講義正式版摘要

1、交 通 大 學博士尼莫地平促進大鼠面神經損傷修復的作用及機制研究EFFICACY AND MECHANISM OF NIMODIPINE ON IMPROVINGFUNCTIONAL RECOVERY OF INJURED FAL NERVE IN RATS作者：達指導教師：學科專業(yè)：外科學（神經外科）培養(yǎng):交通大學附屬新華醫(yī)院期：2015 年 4 月 29 日號：0127229216答辯日學生學交 通 大 學博士尼莫地平促進大鼠面神經損傷修復的作用及機制研究EFFICACY AND MECHANISM OF NIMODIPINE ON IMPROVINGFUNCTIONAL RECOVERY

2、 OF INJURED FAL NERVE IN RATS作者：達申請學位：博士指導教師：學科專業(yè)：外科學（神經外科）研究方向：顱神經疾病的診治培養(yǎng):交通大學附屬新華醫(yī)院答辯日期：2015 年 4 月 29 日資助項目：市科委（No. 11JC1408600）交通大學性本人鄭重：所呈交的，是本人在導師的指導下，獨立進行研究工作所取得的成果。除文中已經注明的內容外，本論包含任何其他個人或集體已經或撰寫過的作品成果。對本文的研究做出重要貢獻的個人和集體，均已在文中以明確方式標明。本人完全本的法律結果由本人承擔。作者簽名：日期：年月日交通大學使用書本作者完全了解學校有關保留、使用的規(guī)定，同意學校保留

3、并向國家有關部門或機構送交的復印件和，允許被查閱和借閱。本人交通大學可以將本的全部或部分內容編入有關數(shù)據(jù)庫進行檢索，可以采用影印、縮印或掃描等保存和匯編本。，在年后適用本書。本屬于不。（請在以上方框內打“”）作者簽名：指導教師簽名：日期：年月日日期：年月日摘要背景和目的面神經損傷嚴重影響患者的生活及社交能力，而臨床療效欠佳。尼莫地平作為一種 L 型電壓門控鈣通道阻滯劑，目前主要用于防治蛛網膜下腔后的腦痙攣。近來，臨床研究發(fā)現(xiàn)，預防性運用尼莫地平有益于聽神經瘤術后的面神經功能恢復；動物實驗也發(fā)現(xiàn)其可通過促進神經元存活及軸突生長來改善神經功能，然而機制尚不明了。另外，關于該藥是否可通過促進髓鞘修復

4、來實現(xiàn)上述作用，目前尚無相關研究。該研究從以下兩方面探討尼莫地平促進面神經損傷修復的具體機制：（1）是否促進面神經髓鞘的修復；（2）如何對神經元胞體和軸突再生起到保護和促進作用。希望通過本研究，進一步明確尼莫地平促進面神經修復的作用及其機制，為將來開展相關臨床試驗提供理論依據(jù)。方法第一部分首先采用周圍神經定量損傷鉗對面神經顱外段主干進行定量擠壓傷，并綜合各檢測確定合適參數(shù)。在第二部分，分別于術后各時間點對藥物組及干預組大鼠作行為學及電生理學檢測；取材面神經主干行 H-E、神經髓鞘固藍（luxol fast blue，LFB）染色；并通過免疫組化檢測髓鞘堿性蛋白（myelin basicprot

5、eBP）、磷酸化 p38 MAPK（p-p38）和鈣結合蛋白 S-100 在雪旺細胞的抗原表達。在第三部分，于術后 20 天取材面神經核團，對其行尼氏染色及 TUNEL 檢測；觀測熒光金逆行標記的神經元；免疫組化檢測面神經核團內 RhoA、磷酸化 ROCK2（p-ROCK2）的抗原表達；通過 Western blot 和 RT-PCR 檢測上述分子在面神經主干的表達。結果第一部分：對大鼠面神經顱外段主干行 50g/90s 卡壓傷，可為后續(xù)試驗提供合適的動物模型。第二部分：行為學、電生理學及組織學結果證明尼莫地平可促進神經結構和功能的修復。LFB 染色及 MBP 免疫組化證明尼莫地平可促進髓鞘修

6、復。S-100 抗原表達的增多，提示尼莫地平的作用機制與增強雪旺細胞的鈣緩沖能力有關。p-p38 抗原提示其介導的信號通路可能并未涉及。第三部分：尼氏染色及 TUNEL 檢測提示本模型所致大鼠面神經顱外段主干卡壓傷并未造成神經元的。藥物組 FG 逆行標記的神經元數(shù)量明顯增多，表明軸突再生的加強。免疫組化發(fā)現(xiàn)尼莫地平可下調損傷后神經元內 RhoA 和 p-ROCK2 的抗原表達。Western blot 和 RT-PCR 結果也表明，尼莫地平可在轉錄和翻譯水平對RhoA/ROCK 通路起到抑制作用。結論本研究證明尼莫地平可明顯促進大鼠面神經損傷后的髓鞘修復，其機制與上調損傷后雪旺細胞S-100的

