Animals

The experimental animals were clean-grade Sprague–Dawley (SD) male rats (8 weeks old, 200 – 250 g) purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd [Production License No.: SCXK (LU) 2019–0003] and raised in the Laboratory Animal Room of Key Laboratory of Xin’an Medicine Ministry of Education under controlled conditions (temperature: 24 ± 2 ℃, relative humidity: 50 – 60%, natural light 12 h alternating light and dark). The animals are free to eat and drink water. All experiments were conducted after 1 week of adaptive feeding. This study was performed according to the Guide for the Care and Use of Laboratory Animals of the Chinese National Institutes of Health. Approval for the study was granted by the Committee for the Care and Use of Research Animals of the Anhui University of Chinese Medicine (Approval No. AHUCM-rats-2022125).

Model replication

Rats were fasted and deprived of water for 12 h before MIRI model preparation. Rats were positioned supine on an operating table and given 2% isoflurane (1 L/minute) to induce anaesthesia. The limbs and head were fixed and electrocardiography (ECG) was performed from standard limb leads II. The left anterior descending coronary artery, which is situated between the left atrium appendix and the pulmonary artery cone, was exposed after the skin was cut from the left third to fourth intercostal space, the muscular tissue was separated, the chest was opened, and the pericardium was separated. The left anterior descending coronary artery was ligated with a sterile No. 6–0 suture needle (depth of entry: 0.5 mm) 1 – 2 mm outside the starting point of the branch. After ligation, the ischemic myocardial wall became cyanotic and bulging, and the ST-segment of the standard limb lead II was bowed and elevated, suggestive of the presence of myocardial ischemia. After 30 min, the ligature was loosened, the chest cavity was closed, gases were dischared, and the ischemic coronary artery was reperfused for 120 min. When the ECG displayed ST-segment or T-wave elevation and a reduction in ST-segment elevation of greater than 50% following reperfusion, MIRI modelling was deemed successful. In the sham-operation group, the left anterior descending coronary artery was not ligated after the chest was opened, and only one puncture with a needle at the corresponding site was performed. Rats with an abnormal ECG before model replication, those that died during the experiment, and those for which modeling was unsuccessful were excluded from the study.

EA pretreatment

The Shenmen (HT7) and Tongli (HT5) acupoints of the Hand Shao Yin heart meridian were selected for EA stimulation. Disposable sterile acupuncture needles (0.3 × 25 mm; Jiangsu Tianxie Acupuncture and Moxibustion Instrument Co., Ltd) were routinely sterilized before needling and then directly inserted 2–3 mm into the points. After needling, the “Shenmen” point was connected to the positive pole of the EA device (HANS-200A/100B; HANS, Beijing, China), and the “Tongli” point was connected to the negative pole,and were stimulated with a continuous wave of current at a frequency of 2 Hz and an intensity of 1.5 mA [22, 23]. The fake electroacupuncture (fEA) group chose non-acupuncture points in the tail of rats with the same electroacupuncture parameters [24]. The stimulation was repeated once a day for 30 min for 7 days. All EA operations were performed under isoflurane-induced anaesthesia, and the group that did not require EA was anaesthetised for the same duration using isoflurane.

Animal grouping and experimental design

To clarify the mechanism of action associated with the impacts of EA pretreatment on MIRI, the experiment was divided into three parts.

Experiment I

In the first part, 72 SD rats were randomly divided into four groups (Sham, Model, EA, and fEA) with 18 rats in each group. By observing the relevant indicators, it was determined which neurons were the key target of PVN in the process of EA pretreatment to alleviate MIRI.

Experiment II

To further clarify whether CRH neurons are engaged in the anti-MIRI effect of EA pretreatment, we performed fiber-optic recording experiments on CRH neurons within the PVN. Eighteen SD rats were randomly divided into Sham, Model, and EA groups, with 6 rats per group. For virus injection, rAAV-CRH-CRE-WPRE-hGH-polyA and rAAV-EFla-DIO-GCaMP6s-EGFP-WPRE-pA were mixed in a 1:2 ratio and then injected unilaterally into the PVN (250 nL, 50 nL/min). In the Sham group, no model replication was carried out, and fiber-optic recordings were made directly 21 days after virus expression. In the Model group, modeling replication was carried out 21 days after virus expression and fiber-optic recordings were made immediately after the completion of modeling. In the EA group, EA pretreatment was performed on 14 day after virus expression for 7 consecutive days, and model replication was performed at the end of EA; fiber-optic recordings were made immediately after model replication. After the completion of the experiment, the rats in each group were perfused and the brains were removed to observe the virus injection and optical fiber embedding sites.

