術前局部麻醉劑處理及糖尿病對大白鼠正中神經損傷後之影響

Abstract

Part 1. 術前局部麻醉劑處理對大白鼠正中神經損傷後之影響 中文摘要 由前人的研究已經證實在周邊神經損傷後，對於受損的神經給予局部麻醉劑利路卡因鹽酸鹽(lidocaine)可有效的抑制因傷害所造成的異位性放電(ectopic discharges)。然而關於術前給予局部麻醉劑lidocaine對神經傷害所造成的異位性放電的影響仍沒有充分的研究。因此本實驗利用正中神經截斷或是慢性纏繞傷害動物模式，並配合活體紀錄複合性動作電位、電刺激、免疫組織化學染色以及給予拮抗性藥物、動物行為測試等實驗方式來探討術前給予局部麻醉劑對於：（1）正中神經截斷前後與電刺激前後異位性放電(cetopic discharges) 的影響；（2）截斷正中神經同側之楔狀神經核（cuneate nucleus，CN）內，神經胜肽Y (neuropeptide Y，NPY)免疫反應陽性神經纖維與c-Fos免疫反應陽性神經元表現量的變化；（3）若神經胜肽Y免疫反應陽性神經纖維與c-Fos免疫反應陽性神經元在楔狀神經核中的表現量受到術前給予局部麻醉劑的影響，接著再以拮抗劑處理來探討神經胜肽Y與c-Fos表現量之間的相互關係；（4）利用慢性纏繞傷害動物模式檢測術前給予局部麻醉劑可否對動物神經病變疼痛產生緩解。 首先我們在正中神經上局部給予生理食鹽水以及不同濃度的lidocaine（1%, 5%, or 10%），藥物處理15分鐘後再將正中神經截斷。並以電生理方式分別記錄各組別在藥物注射後、神經截斷後與電刺激前後不同時間點的複合性動作電位，來比較異位性放電的變化。結果顯示生理食鹽水實驗組異位性放電的頻率（frequency）相較於注射後、神經截斷後與電刺激前後都有顯著的增加，並且在神經截斷後達到最高峰。但在lidocaine處理的組別中，因正中神經截斷所誘發異位性放電頻率，則會隨著lidocaine濃度的增加而有減少的趨勢。 接著以免疫組織化學染色偵測楔狀神經核中神經胜肽Y免疫反應陽性神經纖維與c-Fos免疫反應陽性神經元的表現量。神經胜肽Y免疫反應陽性神經纖維的表現量在電刺激後受損正中神經同側楔狀神經核中，相較於未給予電刺激的對側，在各實驗組別中都有顯著的減少；而比較不同藥物實驗組間的差異可發現，術前給予lidocaine處理可隨濃度增加而減少神經胜肽Y免疫反應陽性神經纖維在同側楔狀神經核中的表現。另一方面，在正中神經未受損傷或是正中神經損傷後未受到電刺激的組別，都無法以免疫染色的方式偵測到c-Fos免疫反應陽性神經元；而比較各術前處理實驗組，c-Fos免疫反應陽性神經元表現量在生理食鹽水實驗組顯著高於其他組別，且c-Fos免疫反應陽性神經元在楔狀神經核中的表現量也隨lidocaine濃度增加而減少。 在發現神經胜肽Y免疫反應陽性神經纖維與c-Fos免疫反應陽性神經元在術前給予lidocaine處理後，兩者在楔狀神經核中的表現量都會隨lidocaine濃度增加而減少。我們以線性迴歸分析發現在各實驗組楔狀神經核中因電刺激而減少的神經胜肽Y免疫反應陽性神經纖維百分比與c-Fos免疫反應陽性神經元兩者間有明顯的正相關 (r = 0.77, P<0.005)。接著以神經胜肽Y受體的拮抗劑([D-Tyr27, 36 D-Thr32]-NPY (27-36))來檢測神經胜肽Y釋放與c-Fos之間的關係。結果發現在楔狀神經核吻端使用[D-Tyr27, 36 D-Thr32]-NPY (27-36)處理後，電刺激雖可使楔狀神經核中神經胜肽Y免疫反應陽性神經纖維下降，但電刺激所誘發之c-Fos免疫反應陽性神經元相較於未給予拮抗劑處理的組別明顯下降(p<0.05)。最後利用慢性纏繞傷害動物模式檢測術前給予局部麻醉劑是否可對正中神經損傷後所產生的神經病變疼痛有所影響，並評估神經病變疼痛行為與楔狀神經核中c-Fos的表現量是否有所關聯，結果發現術前給予局部麻醉劑可延緩並降低機械性痛覺敏感（mechanical allodynia）的產生，並且楔狀神經核中c-Fos的數量與機械性刺激的閥值呈顯著負相關(r = -0.80, P < 0.005)，這樣的結果暗示或許楔狀神經核中c-Fos的數量可作為評估正中神經損傷後對機械性刺激痛覺敏感的指標。 Part 2. 誘發糖尿病對大白鼠正中神經截斷後之影響。 中文摘要 在本研究中，我們利用鏈佐菌素（streptozotocin，STZ）誘導的糖尿病大白鼠來檢測正中神經截斷對神經胜肽Y（NPY）在背根神經節（DRG）與楔狀神經核（cuneate nucleus，CN）中表現的影響。在糖尿病組無法偵測到神經胜肽Y免疫反應，但在糖尿病合併正中神經截斷後，背根神經節與楔狀神經核中分別可以偵測到大量的神經胜肽Y免疫反應陽性神經元與神經纖維。除此之外，糖尿病合併正中神經截斷(DMNT)，神經胜肽Y在背根神經節與楔狀神經核中的表現趨勢十分一致，都是在正中神經截斷後兩週達到最高峰。 在電刺激截斷正中神經之後，同側楔狀神經核中的神經胜肽Y免疫反應纖維會減少並出現大量的c-Fos免疫反應神經元。c-Fos免疫反應神經元在糖尿病合併正中神經截斷後楔狀神經核中表現量同樣在兩週達到高峰，與神經胜肽Y免疫反應纖維在楔狀神經核中的表現趨勢相同。但這樣的結果與我們實驗室先前所發表，非糖尿病大白鼠背根神經節中神經胜肽Y免疫反應陽性神經元，楔狀神經核中神經胜肽Y免疫反應陽性神經纖維與c-Fos免疫反應陽性神經元，在正中神經截斷後四週達到高峰，有顯著的差異。 