Study On The Effects Of Neural Mechanisms On Acute Myocardial Ischemia Induced By Coronary Artery Occlusion In Rats

Cardiovascular disease (CVD), especially coronary artery disease (CAD), is a major threat to the health of human people. Despite progress in new treatment strategies developed in the past few decades aimed at different pathomechanisms of CAD, morbidity and mortality due to CAD and its complications remains a clinical reality. In addition to the severity of ischemia, the body's innate ability to adapt injury is an important factor which influences the prognosis of CAD. Under normal conditions, the activity of heart is under the regulation of neural mechaisms.. During the periods of acute myocardial ischemia, exaggerated norepinephrine (NE) released from sympathetic nerve endings increases oxygen demand by stimulating heart rate and contractility and decreases oxygen supply by constricting coronary vessels. This vicious cycle accelerates the progression of cell damage in ischemic myocardium and potentates the arrhythmogenicity of NE. In addition to sensing tissue ischemia and initiating cardiac nociception, the regulatory function of the rich sensory innervation of the myocardium and the coronary vascular system is unknown. In this research, we want to investigate the effects of neural factors on myocardial ischemia induced by coronary artery occlusion through nerve block or selective chemodenervation of sensory afferents. There are three parts in this research: Firstly, the changes of substance P and its mRNA in myocardium and dorsal root ganglion during coronary artery occlusion were observed. Secondly, the effects of thoracic epidural anesthesia with ropivacaine on the myocardial injury induced by coronary artery occlusion were investigated. Finaly, the effects of capsaicin-sensitive sensory afferent neurons on myocardial injury and ventricular arrhythmias during coronary artery occlusion were studied with denervating animals.Part I Changes of Substance P and its mRNA in Myocardium and Dorsal Root Ganglion during Coronary Artery Occlusion in RatsObjective: The purpose of this study was to investigate the changes of substance P (SP) and its mRNA (preprotachikinin mRNA,β-PPT mRNA) in myocardium and dorsal root ganglion (DRG) after coronary artery occlusion.Methods: Healthy adult male SD rats were selected for these experiments. 48 rats were randomly divided into two groups: group control (group CON), with the same scheme of surgery but without CAO; group coronary artery occlusion (group CAO). Then group CON and group CAO were divided into 4 subgroups: 0.5h, 1h, 3h and 6h (n=6). Then rats were sacrificed at scheduled time points, and the samples of myocardium in ischemic area and DRG from T1-5 segments of DRGS were collected for immunohistochemistry (IHC) and ELISA. Another 12 rats were divided into group CON and group CAO (n=6). After CAO 0.5h, the samples of myocardium in ischemic area and DRG from T1-5 segments of DRGS were collected for Real time RT-PCR.Results:1,The expression of SP in myocardium and DRG was significantly increased after CAO as compared with the baseline in group CON (P<0.05). There was no significant difference in expression of SP between different time points (P>0.05).2,The levels ofβ-PPT mRNA in DRG and myocardium were significantly elevated after CAO compared with group CON (P<0.05), but the level of myocardium is significantly lower than that in DRG (P<0.001).Conclusion: CAO could evoke significant in SP and its mRNA in myocardium and DRG in rats, and the results might suggest that neurogenic mechanism may participate in the path-physiological proccess of myocardium ischemia.Part II Effects of Thoracic Epidural Anesthesia with Ropivacaine on Myocardial Injury Induced by Coronary Artery Occlusion in RatsObjective: The purpose of this study was to investigate the effects of thoracic epidural anesthesia with ropivacaine on cardiomyocytes apoptosis and content of TNF-αin ischemia area during the left anterior descending branch of coronary artery occlusion.Methods: Seventy-two healthy adult male rats were randomly divided into three groups: group control (group CON), with the same scheme of surgery without CAO; group coronary artery occlusion (group CAO) and group thoracic epidural anesthesia (group TEA). Each group was divided into 3 and 6 hours time points, twelve animals for each time point. Six animals were used for terminal deoxynucleotidyl transferase(TdT)一mediated biotinylated dUTP nickend—labe ling (TUNEL),caspase-3 activity respectively. After the success of intrathecal catheterization, the left anterior descending branch of coronary artery was occluded to establish the acute myocardial ischemia model. At the scheduled time of ischemia the rats were killed, the samples of myocardium and serum were harvested for further research. The apoptotic cardiomyocytes and activity of caspase-3 were tested by TUNEL method, metric assay method respectively. The level of TNF-αwas tested by ELISA methods.Results: In group CAO, the rate of apoptosis of cardiomyocytes,caspase-3 activity,TNF-αwere significantly increased (P<0.05). In group TEA, parameters were decreased significantly after 3,6h of ischemia (P<0.05) but higher significantly than that of group CAO.Conclusion: Upper thoracic segment nerve block can attenuate myocardial injury induced by coronary artery occlusion, which partly through inhibiting the generation of TNF-α. The findings may suggest that neural mechanisms are involved in the pathology of acute myocardial ischemia/infarction.Part III Selective Sensory Denervation by Capsaicin Aggravate Myocardial Injury and Ventricular Arrhythmias after Coronary Artery Occlusion in RatsObjective: The purpose of this study was to investigate the role of capsaicin-sensitive sensory neurons on the arrhythmia and myocardial injury evoked by coronary artery occlusion.Methods: Healthy adult male SD rats were selected for these experiments. All rats were randomly divided into two groups: group vehicle, group capsaicin. The rats in group capsaicin were treated with capsaicin subcutaneously in the sequence of 10, 30, and 50 mg/kg single daily doses for 3 days for selective chemodenervation of capsaicin sensitive sensory nerves. The animals in group vehicle were treated with equivalent amounts of the solvent of capsaicin. Seven days after the last injection when depletion of peptide-containing myocardial sensory nerves is already complete.Then the left anterior branch of coronary artery of all animals were occluded and were divided into two subgroups: CAO3h,CAO6h. The lead II was used for monitoring the changes in ECG. Then rats were sacrificed at scheduled time points, and the samples of myocardium in ischemic area were collected for capsase-3 activity assay,TUNEL staining and SP (CAO6h) assay. The other twelve animals (6 animals each group) were selected for left ventricular developed pressure (LVEDP) assay in isolated rat heart with Langendorff apparatus.Results:1,Baseline withdrawal latency prior to vehicle or capsaicin treatment did not differ significantly between groups (P>0.05). In the vehicle group, the paw withdrawal latency was not significantly altered by vehicle injection (P>0.05). In capsaicin treated rats the paw withdrawal latency was significantly increased (P<0.01).2,In capsaicin treated animals, the level of SP in the samples of myocardium in ischemic area after CAO 6h was significantly decreased (P<0.05).3,In capsaicin treated animals, the incidence of ventricular arrhythmias, including premature ventricular contraction,ventricular tachycardia and ventricular fibrillation during the first 30min of CAO were significantly increased (P<0.05).4,In capsaicin treated animals, caspase-3 activity and the rate of apoptosis of cardiomyocytes induced by CAO were significantly increased (P<0.05).5,Baseline LVDP prior to CAO did not differ significantly between two groups (P>0.05). In capsaicin treated rats, LVDP after CAO was significantly decreased in isolated rat hearts (P< 0.05).Conclusion:In capsaicin treated animals, myocardial injury and ventricular arrhythmias were aggravated after CAO. These results might suggest that capsaicin-sensitive sensory neurons may protect myocardial injury during the process of acute myocardium ischemia.