Effect Of Intrathecal Morphine Preconditioning On Spinal NGF-TRPV1 Sensitization After Myocardial Ischemia And Its Mechanism

Objective Cardiovascular disease is the main reason of disability and premature death worldwide, and it is mostly associated with ischemic heart disease. Restoring the tissue perfusion as soon as possible after acute myocardial ischemia is the most effective treatment measure, but reperfusion itself can also aggravate the myocardial damage, namely the ischemia- reperfusion injury(IRI). Transient receptor potential vanilloid type 1(TRPV1) is a kind of nonselective cation channel which plays great role in sensing, transmitting, and regulating noxious stimulation. Combination of nerve growth factor(NGF) with its receptor can sensitize TRPV1, and increase the sensitivity of the primary afferent neurons to noxious stimulus. The increased NGF after myocardial ischemia sensitizes TRPV1 receptors on primary afferent neurons, thus increases cardiac sympathetic nerve activity. The excessive activation of sympathetic nerve will increase myocardial oxygen consumption and affect the myocardial oxygen supply and demand equilibration and increase myocardial injury. Inhibition of the neurotransmitter release induced by the TRPV1 activation after myocardial ischemia could protect the myocardium against IRI by avoiding the excessive activation of sympathetic system. A large number of studies have confirmed that ischemic preconditioning(IPC) can protect the myocardium against IRI. Our previous study confirmed that intrathecal morphine preconditioning( ITMP) can activate central opioid receptors, so as to simulate the cardioprotective effects of IPC. However, the mechanism of activation of central opioid receptor by ITMP to protect myocardium from IRI has not been elucidated clearly. This study aims to discuss the effect on spinal NGF-TRPV1 sensitization after myocardial ischemia and mechanism, so as to clarify the mechanism of ITMP against IRI.Methods Healthy male Sprague-Dawley rats weighing 250~350 g, in which intrathecal catheters were successfully placed without complications were randomly divided into 6 groups(n = 9 each): Sham-operated group(SHAM group), ischemia reperfusion group(IR group), ischemic preconditioning group(IPC group), intrathecal morphine preconditioning group(ITMP group), μ receptor antagonist CTOP + ITMP group(CTOP + ITMP group), CTOP control group(CTOP group). Myocardial ischemia was induced by 30 min of occlusion of anterior descending branch of left coronary artery followed by 120 min of reperfusion in all groups, except for the SHAM group which only did sham operation. IPC were produced by three cycles of 5 min ischemia and 5 min reperfusion before 30 min myocardial ischemia, ITMP were produced by three cycles of 5 min intrathecal injection of morphine(3 ug·kg-1, 10 ul) and 5 min intermission before myocardial ischemia, IR were achieved by intrathecal injection of saline in the same way, CTOP + ITMP and CTOP were prepared by intrathecally administering CTOP(1 ug·ul-1,10 ul) 10 minutes prior to ITMP and 40 minutes prior to ischemia respectively. During the experiments, all rats’ vital signs were closely observed and written down, especially for the incidence of arrhythmia. The rats were sacrificed at the end of 120 min of reperfusion, and myocardial specimens were obtained for determination of myocardial infarct size as the percentage of area at risk(IS/AAR)(n = 6); and DRG specimens were obtained for double immunofluorescence to test the coexpression of TRPV1 and μ receptor in DRG, also for Western Blot to measure the expression of NGF and TRPV1(n = 3); and myocardial specimens, DRG specimens, spinal cord specimens were obtained for immunohistochemistry to measure the expression of NGF and TRPV1(n = 3).Results 1. Cardioprotection induced by ITMPBoth ITMP and IPC can protect the myocardium against IRI, and reduce the myocardial infarction and the incidence of arrhythmia. Intrathecal injection of μ receptor blockers CTOP before ITMP could block the cardioprotection induced by ITMP, suggesting the central μ receptor play a role in the cardioprotection induced by ITMP.2. Effect of ITMP on the coexpression of TRPV1 and μ receptor in rat DRG after myocardial ischemia.Both ITMP and IPC can significantly reduce the rise of TRPV1 expression in DRG, and increase μ receptor expression, thus reduce the coexpression of TRPV1 and μ receptor in rat DRG after myocardial ischemia. Intrathecal injection of μ receptor blockers CTOP before ITMP can block the effect of ITMP on the expression of TRPV1 and coexpression of TRPV1 and μ receptor in rat DRG.3. Effect of ITMP on the expression of NGF in rat DRG, spinal dorsal horn and myocardium after myocardial ischemia.Both ITMP and IPC can significantly reduce the rise of NGF in rat DRG, spinal dorsal horn and myocardium after myocardial ischemia. Intrathecal injection of μ receptor blockers CTOP before ITMP coud block the effect of ITMP on the expression of NGF.4. Effect of ITMP on the expression of TRPV1 in rat DRG, spinal dorsal horn and myocardium after myocardial ischemia.Both ITMP and IPC can significantly reduce the rise of TRPV1 in rat DRG, spinal dorsal horn especially the lamina Ⅰ,Ⅱ and myocardium after myocardial ischemia. Intrathecal injection of μ receptor blockers CTOP before ITMP could block the effect of ITMP on the expression of TRPV1.Conclusions The increased expression of NGF after myocardial ischemia could aggravate the myocardial damage by increasing the expression of TRPV1, sensitizing TRPV1 and rising the sensitivity of the afferent neurons to noxious stimulus; The cardioprotective effect of ITMP might be induced by activating the central opioid receptor and reducing the expression of NGF and TRPV1, whereafter, suppressing the sensitization of NGF-TRPV1 and reducing noxious stimulus.