HCN通道在神经病理性痛及焦虑抑郁中的作用和机制研究
发布时间：2019-04-03 07:26
【摘要】：慢性神经病理性痛和其伴发的焦虑抑郁产生机制复杂.最新研究发现外周神经损伤后,超极化激活的环核苷酸门控通道(HCN)表达明显改变,同时与焦虑、抑郁关系密切,但HCN通道在神经病理性痛中的作用目前尚不清楚.本研究拟探讨三个问题：1.侧脑室给予HCN通道阻断剂ZD7288是否可以缓解大鼠坐骨神经分支阻断模型(SNI)术后的疼痛,并改善动物焦虑、抑郁行为?2.SNI术后脑内HCN通道的变化如何,HCN通道的辅助亚基含Rab8b的TPR结构域相互作用蛋白(TRIP8b)是否参与调节?3.给药后脑内γ-氨基丁酸((ABA)的变化和其机制是否与谷氨酸脱羧酶(GAD)相关?方法： 128只Wistar Kyoto(WKY)大鼠(抑郁大鼠)分为三部分进行实验.首先选取40只WKY大鼠随机分为Veh、ZD7288(2.5μg/kg)、ZD7288(5μg/kg)、 ZD7288(10μg/kg)四组,通过自发活动实验观察ZD7288侧脑室给药后大鼠的自身活动.其次28只WKY大鼠随机分为Sham、ZD7288(5μg/kg)、ZD7288(10μg/kg)、 Saline四组,通过开场实验、高架十字迷宫实验、强迫游泳实验观察侧脑室单次给予ZD7288后抑郁大鼠的焦虑、抑郁情绪.最后60只WKY大鼠随机分为Sham、SNI、 SNI+ZD7288(5μg/kg)、SNI+ZD7288(10μg/kg)、SNI+Saline五组,通过机械性缩足反射实验、热辐射潜伏期实验观察ZD7288侧脑室连续给药14天痛阈的变化,通过开场实验、高架十字迷宫实验、悬尾实验观察大鼠神经病理性痛伴随的焦虑、抑郁情绪。行为检测后取材,通过免疫荧光染色观察HCN通道的表达,同时观察HCN与锥体神经元、中间神经元的共定位,通过western blot方法检测丘脑和海马内HCN通道、辅助亚基TRIP8b和GAD的表达,通过高效液相色谱检测大鼠脑内GABA的含量,并通过全细胞膜片钳记录ZD7288(10μM)灌流后微小抑制性突触后电流(mIPSCs)。结果：侧脑室给予2.5 μg/kg、5μg/kg、10μg/kg三种剂量ZD7288都不影响大鼠自发活动；侧脑室单次给予ZD7288 10μg/kg可以增加抑郁大鼠在高架十字迷宫开放臂的停留时间百分比,5μg/kg、10μg/kg两组抑郁大鼠强迫游泳实验自主活动时间延长；抑郁大鼠SNI术后侧脑室连续14天给予ZD7288 5μg/kg、10μg/kg可以不同程度提高机械痛阈,以10μg/kg作用更明显.在悬尾实验中,10μg/kg组自主活动明显增力口。Western blot结果显示丘脑内HCN2的表达明显增加,给予ZD7288后HCN2的表达有不同程度下降,但是辅助亚基TRIP8b变化不明显.在海马内,SNI术后HCN1表达上升,10μg/kg组HCN1表达明显下降,并且TRIP8b的表达与HCN1呈相反趋势.同时5μg/kg、10μg/kg两组丘脑和海马中GAD的表达与SNI组相比显著升高。高效液相色谱结果显示10μg/kg组丘脑中GABA表达明显升高,5μg/kg、10μg/kg两组海马中GAB A的表达显著增加.全细胞膜片钳结果表明海马脑片在给予ZD7288灌流后对mIPSCs没有增频增幅作用.结论：侧脑室给予ZD7288可以对WKY大鼠产生镇痛、缓解焦虑抑郁的作用,这可能是通过调节HCN通道的表达和兴奋性从而影响了GAD的表达,进而改变GABA的水平介导的.
