辣椒素(capsaicin)造成之周邊神經病變:小鼠後腳掌重複施打辣椒素引起之表皮神經消失及功能性變化

Abstract

神經毒素造成的神經病變(toxic neuropathy)是周邊神經病變起因之一，而大多數神經毒素會同時影響大直徑及小直徑周邊神經，不易觀察小直徑感覺神經受到損傷時的變化。辣椒素(capsaicin)，是辣椒中引起辛辣感的主要成分，接觸辣椒素短期內會造成小直徑感覺神經元興奮，個體對外界刺激則產生過度敏感的症狀。辣椒素重複施用，則造成個體對熱刺激的敏感性降低，因而辣椒素常在臨床上被用於減輕神經性疼痛患者的痛楚，但是辣椒素引起敏感性降低的機制仍未有定論。此外重複使用辣椒素，是否影響個體對機械性刺激的反應，目前無一致結論。本實驗即試圖以周邊重複施用辣椒素建立小直徑周邊神經病變模式，並了解辣椒素對於個體行為、感覺神經纖維表型的表現產生何種影響。 使用成熟ICR公鼠，於單側腳掌連續7日皮下施打10 μl 1% 辣椒素，對側施打等量對照組溶液，在施打第7日起，每周觀察小鼠對熱刺激、機械性刺激反應，分別於第7日及第42日犧牲小鼠，以神經標的抗體protein gene product 9.5 (PGP9.5)、calcitonin gene-related peptide (CGRP)、substance P (SP)進行免疫組織化學染色及電子顯微鏡觀察。 研究結果發現，重複辣椒素施打側的腳掌表現PGP9.5的表皮神經密度相較於對側下降31%，表現神經胜肽CGRP、SP的表皮神經密度分別下降60%以及97%。伴隨著表皮神經密度下降，施打辣椒素側的腳掌對於熱刺激以及機械性刺激均出現敏感性降低的現象。使用電子顯微鏡觀察辣椒素對內側蹠神經(medial plantar nerve)中無髓鞘軸突(ummyelinated axons)構造的變化，許旺氏細胞包含的無髓鞘軸突數目略為下降，軸突出現瓦勒式退化(Wallerian degeneration)特徵的比例較施打對照組溶液側顯著上升。 為了解停止施打辣椒素是否造成功能及神經末梢表型的回復，觀察小鼠停止施打藥物後的行為變化。施打辣椒素第42日，該側腳掌對熱刺激及機械性刺激的反應回復正常。以免疫組織化學染色觀察表皮PGP9.5、CGRP、SP神經纖維的表現情形，施打辣椒素第42日表現PGP9.5與CGRP的表皮神經密度與對側相比無顯著差異，但表現SP的表皮神經密度仍顯著低於對側腳掌，下降78%。 由實驗結果得知，周邊重複辣椒素施打能造成小直徑神經病變，使得個體對外界刺激敏感性降低的功能退減以及表皮神經去支配，周邊神經退化則是造成神經病變的原因之一。此一神經病變可經由停止施打辣椒素逐漸回復個體功能，但辣椒素對於不同表型的表皮神經再支配能力影響不一致。

Environmental chemical pollutants and certain medication for chemotherapy are the inducers of toxic neuropathy, which affect the structure and functions of peripheral nerves. Most neurotoxins affect both large and small-diameter neurons, rendering confusion when trying to elucidate the specific effects on different types of neurons. Capsaicin (8-methyl-N-vanillyl-6-noneamide), the main ingredient in hot chili peppers, evoked the pungent sensation on subjects. Meanwhile, it could be applied topically to alleviate pain in patients with neuropathic pain. In this study, we asked whether repeated local injection of capsaicin could serve as a model of small-fiber neuropathy and how behavioral changes developed. 10 μl Capsaicin (1%) was injected daily into one side of the hindpaw of mice for 7 days; the other side was injected with vehicle solution. Using immunuhistochemistry, pan-neuronal marker protein gene product 9.5 (PGP9.5) (+) epidermal nerve density was reduced by 31%, peptidergic neuron marker calcitonin gene-related peptide (CGRP) (+) nerve density by 60%, and substance P (SP) (+) nerve density by 97% after capsaicin treatment on post-injection day 7 (PID7). Accompanying with denervation of epidermis, animals with capsaicin treatment exhibited marked reduction of sensitivity to thermal and mechanical stimuli on PID7 compared with the contralateral side. Unmyelinated axons of medial plantar nerve showed signs of Wallerian-like degeneration under capsaicin treatment. To assess whether capsaicin-induced neuropathy could be reversed, capsaicin injection ceased at PID7. Animals were kept alive and their behaviors were evaluated weekly until PID42. On PID42, both mechanical and thermal responses returned to normal. However, there was difference in the pattern of skin reinnervation among epidermal nerves of different phenotypes. Epidermal nerve density of SP (+) nerve fibers was reduced by 78%, whereas PGP9.5 (+) and CGRP (+) nerve fibers nearly replenished on PID42. These findings provided a model of capsaicin-induced neuropathy and indicated different vulnerability to capsaicin among different types of epidermal nerves.