正中神經截斷後背根神經節中神經生長因子及其相關因子的表現變化

Abstract

Nerve growth factor（NGF）為神經滋養物質（neurotrophin）家族的一員，根據許多研究得知周邊神經損傷後，它往往與背根神經節中神經元表現型的改變，乃至於功能上的調整有所關聯。雖然已有相當多的研究以後肢周邊神經損傷的模式為題材，報導有關神經損傷後NGF、及其相關接受器和神經胜肽的變化，但是與正中神經損傷有關的研究則相當缺乏。因此本研究以正中神經截斷的神經瘤模式為主，並運用免疫染色方法、西方免疫轉漬法、電子顯微鏡及藥物的處理等方式，來探討背根神經節中，神經生長因子（nerve growth factor, NGF）及其接受器，TrkA及p75，還有其他相關的神經胜肽，如substance P及CGRP的表現變化情形。 我們將實驗動物進行正中神經截斷後，爾後將它們分別存活為1天、3天、1週、2週及四週不同的時間點及正常神經未損傷等組別，探討其第六頸椎背根神經節當中，神經生長因子及神經胜肽的變化。在NGF部分，利用免疫染色可以發現在手術後一天可以看到NGF的表現有增加的趨勢，三天開始下降，到一週相較於正常其表現量有很明顯的下降，而在兩週時則有回升的趨勢；以西方免疫轉漬法分析的結果，則發現NGF蛋白的表現量在一週時下降，而兩週時則上升甚至比正常組來得高，但是到四週又下降。在TrkA的表現方面，可以發現在手術後三天相較於正常組有上升的趨勢，持續增加維持到一週，而到兩週時下降的幅度最為明顯，至四週雖有些回升但是仍然處於低表現量狀態。在p75免疫染色方面，發現其表現量似乎在手術後各個時間點之間並沒有顯著的差異；但以西方免疫轉漬法分析，其結果則發現在正中神經截斷後一週，其表現量即有上升的情形，到兩週時達到高峰，爾後在四週則下降漸趨於正常組。另外我們探討背根神經節中NGF表現的神經元，分析及比較在不同的神經損傷點其細胞大小的分佈情形是否有所改變時，結果發現在手術後一週組相較於正常組，具NGF表現的神經元屬於中大型的比例已有明顯增加的趨勢。另外分析正中神經截斷後substance P及CGRP的表現變化情形方面，結果顯示神經損傷後一週，背根神經節中substance P及CGRP的表現量相較於正常組都有明顯地減少，這趨勢與NGF的表現變化頗為相似。除此之外，我們注射螢光金神經追蹤劑至截斷損傷的正中神經近端處，藉此標誌背根神經節中受損的神經元，並配合免疫染色法檢視NGF、TrkA、p75及相關的神經胜肽的神經元而來進行雙重標誌。經計量分析後，結果顯示在手術後四週組相較於一週組，雙重標誌的神經元占各免疫反應神經元的比例都有明顯下降的情形，由此推測這也許與NGF等各免疫反應細胞來自非受損神經元的數量，隨著神經損傷時程延長而增加所導致的結果。例如我們在電子顯微鏡下發現，背根神經節神經元周圍的satellite cell也有表現NGF及TrkA，這也許可以間接證實這些新合成的NGF代償神經截斷後所減少的數量。 最後我們在正中神經截斷手術前分別使用局部麻醉劑lidocaine及神經活性阻斷劑Tetrodotoxin等藥物處理，爾後分析及比較神經損傷後四週，背根神經節中NGF及TrkA的表現變化。結果顯示，術前藥物處理組相較於未處理組其NGF及TrkA的表現量並沒有顯著的差異，這結果不符合我們的預期。於是我們後續將以神經損傷後一週為觀察的時間點，再評估術前藥物處理對於神經滋養物質及神經胜肽的影響，以期待可以瞭解術前藥物的處理與緩解神經受損造成的神經病變疼痛之間的關係。

Nerve growth factor (NGF) is believed to play a critical role in altering the phenotypic and functional properties of dorsal root ganglion cells after peripheral nerve injury. It is well known that expression of NGF and pain-related neuropeptide in the primary afferent neurons change after peripheral nerve injury of lower limb, but remains uncertain following median nerve injury. In this study, immunocytochemical technique and western blot methods were used to investigate the expression and distribution of NGF, TrkA, p75, substance P and CGRP in the C6 dorsal root ganglion (DRG) at various time points following complete median nerve transection. Quantitative examination showed that the percentage of NGF immunoreactivity (NGF-IR) neurons in the C6 DRG increased at 1 day postoperation (PO) but decreased dramatically at 1 week (24.1%) PO compared with control (32.4%), and it recovered at 2 weeks (26.2%) PO. NGF western blot analysis and immunocytochemistry showed a similar expression pattern, except for the result at 2 weeks. The percentage of TrkA-IR neurons in the DRG increased at 1 week (36.7%) PO compared with control (31.3%) and significantly decreased at 2 weeks (20.2%), then it recovered at 4 weeks (22%). The percentage of p75-IR neurons had no manifest change between various time points, but we found that the p75-IR glia cell surrounding large-diameter neurons was significantly increased with post-injury times. Following p75 western blot analysis showed that the p75 protein level was increased at 1 week PO and peaked at 2 weeks PO. On the other hand, the cell size distribution of NGF-IR neuron was examined. It displayed that the proportions of medium-large neurons increase at 1 week (14.09%) compared with control (8.07%). In addition, we examined the percentage of substance P and CGRP immunoreactive DRG neurons after median nerve transection. The percentage of substance P-IR and CGRP-IR neurons dramatically decreased at 1 week PO and recovered at 2 weeks. These expression patterns are similar to that of NGF. With Fluorogold (FG) injection into the proximal end of transected median nerve, we examined the percentage of NGF-IR, TrkA-IR, substance P-IR and CGRP-IR neurons labeled with FG, respectively, was decreased at 4 weeks compared with that at 1 week. Taken together, the results in this study suggest that the above-mentioned four immunoreactive neurons derived from uninjured neuronal element increase as the extent of nerve injured course. Moreover, under electron microscopy examinations, we detected that several satellite cells surrounding the neurons were expressed NGF and TrkA, it maybe provided evidence to support such implication. Finally, we also examined the expression of NGF and TrkA in the DRG by pre-emptive lidocaine (local analgesic) and tetrodotoxin (TTX, electrical activity blocker) treatments on the median nerve prior to its transection. At 4 weeks PO, there was no significant difference in the expression of NGF and TrkA between pre-empitve and control groups. In the future, we will examine the effect of pre-emptive treatment the on the expression of NGF and TrkA in the DRG at 1 week PO.