[Abstract]:The mechanism of chronic neuropathic pain and its associated anxiety and depression is complicated. Recent studies have found that hyperpolarization-activated cyclic nucleotide gated channel (HCN) expression changes significantly after peripheral nerve injury, and is closely related to anxiety and depression. However, the role of HCN channel in neuropathic pain is still unclear. This study aims to explore three issues: 1. Can the intracerebroventricular administration of ZD7288, a HCN channel blocker, relieve the pain and improve the anxiety and depression behavior of the rat model of sciatic nerve branch occlusion after (SNI)? how about the changes of HCN channel in the brain after 2.SNI? Does the Rab8b-containing TPR domain interacting protein (TRIP8b) of the auxiliary subunit of the HCN channel participate in the regulation? 3. Is the change of 纬-aminobutyric acid (ABA) and its mechanism related to glutamic acid decarboxylase (GAD) after administration? Methods: 128 Wistar Kyoto (WKY) rats (depression rats) were divided into three parts. At first, 40 WKY rats were randomly divided into four groups: Veh,ZD7288 (2.5 渭 g / kg), ZD7288) (5 渭 g / kg), ZD7288 (10 渭 g / kg). Secondly, 28 WKY rats were randomly divided into four groups: Sham,ZD7288 (5 渭 g / kg), ZD7288) (10 渭 g / kg), Saline) group. The anxiety and depression of depression rats were observed by open field experiment, elevated maze test and forced swimming test after single administration of ZD7288 in lateral ventricle. Finally, 60 WKY rats were randomly divided into five groups: Sham,SNI, SNI ZD7288 (5 渭 g / kg), SNI ZD7288) (10 渭 g / kg), SNI Saline) group. The changes of pain threshold of ZD7288 were observed by mechanical foot contraction reflex test and thermal radiation latency test for 14 days after continuous administration of ZD7288. The anxiety and depression associated with neuropathic pain in rats were observed by opening experiment, elevated maze test and tail suspension test. The expression of HCN channel was observed by immunofluorescence staining, and the co-localization of HCN with pyramidal neurons and intermediate neurons was observed. The expression of HCN channel, auxiliary subunit TRIP8b and GAD in thalamus and hippocampus were detected by western blot method. The content of GABA in rat brain was determined by high performance liquid chromatography (HPLC), and the minimal inhibitory postsynaptic current (mIPSCs).) was recorded by whole cell patch clamp after ZD7288 (10 渭 M) perfusion. Results: administration of 2.5 渭 g / kg, and 5 渭 g / kg, 10 渭 g / kg of ZD7288 in lateral ventricle did not affect the spontaneous activity of rats. Single administration of ZD7288 10 渭 g / kg in lateral ventricle increased the percentage of open arm of depression rats in elevated maze, and prolonged the autonomous activity time of forced swimming in 5 渭 g / kg, 10 渭 g / kg group. Administration of ZD7288 5 渭 g / kg, 10 渭 g / kg into lateral ventricles of depressed rats after SNI for 14 days could increase the mechanical pain threshold in varying degrees, and the effect of 10 渭 g / kg was more obvious than that of 10 渭 g / kg. In tail suspension test, the results of. Western blot showed that the expression of HCN2 in thalamus was significantly increased in 10 渭 g / kg group, and the expression of HCN2 was decreased in different degrees after ZD7288 administration, but the change of auxiliary subunit TRIP8b was not obvious. In hippocampus, the expression of HCN1 increased after SNI, and the expression of HCN1 decreased significantly in 10 渭 g / kg group, and the expression of TRIP8b was opposite to that of HCN1. At the same time, the expression of GAD in thalamus and hippocampus in 5 渭 g / kg, 10 渭 g / kg group was significantly higher than that in SNI group. The results of high performance liquid chromatography (HPLC) showed that the expression of GABA in thalamus and GABA in hippocampus of 10 渭 g / kg group and 5 渭 g / kg, 10 渭 g / kg group were significantly increased. Whole-cell patch clamp results showed that hippocampal slices did not increase the frequency of mIPSCs after perfusion with ZD7288. Conclusion: intracerebroventricular administration of ZD7288 can induce analgesia and relieve anxiety and depression in WKY rats, which may be mediated by regulating the expression and excitability of HCN channel, thus affecting the expression of GAD and then changing the level of GABA.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614
