腺病毒载体介导的CTLA4Ig基因追踪臂丛神经通路的研究

目的 利用腺病毒载体(Ad)介导的CTLA4Ig基因示踪大鼠臂丛神经轴突及其运动和感觉神经元,探讨转基因产物CTLA4Ig蛋白作为神经通路示踪剂的可行性及其特点.方法 经正中神经或尺神经或肌皮神经近断端导入携带CTLA4Ig或增强型绿色荧光蛋白(EGFP)基因的Ad(各2×107pfu).在转染后3周内6个不同时间点取标本,行CTLA41g免疫荧光染色,观察标记的运动及感觉神经元所在脊髓和脊髓后根神经节(DRGs)节段、神经根及轴突被标记范围及时间,并与EGFP基因的示踪结果比较.结果 由正中神经、尺神经和肌皮神经所标记的神经元分别在C6～Th1、c7～Th1及C5～C7节段;其相应的神经根及神经干近段轴突亦被标记.CTLA4Ig标记的神经元在转染3周后消失,所标记神经干轴突在转染5周后消失;而EGFP所标记的神经元和神经干分别2周、3周后消失.结论 CTLA4Ig基因能在Ad介导下经外周逆行性、特异性示踪臂丛神经及其靶运动和感觉神经元.     

大鼠臂丛放射性损伤后脊髓前角运动神经元病理变化及c-fos基因表达

目的模拟乳癌放疗方案，观察实验大鼠臂丛神经受照射后相应脊髓前角运动神经元的病理改变及c—fos基因表达。方法采用剂量分割照射方案，建立大鼠臂丛神经放射性损伤动物模型。将加只SD大鼠随机分成臂丛神经损伤组（实验组）和正常对照组，实验组又根据损伤后取材时间的不同分成损伤后3、5、7和9周4个时间组，每个时间组和对照组各4只大鼠，每个时间组以左侧为照射侧，右侧为对照侧，采用日照射2Gy／次，每周5次深部X线照射，累计剂量达到30Gy后，分别于末次照射后3、5、7和9周取材。检测两侧脊髓前角运动神经元形态及数目变化并以免疫组织化学染色法检测c—fos在前角运动神经元中的表达。结果大鼠臂丛神经受照射后3周，实验组照射侧脊髓前角运动神经元存活数目随时间延长而明显减少，神经元内有明显的c—fos染色，c—fos阳性细胞数渐减。照射后9周，c-fos阳性细胞偶见。结论大鼠臂丛神经受大剂量X线照射后可使大鼠脊髓前角运动神经元存活数目明显减少；与脊髓神经元凋亡相关的c—fos基因呈阳性表达，表明原癌基因的表达在臂丛神经放射损伤的凋亡、损伤和修复过程中起重要作用。     

大鼠臂丛神经根性回植后脊髓病理改变和轴突再生

目的 探讨臂丛神经根性撕脱后神经根再植入脊髓的可行性。方法 采用大鼠颈5-7，神经根性撕脱伤实验动物模型，伤后将C5-7，神经根即刻植入脊髓。分别于神经根植入后3周、3个月、6个月取材。应用组织病理活检、免疫组化技术及神经示踪技术，对神经中枢及吻合口下段神经干检查。观察脊髓前角运动神经元和神经元内尼氏体数目和形态的改变；周围神经纤维再生数目、距离，轴索和髓鞘发育情况。结果 臂丛神经根性撕脱伤对动物生长和存活有较大的影响。脊髓前角运动神经元数目在3个月内持续减少，3个月后趋于稳定，6个月时脊髓前角大型运动细胞坏死比率在40％左右，残存的神经元多为受损的神经元，尼氏体减少或消失。脊髓前角运动神经元再生轴突可重新生长入周围神经干，再生神经纤维轴索较细，大部分髓鞘发育不完全，轴突再生距离较短，肌皮神经6个月内无神经纤维再生。结论 臂丛神经根性撕脱伤，神经根回植入脊髓后，脊髓前角运动神经元坏死比率为40％左右，残存神经元多为受损神经元，再生神经纤维表现为动力不足和发育不全，对终末器官功能恢复没有意义。     

