鞘内吗啡预处理通过调控心肌缺血后脊髓神经元兴奋性发挥心肌保护作用的机制研究
发布时间：2018-09-01 13:57
【摘要】：背景与目的缺血性心脏病是我国目前致死率和致残率最高的疾病之一。虽然及时恢复缺血心肌血流灌注是治疗的重要手段,但心肌缺血后最终心肌梗死面积大约一半以上是由缺血再灌注损伤(ischemia reperfusion injury,IRI)所致。心肌缺血再灌注过程中局部微环境产生的缓激肽、腺苷、氢离子等可兴奋心肌缺血敏感性传入神经元,并将伤害感受信号经背根神经节(dorsal root ganglia,DRG)和相应节段脊髓上传至高级中枢。脊髓背角浅层尤其是脊髓背角II层又称胶状质SG(substantia gelatinosa,SG),是伤害性信息向中枢传递的初级门户,主要由兴奋性中间神经元组成,对伤害性刺激信息的传入与整合具有重要的调控作用。心肌缺血引起相应节段脊髓神经元兴奋,进而通过交感反射释放神经递质加重心肌细胞损伤。因此,调控脊髓背角神经元兴奋性,抑制神经递质的释放,可能会减轻缺血性心肌损伤。本课题组前期的研究发现,鞘内吗啡预处理(intrathecal morphine preconditioning,ITMP)可明显减轻心肌缺血后损伤。然而,ITMP对远端心脏发挥保护作用的神经机制尚未阐明,本研究拟探讨ITMP对抗心肌IRI的神经信号调控机制。方法SD雄性大鼠,体重180~280 g,成功建立鞘内置管模型。本实验分两个系列进行研究。第一部分:探讨ITMP对大鼠心肌缺血后脊髓SG神经元兴奋性的神经调控机制。实验随机分为6组(n=6),假手术组(SHAM组)、缺血再灌注组(IR组)、鞘内吗啡预处理组(ITMP组)、δ受体阻断剂NTD+ITMP组(NTD+ITMP组)、κ受体阻断剂nor-BNI+ITMP组(nor-BNI+ITMP组)和μ受体阻断剂CTOP+ITMP组(CTOP+ITMP组)。所有组除SHAM组外均以结扎冠状动脉左前降支缺血30 min、再灌注120 min的方法建立心肌缺血再灌注损伤模型。ITMP组在心肌缺血前30 min经鞘内泵注吗啡(3μg·kg-1,10μl)5 min,停止5 min,共3个循环;I/R组给予等容量生理盐水。NTD+ITMP组、nor-BNI+ITMP组以及CTOP+ITMP组分别于吗啡预处理前10 min鞘内分别注射NTD(1 ug·ul-1,10 ul)、nor-BNI(1 ug·ul-1,10 ul)、CTOP(1 ug·ul-1,10 ul)。于再灌注10 min结束时,急性分离大鼠T2~T6脊髓,制备脊髓组织切片,采用全细胞膜片钳技术对大鼠SG神经元进行动作电位(action potential,AP)记录,包括SG神经元静息电位(resting potential,RP)、动作电位阈值(threshold of action potential,APT)、动作电位峰值(peak of action potential,APP)、动作电位时程[用半峰时间(APD50)表示],记录步阶电流40、60、80、100 p A诱发的动作电位个数。第二部分:研究ITMP对心肌缺血后损伤及相关神经递质释放的影响。实验分组以及动物模型建立同第一部分研究。实验过程中记录心律失常的发生率。于再灌注120 min时处死大鼠,取心肌组织,测定梗死区(IS)体积与缺血危险区(ARR)面积,计算IS/ARR比值;Western blot法检测T2~T6脊髓和DRG组织中c-fos蛋白表达;免疫荧光观察T2~T6脊髓背角P物质(substance P,SP)、降钙素基因相关肽(calcitonin gene related peptide,CGRP)和内吗啡肽2(endomorphin 2,EM-2)的释放水平以及DRG中的SP和CGRP的表达情况。结果第一部分:探讨ITMP对大鼠心肌缺血后脊髓背角SG神经元兴奋性的神经调控机制结果发现,心肌缺血后大鼠脊髓背角SG神经元兴奋性增强,表现为APT降低,APP增加,相同步阶电流刺激电流下诱发的AP个数增多;而ITMP显著逆转IR组大鼠SG神经元的兴奋性升高,表现为APT升高,APP降低,相同步阶电流刺激下诱发的AP个数减少。各组大鼠脊髓背角SG神经元RP和APD50差异无统计学意义。与ITMP组相比,通过预先分别给予δ、κ、μ三种阿片受体阻断剂可以取消SG神经元兴奋性的降低,表现为APT降低,APP增高,相同步阶刺激下诱发的AP个数增加。第二部分:研究ITMP对心肌缺血后损伤及相关神经递质释放的影响1.ITMP介导的大鼠心肌保护效应:ITMP可明显降低心肌梗死体积和心律失常发生率,且NTD、nor-BNI和CTOP完全阻断ITMP的心肌保护作用。2.ITMP对大鼠心肌缺血后脊髓SP、CGRP和EM-2表达水平的影响:免疫荧光结果显示,SP、CGRP和EM-2免疫阳性产物在脊髓背角浅层存在共表达。心肌缺血后脊髓背角SP和CGRP大量表达,ITMP可以显著抑制心肌缺血后脊髓背角SP和CGRP的表达上调;EM-2在心肌缺血后脊髓背角的表达无明显变化,而ITMP可以显著上调心肌缺血后EM-2的表达。3.ITMP对大鼠心肌缺血后DRG内SP和CGRP表达水平的影响:免疫荧光结果显示心肌缺血后,DRG内SP和CGRP表达均显著增加,ITMP可显著减少心肌缺血再灌注损伤诱导的DRG内SP和CGRP的表达增加。4.ITMP对大鼠心肌缺血后脊髓背角和DRG内c-fos表达的影响:Western blot结果证明,心肌缺血后大鼠脊髓背角和DRG内c-fos明显增多,ITMP可显著抑制心肌缺血后大鼠脊髓背角和DRG内c-fos表达的增多;三种中枢阿片受体阻断剂能取消ITMP的抑制效应。结论ITMP对在体大鼠心肌缺血后损伤具有保护作用。其可能的神经调控机制:1.ITMP心肌保护作用与降低心肌缺血后大鼠脊髓SG神经元的兴奋性,减轻心脏伤害感受反应,调控脊髓水平伤害感受信号有关,δ、κ、μ三种中枢阿片受体均参与了这种保护作用。2.ITMP心肌保护作用与抑制心肌缺血后脊髓SP和CGRP等神经递质的过度释放,促进EM-2在脊髓水平的释放,降低神经元兴奋性有关。
[Abstract]:BACKGROUND & OBJECTIVE Ischemic heart disease is one of the most lethal and disabled diseases in China at present. Although timely recovery of ischemic myocardial perfusion is an important means of treatment, more than half of the final myocardial infarction area after myocardial ischemia is caused by ischemia reperfusion injury (IRI). Bradykinin, adenosine and hydrogen ions produced in the local microenvironment during reperfusion excite myocardial ischemia-sensitive afferent neurons and upload nociceptive signals via dorsal root ganglia (DRG) and corresponding segments of the spinal cord to the higher central nervous system. Gelatinosa, SG, is