[Abstract]:The mechanism of chronic neuropathic pain and its associated anxiety and depression is complicated. Recent studies have found that hyperpolarization-activated cyclic nucleotide gated channel (HCN) expression changes significantly after peripheral nerve injury, and is closely related to anxiety and depression. However, the role of HCN channel in neuropathic pain is still unclear. This study aims to explore three issues: 1. Can the intracerebroventricular administration of ZD7288, a HCN channel blocker, relieve the pain and improve the anxiety and depression behavior of the rat model of sciatic nerve branch occlusion after (SNI)? how about the changes of HCN channel in the brain after 2.SNI? Does the Rab8b-containing TPR domain interacting protein (TRIP8b) of the auxiliary subunit of the HCN channel participate in the regulation? 3. Is the change of 纬-aminobutyric acid (ABA) and its mechanism related to glutamic acid decarboxylase (GAD) after administration? Methods: 128 Wistar Kyoto (WKY) rats (depression rats) were divided into three parts. At first, 40 WKY rats were randomly divided into four groups: Veh,ZD7288 (2.5 渭 g / kg), ZD7288) (5 渭 g / kg), ZD7288 (10 渭 g / kg). Secondly, 28 WKY rats were randomly divided into four groups: Sham,ZD7288 (5 渭 g / kg), ZD7288) (10 渭 g / kg), Saline) group. The anxiety and depression of depression rats were observed by open field experiment, elevated maze test and forced swimming test after single administration of ZD7288 in lateral ventricle. Finally, 60 WKY rats were randomly divided into five groups: Sham,SNI, SNI ZD7288 (5 渭 g / kg), SNI ZD7288) (10 渭 g / kg), SNI Saline) group. The changes of pain threshold of ZD7288 were observed by mechanical foot contraction reflex test and thermal radiation latency test for 14 days after continuous administration of ZD7288. The anxiety and depression associated with neuropathic pain in rats were observed by opening experiment, elevated maze test and tail suspension test. The expression of HCN channel was observed by immunofluorescence staining, and the co-localization of HCN with pyramidal neurons and intermediate neurons was observed. The expression of HCN channel, auxiliary subunit TRIP8b and GAD in thalamus and hippocampus were detected by western blot method. The content of GABA in rat brain was determined by high performance liquid chromatography (HPLC), and the minimal inhibitory postsynaptic current (mIPSCs).) was recorded by whole cell patch clamp after ZD7288 (10 渭 M) perfusion. Results: administration of 2.5 渭 g / kg, and 5 渭 g / kg, 10 渭 g / kg of ZD7288 in lateral ventricle did not affect the spontaneous activity of rats. Single administration of ZD7288 10 渭 g / kg in lateral ventricle increased the percentage of open arm of depression rats in elevated maze, and prolonged the autonomous activity time of forced swimming in 5 渭 g / kg, 10 渭 g / kg group. Administration of ZD7288 5 渭 g / kg, 10 渭 g / kg into lateral ventricles of depressed rats after SNI for 14 days could increase the mechanical pain threshold in varying degrees, and the effect of 10 渭 g / kg was more obvious than that of 10 渭 g / kg. In tail suspension test, the results of. Western blot showed that the expression of HCN2 in thalamus was significantly increased in 10 渭 g / kg group, and the expression of HCN2 was decreased in different degrees after ZD7288 administration, but the change of auxiliary subunit TRIP8b was not obvious. In hippocampus, the expression of HCN1 increased after SNI, and the expression of HCN1 decreased significantly in 10 渭 g / kg group, and the expression of TRIP8b was opposite to that of HCN1. At the same time, the expression of GAD in thalamus and hippocampus in 5 渭 g / kg, 10 渭 g / kg group was significantly higher than that in SNI group. The results of high performance liquid chromatography (HPLC) showed that the expression of GABA in thalamus and GABA in hippocampus of 10 渭 g / kg group and 5 渭 g / kg, 10 渭 g / kg group were significantly increased. Whole-cell patch clamp results showed that hippocampal slices did not increase the frequency of mIPSCs after perfusion with ZD7288. Conclusion: intracerebroventricular administration of ZD7288 can induce analgesia and relieve anxiety and depression in WKY rats, which may be mediated by regulating the expression and excitability of HCN channel, thus affecting the expression of GAD and then changing the level of GABA.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R614
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