| BACKGROUNDAccording to the theory of traditional Chinese medicine (TCM), Neiguan (PC 6) is the collateral-point of the Pericardium Meridian of Hand-Jueyin, communicates with Yinwei Vessel; while Gongsun (SP 4) is the collateral-point of the Spleen Meridian of Foot-Taiyin and communicates with the Thoroughfare Vessel, both of which have a close relation to the heart, chest and the stomach. So, in clinic, they are used together to treat disorders of the heart, chest, stomach and abdomen. Clinical and experimental researches demonstrated that acupuncture of Neiguan (PC 6) could effectively relieve clinical symptoms and signs including angina pectoris, oppressed feeling in the chest etc., improve ECG-ST, left-cardiac performance, raise cardiac index, stroke volume, ameliorate blood rheology, lower blood triglyceride, serum endothelin and norardrenaline levels, suppress the activity of blood platelet, and regulate activity of the cardiac vegetative nerve. Experimental results of our lab and other laboratories showed that acupuncture of "Neiguan"(PC 6) of the cat, rabbit and rat in acute myocardial ischemia (AMI) model could reduce the area of the myocardial ischemia, raise the fluidity of the erythrocytic membrane, improve cardiac hemodynamics, myocardial microcirculation, substance metabolism and energy supply and protect myocardial untrastructure. "Gongsun" (SP 4) combined with "Neiguan" (PC 6) is often used to treat disorders of the gastrointestinal tract such as vomitus gravidarum, peptic ulcer, abdominal pain, digestive tract responses to chemotherapy for cancers, etc.. These two acupoints might improve functions of the vegetative nerve system and regulate secretions of various gastrointestinal hormones.However, most evidence obtained so far was based on the researches in the so called "one to one " mode, that is, responses of a single internal organ to EA of one acupoint were observed. Scarce work was performed to investigate the multiple organs' reactions to acupuncture stimulation of one acupoint. We had reported that EA stimulation of "Neiguan" (PC 6) produced relatively specific regulation on the functions of the heart. It is generally accepted in clinic that the combination of two points or more induces synergetic effects. Therefore, we simultaneouslyobserve the pathological changes of both heart and stomach in rats with myocardial ischemia during EA stimulation of "Neiguan"(PC 6) and "Gongsun"(SP 4) and aim to explore its underlying mechanisms.MATERIALS AND METHODS1. Electrophysiological study40 male Wistar rats were used in this work. They were fasted 24 hours prior to each test and then anesthetized intraperitoneally with the mixture solution of 1.5% chloralose (50mg/kg) and 25% urethane (420mg/kg). The rats were cannulated and ventilated artificially. After full exposure of the gastric antrum, a pair of stainless steel needle electrodes (with a 0.3 cm distance between them) were embeded under the serosa about 0.5 cm to the pylorus. The cervicothoracic lead ECG was recorded.These 40 rats were randomly divided into normal control group (thoracotomy without coronary artery ligation and without EA), model group (thoracotomy with coronary artery ligation, and without EA), "Neiguan" group (thoracotomy with coronary artery ligation and EA), "Gongsun" group (thoracotomy with coronary artery ligation and EA) and Neiguan +Gongsun group (thoracotomy with coronary ligation and EA), with 8 animals in each. Two filiform needles (being about 3mm apart) were inserted into each acupoint area and connected to a HANS device. The stimulation parameters were frequency of 2/15 Hz, current strength of 3 mA and a duration of 30 min.Before thoracotomy, 10 min recordings of normal electrogastrogram (EGM) and ECG were made in all the rats and used as the basic control in each rat. Following artificial ventilator and a 2-3 minutes' break after the thoracotomy, the EGM and ECG were recorded by using a Polygraph System (RM 6000, Nihon Kohden). Acute myocardial ischemia (AMI) model was established by occlusion of the descending anterior branch (DAB) of the left coronary artery for 30 min followed by reperfusion. In EA groups, EA stimulation was given following a significant elevation of ECG-ST segment. The bioelectrical signals were fed to the biophysical amplifiers and then fed to Powerlab/8S data acquisition and analysis system (ADInstrument Corp.) and finally analyzed with Chart5.0 software.All the data collected in this work were expressed as mean ± standard deviation (X±s) and analyzed with one-way analysis of variance (ANOVA) for comparisons among groups and the different time courses within each group. T test was used to compare data between two groups and those between two time courses in each group.PO.05 was considered significant. 2. Histochemical studyAfter the physiological test, transcardiac perfusion of normal saline and 4% paraformaldehyde was performed in 4 rats of each group, followed by collection of the tissues of the gastric antrum, duodenum, basis cordis, dorsal root ganglia, stellate ganglia, celiac ganglia, and the spinal cord of C7-T2 and T12-L2 segments and the celiac ganglia. These tissues, frozen, were cut into sections (20Wn thick) and mounted to glass slides separately. After nicotinamide adenine dinucleotide hydrogen phosphate-diaphorase (NADPH-d) histochemistry [for displaying the activity of nitric oxide synthase (NOS)] of the slices, examination was carried out under the microscope and the optical density measured with an image analysis system (MSP -2000 Spectrometer). RESULTS 1. Electrophysiological part1.1 ECG-ST segment:After ligation of DAB of the left coronary artery, ECG-ST segment elevated evidently in rats of model and 3 EA groups (P<0.05). At 30 min of ligation, the elevated ECG-ST segments declined to varying degrees in 3 EA groups. After release of the ligation, ECG-ST segment of model group did not restore to the control level and a significant difference still existed when compared with the basic values of pre-ligation and normal control group (PO.05). Whereas the ECG-ST segment in the 3 EA groups restored quickly with "Neiguan" group being the best, that is, there had no significant difference in comparison with the basic value of the same group and that of normal control group 20min after termination of EA (P>0.05). The second was Neiguan +Gongsun group, its ST segment restored to the control level at 30 min after termination of EA and there was no significant difference in comparison with normal control group. In "Gongsun" group, there was no significant difference between ST segment at 30 min after termination of EA and the basic control value, but the ST segment value was higher than that of normal control group (P<0.05). These data indicated that EA stimulation of "Neiguan", "Gongsun" and "Neiguan" + "Gongsun" points might obviously accelerate the recovery of myocardial ischemia with the order of the therapeutic effect being better in "Neiguan" group, good in "Neiguan"+"Gongsun" group and slightly poor in "Gongsun" group.1.2 Effect of EA on HRVFollowing occlusion of DAB of the coronary artery, all values of LF, HF and LF/HF were increased markedly (PO.05). And at 30 min of ligation, they increased further in model group, decreased obviously and restored near the normal level in the EA groups (P>0.05) and almost recovered in "Neiguan" group. After termination of EA, LF, HF and LF/HF of the 3 EA groups maintained near the levels of the basic control of each group and normal control group (P>0.05), but were significantly lower than those of model group (PO.05). LF in "Neiguan" group recovered more quickly which restored to the control level 30 min during ligation and 20min after the termination of EA. Besides, LF/HF of "Neiguan" group became normal 30 min after the termination of EA. On the contrary, LF and LF/HF in "Neiguan"+"Gongsun" group and "Gongsun" group didn't restore to their individual control levels, except HF. These data demonstrated that EA might markedly suppress disorder of the vegetative nerve induced by myocardial ischemia and restore the sympatho-vagal balance. The efficacy was in such a sequence as "Neiguan" group> "Neiguan"+"Gongsun" group > "Gongsun" group. 1.3 Effect of EA on EGGAfter ligation of the coronary artery, the amplitude and frequency of the slow waves of EGG decreased remarkably in model and EA groups (PO.05). At 30 min post the ligation, they were further decreased in model group but recovered to or near the control level in EA groups (P>0.05). Unlike the model group, the amplitude and frequency of the slow waves of EGG in 3 EA groups roughly restored to the control level after cessation of EA (P>0.05). Although there existed no significant statistical difference among the three different EA groups, with respect to the promotion of recovery of the amplitude and frequency of the slow waves of EGG, EA stimulation of "Gongsun" point produced the best effect, followed by the "Neiguan"+ "Gongsun" and "Neiguan" point respectively. There presented a similar tendency for the amplitude and frequency of the fast waves of EGG in the five groups. 2. Histochemical studyNADPH-d/NOS positive neurons and their processes were in blue. The general tendency was that stronger NADPH-staining cells were found in normal control group whereas light staining cells in model group, while the staining intensity of NADPH positive cells of acupuncture groups was between them. In the three EA groups, the intensity of NADPH-d staining in 3 EA groups was near that of normal control group.2.1 Heart:The beaded NOS positive products were mainly distributed in the walls of the larger blood vessels of the basis cordis. In normal control group, the positive products were in deep blue; in model group, they were obviously lighter, and in EA groups, the positive substances stained much darker than that of model group, in particular, those of "Neiguan" group were quite close to those of normal control group.2.2 Stomach and the duodenum:The NOS positive substances were mainly distributed in the mucous and submucous layers of the gastric antrum and duodenum. Most of the NOS positive neurons were round and ellipsoid, and fewer were irregular polygon. The cytoplasm was uniformly deep blue and the nucleus was light. In normal control group, more NOS positive neurons with intense staining were located in the gastric antrum and duodenum. In model group, the number of the neurons remarkably reduced with light staining. In 3 EA groups, the number of the positive neurons increased markedly compared with that of model group, and furthermore, the staining was much darker with the deepest in the "Gongsun" group that was near the normal control level.2.3 Stellate ganglia and the celiac ganglia:The NOS positive neurons were evenly distributed in the stellate and celiac ganglia. The neuronal bodies and the fibers were in dark blue with uniform staining of the cytoplasma and rare staining of the nucleus. Most neurons were round and ellipsoid. In normal control group, there were more NOS positive neurons in dark blue. In model group, there were fewer neurons with lighter staining than that of normal control group. In EA groups, the NOS positive neurons staining was darker than that of model group, in which the neurons of the stellate ganglia had dark staining in "Neiguan" group and "Neiguan"+"Gongsun" group, and neurons of the celiac ganglia had dark staining in "Gongsun" group and "Neiguan"+"Gongsun" group, that approximated to that of normal control group. 2. 4 C2-T2 the T12-L2 spinal dorsal ganglia:NOS positive neurons with uniform staining in dark blue, round or elliptic, were scattered in the ganglia. In blank group, there were more stained neurons in dark blue. In model group, there were fewer neurons with lighter staining than that of normal control group. In EA groups, the NOS positive neurons were stained somewhat darker than that in model group, in which the neurons of the C7-T2 spinal dorsal ganglia had dark staining in "Neiguan" group and "Neiguan'VGongsun" group, and neurons of... |
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