实验性脊髓缺血性损伤后神经元病变的神经丝免疫组织化学研究

目的：观察实验性兔腰髓缺血４０ｍｉｎ和再灌流４ｈ的脊髓运动神经元胞体和轴突的病理学改变。方法：用免疫组织化学方法观察神经丝（ｎｅｕｒｏｆｉｌａｍｅｎｔｓ，ＮＦ）抗体特异标记神经元胞体和轴突，并对其结果进行图像分析。结果：缺血４０ｍｉｎ，脊髓前角运动神经元胞体和轴突内神经丝反应异常增强，胞体内神经丝分布紊乱聚集。再灌流４ｈ，脊髓前角运动神经元胞体和轴突内神经丝反应阳性，胞体内神经丝散乱、稀疏、崩溃和溶解，轴突肿胀、扩大、消失。图像分析了脊髓前角运动神经元胞体内神经丝的面积、灰度和脊髓前索内轴突的数量，其结果具有明显的统计学意义。结论：神经丝免疫组织化学方法能更清楚地显示脊髓运动神经元胞体和轴突的病理学改变，脊髓损伤后细胞骨架紊乱在神经元的病理发病机制中起重要作用。     

臂丛损伤神经干细胞脊髓前角移植后分化情况及对运动神经元的保护作用

目的观察臂丛根性撕脱伤后将脊髓源性神经干细胞(neuralstemcell,NSC)移植于脊髓前角后的存活、分化情况及对脊髓前角受损运动神经元的保护作用。方法取新生鼠脊髓,分离获得脊髓源性神经干细胞,体外培养、扩增、鉴定、5溴2-脱氧尿苷(BrdU)标记。取SD大鼠60只,随机分成实验组、对照组和单纯组。从后路制备C5~C7臂丛神经根性撕脱伤动物模型。实验组移植神经干细胞于C6脊髓前角,对照组移植灭活神经干细胞,单纯组不作移植。术后1、2、4、8、12周取脊髓标本进行组织学与免疫组化染色观察。结果神经干细胞移植入脊髓后能存活、分化;臂丛根性撕脱伤后脊髓前角运动神经元数目明显减少;实验组神经干细胞移植后2、4、8、12周各个时间点运动神经元的存活率均高于对照组和单纯组。结论臂丛根性撕脱伤脊髓前角神经干细胞移植后能存活并分化为神经元及星型胶质细胞,脊髓源性神经干细胞移植能明显减少前角运动神经元的继发性死亡,对脊髓前角受损运动神经元有保护作用。     

雪旺细胞源神经营养因子预防臂丛神经根性撕脱伤所致运动神经元死亡

目的　研究雪旺细胞源神经营养因子（ Ｓ Ｄ Ｎ Ｆ）对臂丛神经根性撕脱伤所致前角运动神经元死亡的保护作用。方法　选成年 Ｓ Ｄ 大鼠,制成颈６、７神经根性撕脱伤动物模型,损伤处定期应用 Ｓ Ｄ Ｎ Ｆ,３ 周后观察损伤侧脊髓前角运动神经元的成活率和形态学变化,以及一氧化氮合酶（ Ｎ Ｏ Ｓ）表达的情况。结果　对照组６８６％ 的脊髓前角运动神经元死亡,成活神经元胞体明显萎缩,同时表达 Ｎ Ｏ Ｓ神经元增多;实验组脊髓前角运动神经元的死亡率较对照组降低３５％ ,成活神经元胞体无萎缩,表达 Ｎ Ｏ Ｓ神经元未见增多。结论　 Ｓ Ｄ Ｎ Ｆ对臂丛神经根性撕脱伤所致运动神经元死亡有明显的保护作用,一氧化氮在臂丛神经根性撕脱伤所致运动神经元死亡中起一定作用。     

雪旺细胞源神经营养因子对脊神经根性撕脱所致运动神经元死亡的保护作用

目的研究雪旺细胞源神经营养因子（ＳＤＮＦ）对臂丛神经根性撕脱所致前角运动神经元死亡的保护作用。方法于颈神经根性撕脱处应用ＳＤＮＦ,３周后观察损伤侧脊髓前角运动神经元的存活率和形态学变化以及ＮＯＳ表达的情况。结果对照组６８．６％的神经元死亡,存活神经元胞体明显萎缩,同时表达ＮＯＳ神经元增多;实验组的死亡率较对照组降低３５％,存活神经元胞体无萎缩,表达ＮＯＳ神经元未见增多。结论ＳＤＮＦ对脊髓前角运动神经元死亡有明显的保护作用,ＮＯ在臂丛神经根性撕脱所致运动神经元死亡中起一定作用。     