the primary portal of nociceptive information to the central nervous system. It is mainly composed of excitatory intermediate neurons. It plays an important role in regulating the input and integration of nociceptive stimulus information. Myocardial ischemia induces excitation of spinal neurons in corresponding segments, and then releases neurotransmitters through sympathetic reflex to aggravate myocardial injury. Previous studies have shown that intrathecal morphine preconditioning (ITMP) can significantly reduce myocardial ischemic injury. However, ITMP plays a protective role in the distal heart. Methods SD male rats, weighing 180-280 g, were successfully established intrathecal catheterization model. This study was divided into two series. Part I: To investigate the neural regulation mechanism of ITMP on the excitability of spinal SG neurons after myocardial ischemia in rats. Six groups (n=6), sham operation group (SHAM group), ischemia-reperfusion group (IR group), intrathecal morphine preconditioning group (ITMP group), delta receptor blocker NTD+ITMP group (NTD+ITMP group), kappa receptor blocker nor-BNI+ITMP group (nor-BNI+ITMP group) and mu receptor blocker CTOP+ITMP group (CTOP+ITMP group). All groups except SHAM group were ligated with 30 mi left anterior descending coronary artery ischemia. The model of myocardial ischemia-reperfusion injury was established by reperfusion for 120 minutes.Morphine was injected intrathecally 30 minutes before myocardial ischemia in ITMP group for 5 minutes and stopped for 5 minutes.The same volume of saline was given to I/R group.NTD+ITMP group,nor-BNI+ITMP group and CTOP+ITMP group were injected intrathecally 10 minutes before morphine preconditioning respectively. NTD (1 ug.ul-1,10 ul), nor-BNI (1 ug.ul-1,10 ul), CTOP (1 ug.ul-1,10 ul). At the end of 10 minutes of reperfusion, the T2-T6 spinal cord of rats was separated and the spinal cord tissue sections were prepared. The action potential (AP) of SG neurons, including resting potential (R-P) of SG neurons, were recorded by whole-cell patch clamp technique. P, threshold of action potential (APT), peak of action potential (APP), action potential duration (expressed as APD50), and the number of action potentials evoked by step current 40, 60, 80, 100 P A were recorded. Part II: To study the effects of ITMP on myocardial ischemia injury and related neurotransmitter release. The incidence of arrhythmia was recorded during the experiment. Rats were sacrificed at 120 minutes after reperfusion. The infarct area (IS) and the area of ischemic risk area (ARR) were measured and the ratio of IS to ARR was calculated. The expression of c-fos protein in spinal cord and DRG tissues of T2-T6 was detected by Western blot. The release levels of substance P (SP), calcitonin gene related peptide (CGRP) and endomorphin 2 (EM-2) and the expression of SP and CGRP in DRG were observed by immunofluorescence. Results Part I: To investigate the excitability of ITMP on SG neurons in spinal dorsal horn after myocardial ischemia in rats. The results showed that the excitability of SG neurons in spinal dorsal horn was enhanced after myocardial ischemia, which was manifested by the decrease of APT, the increase of APP and the increase of the