Experiment III

To clarify the role of CRH neurons in the anti-MIRI effects mediated by EA pretreatment, 60 SD rats were randomly divided into five groups mCherry + Sham, mCherry + Model, mCherry + EA + Model, hM4Di + Model, and hM3Dq + EA + Model with 12 rats in each group. For virus injection, rAAV-CRH-CRE-WPRE-hGH-polyA and rAAV-EF1a-DIO-hM3Dq(hM4Di)-mCherry-WPREs were mixed in a 1:2 ratio and injected bilaterally (250 nL, 50 nL/min) into the PVN. In the hM4Di + Model group, the model was prepared on day 8 after intraperitoneal injection of clozapine N-oxide (CNO) for 7 consecutive days after 21 days of virus injection; in the hM3Dq + EA + Model group, CNO was injected intraperitoneally for 7 consecutive days after 21 days of virus injection, and EA was performed 40 min after the completion of the CNO injection, and the model was replicated on day 8 after the end of the EA; and in the remaining groups, an equal amount of CNO was injected. The anesthesia was induced by isoflurane, and the anesthesia time of each group was kept the same. At the end of the experiment, samples were taken to test the relevant indexes.

Virus injection and fiber-optic burial

Virus injection: rats were fixed on a stereotactic device after isoflurane-induced anesthesia. An incision was made to expose the skull and holes were drilled in the target brain area based on the PVN coordinates ([relative to Bregma]; anteroposterior [AP]: − 1.5 mm, rostrolateral [RL]: ± 0.35 mm, dorsoventral [DV]: − 7.6 mm;), with the location of the nucleus pulposus, determined from the fontanel, serving as the base point. Virus (250 nL) was injected bilaterally at a rate of 50 nL/min and the needle was left in place for 10 min after completion of injection on both sides; after injection, seamed the incision with sterile suture. Postoperative continuous intraperitoneal injection of penicillin for 3 days to prevent infection. Fiber-optic burial: after unilateral injection of the virus the fiber was added via a fiber optic gripper and buried about 0.2 mm above the virus injection site. The entire surface of the skull was covered with instantaneous adhesive. Then covered with dental cement, ear tagged and documented experimentally, and ensure free access to water and feed.

ECG recording

The electrocardiographic signals were acquired by PowerLab standard leads II. Then using LabChart 8 to analyse the ST-segment displacement values and the low-frequency (LF,0.04–0.15 Hz) /high-frequency (HF,0.15–0.4 Hz) ratios (LF/HF), respectively, for the first 5 min of the preparation of the MIRI model in the rats, for the 30 min of myocardial ischemia, and for the 120 min of reperfusion. Heart rate variability (HRV) at 120 min of reperfusion was analyzed using the Kubios HRV analysis software.

Enzyme-linked immunosorbent assay

The standards were prepared, spiked and incubated in strict accordance with the instructions of the ELISA kits. The absorbance values were measured by the enzyme marker and the serum levels of norepinephrine (NE) and creatine kinase isoenzyme MB (CK-MB) in the samples of each group were calculated according to the generated standard curve.

TTC-evans blue double staining

After the surgery was completed, the thoracic cavity of the rats was opened, and 1 mL of 2% Evans blue was injected into the apical part of the heart, which was allowed to perfuse the whole body with the blood, and the heart was removed after the limbs turned blue. Excess dye was washed off the hearts with pre-cooled saline, excess water was absorbed with filter paper, and the hearts were stored at – 20 ℃. After freezing, the heart muscle was cut into five 1-mm-thick slices at the ligation site. Sections were placed in TTC solution and incubated in an oven at 37 ℃ for 15 min, protected from light. Images were taken using a digital camera. The extent of myocardial infarction was calculated as the proportion of myocardial infarction area within the risk area, and the extent of risk was calculated as the proportion of the risk area within the total area of the cardiac section.

Measurement of sympathetic electrical activity

Detection of sympathetic nerve discharges in rats using in vivo electrophysiologic techniques. The rat’s neck was depilated, the skin was incised along the anterior midline, and the muscle was separated layer by layer to locate the left carotid artery, which was separated from the trachea. A clear sympathetic ganglion can be seen. Hooked off the sympathetic nerve below the sympathetic carotid ganglia (SCG) and placed a cling film containing paraffin below it at a temperature of 36 ℃ to isolate surrounding tissues and maintain the activity of the sympathetic nerve. A glass separation needle was used to hook the nerve, and a platinum wire microelectrode was positioned below it at a 30° angle. The negative electrode was placed subcutaneously on the same side of the neck. The signal should be amplified using AM-300 preamplifiers (A-M Systems, Sequim, WA, USA), and it should be collected using LabChart 8.0 software (ADInstruments, China) and the PowerLab data acquisition system. Utilizing the LabChart 8.0 Spike Histogram Module, analyze the spike frequency (gain, 1000 Hz; low cut filter, 100 Hz; high-cut filter, 1000 Hz).

