【作者】 林宏；

【导师】 鞠躬；

【作者基本信息】 第四军医大学 ， 神经生物学， 2001， 博士

【摘要】 经颅重复性磁刺激（rTMS）作为一种研究手段已被广泛应用于人类脑生理功能许多方面如：运动功能、视觉、语言和脑疾病状态下的病理生理改变等的研究。近年来rTMS更是在治疗一系列的神经精神疾患如帕金森病、抑郁等上取得了较好的效果，但其治疗机制尚不明了，而且这些神经精神疾病又被认为是神经环路和神经元在细胞及分子水平上的功能失调，因此探讨rTMS所致的磁刺激对中枢神经系统中神经元基因活性、神经介质或/和调质、结构变化等的调控作用有着重要的意义。 基底节环路对来自皮质的信号加以处理，从而使自主运动得以正确地完成，帕金森病时黑质致密部多巴胺神经元的变性导致整个基底节结构的功能出现变化，特别是对基底节输出核团的影响更加明显；基底节还具有如同海马相似的对感觉运动信息门控功能，参与了认知、记忆等行为活动。 本研究首先用免疫组织化学方法观察了长程rMS对正常及6-OHDA损毁大鼠纹状体（ST）FosB蛋白表达的影响，以了解rTMS是否对纹状体神经元基因活性产生影响及多巴胺能在其中可能的作用。结果发现，rTMS后能够引起大鼠纹状体区域明显的FosB蛋白表达，这种表达广泛分布于尾壳核（CP）及苍白球（GP），尤以腹侧纹状体（VST）及尾侧的壳核明显。6-OHDA损毁后予以rTMS，损毁侧FosB蛋白表达未出现减少，且尾壳核的背外侧部表达明第四军医大学博士论文显上调。对照组动物仅损毁侧有少量的下。sB表达外，纹状体内未见F。sB阳性标记。 我们还观察到，lMS后大鼠海马齿状回及CAI区表现出明显增多及染色增强的F。sB免疫阳性产物，我们认为这些阳性标记更可能是F。SB的稳定变异型 A FOSB，CAZ、CA3区则未出现hSB免疫阳性产物；假刺激组动物仅海马齿状回表现出少许淡染的 F。SB免疫阳性产物，CA、CAZ、CA3区则无 F。SB免疫阳性产物出现。 酪氨酸羟化酶（TH）是基底节环路中重要的神经介质，在皮质纹状体长时程抑制（LTD）的产生上发挥着作用。帕金森病基底节输出核团特别是苍白球外侧部多巴胺（DA）明显下降，由于内源性 DA减少，导致皮质内抑制能力的下降可以出现运动功能的异常。低频rTMS能够使皮质的兴奋性降低也己得到证实。我们应用免疫组织化学法检测了 rTMS对苍白球及黑质（SN）等部位TH免疫阳性产物表达的影响。结果观察到，较之假刺激组动物，rTMS后大鼠 GP的 TH免疫阳性神经突起明显增多，尤以苍白球外侧部（GPe）明显；两组动物黑质致密部（SNC）和网状部（SNR）TH 阳性标记的细胞数目未见明显差别，但 rTMS组 TH阳性细胞染色加深，且 SNR和黑质外侧部（SNL）的TH阳性突起数量有所增加。 神经生长因子能保护及减少神经元的丢失或／和增强存活神经元的功能，新近的报道表明BDNF不仅控制着神经元的生长和生存，而且还具有新的功能一促进多巴肢D3受体的表达，调节神经元对多巴胺这种神经递质的反应。神经变性疾病如Alzheimer、Parkinson病时BDNF下降，而应用BDNF后出现了行为学上的显著改善。本研究中我们对fYMS后正常及6－OHDA损毁侧大鼠基底节BDNF免疫阳性标记物的改变进行了观察，结果显示，rTA4S后正常侧大鼠尾、壳核表现出明显增强的BDNF表达，与假刺激组相比，大量颗粒状阳性BDNF标记物集中于rTMS后大鼠的尾、壳核；苍白球细胞表现出更强的胞浆 4 第四军医大学搏士论文 及突起染色，且BDNF免疫阳性反应物的数量增多，特别是与纹状体相邻的区 域明显：BDNF在SNC和SNR阳性标记的神经元胞浆的着色深度亦明显增强。 注射6OHDA15d后，假刺激组大鼠注射例尾、壳核、苍白球及黑质阳性着色 的胞体和突起从数量和染色的深度上较对照侧明显减少和减弱；riMs组动物 则仍可观察到与对照侧类似的损毁侧尾、壳核密集的颗粒状BDNF阳性反应物 表达及大量胞浆深染的阳性细胞集中于苍白球和黑质。 氧化应激虽然不是唯一因素，但在lzhime和Parknson病中均发现了线 粒体电子传递链的异常，提示其在神经变性疾病神经元的损伤及死亡过程中发 挥着作用。本研究中，应用源自于人神经母细胞瘤的 TE671细胞，MTT法观 察了fi34s对培养的TE671细胞生存力的影响，rTMS处理组MTT法测定的 光密度（OD）值为1．ZI 99土0．11，假刺激组OD值为0．9928士0．互吕，Prto刀卜 将rTMS组和假刺激组TE671细胞与过氧化氢共培养，观察rTMS对细胞毒性 反应的影响发现，MTT法测定的两组OD值分别为0．5884土0．ZI和0．3287土 0．23，P＜0刀1。 尽管rTMS被认为是一种安全的手段而可以应用于临床，但从细胞和结构 水平上探讨其可能的伤害性效应的报道尚且不多。我们利用免疫组织化学法检 测 GPe及 SN等部位高分子量微管相关蛋白（HMW MAPZ）的免疫阳性产物。更多还原

