大白鼠背根神經節中P2X3與其他疼痛相關因子在正中神經損傷後的變化

Changes in expression of P2X3 and pain-related factors in the dorsal root ganglion after median nerve injury

林子瑜(Tzu-Yu Lin)

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/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10016

摘要

從先前的許多研究得知，在周邊神經損傷或組織發炎的情況下，背根神經節 中的P2X3在神經損傷後所誘發的神經病變性疼痛扮演了重要的角色，神經損傷側會對傷害性機械或熱刺激產生痛覺過度敏感的反應。然而，對於正中神經損傷後P2X3於背根神經節中的表現分佈情形以及其在痛覺機制中扮演的角色等相關研究則仍非常貧乏。因此本研究利用免疫組織化學染色的方法來探討實驗大白鼠施以正中神經截斷或慢性纏繞性傷害後不同的存活時程，其P2X3免疫反應神經元在第六頸髓背根神經節中的表現量變化情形；並更進一步利用雙重免疫螢光染色的方式觀察施以慢性纏繞性傷害的實驗大白鼠，其第六頸髓背根神經節中P2X3與NPY、galanin、SP、VR1、Nav1.8、和GAP43的相關性。最後，我們再進行正中神經慢性纏繞性傷害前使用局部麻醉劑處理，進而比較P2X3與NPY的變化。
　　實驗結果顯示， P2X3免疫反應神經元在背根神經節中主要表現在小型至中型的神經元，而且當正中神經截斷四週後，P2X3免疫反應神經元數量會顯著低於正常大白鼠組別；然而在正中神經慢性纏繞性傷害模式中卻發現神經損傷四週後，P2X3免疫反應神經元在背根神經節中表現量則會顯著高於正常大白鼠與損傷兩週後組別，顯示P2X3可能與神經慢性纏繞性傷害模式誘發的疼痛敏感具有相關性。而在雙重免疫螢光染色的結果可觀察到，神經傷害四週後，P2X3免疫反應神經元中也具有NPY、galanin、SP或GAP43免疫反應的表現，其數量皆有顯著增加；P2X3免疫反應神經元中具有VR1免疫反應的數量與正常大白鼠組別相比較，卻無顯著差異；再者，P2X3免疫反應神經元中具有Nav1.8鈉離子通道蛋白免疫反應的數量則在神經傷害後顯著下降。另外，在神經傷害兩週及四週後，同時具有P2X3與NPY免疫反應神經元佔該背根神經節的百分比，皆顯著高於正常大白鼠組別；同時具有P2X3與galanin免疫反應的神經元在背根神經節之百分比，於傷害兩週後有上升情形，但在第四週與正常大白鼠組別達到統計上的差異；同時具有P2X3與SP、VR1或GAP43免疫反應的神經元在背根神經節之百分比，在傷害四週後有顯著增加；P2X3與Nav1.8免疫反應的百分比，則於傷害四週後有增加趨勢，但與正常大白鼠組別未達顯著差異。
　　正中神經術前給予生理食鹽水、1%與5%局部麻醉劑時，發現注射生理食鹽水後，P2X3免疫反應神經元中同時具有NPY免疫反應的數量以及在背根神經節中的百分比皆有顯著地增加，並隨著局部麻醉劑注射濃度提高而顯著下降。
　　綜合上述，正中神經慢性纏繞性傷害P2X3免疫反應數量的增加與其他疼痛相關因子和神經生長因子在背根神經節共同存在時程及數量變化，透過本篇研究結果，或許可以提供慢性纏繞性傷害所誘導神經病變性疼痛可能誘發機制的基礎。

關鍵字

P2X3 ； 背根神經節 ； 正中神經 ； 神經胜肽神經生長相關蛋白質

並列摘要

Previous studies showed that with peripheral nerve injury or tissue inflammation, P2X3 receptors in the dorsal root ganglions (DRG) may contribute to nerve injury-induced neuropathic pain and cause hyperalgesia to thermal or mechanical noxious stimuli on the ipsilateral side. However, for the mechanisms and distributions of P2X3 receptors in DRG neurons after the median nerve being injured are still unclear. In this study, we used immunohistochemistry (IHC) staining to examine the changes in the proportion of P2X3-like immunoreactive (LI) neurons in the six cervical dorsal root ganglions (C6 DRG) after complete median nerve transection (CMNT) and chronic constriction injury (CCI) and compared nerve-injured rats with naïve ones at different time points. Furthermore, we also used double immunofluoresence (DIF) staining to investigate the number of P2X3 as well as neuropeptide Y (NPY), galanin, substance P (SP), vanilloid receptor subtype 1 (VR1), Nav1.8 and GAP43 in the C6 DRGs of CCI and naïve rats.
　　The result showed that the expression of P2X3 was found predominantly in a subpopulation of small- to medium-sized DRG neurons. Four weeks after CMNT, the proportion of P2X3-LI neurons in C6 DRG was lower than naïve rats. On the other hand, P2X3-LI neurons were up-regulated after four weeks of CCI, comparing to naïve and two weeks of CCI rats, suggesting that a correlation exists between P2X3 and CCI-induced abnormal nociceptive sensitivity. With DIF labeling, we found that the proportion of P2X3-LI neurons, which also expressed NPY, galanin, SP or GAP43, was significantly increased after four weeks of CCI. P2X3-LI neurons for VR1 were similar between naïve and CCI rats. In contrast, P2X3-LI neuron profiles contained with Nav1.8, which were significantly reduced four weeks after CCI. Likewise, the percentage of co-expressing P2X3-LI and NPY-LI, galanin-LI, SP-LI, VR1-LI or GAP43-LI four weeks after CCI was highly up-regulated than that in naïve rats. Moreover, no significant difference between four weeks after CCI and naïve groups on co-expressing P2X3-LI and Nav1.8-LI was observed.
　　With saline, 1% and 5% lidocaine (LDC) injection into the median nerve before CCI, we found that the percentage of P2X3-LI neurons for NPY was significantly increased after saline injection while it decrease remarkably with LDC treatment relating to dose-dependent. In saline group, the percentage of co-expressing P2X3-LI and NPY-LI was significantly increased than that in other groups.
　　Our present result shows that the number of P2X3 as well as other pain-related factors or nerve growth factor in DRGs changes with time. We hope that our findings would provide a framework of the mechanism of CCI-induced neuropathic pain.

並列關鍵字

P2X3 ； dorsal root ganglions ； median nerve ； neuropeptides ； nerve growth associated protein

參考文獻

Abdi S, Lee DH, Chung JM (1998) The anti-allodynic effects of amitriptyline, gabapentin, and lidocaine in rat model of neuropathic pain. Anesth. Analg. 87:1360-1366.

Abdulla FA, Smith PA (2002) Changes in Na+ channel currents of rat dorsal root ganglion neurons following axotomy and axotomy-induced autotomy. J. Neurophysiol. 88:2518-2529.

Akopian AN, Sivilotti L, Wood JN (1996) A tetrodotoxin-resistant voltage-gated sodium channel expressed by sensory neurons. Nature 379:257-262.

Averill S, Inglis JJ, King VR, Thompson SWN, Cafferty WBJ, Shortland PJ, Hunt SP, Kidd BL, Priestley JV (2008) Reg-2 expression in dorsal root ganglion neurons after adjuvant-induced monoarthritis. Neuroscience 155:1227-1236.

Barclay J, Patel S, Dorn G, Wotherspoon G, Moffatt S, Eunson L, Abdel’al S, Natt F, Hall J, Winter J, Bevan S, Wishart W, Fox A, Ganju P (2002) Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain. J.Neurosci. 22:8139-8147.