托吡酯相关的小鼠泌汗障碍模型建立和机制初步研究

发布时间：2018-02-22 07:39

本文关键词： 托吡酯泌汗障碍外泌汗腺乙酰胆碱酯酶胆碱乙酰转移酶毒蕈碱M3受体 GABA能神经元　出处：《第四军医大学》2007年博士论文　论文类型：学位论文

【摘要】： 托吡酯(topiramate,TPM)是一种新的抗癫vN药,其化学结构为2,3:4,5-双-O-(1-甲基亚乙基)-β-右旋-氨基磺酸吡喃果糖,在多种癫vN发作类型治疗中有效,包括局灶性癫vN发作、原发性强直阵挛发作、Lennox-Gastaut综合征等[1-2]。其作用机制有:1.可阻断神经元持续去极化导致的反复电位发放,此作用与使用TPM后的时间密切相关,表明TPM可以阻断钠通道[3];2.可以增加γ-氨基丁酸(GABA)激活GABAA受体的频率,加强氯离子内流,增强抑制性中枢神经递质的作用[4,5];3.降低谷氨酸AMPA受体的活性[6,7],抑制谷氨酸和天冬氨酸的释放[8],降低兴奋性中枢神经递质的作用;4.抑制碳酸酐酶,特别是其同工酶CA II和CA IV [9];5.对K+通道电流的调节作用[10];6.抑制神经元L-型电压激活钙离子通道[11]。最近陆续报道TPM在治疗儿童癫vN时出现少汗或无汗为主要表现的对热不耐受现象[12-17],该现象是可逆的,当停用TPM时,少汗表现消失。泌汗障碍发生率一般在10%-20%左右,但有研究发现未出现临床症状者,其发汗试验提示有出汗量下降,下降人数超过测试总人数的50%。这个现象提示TPM相关的泌汗障碍存在普遍性。目前TPM引起泌汗障碍的机制尚不清楚。根本的原因是汗液的调节机制尚处于研究中。有研究提示泌汗障碍与TPM对碳酸酐酶亚型抑制有关,该抑制作用减低了汗液原液的分泌,减少汗液的形成从而导致泌汗率下降。但作为一个酶类,其调节存在一个过程,机体并不能快速的调节此过程,因此这种假设还有待证实。在小鼠汗腺分泌细胞的管腔侧和基底侧均存在AQP5表达,有实验发现该蛋白参与了汗液的分泌过程。但也有研究提出该蛋白与泌汗没有联系。因此,泌汗障碍的原因有待进一步研究。泌汗通路起始于丘脑下部视前区,经脑干侧索中央下行,到达脊髓的中间外侧柱,位于中间外侧柱的节前神经元发出纤维到达同侧的交感神经节,换元后发出节后纤维支配汗腺器官。人类在高温或运动时的热驱散主要依靠小汗腺的蒸发而带来的散热作用。因此,汗液的产生对热耐受程度产生重要的影响,出汗可以防止运动中出现过热现象。汗腺由交感节后神经元轴突支配,不同于一般的交感神经节后递质,汗液的分泌以乙酰胆碱为主要神经递质。啮齿类动物的汗腺主要位于足垫和足指(趾),小汗腺的出汗可以由拟胆碱剂、肾上腺素等注射或热刺激运动刺激激发。TPM是如何引起在儿童的泌汗障碍的,是否与对胆碱能系统的作用有关系?实验以此着手展开研究。本研究首先采用硅胶印迹模型观察了TPM在小鼠引起的泌汗功能情况。其次在此模型基础上应用HE染色法和电镜下观察TPM引起的泌汗障碍是否与汗腺结构变化有关系;采用免疫组化染色观察支配汗腺的一般神经末梢、胆碱能神经、肾上腺能神经及胆碱能M3受体的表达变化。并结合蛋白印迹和免疫组织化学观察胆碱酯酶在汗腺的表达,进行胆碱酯酶活性测定。此外对GABA神经元参与脊髓和丘脑下部区域的交感活动进行了形态上的初步的探索。实验结果如下: (1)2周龄的幼鼠每天经灌胃给予TPM(80mg/kg),连续给药一个月后和停药后一周,对小鼠进行匹罗卡品刺激发汗试验,发现TPM处理后小鼠与对照组小鼠相比其分泌汗腺数目下降17%,单位汗腺的泌汗量下降40%。光镜结构和汗腺的超微结构未见明显改变。 (2)与对照相比,TPM处理对汗腺腺周一般神经支配PGP9.5、胆碱能神经支配标志物ChAT、肾上腺能标志物TH神经支配面积无明显影响;对胆碱能毒蕈碱M3受体表达以及对星状神经节神经递质表达无明显影响。单个汗腺分泌部的面积无明显变化。 (3)TPM处理后汗腺AChE免疫荧光及蛋白免疫印迹表达结果与对照相比无明显差异,汗腺组织匀浆后提示TPM对AChE活性无明显影响。 (4)交感节前神经元存在ChAT和GAD65免疫荧光双标记。热激活后,丘脑下部热敏感神经元与GABA能神经元存在双标记。从这些结果中可以初步得出以下结论: (1)TPM(80mgkg-1d-1)较长时期给药在一定程度上降低了小鼠的泌汗功能,停药后泌汗功能恢复。 (2)TPM导致的泌汗障碍没有影响到小鼠汗腺的面积大小和形态、超微结构。 (3)长期的TPM处理可降低小鼠外泌汗腺对匹罗卡品的分泌反应性,TPM未影响到小鼠汗腺的交感神经发育和支配、汗腺周围的胆碱能毒蕈碱M3受体表达,对汗腺部位的AChE表达及其活性也无明显影响。 (4)GABA能神经元可能参与节前胆碱能神经元调节、GABA能神经元参与丘脑下部对高温的反应。这种参与活动可能与TPM泌汗障碍相关。