接著我們分析神經滋養素-3在正中神經截斷後背根神經節中表現的情形，非糖尿病大白鼠背根神經節中,神經滋養素-3免疫反應陽性神經元(neurotrophin-3 like immunoreactivity neurons，NT-3-LI neurons)所占百分比在正中神經截斷後開始降低，並在正中神經截斷後四週達到最低點。而糖尿病大白鼠背根神經節中，神經滋養素-3免疫反應陽性神經元所占百分比在正中神經截斷後也開始下降，但所占百分比最低點提前於正中神經截斷後兩週，之後開始逐漸回升。緊接著我們選定非糖尿病與糖尿病大白鼠，正中神經未截斷與正中神經截斷後神經滋養素-3免疫反應陽性神經元所占百分最低的時間點(非糖尿病四週 糖尿病兩週)，以免疫螢光雙重標誌方式分析神經滋養素-3免疫反應陽性神經元在背根神經節中細胞大小分佈的情形，結果發現正中神經截斷後無論在非糖尿病或是糖尿病組，神經滋養素-3免疫反應陽性在中大型的神經元(A-type) 所佔比例均顯著下降。我們先前的研究已指出神經胜肽Y在背根神經節中只會在中大型的神經元表現，而是否因為神經截斷後神經滋養素-3免疫反應陽性神經元佔中大型神經元比例下降而誘發神經胜肽Y在背根神經節中大量表現？是我們下ㄧ步需要釐清的。我們使用螢光雙重標示偵測正中神經截斷四週、糖尿病正中神經截斷兩週在背根神經節中神經滋養素-3與神經胜肽Y是否會雙重標誌於背根神經元，結果發現只有極低比例的背根神經元現神經滋養素-3與神經胜肽Y雙重標誌。 綜合以上結果，我們可以推測在糖尿病大白鼠正中神經截斷後，背根神經節中神經胜肽Y免疫反應陽性神經元提早大量表現，或許是因為誘發糖尿病後神經滋養素-3在背根神經節中中大型神經元表現量下降所導致。

Part 1. Effects of pre-emptive lidocaine treatment on rats following median nerve injury ABSTRACT Following peripheral nerve injury, lidocaine application has been demonstrated to suppress injury discharges. However, there is very little information about the effects of lidocaine pre-treatment. The aim of the present study was to examine the effects of pre-treatment with lidocaine on injury discharges of the nerve, and neuropeptide Y (NPY) and c-Fos expression in the cuneate nucleus (CN) after median nerve transection (MNT). Rats received either saline or 1%, 5%, or 10% lidocaine applied topically to the median nerve before nerve transection. Electrophysiological recording was used to examine the changes in injury discharges of the nerve at post-injection, transection, pre- and post-electrical stimulation stages in the different groups. Sequential immunohistochemistry was also used to identify the number of NPY-like immunoreactive (NPY-LI) fibers and c-Fos-LI cells in the corresponding CN. An increasing frequency of injury discharges was observed at all stages in the pre-saline group, which were suppressed by lidocaine pre-treatment in a dose-dependent manner. Lidocaine pre-treatment also attenuated the number of injury-induced NPY-LI fibers and c-Fos-LI neurons within the CN in a dose-dependent manner. Furthermore, expression of c-Fos-LI neurons in the CN was significantly reduced by an NPY receptor antagonist, indicating that NPY modulated c-Fos expression following MNT. In chronic constriction injury (CCI) model, lidocaine pre-treatment dose