【Abstract】 Repetitive transcranial magnetic stimulation (rTMS) has been widely used as a research tool to study aspects of human brain physiology including motor function, vision, language and pathophysiology of brain disorders. In recent years, rTMS has also been used as a therapeutic tool in a variety of neuropsychiatry disorders such as Parkinson disease and depression that were conceptualized in term of a dysfuction of neuronal circuits and neurons at a cellular and molecular level. However, the mechanism is unclear. To understand how magnetic stimuli induced by rTMS interact with central nervous system such as neuronal gene activity, neurotransmitter or modulator regulation and structure alterations are necessary.The basal ganglia circuitry processes the signals flow from the cortex, allowing the correct execution of voluntary movements. In Parkinson’s disease, the degeneration of dopaminergic neurons of substantia nigra pars compacta triggers a cascade of functional changes affecting the whole basal ganglia network especially the output nuclei of the circuit. Another possible function of the basal ganglia is gating sensorimotor processing as hippocampus plays, and involves in controlling many behavioral activities such as cognition and memory.In present study, we firstly observe the effect of long-term rTMS on FosB expressions in normal and 6-OHDA lesioned striatum immunohistochmically. Theresult shows, high FosB expressions are induced in the striatum of rTMS treated rats, especially in the rostral ventral striatum and caudal putamen. There is an apparent upregulation of FosB expression in the dorsalateral caudate putamen of 6-OHDA lesioned side after rTMS. In controlled animals, striatal cells do not express FosB- like immunoreactivity except few positive neurons appear in 6-OHDA lesioned side.We also find that high and deeply stained FosB expressions which most possibly are the stable variants of FosB?A FosB are induced in hippocampus of experimental rats, mainly locate in the granule cell layer of dentate gyrus and pyramidal cell layer of CA1 area. No FosB immunoreactive nuclei can be seen in CA2 and CA3 areas. In controlled animals, few and slightly stained FosB positive neurons are found in the granule cell layer of dentate gyrus, but no FosB expressions appear in CA1, CA2 and CA3 areas.Tyrosine hydroxylase (TH) is an important neurotransmitter in basal ganglia circuitry, and plays the role in inducing corticostriatal long-term depression (LTD). In Parkinson’s disease, endogenous dopamine in basal ganglia output nucleus especially external globus pallidus reduces, which induces the ability of intracortical inhibition decrease and then abnormal movement turns up. It has been approved that low frequency of repetitive transcranial magnetic stimulation can depress the motor cortex excitability. In current study, we investigate the changes of TH immunostaining in globus pallidus (GP) and substantia nigra (SN). Compared with that in control group, high TH positive projections are induced in the GP of experimental rats, especially in the external part of GP (GPe). No significant difference of the numbers of TH-positive cells in substantia nigra pars compacta (SNC) and pars reticulata (SNR) in both groups, but TH-positive projections inSNR and lateral substantia nigra (SNL) of rTMS group animals increase than that in sham stimulation group and cells stain darkly in rTMS group.Newly report showed that brain derived neurotrophin factor (BDNF) not only controlled the growth and survival of neurons, but also had another task: boosting the expression of dopamine D3 receptors that allowed neurons to respond to the neurotransmitter dopamine. BDNF reduced in neurodegenerative disorders such as Alzheimer disease and Parkinson’s disease, and significant behavioral improvement could be induced when BDNF were applied. The effects of long-term of rTMS on the BDNF expression in normal and 6-OHDA lesioned basal ganglia are studied in present research. Significant increase of large BDNF immunoreactive gr更多还原