number of AP induced by synchronous current stimulation, while ITMP significantly reversed the increase of excitability of SG neurons in IR group, which was manifested by the increase of APT, the decrease of APP and the induction of synchronous current stimulation. There was no significant difference in RP and APD50 of SG neurons in the spinal dorsal horn of each group. Compared with ITMP group, the decreased excitability of SG neurons could be cancelled by pretreatment with delta, kappa, and mu opioid receptor blockers respectively. The decrease of APT, the increase of APP and the increase of AP induced by synchronous stimuli were observed. Effects of ITMP on myocardial ischemia injury and related neurotransmitter release 1. ITMP-mediated myocardial protective effect: ITMP can significantly reduce myocardial infarction volume and arrhythmia incidence, and NTD, nor-BNI and CTOP completely block the myocardial protective effect of ITMP. 2. ITMP on the expression of SP, CGRP and EM-2 in spinal cord after myocardial ischemia in rats Immunofluorescence showed that SP, CGRP and EM-2 immunoreactive products co-expressed in the superficial layer of the spinal dorsal horn. After myocardial ischemia, SP and CGRP were overexpressed in the spinal dorsal horn. ITMP could significantly inhibit the up-regulation of SP and CGRP expression in the spinal dorsal horn after myocardial ischemia. EM-2 expression in the spinal dorsal horn after myocardial ischemia did not change significantly, but ITMP could significantly up-regulate the Regulation of EM-2 expression after myocardial ischemia. 3. Effect of ITMP on SP and CGRP expression in DRG after myocardial ischemia in rats: Immunofluorescence results showed that SP and CGRP expression in DRG increased significantly after myocardial ischemia. ITMP could significantly reduce the expression of SP and CGRP in DRG induced by myocardial ischemia-reperfusion injury. Effects of three central opioid receptor blockers on the expression of c-fos in dorsal horn and DRG: Western blot showed that the expression of c-fos in spinal dorsal horn and DRG increased significantly after myocardial ischemia in rats. ITMP significantly inhibited the expression of c-fos in spinal dorsal horn and DRG after myocardial ischemia in rats. Postoperative neuroprotective effects of ITMP on myocardial ischemia are related to decreasing excitability of spinal SG neurons, reducing cardiac nociceptive response, and regulating nociceptive signals at spinal cord level. Three central opioid receptors, delta, kappa and mu, are involved in this protective effect. 2. The protective effect is related to inhibiting the excessive release of neurotransmitters such as SP and CGRP in the spinal cord after myocardial ischemia, promoting the release of EM-2 at the spinal cord level and reducing the excitability of neurons.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614
本文编号：2217414
