Mechanisms Of Intrathecal Morphine Preconditioning Exerting Cardioprotective Effects By Modulating Spinal Neural Excitability After Myocardial Ischemia

Objective Ischemic heart disease is one of the diseases with the highest rate of mortality and disability in our country currently.The immediate reperfusion for ischemic myocardium is an important method,however,over half of the infarct size is caused by the ischemia-reperfusion injury(IRI)ultimately.The acidic materials,adenosine and bradykinin as well from myocardium during reperfusion excites cardiac afferent sensory neurons and deliver the nociceptive signal to the advanced central nervous system from dorsal root ganglia and relevant spinal cord segments.The so-called substantia gelatinosa(SG)is the primary gateway for the nociceptive signal transmitting to the central nervous system,which consists of excitatory intermediary neurons to regulate the afferent and integration of the nociceptive signals.Myocardial ischemia excites the relevant spinal cord neurons and subsequently aggravates the cardiomyocytes injury by the neurotransmitter release due to the sympathetic nerve reflex.Therefore,inhibiting the excitation of the spinal cord neurons and excessive stimulation of cardiac sympathetic nerve is able to protect the myocardium.The study in our lab showed that intrathecal morphine preconditioning(ITMP)attenuated the ischemia-reperfusion injury significantly.However,the nerve mechanism of this protection is still unknown,and this study was designed to investigate the mechanism of ITMP regulating the signal to deliver the cardiprotection against myocardial IRI.Methods Intrathecal catheter placement model was established in male SD rats,weighing 180-280 g.This study consisted of two parts for the study,in part one,which was to investigate the alteration of SG neuron excitability and the mechanism of ITMP regulating the SG neuron excitability in spinal cord after ischemia,the rats were divided into six groups according to a randomized number table(n=6): SHAM group.Ischemia-reperfusion group(IR),ITMP group,d-receptor antagonist NTD+ITMP(NTD+ITMP),k-receptor antagonist nor-BNI+ITMP(nor-BNI+ITMP),m-receptor antagonist CTOP+ITMP(CTOP+ITMP).The left anterior descending branch of coronary artery was ligated for 30 min followed by 120 min of reperfusion,except for the SHAM group,to establish the IRI model.In ITMP group,the rats were intrathecally injected with morphine(3 μg·kg-1,10 μl)for 5 min intermitted with 5min pause,3 cycles in total 30 min prior to ischemia.The rats in IR group were infused with the same volume of saline.And the rats were injected with NTD(1 ug·ul-1,10 μl),nor-BNI(1 ug·ul-1,10 μl)and CTOP(1 ug·ul-1,10 μl)10 min before morphine preconditioning,respectively in the groups of NTD+ITMP,nor-BNI+ITMP and CTOP+ITMP.The relevant spinal cord of T2-T6 was acquired immediately at the end of10 min of reperfusion and produced into slices for recording the action potential(AP)of SG neurons via whole-cell patch clamp technique,including the resting potential(RP),threshold of action potential(APT),peak of action potential(APP)and duration of action potential(APD50).The number of action potential provoked by step currency at four levels(40,60,80 and 100 p A)were also gathered.In part two,which was to study the effect of ITMP on IRI and the release of relevant neurotransmitters,the rats were also divided into six groups mentioned above.The rats were sacrificed at the 120 min of reperfusion to calculate the infarct size(IS)and the area at risk(AAR)as well as the ratio of IS/AAR for the hearts.The expression of c-fos in the spinal cord of T2-T6 and DRG were detected by western blot.The release of the substance P(SP),calcitonin gene related peptide(CGRP)and endomorphine-2(EM-2)in the spinal cord of T2-T6 was observed through immunofluorescence.Also,the double immunofluorescence was carried out to determine the co-existence of the SP and CGRP.Results Part one Investigation on the alteration of SG neuron excitability and the mechanism of ITMP regulating the SG neuron excitability in spinal cord after ischemiaThe results showed the excitability of SG neurons in spinal dorsal horn was increased significantly induced by IRI,the APT was repressed while the APP was elevated,and the number of AP provoked the same step current was increased.ITMP markedly revered this alteration.Further,compared to the group of ITMP,pre-injections of the three opioids receptors antagonists abolished the ITMP mediated effect.Part two Study the effect of ITMP on IRI and the release of relevant neurotransmitters1.The ITMP-induced cardioprotection in SD rats: The myocardial infarct size and the arrhythmia were reduced significantly by ITMP,and this protective effect was abolished by NTD,nor-BNI and CTOP.2.The impact of ITMP on the expressions of SP,CGRP and EM-2 in spinal cord after myocardial ischemia: Double immunofluorescence showed the co-existence of SP and EM-2 which mainly located in the superficial laminae of spinal dorsal horn.The strong expression of SP in the spinal dorsal horn after ischemia was inhibited markedly by ITMP.And ITMP significantly up-regulated the expression of EM-2 which did not alter distinctly after ischemia.3.The effect of ITMP on the expression of SP and CGRP in DRG after myocardial ischemia: Double immunofluorescence showed the significant co-existence of SP and CGRP in DRG.The IRI induced increase of SP and CGRP were significantly attenuated by ITMP.4.The effect of ITMP on the post-IRI level of c-fos in spinal cord and DRG: The western blot showed that ITMP restrained the IRI induced increase of c-fos in spinal cord and DRG,and pre-injections of NTD,nor-BNI and CTOP blocked this restraint.Conclusion ITMP conferred cardioprotection against myocardial ischemia and reperfusion injury in SD rats.And the possible mechanisms were as following:1.The ITMP delivered cardioprotection weas mediated by repressing the excitability of SG neurons in spinal cord and the cardiac response to nociception,as well as regulating nociceptive signal of spinal cord after ischemia,in addition,d,k and m-receptors were all involved in this protection.2.ITMP conferred cardioprotection was induced by inhibiting the over release of SP and CGRP,up-regulating the release of EM-2 at the spinal level,as well as restraining the excitability of spinal neurons.