【作者】 吴可晚；

【导师】 陈琳； 蔡佩琴； 虞聪； 彭峰； 杨剑云；

【作者基本信息】 复旦大学 ， 手外科学， 2010， 硕士

【摘要】 第一部分颈交感神经不同功能状态对周围神经慢性卡压伤的影响交感神经属于自主神经系统,但从解剖学上分,交感神经节后纤维是周围神经组成部分。在正常生理状态下,交感神经系统与周围感觉神经系统极少发生解剖学上的直接联系,周围感觉传入神经及其末梢感受器也极少受交感神经影响(Janig & Koltzenburg,1991)。但在病理条件下(如周围神经慢性卡压伤),由于神经瓦勒变性,产生神经生长因子、神经营养因子,诱导交感神经芽生,形成交感-感觉耦联作用,或通过影响周围神经血供,参与了神经病理性疼痛的形成和维持。周围神经急性切断伤后,交感封闭促进其再生,而交感切除则不利于其再生,提示交感神经对周围神经损伤后再生的影响,可能与交感神经功能状态有关。既往有关交感神经对周围神经功能影响的研究主要针对慢性疼痛和神经急性损伤后再生方面,那么,交感神经对受慢性卡压伤的周围神经损伤程度,以及去除卡压后神经功能恢复有无影响?与交感神经功能状态是否有关?目前尚无这方面的文献报道。本研究旨在探讨交感神经不同功能状态对受慢性卡压伤的周围神经损伤程度的影响,以及慢性卡压松解后,交感神经不同功能状态对周围神经功能恢复的影响。实验一：交感神经不同功能状态对受慢性卡压的周围神经损伤的影响实验材料一、实验动物雄性体重约200g SD大鼠32只,由复旦大学实验动物科学部提供。二、主要试剂Trizol购于invitrogen公司,RevertAid First Strand cDNA Synthesis Kit购自Fermentas公司,FastStart Universal SYBR Green Master(ROX)购自Roche公司,P物质、S100b以及GAPDH引物由上海生工生物工程有限公司合成。三、实验仪器手提式高速电动离散器(美国biospec),4℃离心机,紫外分光光度仪,Opticon-2 PCR循环仪,Leica FW4000图像分析系统,Medtronic肌电图仪。实验方法将32只SD大鼠分成A1、B1、C1三个实验组和一个正常对照组D,每组8只。颈正中入路,左侧为实验侧,显露臂丛下干,参照Machinnon坐骨神经慢性卡压伤模型制作方法制作大鼠左臂丛下干慢性卡压伤动物模型。C1组在制作下干卡压模型同时切除左侧颈中神经节。B]组造模术后2个月,以0.25%布比卡因0.5ml作颈中神经节阻滞,每日一次,共1个月。三组造模术后满3个月,去除卡压,检测骨间肌CMAP,记录波幅和潜伏期。取卡压远段神经计数有髓纤维和检测S100bmRNA表达水平,取C8T1背根节检测P物质mRNA表达水平。实验结果1、去卡压后A1(完全交感功能)、B1(抑制交感功能)、C1(去除交感)三个实验组和对照组D行电生理检测其左侧第一骨间肌CMAP,潜伏期均数A1>B1>C1>D,分别为：(4.8±0.9)ms、(4.3±0.6)ms、(3.9±0.5)ms、(2.8±0.2)ms,经统计学分析,A1、B1、C1三组潜伏期间的差异有统计学意义(P=0.047)。波幅均数A1<B1<C1<D,分别为：(2.2±1.1)mv、(2.6±0.8)mv、(3.9±1.1)mv,(8.6±2.0)mv,经统计学分析,A1、B1、C1三组波幅有显著性差异(P=0.011)。2、卡压远段有髓纤维数A1<B1<C1,三组分别为：(3583±540)、(4156±746)、(5098±742),且均小于正常(7934±868)。经方差分析,A1、B1、C1三组差异有统计学意义(P=0.001)。3、卡压远段神经检测的S100bmRNA表达相对定量A1>B1>C1,分别为：(8.05±3.47)×10-3、(5.95±2.51)×10-3、(2.69±0.92)×10-3,经方差分析,三者有著性差异(P=0.001),且均显著高于正常表达水平(0.52±0.15)×10-3,(P=0.000)。4、卡压侧C8T1背根节中SP mRNA表达水平相对定量A1>B1>C1,分为：(3.62±0.83)×10-2、(2.95±0.72)×10-2、(2.24±0.73)×10-2,经方差分析,三者有显著性差异(P=0.006),且均高于正常水平(1.21±0.28)×10-2。结论1、周围神经卡压性疾病的发展除机械卡压因素外,交感神经在疾病的发展过程中也起着重要作用；2、异常的交感神经功能越强,则卡压引起的疼痛症状越重,卡压神经结构性损伤也越大,去除病态的交感神经有利于卡压损伤程度的减轻；3、过高表达的P物质和S100b蛋白,可能是交感神经参与卡压性疾病发展的介质之一。实验二、交感神经不同功能状态对周围神经卡压后神经功能恢复的影响实验材料一、实验动物雄性体重约200g SD大鼠24只,由复旦大学实验动物科学部提供。二、主要试剂Trizol购于invitrogen公司,RevertAid First Strand cDNA Synthesis Kit购自Fermentas公司,FastStart Universal SYBR Green Master(ROX)购自Roche公司,P物质、S100b以及GAPDH引物由上海生工生物工程有限公司合成。三、实验仪器手提式高速电动离散器(美国biospec),4℃离心机,紫外分光光度仪,Opticon-2 PCR循环仪,Leica FW4000图像分析系统,Medtronic肌电图仪。实验方法将24只SD大鼠分成A2、B2、C2三组,每组8只。颈正中入路,左侧为实验侧,显露臂丛下干,参照Machinnon坐骨神经慢性卡压伤模型制作方法制作大鼠左臂丛下干慢性卡压伤动物模型。卡压满3个月,A2组去除卡压,不处理颈交感神经,继续饲养1个月；B2组去除卡压后,以0.25%布比卡因行颈中神经节封闭,每日1次,共1个月；C2组去除卡压同时切除颈中神经节及其支配臂丛的分支,继续饲养1个月。三组动物在去卡压后1个月,检测骨间肌CMAP,记录波幅和潜伏期。取卡压远段神经计数有髓纤维和检测S100bmRNA表达水平,取C8T1背根节检测P物质mRNA表达水平。实验结果1、A2(完全交感功能)、B2(抑制交感功能)、C2(去除交感)三组行电生理检测左侧骨间肌CMAP的潜伏期A2>B2>C2,分别为：(3.8±0.6)ms、(3.6±0.4)ms、(3.2±0.3)ms,波幅A2<B2<C2,分别为：(3.2±1.1)mv、(3.6±1.3)mv、(4.3±1.8)mv。经方差分析,三组潜伏期有显著性差异(P=0.04),波幅差异无统计学意义(P=0.244)。2、卡压远段有髓纤维数A2<B2<C2,三组分别为：(5374±642)、(6276±819)、(7162±973),经方差分析,差异有统计学意义(P=0.001)。3、卡压远段神经检测的S100bmRNA表达相对定量A2>B2>C2,分别为：(5.39±1.19)×10-3、(1.76±0.32)×10-3、(1.03±0.39)×10-3,经方差分析,三者有著性差异(P=0.001)。但仍高于正常水平(0.52±0.15)×10-3。4、卡压侧C8T1背根节中SP mRNA表达水平相对定量A2>B2>C2,分为：(3.16±1.03)×10-2、(2.36±0.68)×10-2、(1.49±0.80)×10-2,经方差分析,三者有显著性差异(P=0.003)。