[Abstract]:Topiramate (topiramate, TPM) is a new antiepileptic drug vN, the chemical structure of 2,3:4,5- double -O- (1- methylethylidene) - beta - - amino acid fructophyranose, in a variety of vN types in the treatment of epilepsy seizures, including focal epilepsy vN seizures, primary clonic seizures the mechanism of [1-2]. Lennox-Gastaut syndrome: 1. blocked repeatedly issued potential long-lasting depolarization of neurons caused by this effect and the use of TPM is closely related to the time, showed that TPM could block the sodium channel [3]; 2. can increase the gamma aminobutyric acid (GABA) GABAA receptor activation frequency, strengthen chloride influx enhancement of [4,5] inhibitory neurotransmitters; 3. [6,7] decreased the activity of glutamate AMPA receptors, inhibit the release of glutamate and aspartate [8], reduce the excitability of the central neurotransmitter role; 4. inhibition of carbonic anhydrase, especially the CA II and CA IV [9] isozyme 5. regulation of K+ channel current [10]; 6. inhibition of neuron L- type voltage activation of calcium channel [11].
TPM has been recently reported on heat intolerance [12-17] Hypohidrosis or anhidrosis mainly in the treatment of children with epilepsy vN, this phenomenon is reversible, with the cessation of TPM, less was disappeared. Hypohidrosis occurred at around 10%-20%, but the study found no symptom, the sweat test indicate sweating decline, decline in the number of exceed the total number of test 50%. suggesting that TPM related Hypohidrosis exists.
The mechanism of TPM induced Hypohidrosis is unclear. The basic reason is that the research is still in the adjustment mechanism in the sweat. Some studies suggest that Hypohidrosis and TPM on inhibition of carbonic anhydrase isoforms, the inhibitory effect of reduced secretion of perspiration, reduce the formation of resulting sweat sweat. But as the rate of decline a regulating enzymes, there is a process of adjusting the process of the body were not fast, so this hypothesis remains to be proven. The secretion of luminal side cells and basal side of the mouse sweat gland of AQP5 were detected, experiments have revealed that the protein involved in the secretion process of sweat. But there are also presented the urinary protein and no sweat contact. Therefore, the reason for Hypohidrosis needs further study.