颈7神经不同平面切断对神经元影响的实验研究

目的 研究颈7神经不同平面切断及不同时期对神经元逆行性退变的影响,为临床选择合适的时间和方法保护受损神经元提供实验依据.方法 36只SD雌性大鼠随机分为三组:对照组、神经根部切断组和神经支部切断组.利用True Blue逆行示踪技术对各组大鼠背根神经节感觉神经元和脊髓前角运动神经元精确计数,并利用神经元特异性烯醇化酶(neuron specific enolase,NSE)免疫荧光双标技术检验标记神经元的活性.结果 术后1周,神经根部切断组和神经支部切断组较对照组无明显的感觉和运动神经元数量变化.术后16周,神经根部切断组和神经支部切断组较对照组感觉和运动神经元减少,其中神经根部切断组较神经支部切断组减少更明显(P＜0.05).且各组True Blue标记神经元全部被NSE免疫荧光双标.结论 颈7神经根部切断和神经支部切断均发生感觉和运动神经元逆行性退变,且不同平面切断对神经元退变的影响有显著性差异.提示臂丛神经根性损伤后早期修复受损神经有利于防止神经元退变.     

NT-3基因修饰许旺细胞与神经干细胞联合移植促进大鼠全横断脊髓受损伤神经元的存活及其轴突再生

目的探讨神经营养素-3基因修饰许旺细胞（NT-3-SCs）与神经干细胞（NSCs）联合移植对大鼠因脊髓全横断而受损伤的神经元存活及其轴突再生的作用。方法将NT-3-SCs及LacZ报告基因修饰SCs（LacZ-SCs）与NSCs联合移植到全横断性脊髓损伤处，60d后在脊髓横断处尾侧注射荧光金（FG）进行逆行标记。第67天，取材进行组织学检测大脑皮质体感区、红核及脊髓横断处头侧FG标记神经元；大脑皮质体感区、红核和脊髓背核内存活的神经元以及脊髓损伤处再生的轴突。结果大脑皮质体感区、红核及脊髓L1背核内存活的神经元由多到少依次为NT-3-SCs与NSCs联合组、LacZ—SCs与NSCs联合组和实验对照组。NT-3-SCs与NSCs联合组和LacZ—SCs与NSCs联合组的大脑皮质体感区、红核和脊髓横断处头侧有FG标记神经元；脊髓横断处及其附近组织有5-HT、CGRP和SP阳性神经纤维。结论NT-3-SCs与NSCs联合移植能够促进脊髓全横断损伤后神经元的存活以及轴突再生。     

脊髓内注射NT3重组腺病毒对前角神经元的保护作用

目的 观察脊髓内注射神经营养素3（Neurotrophin-3，NT3）重组腺病毒对坐骨神经损伤后脊髓前角运动神经元的保护作用。方法 制作大鼠坐骨神经夹伤模型，12只大鼠随机分为治疗组与对照组，治疗组大鼠在立体定位仪上于脊髓腹角给予神经营养素3重组腺病毒（Adeno-NT3），对照组给予生理盐水。术后不同时间通过CTB逆标观察脊髓相应节段运动神经元再生的数目；通过尼氏染色计数并计算运动神经元的存活百分率。结果 给予Adeno—NT3组与对照组相比，相应节段运动神经元再生数目和存活百分率明显增加，而且再生神经元增加的比例高于存活神经元。结论 Adeno-NT3对坐骨神经损伤后的脊髓前角运动神经元具有保护作用。     

Newly-formed axonal branches of rat sciatic neurons sprouting in the spinal cord after peripheral axotomy.

A retrograde method of nerve tracing using a recombinant adenovirus was applied to experimental regeneration of peripheral nerves to study the sprouting position of the regenerating axon. This enabled us to see the entire length of the axons on a whole-mount neural specimen. The peroneal nerve was transsected and infected with this virus, and the tibial nerve was transsected and sutured in eight Wistar rats. Four to five weeks later, labelled axons appeared in the tibial nerve, some of which could be traced from the tibial nerve to the spinal cord without making a connection with other labelled fibres. Control experiments negated the possibilities of transneuronal immigration or contamination of the virus. When the peroneal and tibial nerves were double-labelled with fluorescent tracers four weeks after their transsection, double-labelled motor neurons appeared. Based on these findings, we conclude that regenerating branches do sprout in the spinal cord after axotomy.     

