2.1 Chemicals and reagents
Rabbits anti-mouse 5-HT antibody and goat anti-rabbit secondary antibody were purchased from Invitrogen and Abcam (MA1-81821 and ab150077; California USA and Cambridge UK respectively).
2.2 Animals groups and Models
All experimental procedures comply with the guidelines of National Institutes of Health (NIH) “Guide for the Care and Use of Laboratory Animals and the legislation of the People’s Republic of China for the use and care of laboratory animals. The experimental protocols were approved by the Animal Experimentation Ethics Committee of the Hubei University of Traditional Chinese Medicine, China. A total of forty 3-month-old female Sprague–Dawley (SD) rats, weighing 200 g to 220 g, were provided by Tongji Medical College of HUST (SCXK (E) No. 2016-0057). In accordance with the random number table, rats were divided into four groups which are normal, model, EA and sham EA groups, with ten rats in each group. Rats in the normal group received 3 mg/kg·d of physiologic saline injection into the back skin, whereas the other rats were injected with isoproterenol hydrochloride (ISO), as previously described .
2.3 EA stimulation
The acupuncture stimulation involved the insertion of stainless steel needles (0.2 mm in diameter and 13.0 mm in length) into the acupoint PC6 at a vertical depth of 2-3 mm in groups EA and sham EA. Needles were manually twisted for one minute at a frequency of about 2 Hz before EA treatment. Rats in EA group received EA stimulation on bilateral acupoint PC6 with the following stimulus parameters: continuous-wave, frequency of 2 Hz, and current intensity of 1 mA, while rats in sham EA group received no electric current. They were treated for 20 min every day for 14 days.
2.4 Evaluation of regression of cardiac ischemia and cardiac hypertrophy
Reversion of cardiac hypertrophy was assessed by ratio of heart weight/body weight (HW/BW) and ratio of left ventricular weight/body weight (LVW/BW). Regression of cardiac ischemia was recorded by electrocardiogram (ECG) recording electrode resettlement as performed previously . For recording the standard Ⅱ-lead ECG, we used BL-420 physiological function experiment system. Its negative and positive stainless steel electrodes were placed beneath the skin close to bilateral left and right forelimb horizontal grain respectively, and the reference electrode was placed beneath the skin of the right hind limb. In process of recording, we selected a resolution ratio of 500nv/mv, chart speed of 50ms/div, and taken ten cardiac cycle into calculation.
2.5 Ultrastructure observation
Rats were quickly decapitated after anesthesia with intraperitoneal injection of 10% chloral hydrate (100 g/0.3 ml), and cardiac tissues were separated. The central ventricle muscle of left ventricular free wall was removed and chopped into 1mm×1mm×1mm size, fixed in 2.5% glutaraldehyde for 48 hours, then in 1% osmium tetrachloride for 1.5 hour, dehydration, embedding, sectioning, and examined on Hitachi H-600 transmission electron microscopy (Hitachi Company, Tokyo, Japan).
2.6 Infarct size measurement
Rats were euthanized and hearts were removed after 2 weeks modeling. In order to histological assessment, a small fragment from each heart was fixed in 10% buffered formalin solution and dehydrated with alcohol and xylene. After dehydration, each sample was embedded in paraffin wax and divided into slides of 5 μm thickness with a Leica rotation microtome. Groups of slides were stained with hematoxylin and eosin (H&E) based on standard methods. Areas of myocardial infarction were observed using ImageJ software (NIH, Bethesda, MD, USA).
2.7 Sucrose preference test
Rats were single placed in a quiet room with two bottles of sucrose solution (1%, w/v) for the first 24 h period. Then one bottle was replaced with tap water for the second 24 h period. After this initial adaption phase, rats were deprived of food and water for 24 h and then permitted access to two bottles for 3 h, one containing 100 ml of 1% sucrose solution and the other 100 ml of tap water. Sucrose preference was defined as the ratio of consumption of sucrose solution and the consumption of both pure water and sucrose solution during the test phase, and calculated as follows: percentage (%) = total sucrose consumption / (total sucrose consumption + total water consumption) × 100%.
2.8 Forced swim test
Briefly, each rat was subjected to pre-swimming habituation for 15 minutes on day 15 and tested 24 hours later. Rats were individually placed into plexiglas cylinders (internal diameter 21 cm; height 50 cm) filled with water (depth 30 cm; 25 °C) for 5 min of forced swimming in the training session. Time spent on swimming, striving, and immobile and the latency to immobility were recorded, using a swimming analysis system (EthoVision XT, Noldus, the Netherlands) to analyze.
2.9 Open-field test
Briefly, rats were placed individually at center of the field and were allowed to explore any areas freely in open field test. Then, behavior was recorded using a detection system (JLBehv-LAR-1; Shanghai Jiliang Software Technology Co., Ltd., Shanghai, China). The total and central distances explored during the 5-minute test were used to analyzed the behaviors.
2.10 Determination of gastric residual and gastrointestinal transit rates
Before commencing the experiment, animals were deprived of food for 24h but allowed free access to water. Each rat was received 1 mL/100 g nutritional black semi-solid paste and was sacrificed 20min later. Afterward, opening the abdominal cavity, stomach and small intestine were excised after ligation of the pylorus and the cardia, then the stomach was obtained, dried, and weighed. It was cut along the greater curvature, the contents were rinsed, and the stomach was weighed again. The difference in gastric weight showed the weight of the gastric residues. The gastric residual rate was calculated as follows: gastric residual rate (%) = (total gastric weight − gastric net weight) / intragastric volume × 100.
Immediately after excision of the stomach, the small intestine (from the pylorus to the ileocecum) was completely freed from its mesenteric attachments. Its total length and the carbon propulsion distance were measured by a meter scale. The gastrointestinal transit rate was calculated as follows: intestine propulsion rate (%) = carbon propulsion distance /total length of small intestine × 100%.
2.11 Immunofluorescence staining of 5-HT
The rat brain and terminal ileum was removed and sections (5 μm thick) were obtained consecutively. The sections were washed with phosphate-buffered saline (PBS), and processed with green-fluorescent Alexa Fluor 488 (1 : 500) to incubate. The nuclear marker DAPI was used. The fluorescence microscope (Leica, Germany) was used to observed and imaged these sections. SABC method was used to perform immunohistochemical staining of 5-HT protein. Positive staining of protein was shown in the cytoplasm. NeuN-positive cells were counted in three random images from each section by a technician blinded to this study.
2.12 Statistical analysis
Data were presented as mean ± standard error of mean (S.E.M) in this study. Unpaired student’s t-test was used to analyze two sets of data. Differences among groups were tested using one way or repeated measures analysis of variance (ANOVA). The Tukey–Kramer test was used to differentiate means. A P-value of less than 0.05 was considered statistically significant.