结论1、在周围神经卡压性疾病的治疗过程中,除去除机械性卡压因素外,降低异常功能的交感神经的兴奋性,有利于受卡压的周围神经再生和功能恢复,疼痛症状消退。2、异常的交感神经功能越低,则去卡压后受卡压的周围神经功能恢复越好。3、降低异常功能的交感神经兴奋性促进慢性卡压后神经再生和功能恢复、疼痛缓解可能是通过减少P物质和S100b蛋白等介质引起的炎症反应和损伤作用,创造一个有利于神经功能恢复的生理环境而起作用。第二部分交感神经功能状态临床检测方法的改良交感神经皮肤反应(sympathetic skin response,SSR)是目前临床上检测交感神经功能的一种无创、简便、可靠的手段。手掌部为最常采用的检测部位,电刺激为最常采用的刺激方法。然而手掌部记录不能反应单神经通路上交感传出功能状态,因此,本实验将检测部位进行适当改良,以尺、正中神经绝对支配区为检测部位,研究绝对支配区SSR特点,并建立正常参考值。研究对象与方法一、研究对象1、健康自愿者60例,其中男31例,女29例,年龄18～54岁,平均32.4岁。30例检测右侧,30例检测左侧。2、尺神经损伤者10例,其中男6例,女4例,平均年龄32.3岁。右侧7例,左侧3例,损伤时间为3个月以内。3、正中神经完全损伤10例,男7例,女3例,平均年龄35.1岁。左右侧各5例,损伤时间为3个月以内。4、正中神经和尺神经均损伤共3例,男2例,女1例,平均年龄28岁,损伤部位均为腕部,损伤时间1个月。二、研究方法1、检测部位：掌心、示指腹和小指腹。2、条件要求：室温、明亮、安静环境,受试者放松,皮温32～36℃。3、检查仪器：神经肌电图仪。4、检查方法：直流方波0.2ms,强度10～30mA,电刺激对侧正中神经,表面电极记录SSR的潜伏期和波幅。结果1、左右手SSR检查结果：掌心、示指指腹和小指指腹SSR潜伏期和波幅左右侧差异无统计学意义(P>0.05)。2、不同性别SSR检查结果：掌心、示指和小指记录的SSR的潜伏期平均值男性较女性长,但差别无统计学意义(P>0.05)。波幅均值男性较女性大的趋势。3、正常者掌心、示指腹和小指腹的SSR的潜伏期分别为：(1.38±0.15)s、(1.54±0.22)s、(1.59±0.23)s,波幅分别为：(3.78±2.37)mv、(2.26±1.52)mv、(2.46±1.84)mv。示、小指腹记录的SSR的潜伏期较掌心部位长,波幅较掌心部位低(潜伏期P<0.01,波幅P<0.01)。示指腹和小指腹记录的SSR的潜伏期和波幅,两部位比较,差异无统计学意义(P>0.05)。4、正中神经损伤后,其绝对支配区(示指腹)SSR消失,掌心部位SSR潜伏期略延长,与正常者相比,差异无统计学意义,但波幅明显变低(P=0.000),小指腹SSR不受影响。尺神经损伤后,其绝对支配区(小指腹)SSR消失,掌心部位SSR不受明显影响,示指腹SSR不受影响。尺神经和正中神经二者均损伤,则手掌部任何部位SSR消失。5、SSR正常参考值：掌心部位潜伏期和波幅分别为：≤1.69s和0～8.52mv；示、小指腹部位SSR的潜伏期和波幅分别为：≤2.00s和0～5.73mv。结论1、手掌部、指腹均可检测到SSR,与掌心部位相比,指腹处SSR潜伏期长,波幅小。左右侧同一部位SSR无差别。女性SSR波幅有较男性低的趋势。2、SSR潜伏期稳定,波幅变异大,潜伏期为可靠指标,波幅为参考指标。潜伏期延长为异常,波幅消失为异常。判定波幅过高是否为异常,要结合潜伏期,综合考虑。3、尺、正中神经通路上的交感纤维支配区在掌部有交叉,而在绝对支配区无交叉支配现象,互不干扰,故而绝对支配区SSR可反应单神经(正中神经或尺神经)通路上的交感传出纤维功能状态。更多还原

【Abstract】 Part I Effect of different functions of sympathetic nerve on chronically compressed peripheral nerveSympathetic nerve belongs to autonomic nerve system. Anatomically, the postganglionic fiber of sympathetic nerve is a component of peripheral nerve. Under a normal physiological condition, directly anatomical connection seldom exists between peripheral nerve and sympathetic nerve system, and peripheral afferent nerves and their terminal receptors are also seldom affected by sympathetic nerve (Janig & Koltzenburg, 1991). However, under pathologic conditions, such as peripheral chronic compressive injury, sympathetic nerve is involved in the development and maintenance of neuropathologic pain via insulting the blood supply of peripheral nerve or developing sympathetic-sensory coupling by sympathetic sprouting due to nerve growth factor or neurotrophic factor which are produced by Wallarian degeneration. Recent experiment research showed that sympathetic nerve blockage promoted regeneration of peripheral nerve when acutely transected, while excision of sympathetic nerve was harmful to peripheral nerve regeneration, which indicates that the effect of sympathetic nerve on the regeneration of acutely injuried peripheral nerve may have something to do with the different functions of sympathetic nerve. Recently, studies on the relationship between sympathetic nerve and peripheral nerve focus on chronic pain and regeneration of peripheral nerve acutely injuried. Then, whether the effect of sympathetic nerve on the injury degree of chronically compressed peripheral nerve and on the