Western blot

After 2 h of reperfusion, cardiac specimens were collected and tested for NE and Tyrosine hydroxylase (TH) protein expression. Samples were lysed in RIPA cell lysate (600 µL containing 0.6 mM PMSF) and then centrifuged at 12,000 rpm for 15 min to collect the supernatant. Gel configuration according to TRAKRA catalog. Add 5X SDS-PAGE protein uploading buffer at 1:4 to the collected protein samples. Heat in a boiling water bath for 15 min to fully denature the proteins. After the sample is cooled to room temperature, the protein sample can be directly sampled into the SDS-PAGE gel spiking wells. Add 5-10 uL per well. Electrophoresis at constant pressure 80 v for about 1 h. Pre-cut filter paper and PVDF membrane of the same size as the gel strip (pre-soaked in methanol for 2–3 min) were immersed in the membrane transfer buffer for 5 min. The membrane transfer device was placed in the order of anode plate, 3-layer filter paper, PVDF membrane, gel, 3-layer filter paper, and cathode plate from top to bottom, with the filter paper, gel, and PVDF membrane precisely aligned, and air bubbles removed at each step. Referring to the instructions of primary antibody, dilute with primary antibody diluent according to the appropriate ratio, and incubate overnight at 4 ℃ with slow shaking. Add the washing solution (PBST), wash for 10 min each time, wash three times in total. Referring to the properties of the primary antibody and the secondary antibody instructions, dilute horseradish peroxidase (HRP)-labeled secondary antibody with secondary antibody diluent according to 1:20,000. Incubate for 1.2 h at room temperature. Add washing solution (PBST) and wash for 10 min each time for a total of 3 times. Refer to the relevant instructions for the use of the ECL luminescent kit to detect proteins. The following antibodies were used: NE (Abcam, ab310335, 1:1000); TH (Abcam, ab315252, 1:3000); GAPDH (Zsbio, TA-08, 1:2000).

Hematoxylin and eosin staining

Myocardial tissue to be tested was placed in paraformaldehyde fixative. Ethanol gradient dehydration: 75% ethanol 1.5 h, 5% ethanol 1.5 h, 95% ethanol 2.5 h, anhydrous ethanol I 0.5 h, anhydrous ethanol II 0.5 h. Xylene transparency: xylene I 15 min, xylene II 30 min. After paraffin embedding, the slices were cut into 5 μm-thick slices, dehydrated by ethanol gradient, and stained with hematoxylin–eosin in the routine procedure.

Echocardiography

After 2 h of reperfusion, the wounds were sealed with absorbable sutures to avoid the influence of the sutures on the measurements. Rats were anaesthetised with inhaled isoflurane, chest hair was removed with depilatory agents, coupling agents were applied, and cardiac function was measured using a digital ultrasound device (Vinno 6 Lab, China) and an 18 MHz echocardiographic transducer. Left ventricular ejection fraction (LVEF), left ventricular short-axis shortening (LVFS), left ventricular end-systolic internal diameter (LVIDs), and interventricular septal (IVSs) thicknesses were then recorded using the M-mode.

Immunofluorescence staining

Rat brain slices were immersed in PBS and washed three times, 5 min each wash. The brain slices were then blocked in a base solution containing 0.5% Tritonx-100 (Biosharp, Anhui, China) and 5% BSA (Spark Jade, Shandong, China) for 1.5 h. Subsequently, the slices were placed in 0.3% Tritonx-100 + 5% BSA base solution, the primary antibody was added dropwise at the ratio of 1:500, and the slices were incubated at 4 ℃ overnight. The next day, after washing three times with PBS, 5 min each wash, the slices were placed in a base solution containing 0.3% Tritonx-100 + 5% BSA, and the secondary antibody was added dropwise at a ratio of 1:500, followed by incubation for 2 h at room temperature, protected from light. After three PBS washes, the slices were sealed with an anti-fluorescence quenching solution containing DAPI. After staining was completed, three randomly selected image were photographed. The quantification of c-Fos-, CRH-, and GABA- positive neurons was performed in Image-Pro Plus 6.0. The following antibodies were used: rabbit anti-c-Fos (Beyotime, Cat. No. AF6489), rabbit anti-CRH (Proteintech, Cat. No. 10944-1-AP), rabbit anti-GABA (SIGMA, A2062-2ML), goat anti-rabbit IgG (Proteintech, Cat.No.SA00001-2), goat anti-rabbit IgG (Beyotime, Cat.No.A0516).

Fiber-optic recording

After the surgery was completed, the rats were placed in special space containers. When they naturally awoke the fiber-optic connecting wire was attached to them and they were allowed to move freely. A multi-channel fiber-optic recording system (Chiaoxingo) was used to couple a 405 nm laser to a 470 nm blue light-emitting diode. Passage through the optical fiber was used to excite GCaMP6s fluorescence with a light intensity of 0.03 mW at the fiber tip. Band-pass filtered light was collected by a photomultiplier, and then an amplifier converted the current output from the photomultiplier to a voltage signal. Neuronal calcium signals were recorded in vivo using customized acquisition software at a frequency of 100 Hz. Further analysis was undertaken on MATLAB mat files to derive calcium signal (fluorescence) changes (ΔF/F), defined as (F − F0)/F0, which were presented as a graph of mean values or a heatmap.

Statistical analysis

GraphPad Prism 8 was used to analyze all the data, and the results were presented as mean ± standard deviation (S.D.). One-way ANOVA with a 95% confidence interval was used to assess the differences between three or more experimental groups. Tukey’s multiple comparison post-hoc test was then performed. Paired or unpaired parameter t-tests were used to examine the differences between the two groups with a 95% confidence interval.