Rerouting peripheral nerves for spinal cord lesions

To provide control of paralyzed limb muscles following spinal cord lesions, peripheral nerves containing motor axons from motoneurons located above a spinal cord lesion could potentially be rerouted to nerves containing motor axons located below the spinal cord lesion. To test this hypothesis in rats, the distal end of a cut tibial nerve, innervated by the L4-6 spinal level, was anastomosed or rerouted to the central end of the cut femoral nerve, innervated by the L3-4 spinal level. Appropriate controls were used. Recovery of lower hind limb motor function was followed at regular intervals, measuring the twitch tension of toe flexion (innervated by the tibial nerve) induced by transcutaneous stimulation of nerve rootlets exiting the spinal cord. After 4-6 months, 54% of motor function returned in the experimental group. Retrograde transport of horseradish peroxidase from the gastrocnemius muscles to spinal motoneuron cell bodies confirmed that the innervation of this group was at a higher level. Furthermore, after an L4 spinal transection, twitch tension responses to spinal cord outlet stimulation remained only in the experimental group. Therefore, a peripheral nerve containing motor axons from above the lesion was rerouted to a distal peripheral nerve to control muscles that would have otherwise been denervated.     

电刺激对鼠坐骨神经横断后脊髓运动神经元凋亡的影响

目的 观察直流电场对鼠坐骨神经横断后脊髓运动神经元凋亡的影响。方法 鼠坐骨神经横断后,实验组在相应的脊髓节段立即置入直流电刺激器,对照组置入无输出电流的刺激器,分掇于术后1、4、7、14、28d取材,应用HE染色,光镜下进行形态学观察,对脊髓运动神经元计数半进行立体定量分析,用TUNEL染色,对脊髓运动神经元中阳性细胞计数。结果 坐骨社会横断后4、7、14、28d,实验组脊髓运动神经元数量明显多于对照组,术后7、14、28d,实验组脊髓运动神经元体积在于对照组,运动神经元TUNEL染色阳性数明显小于对照组。结论 直流电刺激对坐骨神经横断引起的脊髓运动神经元凋亡具有防治作用。     

Adenoviral gene transfer in the peripheral nervous system

Background Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 10⁸ pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves.     

Return of spinal reflex after spinal cord surgery for brachial plexus avulsion injury

Motor but not sensory function has been described after spinal cord surgery in patients with brachial plexus avulsion injury. In the featured case, motor-related nerve roots as well as sensory spinal nerves distal to the dorsal root ganglion were reconnected to neurons in the ventral and dorsal horns of the spinal cord by implanting nerve grafts. Peripheral and sensory functions were assessed 10 years after an accident and subsequent spinal cord surgery. The biceps stretch reflex could be elicited, and electrophysiological testing demonstrated a Hoffman reflex, or Hreflex, in the biceps muscle when the musculocutaneous nerve was stimulated. Functional MR imaging demonstrated sensory motor cortex activities on active as well as passive elbow flexion. Quantitative sensory testing and contact heat evoked potential stimulation did not detect any cutaneous sensory function, however. To the best of the authors' knowledge, this case represents the first time that spinal cord surgery could restore not only motor function but also proprioception completing a spinal reflex arch.     

Effect of electrical stimulation of peripheral nerves on neuropathic pain

STUDY DESIGN: Changes in the electrophysiologic response of spinal dorsal horn neurons elicited by peripheral electrical stimulation were examined. OBJECTIVE: To investigate whether the electrical stimulation of peripheral nerves causes an inhibition of pain at the spinal cord level. SUMMARY OF BACKGROUND DATA: The wide dynamic range neurons studied were known to be excited by primary afferent fibers, not only combined A (delta) and C nociceptive fibers, but also low-threshold mechanoreceptive A (beta) fibers and A (delta) fibers of down hairs. The wide dynamic range neurons are classified as nociceptive neurons. METHODS: Responses of wide dynamic range neurons in the lumbosacral dorsal horn to input from C fibers were studied in urethane chloralose-anesthetized cats. The posterior tibial nerve and sciatic nerve were stimulated simultaneously to examine the effect on the C fiber responses elicited by superficial peroneal nerve stimulation. RESULTS: Simultaneous stimulation of the posterior tibial nerve and sciatic nerve was performed with superficial peroneal nerve C fiber stimulation. CONCLUSIONS: This study demonstrated that electrical stimulation of peripheral nerves leads to inhibitory input to the pain pathways at the spinal cord level.     