function recovery of the peripheral nerve after decompression? And is the effect related to the function state of sympathetic nerve? There still is no literature concerned these issues.The experiments are performed to investigate the effects of different functions of sympathetic nerve on the lesion degree of chronically compressed peripheral nerve and on the recovery of peripheral nerve functions after decompression.Experiment I Effect of different functions of sympathetic nerve on the lesion degree of peripheral nerve chronically compressed Materials1. experimental animals:thirty-two Sprague-Dawley male rats weighted 200g were offered by experimental animal department of Fudan university.2. Main reagents:Trizol reagent(invitrogen), RevertAid First Strand cDNA Kit(Fermentas), FastStart Universal SYBR Green Master(ROX)(Roche), Primers of substance P, S100b and GAPDH were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co.3. laboratory apparatus:portable tissue tearor(biospec),4℃centrifuge, ultraviolet spectrometer, Opticon-2 PCR instrument, Leica FW4000 image analysis system, Medtronic electromyography.MethodsThirty-two rats were divided into three experimental groups (A1, B1,C1) and a normal control group(D group), each group had 8 rats. A mid-anterocervical approach was applied to expose the lower trunk of left brachial plexus which was experimental side. A model of the lower trunk chronic compression was made according to the rat model of sciatic nerve compression designed by Mackinnon. The left middle cervical ganglions of rats in group C1 were resected when the compression models were made. The middle cervical ganglions of the rats in group B1 were blocked once a day for one month with 0.5ml of 0.25% bupivacaine, starting from two months after surgery for model. The lower trunk nerves were decompressed three months after making models. And then, CMAP of the first interosseus was tested by electrograph to record latency and amplitude. Myelinated nerve fibers of the distal part of the compressed lower trunk were analyzed. The expression level of S100b mRNA of the distal part of the compressed nerve and that of substance P mRNA of C8T1 dorsal root ganglion were detected by RT-PCR.Results1. The mean latencies of CMAP of first interosseus (A1>B1>C1>D) were (4.8±0.9)ms、(4.3±0.6)ms、(3.9±0.5)ms、(2.8±0.2)ms, respectively. The difference of latencies among group A1, B1 and C1 was significant (P P=0.047). The mean amplitudes of CMAP(A1<B1<C1<D) were (2.2±1.1) mv、(2.6±0.8) mv、(3.9±1.1)mv, (8.6±2.0)mv, respectively. The difference of amplitudes among group A1,B1 and C1 was significant(P=0. 011).2. The numbers of myelinated nerve fibers of the distal part of compressed trunks (A1<B1<C1) were (3583±540)、(4156±746)、(5098±742), respectively, but all were less than the number of normal control group which was (7934±868). Statistical analysis showed a significant difference (P=0.001) among the three groups of A1, B1 and C1.3. The relative quantitation of S100b mRNA expression (A1>B1>C1) was (8.05±3.47)×10-3、(5.95±2.51)×10-3、(2.69±0.92)×10-3, respectively, with a significant difference among them. The expression levels of S100b mRNA of group A,, B, and C, were distinctively higher than that of control group D, the level of which was (0.52±0.15)×10-3.4. The relative expression levels(A1>B1>C1) of SP mRNA of C8T1DRG were (3.62±0.83)×10-2, (2.95±0.72)×10-2, (2.24±0.73)×10-2, respectively, among which the difference was significant (P=0.006). All of them were more than that of normal control group D, which was (1.21±0.28)×10-2。