The sweat pathway originates in the preoptic area of the hypothalamus, the central lateral funiculus of the brain stem down, reach the intermediolateral column of the spinal ganglion is located in the intermediolateral column of preganglionic neuron fibers from the ipsilateral reach, change the yuan after postganglionic fibers from sweat glands. Human dominated at high temperature or dissipate heat during exercise mainly rely on small sweat glands the evaporation caused by the cooling effect. Therefore, sweat production is an important influence on heat tolerance, sweating can prevent overheating in movement. The sweat glands innervated by sympathetic postganglionic neurons in sympathetic postganglionic axons, different neurotransmitters in general, sweat secretion by acetylcholine as the main neurotransmitter. The main rodent animal sweat glands located in the foot pad and fingers (toes), eccrine sweat by cholinergic agent, epinephrine injection or thermal stimulation of the motor stimuli how.TPM causes in children Whether the secretion of sweat is related to the effect on the cholinergic system? Experiments are conducted to start the study.
In this study, using silica gel blot model to detect the sweat function of TPM in mice. Then based on the model using HE staining and electron microscope observation of Hypohidrosis caused by TPM whether a relationship with the sweat gland structure changes; immunohistochemical staining observation of dominant nerve endings generally sweat glands, cholinergic nerve, adrenal gland adrenergic and cholinergic M3 receptor expression. Combined with Western blot and immunohistochemistry to observe the expression of cholinesterase in sweat, determination of cholinesterase activity. In addition a preliminary exploration on the morphology of GABA neurons in the spinal cord and the subthalamic region of sympathetic activity. The experimental results are as follows:
(1) 2 week old mice per day by intragastric administration of TPM (80mg/kg), administered continuously for a month later and after stopping for a week, the mice were pilocarpine stimulated sweat test, found that TPM treated mice compared with control mice the secretion of sweat glands secrete sweat number decreased by 17%, per unit of sweat gland no ultrastructural 40%. decreased light microscopic structure and sweat glands significantly changed.
(2) compared with the control group, TPM treatment of sweat gland innervation PGP9.5 weeks in general, cholinergic innervation marker ChAT, adrenergic marker TH innervation area had no obvious effect on cholinergic; expression of muscarinic M3 receptor expression and no significant effect on stellate ganglion neurotransmitters. Single sweat glands of the area no obvious change.
(3) the expression of AChE immunofluorescence and protein immunoblot in sweat glands after TPM treatment was not significantly different from that in contrast. After homogenization of sweat glands, TPM showed no significant effect on AChE activity.
(4) there is a double marker of ChAT and GAD65 immunofluorescence in the preganglionic neurons. After heat activation, there is a double mark in the hypothalamic thermosensitive neurons and the GABA neurons.
From these results, the following conclusions can be concluded:
(1) the administration of TPM (80mgkg-1d-1) to a certain extent reduced the function of sweat secretion in mice to a certain extent, and the function of secreting sweat was restored after the drug was stopped.
(2) the secretion of sweat caused by TPM did not affect the size and morphology of the sweat glands of mice and the ultrastructure.
(3) long term TPM treatment can reduce the secretion of reactive mouse eccrine sweat on pilocarpine, TPM did not affect the growth and sympathetic innervation of mouse sweat glands, sweat glands around the cholinergic expression of muscarinic receptors M3, AChE expression and activity of sweat gland has no significant impact.
(4) GABA neurons may participate in the regulation of cholinergic neurons before preganglionic. GABA neurons participate in the hypothalamic response to high temperature. This involvement may be related to TPM's sweating disorder.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R965;R-332

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