[Abstract]:BACKGROUND & OBJECTIVE Ischemic heart disease is one of the most lethal and disabled diseases in China at present. Although timely recovery of ischemic myocardial perfusion is an important means of treatment, more than half of the final myocardial infarction area after myocardial ischemia is caused by ischemia reperfusion injury (IRI). Bradykinin, adenosine and hydrogen ions produced in the local microenvironment during reperfusion excite myocardial ischemia-sensitive afferent neurons and upload nociceptive signals via dorsal root ganglia (DRG) and corresponding segments of the spinal cord to the higher central nervous system. Gelatinosa, SG, is the primary portal of nociceptive information to the central nervous system. It is mainly composed of excitatory intermediate neurons. It plays an important role in regulating the input and integration of nociceptive stimulus information. Myocardial ischemia induces excitation of spinal neurons in corresponding segments, and then releases neurotransmitters through sympathetic reflex to aggravate myocardial injury. Previous studies have shown that intrathecal morphine preconditioning (ITMP) can significantly reduce myocardial ischemic injury. However, ITMP plays a protective role in the distal heart. Methods SD male rats, weighing 180-280 g, were successfully established intrathecal catheterization model. This study was divided into two series. Part I: To investigate the neural regulation mechanism of ITMP on the excitability of spinal SG neurons after myocardial ischemia in rats. Six groups (n=6), sham operation group (SHAM group), ischemia-reperfusion group (IR group), intrathecal morphine preconditioning group (ITMP group), delta receptor blocker NTD+ITMP group (NTD+ITMP group), kappa receptor blocker nor-BNI+ITMP group (nor-BNI+ITMP group) and mu receptor blocker CTOP+ITMP group (CTOP+ITMP group). All groups except SHAM group were ligated with 30 mi left anterior descending coronary artery ischemia. The model of myocardial ischemia-reperfusion injury was established by reperfusion for 120 minutes.Morphine was injected intrathecally 30 minutes before myocardial ischemia in ITMP group for 5 minutes and stopped for 5 minutes.The same volume of saline was given to I/R group.NTD+ITMP group,nor-BNI+ITMP group and CTOP+ITMP group were injected intrathecally 10 minutes before morphine preconditioning respectively. NTD (1 ug.ul-1,10 ul), nor-BNI (1 ug.ul-1,10 ul), CTOP (1 ug.ul-1,10 ul). At the end of 10 minutes of reperfusion, the T2-T6 spinal cord of rats was separated and the spinal cord tissue sections were prepared. The action potential (AP) of SG neurons, including resting potential (R-P) of SG neurons, were recorded by whole-cell patch clamp technique. P, threshold of action potential (APT), peak of action potential (APP), action potential duration (expressed as APD50), and the number of action potentials evoked by step current 40, 60, 80, 100 P A were recorded. Part II: To study the effects of ITMP on myocardial ischemia injury and related neurotransmitter release. The incidence of arrhythmia was recorded during the experiment. Rats were sacrificed at 120 minutes after reperfusion. The infarct area (IS) and the area of ischemic risk area (ARR) were measured and the ratio of IS to ARR was calculated. The expression of c-fos protein in spinal cord and DRG tissues of T2-T6 was detected by Western blot. The release levels of substance P (SP), calcitonin gene related peptide (CGRP) and endomorphin 2 (EM-2) and the expression of SP and CGRP in DRG were observed by immunofluorescence. Results Part I: To investigate