正中神經損傷後一氧化氮參與神經病變疼痛機制之研究

The Role of Nitric Oxide in the Median Nerve Neuropathic Pain

王馨瑩(Hsin-ying Wang)

指導教授 ： 呂俊宏

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/博士(2013年)

https://doi.org/10.6342/NTU.2013.02082

摘要

先前雖有報告指出，周邊神經損傷導致具有一氧化氮合成酶的神經元數量增加，其所釋出的一氧化氮則與神經病變疼痛的誘發有密切的關連，但尚未有研究探討一氧化氮與正中神經損傷病變疼痛之間的關係。在本研究中，我們將探討一氧化氮在正中神經損傷後所扮演的角色。首先，我們檢測大白鼠正中神經截斷後不同存活時程，在第六頸髓背根神經節與楔狀神經核內神經性一氧化氮合成酶的數量變化。結果發現正中神經截斷後第三天，即可在背根神經節及楔狀神經核內觀察到神經性一氧化氮合成酶免疫反應神經元的數量，與正常未損傷組別相比較，已有增多的情形，並且持續至神經截斷後四週數量仍然居高不下。若術前以局部麻醉劑lidocaine處理，在正中神經截斷後四週，背根神經節及楔狀神經核內神經性一氧化氮合成酶免疫反應神經元的數量會明顯地下降。此外，電刺激前給予一氧化氮合成酶抑制劑L-NAME及7-NI後，發現它們可以明顯降低楔狀神經核內神經胜肽Y釋放量與c-Fos免疫反應神經元數量；而給予一氧化氮供給劑SNAP雖然可以顯著增加c-Fos免疫反應神經元數量，但對於神經胜肽Y釋放量卻沒有影響。
我們進一步利用溶血磷脂醯膽鹼（lysophosphatidylcholine, LPC）作為神經髓鞘損傷藥物，探討正中神經以LPC處理後，一氧化氮與神經病變疼痛的關係。研究結果發現，正中神經以4% LPC處理後第五天及第七天，實驗動物手術側前肢掌面對於非傷害性機械刺激及傷害性溫熱覺刺激產生的神經病變疼痛行為達到最顯著的程度。此外4% LPC處理後一週，手術側正中神經纖維髓鞘有剝離脫落、甚至於髓鞘完全退化消失的現象；但是在LPC處理後二及四週，軸突外圍則包覆有完整的髓鞘。進一步探討正中神經施以4% LPC後不同存活時程，背根神經節及楔狀神經核內一氧化氮合成酶免疫反應神經元及c-Fos免疫反應神經元的數量變化。LPC處理後一週，在背根神經節及楔狀神經核內可觀察到神經性一氧化氮合成酶免疫反應神經元的數量增加並且達到最高值。當正中神經施以4% LPC一週後並配合電刺激處理後，其楔狀神經核內c-Fos免疫反應神經元數量也達到最高。腹腔注射NOS抑制劑L-NAME及7-NI後，發現它們可以明顯緩解LPC處理所引起神經病變疼痛症狀；而給予SNAP後，也會加重LPC處理所引起的痛覺過敏。電刺激前給予L-NAME及7-NI後，發現它們可以顯著地降低楔狀神經核內c-Fos免疫反應神經元數量；而給予SNAP則可以有效地增加c-Fos免疫反應神經元數量。
綜合以上結果，正中神經損傷導致神經性一氧化氮合成酶增加，可能促進一氧化氮的產生。釋放出的一氧化氮除了可調控初級傳入神經終末NPY的釋放而興奮突觸後楔狀丘腦投射神經元外，也可能直接影響楔狀丘腦投射神經元內c-Fos的產生；並藉此參與神經病變疼痛的訊息傳遞。

關鍵字

正中神經 ； 楔狀神經核 ； 一氧化氮合成酶神經病變疼痛 ； c-Fos

並列摘要

NO is produced by the increase in the number of nitric oxide (NO)-synthesizing neurons after peripheral nerve injury and mediate the neuropathic pain. However, the direct evidence of NO involvement in the median nerve neuropathic sensation is unavailable. In this study, we investigated the role of NO after median nerve injury. We first examined the temporal changes of neuronal nitric oxide synthase (nNOS) expression in the rat dorsal root ganglion (DRG) and cuneate nucleus (CN) after median nerve transection (MNT). From 3 days to 4 weeks following MNT, the amounts of nNOS like immunoreactive (nNOS-LI) neurons in the DRG and CN significantly increased as compared with those of the normal rats. Four weeks after MNT, the maximums of nNOS-LI neurons in the DRG and CN were attenuated by pre-emptive lidocaine treatment in a dose-dependent manner. After MNT, intraperitoneal administration of L-NAME (Nω-Nitro-L-arginine methyl ester, an inhibitor of NOS) or 7-NI (7-nitroindazole, a specific neuronal NOS inhibitor) suppressed the amount of NPY release from the stimulated terminals and thus attenuated c-Fos expression in the CN; however, SNAP (S-Nitroso-N- acetylpenicillamine; a NO donor) application only increased the number of c-Fos-LI neurons in the CN but not the NPY release level.
Using median nerve demyelination model, we further investigated the role of NO in lysophosphatidylcholine (LPC) induced neuropathy rats followed by the administration of NOS inhibitors and NO donor. We found that at 5 and 7 days after LPC treatment of the median nerve, a maximum effect on the signs of mechanical allodynia and thermal hyperalgesia was induced. One week after 4% LPC treatment, nerve fibers without myelin sheaths were detected in the treated nerve. And the myelin sheath reappeared at the treated nerve 2-4 weeks after 4% LPC treatment. Immunohistochemistry revealed that the amounts of nNOS-LI neurons in both the DRG and CN increased and peaked at 1 week after LPC treatment. Following electrical stimulation of the LPC-treated nerve, the number of c-Fos-LI neurons in the ipsilateral CN also increased by LPC treatment in a dose-dependent manner and peaked at 1 week. Administration of L-NAME or 7-NI 1 week after 4% LPC injection attenuated tactile allodynia and thermal hyperalgesia, but SNAP only exacerbated thermal hyperalgesia. After electrical stimulation of the LPC-treated median nerve, the number of c-Fos-LI neurons in the CN diminished in the L-NAME and 7-NI groups, but increased in the SNAP group.
Taken together, our findings revealed that the number of nNOS dramatically increased after median nerve injury. And the advanced NO, made by the increased nNOS after median nerve injury, might be involved in the maintenance of neuropathic sensation. In addition, NO produced via the upregulation of the amount of nNOS in the CN might increase the neuronal activity of CTN directly or modulate the amount of NPY release from primary afferent terminals and subsequently promote the excitability of CTN and thus up-regulate c-Fos protein expression.

並列關鍵字

median nerve ； cuneate nucleus ； nNOS ； nuropathic pain ； c-Fos

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