2.1. Ethics Statement and Animals. 80 adult male C57BL/6J mice, SPF grade, 10–14 weeks old, weight 23-28g (Jackson Laboratory, Maine, USA, certificate number: 2020062903051) were reared in Duke University Medical Center Animal Center with a 12-hour day/night cycle. The animal room was cleaned at constant temperature and humidity, and the indoor temperature was maintained at 22 ± 0.3°C and the humidity was maintained at 55 ± 5%. Animal experiments were approved by the Duke University Animal Care and Use Committee. The online tool Quickcalcs (http://www.graphpad.com/quickcalcs/) was used to randomly assign animals to groups. 20 mice in each group, A: Sham + VNS group (sham operation plus vagus nerve stimulation); B: pMCAO + VNS group (pMCAO plus vagus nerve stimulation); C: pMCAO + VNI group (pMCAO plus vagus nerve isolation but no stimulation) D: Sham group (sham operation).
2.2. pMCAO Model Establishment. The pMCAO mice model are made as the article described as follows[10]. The mice were anesthetized with isoflurane, intubated, and ventilated. Their rectal temperature was maintained at 37°C ± 0:2°C throughout the procedure. Mice were placed in left lateral position, and a small skin incision was made between the right eye and ear. The temporal muscle was cut slightly using a high temperature loop, and a 3mm long segment of the zygomatic arch was removed. After exposing the skull base and trigeminal nerve branch, a small bone window (3–4 mm2) was drilled on the skull above the MCA. The MCA trunk was lifted with an 8 − 0 needle and permanently ligated with silk suture proximal to the cortical branch to the frontal cortex. The muscle and skin were then closed separately. Animals that did not show an infarct lesion were excluded.
2.3. Vagus Nerve Electrode Implantation[11]. An incision was made in the middle of the neck of the mouse to separate the sternohyoid and sternomastoid muscles longitudinally and is then pulled to the side to expose the common carotid artery and the right vagus nerve located outside the carotid sheath. In order to reduce the collateral circulation compensation and ensure the success rate of the middle cerebral artery ischemia model, we ligated the common carotid artery with silk thread. The vagus nerve is carefully separated from the sur- rounding connective tissue and sympathetic nerve trunk. After the 5mm long VN is exposed, the exposed end of the VN stimulation electrode is wrapped around the nerve in a spiral form and separated from the surrounding tissue by a rubber sheet. Through a continuity test using an ohmmeter, the con- tact between the electrode and the exposed VN was intact. The electrode lead is then passed from the tunnel through the fascia to the back of the skin on the neck. In order to prevent the nerve or electrode from shifting, a suture outside of the electrode was done on the skin of the neck.
Mice in group A and B were stimulated with 500µs width 2mA electric pulses for 10min at 5Hz every day for 10 mins, from 6h after surgery and each day for 7 days, 7 times in total [12]. Mice in group C were operated the same way as mice in group B, but no electrical stimulation was applied. Group D was the simple sham operation group.
2.4 Judgment of model success and vagus nerve stimulation. The criterion for model success is that the blood supply of the ligated blood vessels disappears under the microscope, and the mice develop hemiplegia after surgery (NSS score); The criterion for a successful vagus nerve stimulation is an increase in heart rate variability, animals that did not show an increased heart rate variability were excluded. Mice returned to standard diet after five days of liquid diet.
2.5 Behavioral Test. All evaluations were performed by observers who were blinded to group assignment.
2.5. 1 Adhesive Removal Test. All mice were pretrained on the adhesive removal tasks 3 days prior to the VN surgery. The adhesive removal test measures sensorimotor function as previously described[13]. Two small adhesive dots were placed on forepaws, and the amount of time (seconds) needed to con- tact and remove the sticker from each forepaw was recorded. Recording was then stopped if the animal failed to contact the sticker within 2 min. The test was performed 3 times per mouse, and the average time was used in the analysis before stroke in addition to 7, 14, and 21 days after stroke.
2.5. 2 mNSS Test. Mice were tested and scored for neurological deficits using a modified Neurological Severity Score (mNSS) 7, 14, and 21 days after the onset of ischemia. An 18-point neurological score was employed with slight modifications described before [14]. The score consists of 5 individual clinical parameters, including tasks on motor function, alertness, and physiological behavior, whereby 1 point is given for failure. A maximum NSS of 18 point indicates severe neurological dysfunction with failure at all tasks.
2.6 Ultrasound Doppler Laser Speckle Flow Imaging. The changes of cerebral blood vessel density in different groups of mice at different time points (6h,3 days, 7 days and 14 days) were observed under the Doppler laser speckle imaging system. The target area is the area between the coronal and the chevron sutures.
2.7 Determination of cerebral edema coefficient. The mice were quickly decapitated after inhalation anesthesia, and the brain tissue was taken. The wet weight of each part of the brain tissue was weighed using an electronic analytical balance, and then placed in an oven at 100°C for 24 hours, it was then taken out and quickly weighed and recorded as dry weight. The dry-wet weight ratio was calculated by the dry-wet weight method formula.
2.8 Evans Blue permeability measurement. Under inhalation anesthesia, the external jugular vein was cannulated and injected with Evans Blue dye, and 30 ml of PBS buffer was infused intracardially to eliminate the cerebral circulation of Evans Blue. The hemispheres were isolated and weighed, each isolated hemisphere was homogenized in 2.5 ml PBS, the protein was precipitated, centrifuged, and the EB absorbance of the supernatant was measured using a spectrophotometer at 610 nm after ethanol dilution.
2.9 Double immunofluorescence detection. Neovascular endothelial and glial cells were labeled with CD31/Ki67, GFAP/Ki67 and Iba1/Ki67, and immunofluorescence staining was used to detect angiogenesis and gliosis in the peri-infarct area after pMCAO.
2.10 Enzyme-linked immunosorbent assay. Enzyme-linked immunosorbent analysis was performed as previously described [15] [16] [17]. The concentration of of VEGF, BDNF and eNOS protein content were determined by using ELISA assay kits (Bio-Rad, USA).
2.11 Statistical methods. All data analyses were performed using Prism 8 (GraphPad software). Statistical analysis was assessed by unpaired t-test. Data are presented as mean ± SEM, mean ± SD or median. The significance level was set at P < 0.05.