the excitability of ITMP on SG neurons in spinal dorsal horn after myocardial ischemia in rats. The results showed that the excitability of SG neurons in spinal dorsal horn was enhanced after myocardial ischemia, which was manifested by the decrease of APT, the increase of APP and the increase of the number of AP induced by synchronous current stimulation, while ITMP significantly reversed the increase of excitability of SG neurons in IR group, which was manifested by the increase of APT, the decrease of APP and the induction of synchronous current stimulation. There was no significant difference in RP and APD50 of SG neurons in the spinal dorsal horn of each group. Compared with ITMP group, the decreased excitability of SG neurons could be cancelled by pretreatment with delta, kappa, and mu opioid receptor blockers respectively. The decrease of APT, the increase of APP and the increase of AP induced by synchronous stimuli were observed. Effects of ITMP on myocardial ischemia injury and related neurotransmitter release 1. ITMP-mediated myocardial protective effect: ITMP can significantly reduce myocardial infarction volume and arrhythmia incidence, and NTD, nor-BNI and CTOP completely block the myocardial protective effect of ITMP. 2. ITMP on the expression of SP, CGRP and EM-2 in spinal cord after myocardial ischemia in rats Immunofluorescence showed that SP, CGRP and EM-2 immunoreactive products co-expressed in the superficial layer of the spinal dorsal horn. After myocardial ischemia, SP and CGRP were overexpressed in the spinal dorsal horn. ITMP could significantly inhibit the up-regulation of SP and CGRP expression in the spinal dorsal horn after myocardial ischemia. EM-2 expression in the spinal dorsal horn after myocardial ischemia did not change significantly, but ITMP could significantly up-regulate the Regulation of EM-2 expression after myocardial ischemia. 3. Effect of ITMP on SP and CGRP expression in DRG after myocardial ischemia in rats: Immunofluorescence results showed that SP and CGRP expression in DRG increased significantly after myocardial ischemia. ITMP could significantly reduce the expression of SP and CGRP in DRG induced by myocardial ischemia-reperfusion injury. Effects of three central opioid receptor blockers on the expression of c-fos in dorsal horn and DRG: Western blot showed that the expression of c-fos in spinal dorsal horn and DRG increased significantly after myocardial ischemia in rats. ITMP significantly inhibited the expression of c-fos in spinal dorsal horn and DRG after myocardial ischemia in rats. Postoperative neuroprotective effects of ITMP on myocardial ischemia are related to decreasing excitability of spinal SG neurons, reducing cardiac nociceptive response, and regulating nociceptive signals at spinal cord level. Three central opioid receptors, delta, kappa and mu, are involved in this protective effect. 2. The protective effect is related to inhibiting the excessive release of neurotransmitters such as SP and CGRP in the spinal cord after myocardial ischemia, promoting the release of EM-2 at the spinal cord level and reducing the excitability of neurons.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614
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