7、表達有關；尼莫地平可同時促進大鼠面神經損傷后的軸突再生，這可能是通過抑制RhoA/ROCK來發(fā)揮作用的。：面神經損傷；尼莫地平；髓鞘修復；RhoA/ROCK 信號通路Abstractroduction and ObjectiveFal nerve injuries can cause unilateral fal weakness, with othersymptoms including loss of taste, hyperacusis and decreased salivation andtear secretion, which carries significant advers

8、e sol and functionalconsequen. The current therutic options for fal nerve injuriesinclude steroids, surgicalervention etc. Various pharmacological agentsare also available and have been shown to improve the functional recoveryol nerve after injury, however, none are in clinical use. Nimodipine,an L-

9、type voltage-operated calcium channel （VOCC）antagonist, is a US Foodand Drug Administration (FDA) approved drugt is used as a vasodilatororiginally developed for the treatment of high blood prere and recentlydelayed ischemic deficits in patients with subarachnoid hemorrhage (SAH).In addition, clinic

10、al trials have revealed a benefil effecttnimodipine promotes functional recovery ol nerve following skull base,laryngeal, and maxillofal surgery. Furthermore, several animal studieshave showedt nimodipine can improve survival rate ol motor neuronsand accelerate sproutting and elongation ol axons.The

11、se findings demonstratet nimodipine has shown promise in rodentms and human as asible pharmacological treatment for fal nerveinjury and indicatet nimodipine may have a direct regenerative andrepairing effect on cell bodies and axons. However, the mechanism of thisneuroprotectiveeffectofnimodipinehas

12、 notextensivelystudied.Furthermore, there has not been any studies to investigate thesibilityt nimodipine can promote myelin repair after fal nerve injure, whichis another key pros in peripheral regeneration. It is therefore, the aimof the present study is to investigate the mechanism of nimodipine-

13、mediatedneural repair after fal nerve crush injury in rats based on two aspects,(1) whether nimodipine promotes myelin repaire in demyelinated axons; (2)what is the mechanism of its neuroprotective effects on promoting falneuron survival and axon regeneration.Methodhesection, we constructed a rat mi

14、n which the extracranialportion ol nerve trunk was injured using a specificive injuryforceps withvarious parameter of crush injure. The mwas validate bybehavior, electrophysiological and histopathologicaldetection for thefollowing studies. Rats were dividedo three groups: healthy control,surgery alo

15、ne, and surgery plus nimodipine.he second section, the effect of nimodipine on myelin repair and themechanism was investigated. The ratshe surgery plus nimodipine groupwere administered nimodipine 6 mg/kg/day by oral gavage and underwentbehavior test and electrophysiological study of compound muscle

16、 actionpotential (CMAP) at 3, 10, or 20 days after surgery. Then the fal nervespecimens were harvested and stained using hematoxylin and eosin (HE) andluxol fast blue (LFB). Immunofluorescence (IF) staining was carried out todetect myelin basic protein (MBP), phospho-p38 MAPK (p-p38) and S-100.he th

17、ird section, the neuroprotective effect of nimodipine on neuronsurvival and axon regeneration and the mechanism was investigated. Brainstemsections containing bilateral fal nucleus in each group was harvested 20daystleand was detected andysed by Nissl staining, TUNEL,Fluoro-Gold (FG) retrogradely la

18、beling and IF for RhoA and phospho-ROCK2(p-ROCK2). Western blot and reverse transcriptioR (RT-PCR) wascarriedout to detect the expresof RhoA, p-ROCK2 and ROCK2 in falaxon atboth the translational and transcriptional level 20 daystle.Resultshesection, itis the most suitable to construct the animalmol

19、 nerve injurehe present study as the parameter of crushinjure wasset to 90 secondsfor duration and 50 grams for strength usingtheive injure forceps.hesecond section, by behavior and electrophysiological test, wely demonstratedt nimodipine improved the functional recovery offal nerve after 20 days of

20、 continuous treatment. HE staining indicatedt treatment with nimodipine improved histological repairing andsiblyreduced inflammation. LFB staining and IF for MBP revealedt nimodipineincreased reconstruction of the myelin sheath. Theitive result of IF forcalcium-binding S-100 suggestt the underlying

21、mechanisms might includethe enhancement of the ability of Schwann cells for calcium buffering afterinjure. The immunoreactivity of p-p38 was negative 20 daystle, whichindicatedt the p38 MAPK pathway might not be involved.he third section, Nissle staining and TUNEL detection olnucleus revealed no neu

22、ronal death or apoptosis, suggestingt promotingneuron survival by nimodipine was not involvedhe mechanism,eastin the present m. Nevertheless, nimodipine increased the number ofretrogradely traced FG-itive motor neuronhe fal nucleus 20 daystle, demonstratingt nimodipine could accelerate and improve a

23、xonelongation. The results of IF showedt the immunoreactivity of RhoA andp-ROCK2 is decreasedhe motor neuronal bodyhe surgery plus nimodipinegroup. In fal nerve axon, it was also shownt nimodipine inhabit theactivity of RhoA/ROCK signal pathway at both the translational andtranscriptional level.The results of this study demonstratet nimodipine treatmentameliorated crush injury of the fal nerve in our rat mby promotingre-myelination and provide evidencet the underlyin