Conclusions1. Besides mechanical compression, sympathetic nerve plays an important role in developing of diseases of peripheral nerve compression.2. The effect of sympathetic nerve on the development of peripheral nerve compression is related to the function state of sympathetic nerve, which means the higher excitation of sympathetic nerve, the more pain caused by compression and the more serious lesion of the nerve fibers. To inhibit or remove the effect of unhealthy sympathetic nerve can help to reduce lesion degree of peripheral nerve chronic compression.3. The overexpressed SP and S100b may be the mediators by which sympathetic nerve participate in the development of peripheral nerve chronic compressive diseases.ExperimentⅡEffect of different functions of sympathetic nerve on the function recovery of compressed peripheral nerve after decompression Materials1. experimental animals:Twenty-four Sprague-Dawley male rats weighted 200g were offered by experimental animal department of Fudan university.2. Main reagents:Trizol reagent(invitrogen), RevertAid First Strand cDNA Kit(Fermentas),FastStart Universal SYBR Green Master(ROX)(Roche), Primers of substance P, S100b and GAPDH were synthesized by Shanghai Sangon Biological Engineering Technology & Services Co.3. laboratory apparatus:portable tissue tearor(biospec),4℃centrifuge, ultraviolet spectrometer, Opticon-2 PCR instrument, Leica FW4000 image analysis system, Medtronic electromyography.MethodsTwenty-four rats were divided into three experimental groups of A2, B2, and C2. Each group had 8 rats. A mid-anterocervical approach was applied to expose the lower trunk of left brachial plexus which was experimental side. A model of the lower trunk chronic compression was made according to the rat model of sciatic nerve compression designed by Mackinnon. Three months after compression surgery, the rats of group A2 were decompressed without interfering the middle cervical ganglions and were bred for another month. The rats of group B2 were decompressed, then left middle cervical ganglions were blocked with 0.5ml of 0.25% bupicaine once a day for one month. The middle cervical ganglions of group C2 were resected when decompressed and continued to be breed for one month. One month after decompression, CMAP of the first interosseus was tested by electrograph to record latency and amplitude. Myelinated nerve fibers of the distal part of the compressed lower trunk were analyzed. The expression level of S100b mRNA of the distal part of the compressed nerve and that of substance P mRNA of C8T1 dorsal root ganglion were detected by RT-PCR.Results1. The mean latencies of CMAP of first interosseus (A2>B2>C2) were (3.8±0.6) ms、(3.6±0.4) ms、(3.2±0.3) ms, respectively. The difference