2012-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/642608摘要：多年來我們實驗室致力於研究正中神經損傷所引起病理行為及生化改變，但是有關兼顧神經損傷程度的有效掌控及合理評估行為變化的實驗動物模式，為我們長期探究的重要方向。鑑於神經髓鞘剝離脫落是許多神經損傷模式的共通現象，並且被認為是造成神經病變疼痛的重要因子之一。根據許多文獻指出，溶血磷脂醯膽鹼(LPC)運用於小鼠坐骨神經，會造成其神經髓鞘剝離脫落及產生神經病變疼痛；但是欠缺有關以大鼠為實驗材料，更遑論以正中神經為研究重點的報告。因此本研究計畫的主要目的以LPC 處理正中神經的實驗模式、結合行為測試、免疫細胞化學、西方墨點轉漬法、神經追蹤劑注射、胞外電生理技術、配合藥物使用及電子顯微鏡技術等研究法，預計分三年的研究實驗期來探討背根神經節及楔狀神經核的疼痛相關生化因子，在神經處理LPC 後所產生的改變及其與神經病變疼痛產生的關係。茲將各年度的研究計畫主要目的及方法分別記載如下：《第一年研究計畫》：本年度計畫首先擬以LPC 注射於正中神經後檢視實驗動物行為變化、神經的放電情形、形態及髓鞘鹼性蛋白(MBP)的含量變化。另外利用正中神經截斷手術、神經追蹤劑標誌法及免疫螢光標誌法檢驗ATF3 及GAP-43 是否僅存在於背根神經節中的受傷神經元，然後以這兩者期望借為LPC 處理後神經損傷程度的指標；另外分析LPC 處理後背根神經節及楔狀神經核的疼痛相關生化因子的變化；《第二年研究計畫》：本年度計畫擬以LPC 注射於正中神經後，探討背根神經節nNOS 免疫反應神經元的型態、特性及其與神經病變疼痛的關係。主要分析LPC 處理後一週與正常組別，nNOS分別與NF200、ATF3、NPY、P2X3 及TRPV1 雙重標誌的數量及比例的變化。隨後以nNOS的促進劑或拮抗劑，觀察比較實驗動物神經病變疼痛行為的變化；進而分析在電刺激後，楔狀神經核中c-Fos 免疫反應神經元的數量是否因應有所調整變化；《第三年研究計畫》：本年度研究計畫主要在分析STZ 配合LPC 處理模擬糖尿病神經病變，分析其行為、神經形態及其nNOS 及NPY 在背根神經節與c-Fos 在楔狀神經核的數量及時程變化。另外比較LPC 處理的方法及部位的效果，進而分析以其代謝產物LPA 運用於神經上的效果。另外，探討以局部麻醉劑或LPA1 受體拮抗劑對LPC 所引發實驗動物後續行為、神經形態及其nNOS 及NPY 在背根神經節與c-Fos 在楔狀神經核的數量又有何變化。期望藉由這三年期的計畫，能更直接提供溶血磷脂醯膽鹼處理，造成神經髓鞘剝離脫落與神經病變疼痛之間的關係。<br> Abstract: We have devoted to studing changes in the neuropathic behaviors and biochemical factors aftermedian nerve injury for some years. It is important for us to approach an appropriate animal model forthe comparable damage magnitude and available behavioral assessment both. Because various injurymodels commonly result in demyelination, which seems to be a key mechanism of plasticity inneuropathic pain. Several previous studies have shown that demyelination and neuropathic pain weredetected in the mouse sciatic nerve treated with lysophosphatidylcholine (LPC). To our knowledge, theeffect of LPC treatment on the rat median nerve associated with the contribution to the neuropathicpain still needs to be examined. In the present proposal, we will utilized median nerve treated withLPC model, behavioral test, immunocytochemistry, western blotting, neuronal tracer injection,extracellular electrophysiological recording, drug treatment and electron microscopic study, toinvestigate the changes in pain-relevant factors expression in the dorsal root ganglion (DRG) and CNand relationships between this change and the development of neuropathic pain during three years.The details of the experimental intention and design will to be sequentially itemized in the followingyear, respectively: (The proposal of first year)：In this year proposal, we will first use LPC injectioninto median nerve to assess the effects on the development of neuropathic behaviors, the rate of spikes,morphology and myeline basic protein (MBP) level of the median nerve. Combing median nervetransection with neuronal tracing method and anti ATF3 and/or GAP-43 immunofluorescence labelingwill use to examine both of the two above-mentioned elements whether exist only in the injured DRGneurons. Then, the number of them will be regarded as the damage magnitude following LPCtreatment. Finally, we also will use immunolabeling methods to investigate the alterations in thepain-relevant factors expression in the DRG and CN after LPC treatment. (The proposal of secondyear):We will employ LPC injection into median nerve model along with immunocytochemistry tocharacterize the neuronal nitric oxide synthase-like immunoreactive (nNOS-LI) neurons in the DRGand its role in the neuropathic pain. Then, using double immunofluorescence labeling is going toinvestigate the number and percentage of nNOS-LI colocalized with NF200, ATF3, NPY, P2X3, andTRPV1 at 1 week after LPC treatment, respectively. Furthermore, we will use LPC injection alongwith nNOS inhibitor (L-NAME, 7-NI) or NO donor (SNAP) treatment to examine their effects on thedevelopment of neuropathic pain and expression of c-Fos in the CN. (The proposal of third year):Wewill subsequently utilize STZ treatment along with LPC injection diabetic rats to investigate thechanges in development of neuropathic behavior, morphology of median nerve, and the expressionlevel of nNOS and NPY in the DRG and c-Fos in the CN at various time points after LPC injection.Furthermore, we will compare the efficiency of different LPC delivery methods and sites and withLPA treatment. Subsequently, we will use LPC injection model combined with preemptive lidocaine,LPA1 receptor antagonist (Ki-16425) and blocker (Y-27632) treatment to investigate magnitude ofneuropathic pain, immunoreactive levels of nNOS and NPY in the DRG, expression of c-Fos in the CNat 1 week after LPC treatment.溶血磷脂醯膽鹼正中神經神經髓鞘剝離脫落神經病變疼痛神經性一氧 化氮合成酶神經胜肽Yc-Fos背根神經節楔狀神經核lysophosphatidylcholinemedian nervedemyelinationneuropathic painnNOSNPYc-Fosdorsal root ganglioncuneate nucleus