Effects of Electroacupuncture Applied to Neiguan (PC 6) and Shenmen (HT 7) on Norepinephrine Levels in Serum and in the Paraventricular Nucleus of Hyperlipidemic Rats with Surgically Induced Acute Myocardial Infarction

Experimental Animals and Instrumentation

Experimental animals: After approval of this study by the institutional review board (Animal Experimental Ethical Inspection of Anhui University of Traditional Chinese Medicine [TCM], No. 201108105), 100 healthy Sprague-Dawley (SD) rats were purchased from Nanjing Animal Experimental Animals Co. Ltd, Jiangsu Province (production license number: SCXK (Su) 2010-007). Of these rats, 50% were male and 50% were female; the weight range was 200±20 g. To promote adaptation to the laboratory setting, the animals were fed for 2 weeks under uniform conditions.

Reagents: Kits used included a Rat NE enzyme-linked immunosorbent assay (ELISA; Rapidbio Co.), a Rat NE radioimmunoassay (RIA; Phoenix Co.). Also used were urethane (Caoyang Secondary School Chemical Plant, Shanghai, China) and ultrapure water.

Instrumentation: Instruments used included: a Powerlab 16 physiological recorder (AD Instruments), a PCE-A type program-controlled EA device (Anhui Tianheng Technology Industrial Co. Ltd.), an Allegra 64R refrigerated centrifuge (Beckman), an ELX 800UV absorbance microplate reader (Biotek), a CM1900 cryostat (Leica), and acupuncture needles (Suzhou Medical Supplies Factory).

Assignment

Using a table for randomization, ⁶ 20 rats were selected to be in the sham control group. The remainder of the rats underwent ligation of the distal left anterior descending coronary artery. Of these, 20 rats were randomly assigned to each of the following three groups: the model control group; the group receiving EA at Neiguan (PC 6) and the group receiving EA at HT 7. Surgical deaths and model failures were, of course, excluded.

Model Duplication and Evaluation

To duplicate the hyperlipidemia model, all animals were fed a concentrated fat emulsion (10 mL/kg/day) for a total of 35 days. The dietary supplement was purchased from the Chinese Institute of Food Science. The AMI model was based on a method developed for establishing a model for chronic myocardial ischemia. ⁷ The animals were anesthetized with ether and positioned on an operative stage designed for rats. The skin of the left anterior precordium in each rat was prepared, and sterile drapes were applied in the usual manner. An incision was then made along the left sternum. The left pectoralis major muscle was bluntly dissected from the chest wall with a hemostat, exposing the second through fourth intercostal spaces. At the level where the apical beat was most prominent, the ribs were spread using a right-angle hemostat. The pericardium and anterior wall of the heart were thus exposed. The pericardium was opened and a ligature of 10-0 nylon then placed rapidly at the junction between the middle and distal one-third of the left anterior descending coronary artery. The surgical wound was then closed in layers.

All animals used in the study were treated humanely and in conformance with current standards of ethical animal research practices.

Evidence for a successful model included: duskiness of the apical region of the heart 1–2 minutes after placement of the ligature; persistent tachycardia; ST segment depression, T-wave flattening or T-wave inversion noted on on an electrocardiogram.

Additional confirmation of model success was achieved by laboratory measurement—performed 6 hours after surgery—of elevated troponin I and troponin T levels, indicating myocardial injury. In the animals who were accepted as models, a broad-spectrum antibiotic was administered to prevent wound infection.

Acupuncture Method and Parameter Selection

After restraint of each animal and preparing the skin, EA was performed at PC 6 and HT 7 in the two treatment groups of rats. Stainless steel 28-gauge, 0.5-inch needles were placed perpendicularly into the points, bilaterally, and connections were made to a PCE-A type program-controlled EA device. EA parameters were: stimulus voltage, 5 V; current, 2.0 mA; frequency, 2 Hz; pulse width, 300 μs; needle retention, 20 minutes. Treatments began 2 days after model confirmation and were continued daily for a total of 5 days. Acupoint location was in accordance with a standardized reference for experimental acupuncture. ⁸ Animals in the model control group were restrained in an identical manner each day for the same period but received no acupuncture treatment.

Measurement of PVN NE

The animals were anesthetized after completion of the 5-day course. A whole brain specimens was obtained from each rat and immediately placed on a dissection cold plate. Neuroanatomical structures were identified using a stereotactic map. ⁹ The PVN was located in the hypothalamus and carefully excised by sterile scalpel as a 2 mm×2 mm×2 mm tissue block. Samples were then placed in 3-mL freezing tubes, which were marked and then stored in liquid nitrogen. The duration of the entire tissue-harvesting process was strictly limited to a maximum of 5 minutes for each rat. ELISA assays for NE were subsequently performed, carefully adhering to test kit instructions.

Measurement of Serum NE

While under anesthesia and just prior to brain harvesting, 2–3 mL of blood was drawn from each rat's abdominal aorta, immediately put into a centrifuge tube, and allowed to sit at room temperature for 1–2 minutes. Each samples was then centrifuged at 3000 revolutions per minute for 15 minutes. The supernatant was collected and stored at –20°C. Subsequently, NE levels were measured by the RIA method, carefully adhering to test kit instructions.

Statistics

Continuous variables were considered as mean±standard error of the mean (SEM). SPSS 17.0 for Windows was utilized for data analysis. Pairwise comparison of means was performed using an analysis of variance and the least significant difference methods. p<0.05 for the difference was considered to be statistically significant.