surgery
Rats were first anesthetized with 2% sodium pentobarbital (30 mg/kg) administered by intraperitoneal injection. Next, the right common carotid artery (CCA), internal carotid artery (ICA), and external carotid artery (ECA) of each rat were carefully exposed. The CCA and ECA were then ligated and a small opening was created in the ICA at a distance of 2 mm from the common cervical bifurcation. A nylon embolus was then inserted into her ICA using an 18–22 mm length catheter (Shenzhen Reward Life Technology Co., China) to occlude the right middle cerebral artery (MCA). The CON group rats were anesthetized and then the CCA of the CON group rats was exposed using the procedure described above without MCAO induction.
scalp acupuncture
After MCAO surgery, a needle with a diameter of 0.3 mm was inserted into the left scalp of the rat along the parietal midline and the anterior oblique line of the parietal temple. The acupuncture needles remain in the scalp and rotate at 1 rotation per second for 1 minute, followed by a 4 minute break. These paired steps were then repeated a total of six times during the 30-minute surgery. Acupuncture treatment was performed once a day for 14 days.
Neurological function test
Determination of the modified Zea-Longa score, beam walking test, and screen grasping test were performed to assess the neurological status and motor function of the rats, as shown in Tables 1, 2, and 3. .
TTC staining assay
After the rats were sacrificed, the brains were surgically removed as soon as possible and flash frozen at -80°C for 5 min. Each brain was then cut into 2-mm-thick coronal sections. The sections were then immersed in 2% TTC staining reagent for 10 minutes at 37°C in the dark, then the sections were turned over and incubated for an additional 10 minutes under the same conditions. Brain sections generated from the same brain were then aligned and imaged together. We then calculated the percentage of infarcted brain area within each brain specimen based on the total area of infarcted brain tissue (white brain area) divided by the total area of normal brain tissue (red brain area). did.
Brain edema measurement
The weight of each brain (total weight) was measured immediately after removal from the skull, and all brains were frozen at -80°C overnight, lyophilized, and weighed again to determine the weight of the lyophilized brains. The dry weight was obtained. The difference in brain weight before and after freeze-drying reflects the degree of water retention (edema), as calculated using the following formula: (total weight – dry weight) / total weight × 100%.
Preparation of paraffin sections
After surgical resection, brains were fixed in 4% paraformaldehyde for 24 h at room temperature. Next, the brains were immersed in ethanol to dehydrate, immersed in xylene to be vitrified, and then the vitrified brains were immersed in liquid paraffin and embedded in paraffin. The paraffinized brains were then coronally sliced 2 mm post-bregma into 5-μm-thick sections, and the sections were delipidated and stained with hematoxylin and eosin (H&E), Nissl, or TUNEL staining. H&E and Nissl staining results were obtained by examining the stained brain sections with a light microscope (M8 Microscope, PreciPoint GmbH, Germany), and TUNEL staining results were obtained with a fluorescence microscope (LIONHEART, BioTek, VT; united states of america).
Proteomics analysis
Brains were frozen in liquid nitrogen immediately after surgical resection, and then 50 mg of protein from the right cortex of each rat was extracted using lysis solution and digested with trypsin. Liquid chromatography and tandem mass spectrometry were then performed to identify and compare proteins in the brain tissues of the MCAO and ACU groups using database searches (Maxquant v1.6.15.0) and bioinformatics analysis. Ta.
Fine structure observation
The cortex on the ischemic side was fixed with 2.5% glutaraldehyde (pH 7.4) for 2 to 4 hours at 4 °C, and the samples were rinsed with 0.1 M phosphate buffered saline (PBS, pH 7.4), followed by 1% OsO. Fixed.Four (prepared in 0.1 M PBS) for 2 hours at room temperature. The post-fixed brains were then subjected to graded dehydration, infiltration, and embedding (acetone:embedding medium = 1:1), and the specimens were sliced into ultrathin (60–80 nm thick) sections. Brain sections were then sequentially stained with 2% uranyl acetate for 15 minutes, followed by lead citrate for 15 minutes. The stained sections were then analyzed using a transmission electron microscopy (TEM) system (Hitachi, Japan).
Stereotactic injection of cerebral cortex
After anesthetizing the rats with 2% pentobarbital sodium (30 mg/kg) administered by intraperitoneal injection, the rats’ scalp fur was sterilized and each scalp was then incised to gently expose the bregma. Each rat was then fixed using a Quintessential Stereotaxic Injector (Stoelting Co., IL, USA) with bregma set to 0 (ML: 0.0, AP: 0.0, DV: 0.0) and injected with 2 μL of 25 μM TM. was injected into rats. brain cortex (ML: 2.0, AP: 1.2, DV: 1.5) at a rate of 0.5 μL/min. Meanwhile, rats in other groups were stereotactically injected with the same amount of DMSO. Three weeks after stereotactic injection, MCAO surgery and scalp acupuncture were performed.
Immunofluorescence and immunohistochemical staining assays
Paraffinized brain sections were permeabilized by immersion in 0.3% Triton X-100 for 30 min at room temperature, and then sections were incubated in pH 10.0 Tris-HCl for 10 min at 100 °C. The sections were then blocked by immersion in 5% goat serum for 1 h at 37 °C, followed by immersion in the appropriate primary antibody solution overnight at 4 °C. The sections were then incubated in the appropriate secondary antibody solution for 2 hours at room temperature in the dark. The sections were then photographed with a confocal microscope (Nikon C2 confocal, Japan) or immersed in diaminobenzidine (DAB) for 10 min at room temperature, washed in PBS, immersed in hematoxylin for 0.5 min, and washed with water. After that, I dehydrated it and took a photo. under a light microscope.
statistical analysis
All triplicate data were analyzed by ANOVA test using IBM SPSS Statistics 23 software (IBM, New York, USA) and expressed as mean ± standard deviation (GraphPad Prism 5, GraphPad Software, Illinois, USA). I did. Values of p < 0.05 were considered statistically significant.
Ethics statement
All animal experiments described in this study were conducted in accordance with established international ethical guidelines and approved by the Institutional Animal Care and Use Committee of Changchun University of Traditional Chinese Medicine (Changchun, China IACUC 2021092, 2021) before the start of the experiments. Approved by May 8, 2017).
