| Aim: The current study was to investigate the role of calcitonin gene-related peptide(CGRP) and nerve growth factor (NGF) in mediating the neurogenic reaction in acutemyocardial ischemia (AMI) by exploring the distribution of NGF and CGRP in dorsal rootganglion (DRG), spinal cord and heart of rats; exploring the change in NGF, CGRP and itsmRNA in DRG and heart at different time points after coronary artery occlusion (CAO), and theintervention effects of thoracic epidural anesthesia and pre-treatment with TrkA antagonists andcapsaicin.Background: Although increased evidence indicated the role of CGRP in cardiovascularsystem, the detailed exact regulation mechanisms were not fully established. Recent studiesfound that the concentration of CGRP was increased in myocardium when heart plunged intoacute infarction, indicating CGRP were involved in pathological process of AMI. Little is knownabout the the way of synthesis and release of this neuropeptide in myocardial ischemia, and NGFis one of the most important regulators.Methods: The experiment was performed in four steps. In step one, adult male SD ratswere randomly devided into two groups: Normal group, executed directly after anesthesia; CAOgroup, with the left anterior descending branch of coronary artery occluded under generalanaesthesia. Then rats were killed at 30min after CAO, and the samples were collected forimmunofluorescence (IHC) and hybridization in situ (ISH). The distribution of NGF, CGRP andits mRNA in DRG, spinal cord and heart of the rats were examined, and the innervation of heartwas also observed using silver staining.In step two, rats were randomly devided into two groups: CAO group and Sham group, withthe same scheme of surgery without CAO, were further devided respectively into 15min, 30min,1h and 6h subgroups. Then rats were killed as scheduled at different time points, and the sampleswere collected. MI model was successfully identified by ECG and TTC staining. The mRNA andprotein level of NGF and CGRP in ischemic myocardium and thoracic DRG were examined byreal-time quantitative PCR and ELISA.In step three, rats were randomly devided into three groups: Sham group, CAO group and EA (epidural anesthesia) group, were further devided respectively into 15min, 30min, 1h and 6h subgroups. At 15 minutes before CAO, rats were pre-treated epidurally with 1% ropivacaine 120 g.kg-1 or saline. Then rats were killed as scheduled, and the samples were collected for real-time quantitative PCR and ELISA.In step fore, the time points of 15min and 30min after CAO were chosen respectively for interventional study according to the results in step two. 1. Rats were randomly devided into three groups: Sham group, CAO group and AG879 group. At 15 minutes before CAO, rats were pre-treated epidurally with 10-7mol/L AG879 or saline. 2. Male SD rats were randomly devided into fore groups: Normal-Sham group, Normal-CAO group, Denervated-Sham group and Denervated-CAO group. Capsaicin sensitive sensory afferents denervated modle was prepared by administration of a single dose of capsaicin (50 mg/kg, injected subcutaneouly) to neonatal rats, and identified by radiant heat tail-flick test and heart weight/body weight(%). Then rats were killed as scheduled, and the samples were collected for real-time quantitative PCR and ELISA.Results:1. There is developed local innervational network in heart.2. CGRP was distributed widely but mainly in spinal dorsal horn, epicardial, atrial tissue and vascular endothelial; In DRG, CGRP and its mRNA was mainly expressed in small diameter neurons.3. NGF-positive cells mainly located in epicardium, fat tissue and blood vessels.4. The concentration of CGRP was significantly increased in myocardium and DRG of CAO animals (P <0.05). The expression ofβ-CGRP mRNA was significantly decreased at 15min in DRG of CAO animals (P <0.05);α-CGRP mRNA was up-regulated in CAO 6h group compared with CAO15min group (P <0.05).5. The concentration of NGF was increased markedly in myocardium and DRG of CAO animals (P <0.05). The expression of NGF mRNA in myocardium was significantly increased at 6h in CAO animals. NGF mRNA was up-regulated in myocardium and down-regulated in DRG in CAO 6h group compared with CAO15min group (P <0.05).6. Thoracic epidural anaesthesia attenuates the upregulation of NGF, CGRP and its mRNA in CAO animals (P <0.05), whereas enhances the trend of NGF mRNA in myocardium.7. AG879 attenuates the upregulation of CGRP and its mRNA in CAO animals (P <0.05).8. In denervated animals, heart weight/Body weight (100%) was significantly decreased, and tail flick latencies were significantly extended. Capsaicin pre-treatment down-regulates the expression of CGRP and its mRNA in denervated rats, whereas up-regulates NGF content substantially. Conclusions:1. There is typical distribution of NGF, CGRP in DRG and heart of rats, and CGRP is mainly derived from nerve tissue.2. AMI, as a nociceptive stimulus, activated the neurogenic reactions, and excessive expression of NGF and CGRP were involved in pathophysiological changes in AMI.3. Thoracic epidural anesthesia could partly inhibit the excessive expression of NGF and CGRP induced by ischemia. It may demonstrate that spinal nerves were involved in regulation of NGF and CGRP.4. NGF from DRG might play some role in regulation of CGRP in early stage of AMI.5. Denervation could damage the balance of nerve, and result in a disorder of reactivity to noxious stimuli accompanied with the increase of NGF. Further investigation on the subject is invited.
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