of latencies among group A2, B2 and C2. The mean amplitudes of CMAP(A2 <B2<C2) were (3.2±1.1)mv、(3.6±1.3)mv、(4.3±1.8)mv, respectively. The difference of latencies among group A2, B2 and C2 was significant(P=0.04).the amplitudes difference is not significant(P=0.244).2. The numbers of myelinated nerve fibers of the distal part of decompressed trunks(A2<B2<C2) were (5374±642)、(6276±819)、(7162±973), respectively. Statistical analysis showed a significant difference (P=0.001) among the three groups of A2, B2and C2.3. The relative quantitation of S100b mRNA expression (A2>B2>C2) was (5.39±1.19)×10-3、(1.76±0.32)×10-3, (1.03±0.39)×10-3, respectively, with a significant difference(P=0.001) among them. The expression levels of S100b mRNA of group A2, B2 and C2 were still distinctively higher than normal level that was (0.52±0.15)×10-3.4. The relative expression levels (A2>B2>C2) of SP mRNA of C8T1 DRG were (3.16±1.03)×10-2、(2.36±0.68)×10-2、(1.49±0.80)×10-2, respectively, among which the difference was significant(P=0.003).Conclusions1. During the couse of treating compression diseases, besides removing mechanical compression, decreasing the excitation of abnormal sympathetic nerve does good to alleviating pain, regeneration and function recovery of compressed peripheral nerve when decompressed.2. The lower function of unhealthy sympathetic nerve, the better recovery of peripheral nerve.3. The effect of sympathetic nerve on the pain relieving,nerve regeneration and function recovery of peripheral nerve may via reducing substance P and S100b protein which mediate inflammatory response, so as to maintain a good physiological condition.PartⅡImproved clinical detection of sympathetic nerve functionSympathetic skin response(SSR) is a non-invasive, simple and reliable means of the clinical detection of sympathetic function. the most commonly used test site is palm, and the most commonly used stimulation method is electrical stimulation. However, SSR recorded on palm can reflect the sympathetic efferent function of a single neural pathway such as median nerve or ulnar nerve,therefore, the record site was changed appropriately. The absolutely dominated positions of ulnar and median nerves were taken for the detection sites. SSR characteristics of absolutely dominated area were analyzed and the normal reference values were established.Subjects and methods1. subjects1) 60 healthy volunteers, including 31 males and 29 females, aged 18 to 54 years old, mean age 32.4 years.30 were detected in the right side, the other 30 were detected in the left side.2) Ulnar nerve injury in 10 cases, including 6 males and 4 females, average age of 32.3 years. The right side in 7 cases, left in 3 cases, the injury time within 3 months.3) Complete median nerve injury in 10 cases,7 males and 3 females, average age of 35.1 years. The right side injury in 5 cases, left in 5 cases, the injury time within 3 months.4) Both median nerve and ulnar nerve injury in 3 cases,2 males and 1 female, average age 28 years, are part of the wrist injury, injury time 1 month or less.2. methods1) detection site:the palm, pulp of index finger and little finger.2) Conditions