正中神經截斷後在糖尿病大白鼠背根神經節中鈉離子通道與疼痛相關因子的表現變化

Changes in Expression of Sodium Channels and Pain-related Factors in the Dorsal Root Ganglion after Median Nerve Transection of Diabetic Rats

余明泰(Ming-Tai Yu)

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(2010年)

https://doi.org/10.6342/NTU.2010.02593

摘要

由於神經病變疼痛會嚴重地影響病患的生活品質，而糖尿病病患進行截肢手術的機會相對較高，因此本研究利用免疫組織化學染色的方式探討非糖尿病、短期糖尿病與長期糖尿病大白鼠在正中神經截斷後的不同存活週數下，NaV1.8或NaV1.9免疫反應神經元在第六頸髓背根神經節中所佔百分比的變化，並進一步利用免疫螢光染色的方式觀察正中神經與正中神經截斷後所產生的神經瘤中，NaV1.8或NaV1.9免疫反應的染色情形，最後以免疫螢光雙重標誌的方式探討NaV1.8、NaV1.9與疼痛相關因子： VR1、P2X3、SP與CGRP的表現是否有關聯。
　　在免疫組織化學染色的結果中可以觀察到，NaV1.8主要分布在小型至中型的神經元，經正中神經截斷後一週，背根神經節中NaV1.8免疫反應神經元的百分比會顯著地下降，雖然到神經截斷後三週有回復的情形，但是神經截斷後四週與五週又會持續再下降，至於NaV1.9則主要分布在小型神經元，在正中神經截斷後一週，背根神經節中NaV1.9免疫反應神經元的百分比會顯著地下降並持續至神經截斷後五週。相較於非糖尿病動物，神經截斷後NaV1.8下降的程度會隨著STZ處理的時程越長而越明顯，但是NaV1.9下降幅度卻不會隨著STZ處理的時程長短而有所影響。此外，神經瘤的免疫螢光染色結果中可以觀察到 NaV1.8免疫反應的染色深度與背根神經節中NaV1.8免疫反應神經元的百分比有互補的結果，而NaV1.9免疫反應的染色深度在各組中都沒有明顯的變化。
　　從免疫螢光雙重標誌的結果中可以觀察到，長期糖尿病組在正中神經截斷後三週，NaV1.8免疫反應神經元與P2X3、VR1或SP雙重標誌的百分比都顯著地高於非糖尿病組，而NaV1.8免疫反應神經元與CGRP雙重標誌的百分比則顯著地低於非糖尿病組。相較於非糖尿病組，長期糖尿病組在正中神經截斷後一週與四週，背根神經節中NaV1.9與P2X3雙重標誌神經元的百分比都會顯著地上升；與VR1、CGRP或NF200雙重標誌的百分比則沒有顯著的變化；與SP雙重標誌的百分比則僅在神經截斷後四週顯著低於非糖尿病組。
　　綜合以上所述，正中神經截斷後NaV1.8會從背根神經節重新導向至神經瘤中，而共同存在C型與Ad型神經元中的NaV1.8或NaV1.9與疼痛相關因子或許可以說明在痛覺傳遞的過程中，鈉離子通道與疼痛相關因子具有潛在的交互作用。就目前的研究結果看來，非糖尿病大白鼠與糖尿病大白鼠在正中神經截斷之後所產生的神經病變疼痛與NaV1.8的變化較為相關。希望透過這些結果可以提供了解糖尿病病患在截肢後產生神經病變疼痛的可能機轉。

關鍵字

正中神經截斷 ； 背根神經節 ； 糖尿病神經病變 ； 鈉離子通道

並列摘要

Amputation is a common surgery in patients with diabetes mellitus (DM), and the post-surgical neuropathic pain often impinges on the patients’ quality of life. In this study, we used immunohistochemistry (IHC) staining to examine the changes in the proportion of NaV1.8-immunoreactive (-IR) or NaV1.9-IR neurons in the sixth cervical dorsal root ganglions (C6 DRGs) at different time points after complete median nerve transection (CMNT) of non-diabetic, short-term diabetic and long-term diabetic rats. Furthermore, we also used immunofluorescence (IF) staining to verify the expression patterns of NaV1.8 or NaV1.9 immunoreactivity in the normal median nerve and neuromas following CMNT. Finally, we used IF double labeling to investigate the relationship between NaV1.8/NaV1.9 and pain-related factors: vanilloid receptor subtype 1 (VR1), P2X3, substance P (SP) and calcitonin gene-related peptide (CGRP) in the C6 DRGs at different time points after CMNT of non-diabetic and long-term diabetic rats.
　　The results showed that the expression of NaV1.8 was mainly in small- to medium-sized DRG neurons. Following CMNT, the proportion of NaV1.8-IR neurons in the C6 DRG of non-diabetic rats was lower than that in naïve rats. Moreover, our results also demonstrated that the expression of NaV1.9 was restricted to small-sized DRG neurons and the proportion of them in the C6 DRG significantly decreased after CMNT. Comparing to non-diabetic rats, the reduced level of the proportion of NaV1.8-IR neurons in the C6 DRG after CMNT was based on the streptozotocin (STZ) treatment duration, but not that of NaV1.9-IR neurons. We also found that only NaV1.8 immunoreactivity in median nerve was contrary to that in DRG.
　　Three weeks after CMNT, IF double labeling demonstrated that the proportion of NaV1.8-IR neurons which also expressed P2X3, VR1 or SP in the long-term diabetic DRG was significantly higher, but the percentage of co-localization of NaV1.8 and CGRP was dramatically lower than that of non-diabetic DRG, respectively. In the same way, one or four weeks after CMNT, the percentage of NaV1.9-IR neurons that also expressed P2X3 in the non-diabetic DRG was significantly higher than that of long-term diabetic DRG, and the percentage of NaV1.9-IR neurons which co-localized with VR1, CGRP or NF200 made no significant difference between non-diabetic and long-term diabetic rats. Moreover, the percentage of co-localization of NaV1.9-IR and SP-IR neurons at four weeks after CMNT of non-diabetic rats was significantly higher than that of long-term diabetic rats.
　　In conclusion, NaV1.8 protein may redirect to the neuromas after CMNT. The co-localizations of NaV1.8/NaV1.9 and pain-related factors in C- and A(delta)-type neurons suggested the sodium channels and pain-related factors have the potential for interactions in pain transmission. So far, the evidence showed that NaV1.8 is more complicated than NaV1.9 with neuropathic pain after CMNT of non-diabetic or diabetic rats. We hope the morphological evidence obtained from this study which would be elucidated the mechanisms of the generation of post-surgical neuropathic pain in patients with DM.

並列關鍵字

median nerve transection ； dorsal root ganglion ； diabetic neuropathic pain ； sodium channels

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