dependently delayed and attenuated the development of mechanical allodynia within a 28-day period and also reduced the number of c-Fos-LI neurons within the CN. In addition, the mean number of c-Fos-LI neurons in the CN was significantly negative correlated to the sign of mechanical allodynia following CCI. These data suggest that suppressing injury discharges with lidocaine pre-treatment effectively prevent morphological changes in the CN and attenuate neuropathic pain following median nerve injury. Furthermore, the expression of c-Fos in the CN may be regarded as the scale of mechanical allodynia in rat treated with median nerve injury. Part 2. Effects of diabetes on rats following median nerve injury ABSTRACT In this study we examined the temporal changes in neuropeptide Y (NPY) expression in dorsal root ganglion (DRG) neurons and cuneate nucleus (CN) in streptozotocin (STZ)-induced diabetic rats with or without median nerve transection (MNT). Numerous NPY-like immunoreactive (NPY-LI) neurons and fibers were detected in the DRG and CN in the diabetic MNT (DMNT) rats respectively, but not in those with diabetes alone. Following MNT, the time-course of NPY expression pattern in the diabetic DRG and CN was similar and both peaked at 2 weeks, which was earlier than that in the non-diabetic MNT rats. Consequently, the expression of neurotrophin-3 (NT-3) immunoreactivity in DRG neurons was coincidentally decreased and reached the nadir at 2 weeks in the diabetic MNT rats, which was also earlier than that in the non-diabetic MNT rats. Following electrical stimulation of the transected nerve, the number of NPY-LI fibers became attenuated and the induced c-Fos-LI cells concurrently appeared in the ipsilateral CN. In the diabetic CN, the number of c-Fos-LI cells also peaked at 2 weeks after MNT, which was consistent with the temporal pattern of changes in NPY expression. The results suggest that in diabetes, MNT induced NPY expression via the reduction of NT-3, and electrical stimulation of the injured median nerve evoked the release of NPY and accordingly more c-Fos-LI cells were identified in the CN. Furthermore, this study demonstrated early NPY and c-Fos expression in the diabetic rats after MNT, suggesting that the development of neuropathic signs may be advanced in hyperglycemic rats.