require:room temperature, light, quiet environment, participants relax, skin temperature keeps 32～36℃.3) Equipment:electromyograph.4) Detection methods:contralateral median nerve was stimulated by DC square wave 0.2ms with a current strength of 10-30Am. A surface electrode was used to record the latency and amplitude of SSR.Results1. SSR test results of right and left hands:The difference of the latency and amplitude of SSR tested on palms, index finger pulps and little finger pulps between right and left hands was not significant (P> 0.05).2. Gender and SSR test results:the average latency of SSR recorded on palms, index finger pulps and little finger pulps was longer in men than in women, but the difference was not statistically significant. The mean amplitude was higher in men than in women.3. SSR latencies recorded on palms, index finger and little finger pulps of healthy volunteers were (1.38±0.15) s、(1.54±0.22) s、(1.59±0.23) s, respectively, and the amplitudes of which were (3.78±2.37) mv (2.26±1.52) mv、(2.46±1.84) mv, respectively. The latencies of SSR recorded on index and little finger pulps were longer than on palm, and amplitudes of SSR were lower on index and little finger pulps than on palm. These difference were statistically significant (latency:P< 0.01, amplitude:P<0.01). But the difference of latency and amplitude of SSR between index finger and little finger pulps was not significant (P>0.05).4. when median nerve injuried, SSR of the absolutely dominated region (index finger pulp) disappeared, and the latency of SSR recorded on palm was slightly longer, compared with the normal, the difference was not statistically significant, but the amplitude became much lower (P=0.000), and SSR of the little finger pulp was not affected. while ulnar nerve injuried, SSR of the absolutely dominated region (small finger pulp)disappeared without significant effect on SSR of palm, and SSR of index finger pulp was not affected. when both median nerve and ulnar nerve were injuried, then SSR of any part of the palm disappeared.5. Normal reference value of SSR:SSR of palm:latency≤1.69s and amplitude:0～8.52mv;SSR of index and small finger pulps:latency≤2.00s and amplitude:0～5.73mv.Conclusions1. SSR on both palm and finger pulps can be detected. Compared with SSR of the palm site, the latency of the SSR of finger pulps is longer and the amplitude is lower. there is no statistical difference between SSR of right and left hand. The amplitude of SSR of female is lower than that of male.2. latency is a stable indicator for its stability, while amplitude serves as reference because of its large variability. Overlong latency is abnormal and disappeared amplitude is also abnormal. Whether a high amplitude is abnormal, the latency should be taken into account.3. Palm area innervated by sympathetic nerve which comes from both ulnar and mdian nerve pathways. however, the absolutely dominated region have no cross innervation. Therefore, SSR recorded from the absolutely dominated regions (index and little finger pulps) may reflect the function of sympathetic nerve from a single neural pathway (ulnar nerve or median nerve).更多还原