神經痛是由於神經系統受到損傷而引起的慢性疼痛之一。為了更加了解其作用機制，許多能夠引發神經痛症狀如痛覺過敏和觸感痛的方法已被建立。在諸多實驗模型中，利用小鼠於其坐骨神經分支中產生損傷，即留下部分的分支(Spare Nerve Injury, SNI)，在過去幾十年已經被認為是一個具高再現性的實驗方法。雖然我們已經知道在進行這種手術後，小鼠會患有長期的神經痛，但其中之神經作用機制仍有待確認。關於痛覺的背後機理一直有不同的說法，有可能是受傷的神經元導致疼痛的產生，亦有可能是其周圍健康、保持完整的神經元造成，甚至兩者都有貢獻，因此還需要進一步的實驗來驗證。同時，在這些神經元的軸突中是否有形態上的改變也需要被確認。
在此篇研究中，我們首先使用C57BL/6J的成年公鼠實行各式的SNI手術，並刺激後腳掌的特定區域進而測量其抬腳之閾值，用以當作是否患有神經痛的指標。實驗結果發現:不論我們留下三條坐骨神經分支中的任何一條，都會造成小鼠長達至少兩週的神經痛。而在留下兩條分支的情況下，不管是留下common peroneal和tibial神經分支或是留下common peroneal和sural神經分支，皆會引發神經痛。但若是留下tibial和sural神經分支，則不會產生神經痛，而這個結果亦是符合預期的。除此之外，我們也驚訝地發現由健康神經元所引伸(innervate)的區域較由受傷神經元所引發的區域還來得快產生神經痛。而為了標記背根神經節細胞，我們使用了一種脂溶性的神經元標記物—— DiI，來標記細胞本體。而利用不同形式的SNI也讓我們能初步推斷各個坐骨神經分支神經元之細胞本體是位於哪個背根神經節。我們發現tibial神經分支的細胞核主要位於L4的背根神經節，而sural神經分支的細胞核則主要位於L3和L5的背根神經節。下一階段的實驗會針對特定的背根神經節進行pAAV-hSyn-FLEx-mGFP-2A-Synaptophysin-mRuby質體的注射用以雙重標記在神經痛後保持完整的細胞。整體而言，我們的實驗不僅再次驗證了SNI會引發神經痛，更重要的是研究神經痛時這些細胞型態上的變化以及它們在其中所扮演的角色。這不但可以讓我們更加了解神經痛背後的相關機制，同時也能幫助發展更有效率、更加精準的治療神經痛療法。

Neuropathic pain is one of the chronic pain that originated from the pathology of nervous system. To further investigate the underlying mechanisms of neuropathic pain, several methods have been developed to induce neuropathic pain symptoms such as hyperalgesia or allodynia. Among various kinds of the neuropathic pain animal model, the spared nerve injury (SNI) has been carried out in many studies with high reproducibility and robustness in the past few decades. Although SNI consistently induces allodynia in the mouse model, it remains unclear about the underline neuronal mechanism for chronic neuropathic pain. In addition, based on “the injured afferent hypothesis” and “the intact nociceptor hypothesis”, further studies should be performed to investigate the interaction between intact neurons and injured neurons and their morphological changes under allodynia condition.
In this study, we first measured the withdrawal thresholds in C57BL/6J adult male mice before and after variant kinds of SNI surgeries (i.e., sparing one or two specific branches of the sciatic nerve as one group). Our results showed that sparing one of any terminal branch of the sciatic nerve induces mechanical sensitization on specific areas of the hindpaw plantar skin compared to the sham control. Similar results could be obtained in the spared common peroneal and tibial nerve group, or in the spared common peroneal and sural nerve group. On the contrary, the spared tibial and sural nerve group did not show mechanical sensitization. Moreover, we surprisingly discovered that the region innervated from intact neurons displayed allodynia faster than the denervated region. By injecting DiI, a lipophilic tracer to label dorsal root ganglion (DRG) neurons that remain innervating to the paw after surgery, we found that the cell bodies of tibial nerve were mainly located in L4 DRG, while the cell bodies of sural nerve were mainly located in L3 and L5 DRG. Taken together, our data add to the growing evidence of SNI inducing allodynia. Most importantly, investigating the morphological changes of these DRG cells and their roles in neuropathic pain can provide novel insights and a step forward for understanding more broadly toward the underlying mechanisms of neuropathic pain and for developing more efficient and more accurate treatments on neuropathic pain.

口試委員審定書……………………………………………………………………i
Contents….……………………………………………………………………….…..ii
謝誌……………………………………………………………...………………….vii
摘要…………………………………………………………………………...……viii
Abstract………………………………………………………………………..……...1
Introduction………………………………………..…………………………………3
Statement of purpose……………………………………………….…….…………..8
Material and methods
Animals..…………………………….………………………………..…………….....9
Behavior-- von Frey test……………………………..………………………..………..9
DiI injection.…………………………………………………….……………………10
DiD injection.………………………………………………….……….…………..…11
DAPI staining.…………………………………………….…………….……………11
Images processing. ……………………………………………………….…………..11
Isolation and collection of dorsal root ganglion……………………………...……….12
Quantification of DiI-labeled dorsal root ganglion cells…………………...…………12
Parameters in the fitting curves………………………………………………….……12
Statistical analysis.……………………………………………………………………13
Spinal nerve injection.…………………………………………………...…………...14
Spared nerve injury.…………………………………………………………………..14
Results
1. The plantar skin mechanical sensitivity changes after the spared nerve injury
1.1 Sparing any of the sciatic nerve terminal branches produced significant mechanical sensitivity changes compared to the sham control…………………...….16
1.2 Sparing common peroneal and tibial nerve produced significant mechanical sensitivity changes….………………………………………………...………………17
1.3 Sparing common peroneal and sural nerve also produced significant mechanical sensitivity changes...…………………………………………..……….………….... .17
1.4 Changes in mechanical sensitivity were not observed after sparing tibial and sural nerve..……………………………………………………………………………...…17
1.5 The region innervated from intact neurons displayed allodynia faster than the denervated region..…………………………………………………………………...18
2. Quantification of DiI-labeled dorsal root ganglion neurons after SNI
2.1 The cell bodies of common peroneal and tibial nerves might evenly locate in L3, L4, and L5 DRG.……………………………………………………………………..19
2.2 The cell bodies of common peroneal and sural nerves might mainly locate in L5 DRG.…………………………………………………………………...……….….…19
2.3 The cell bodies of tibial and sural nerves might mainly locate in L4 and L5 DRG.…………………………………………………………………….……………20
2.4 The cell bodies of tibial nerve might mainly locate in L4 DRG.…………………………………………………………………….……………20
2.5 The cell bodies of common peroneal nerve were rarely locate in L3, L4, and L5 DRG.……………………………………………………………………………...…..20
2.6 The cell bodies of sural nerve might mainly locate in L3 and L5 DRG………....20
Discussion……………………………………………………………………………21
Significance of the work………………………………………………………….…24
Appendix I. ……………………………………………………………………….....87
Appendix II. ………………………………………………………………………...88
References…………………………………………………………………………...95

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