周邊神經損傷的病例中，端側神經接合手術的研發具有免犧牲捐贈神經的優點，但復原 後肌肉無力和弱動作電位的限制仍是待克服的最大議題。過去神經損傷的研究中更顯示 鈣離子所激發的多種訊息傳遞路徑與突觸後動作電位的調節息息相關。神經系統中 P/Q 型鈣離子通道(Cav2.1)與鈣離子的互動，決定了神經修復時突觸後電位之震幅大小與周 期長短。前人研究指出Roscovitine為一種細胞週期蛋白依賴型激酶抑制劑，可延長 P/Q 型鈣離子通道(Cav2.1)開啟、減緩活性進而促使鈣離子湧入，導致動作電位大幅增加。 然而 Roscovitine療法應用於神經肌肉的再生機轉與其療效仍待評估。 基於上述的疑問，我們試圖了解鈣離子通道於正常和損傷的動物模式中對於神經肌肉傳 導與調節的影響。實驗設計利用顯微手術動物，測試 Roscovitine是否能增強神經肌肉 傳導，進一步的協調及改善有限的功能。本研究將利用鈣離子指示劑與免疫螢光染色觀 察於小鼠運動神經細胞株(NSC34)探討 Roscovitine是否可藉調節 Cav2.1進而影響鈣離 子流動。研究 Roscovitine於鈣離子通道緩活化的同時，也以鈣離子通道拮抗劑(ω -agatoxins)前處理來反證其功效。 本研究將探討 Roscovitine是否作用於術後大鼠神經末梢的 Cav2.1，促使細胞內鈣離子 達到恆定，進而調節神經肌肉傳導，並得到功能性的改善。複合式動作電位和行為測試 將被用來評估功能恢復程度。共軛焦顯微鏡將協助觀察 Cav2.1於再生運動終板上分布 情形。為了呈現 Cav2.1和鈣離子之間的互動，我們將利用飛行式二次離子質譜儀呈現 鈣離子於再生神經肌肉交接處之離子影像。術後一個月的神經肌肉再生機制將利用定量 即時聚合酶鏈鎖反應、免疫墨點法、免疫組織化學染色與乙醯膽鹼酯酵素活性試驗進行 檢測。 本研究成果除了可以確認 roscovitine是否能改善術後療效和作用機制外，並可以另闢 神經修復新策略以期早日應用於臨床。 In peripheral nerve repair, end-to-side neurorrhaphy (ESN) is proposed as an alternative for end-to-end neurorrhaphy (EEN) for it saves the function of the donor nerve. Using rat ulnar nerve as donor and musculocutaneous nerve as recipient, we recently demonstrated that ESN resulted in slow amplitude of action potential and less than ideal recovery. It’s known that the P/Q-type voltage-dependent calcium channel 2.1 (Cav2.1) of motor end plates (MEPs) plays an important role in regulating Ca2 + release and synaptic transmission within the central and peripheral nervous systems. The Ca2 + -triggered multiple signal transduction pathways are essential to regulate postsynaptic potentials following peripheral nerve injury (PNI). It is indicated that at the regenerating MEPs after ESN, the amplitude and time-course of postsynaptic potentials were greatly influenced by the dynamic interaction of Cav2.1 and Ca2 + . Roscovitine is a calcium channel modifier which could open configuration of Cav2.1, and slow their deactivation kinetics and resulting in a large increase in total calcium entry during motor nerve action potential activity. Roscovitine is currently being evaluated for the treatment of neuromuscular weakness. However, the consequences of roscovitine treatment on the regenerating process of neuromuscular junction following nerve injury remain largely unexplored. To address this question, we will explore the effect of exogenous roscovitine on mouse motor neuron cell line (NSC-34) to find out whether and how it regulate Cav2.1 expression and mediating calcium influx by using calcium indicator and immunohistochemistry. These deactivate effects of roscovitine were reversed by pretreatment with the antagonists of voltage-sensitive calcium channels (ω-agatoxins). On the other hand, we will also use the animal model of ESN as a good paradigm for testing the ability of roscovitine in the processes of PNI relevant behavioral and functional recovery. ESN will be performed microscopically. Compound muscle action potential and behavioral test will be used to assess the functional recovery. The specific localization of Cav2.1 as well as its correlation with the regenerating axons on MEPs was examined by the use of confocal microscopy. To perform the dynamic interaction of Cav2.1 and Ca2 + at the regenerating MEPs, we will concentrate on calcium image at the regenerating neuromuscular junctions by the use of TOF-SIMS. Real-time polymerase chain reaction, immunoblotting, immunohistochemistry, and AchE activity assay will also be used to investigate the mechanism of actions during the first months following ESN. With the smooth processing of this study, the results obtained will not only give us a better understanding for the molecular mechanism of roscovitine in the facilitation of neuro-regeneration, but also shed important lights for clinical use of roscovitine in the promotion of nerve repair following PNI.