A new technique to improve cortical-evoked potentials in spinal cord monitoring. A ratio method of analysis

Systemic effects such as anesthesia, hypotension, hypothermia, and hypoxia affect the cortical evoked responses. We propose, that by sequential stimulation of the median and posterior tibial nerves, and the construction of a ratio from the value of their amplitudes, the systemic effects can be eliminated and thus improve the reliability of the cortical evoked responses. Two groups of scoliosis patients who underwent spinal surgery with instrumentation were analyzed retrospectively. Both groups had spinal cord monitoring using peripheral nerve stimulation and cortical recordings of the somatosensory-evoked response (SER). In Group I, 50 patients were analyzed for changes in posterior tibial nerve response before and after distraction. Wide variability in the response suggested this method to be less reliable in predicting spinal cord conduction deficits. Thirty-eight patients in Group 2 were analyzed using both the median and posterior tibial nerve amplitudes. A ratio of the posterior tibial to median nerve wave amplitude was constructed, thus eliminating any systemic variables. A critical value, alerting the surgeons to possible decreases in spinal cord conduction, was calculated by subtracting one standard deviation from the mean of the postdistraction ratios of the posterior tibial to median nerves (1.20-.633 = .567).     

补阳还五汤促进周围神经损伤后神经元存活的实验研究

目的 研究补阳还五汤对周围神经损伤后脊髓前角运动神经元和脊神经节感觉神经元存活的影响。方法 成年SD大鼠20只，在右侧梨状肌下缘0.5cm处切断坐骨神经，随机分为两组，对照组以生理盐水灌胃，实验组以补阳还五汤灌胃，4W后观察相应节段运动神经元和感觉神经元存活率、神经元胞体直径和面积。结果 4W后相应节段脊髓前角运动神经元存活率：对照组为59．87％，实验组为69．22％（P＜0．01）；脊神经节感觉神经元存活率：对照组为70．99％，实验组为79．13％（P＜0．01）。对照组运动神经元和感觉神经元胞体直径、面积明显小于实验组（P＜0．01）。结论 补阳还五汤能提高周围神经损伤后脊髓前角运动神经元和脊神经节感觉神经元存活率，减轻神经元胞体萎缩程度，有利于周围神经损伤后神经功能的恢复。     

坐骨神经损伤后脊髓前角神经元形态学的观察

目的 研究大鼠坐骨神经损伤后，脊髓前角运动神经元胞体形态学的变化，以探讨其主要死亡性质。方法 切断大鼠右侧坐骨神经，再原位吻合，手术后不同时间取腰4--腰6(L4-L6)节段脊髓，作石蜡切片，通过苏木素—伊红(HE)染色，光镜下观察脊髓前角运动神经元胞体的形态学变化特征；作超薄切片，电镜下观察脊髓前角运动神经元胞体超微结构的变化情况。结果 右侧脊髓前角运动神经元胞体尼氏体和细胞核染色质浓缩(光镜下)；电镜下，细胞膜内陷，将细胞分割成凋亡小体，然后裂解、细胞体消失；而左例脊髓前角运动神经元胞体均一、无变化。结论 坐骨神经损伤后，脊髓前角运动神经元有死亡，死亡性质主要是细胞凋亡。     

Spinal cord monitoring. Electrophysiological measures of sensory and motor function during spinal surgery

Various recording methods were tested in 60 patients who underwent scoliosis surgery to find the most suitable technique for the spinal cord monitoring and to elucidate the neuroanatomic relationship of the evoked potentials recorded by these methods. Responses were recorded from the scalp and spine after stimulation of the tibial nerve or the spinal cord. The potentials from electrodes placed over the muscles and the tibial nerve after stimulation of the spinal cord were also recorded. Epidurally recorded spinal evoked potentials after stimulation of the tibial nerve generally consisted of two major negative peaks, NI and NII, and subsequent multiple waves. NI may be mediated through the spinocerebellar tract, and NII is most likely mediated through the dorsal column. The polyphasic waves are probably conducted through the slower sensory ascending pathways. The potentials recorded from the muscle after spinal cord stimulation may be mediated through the motor tract. Various recording techniques described in this study were mutually complimentary in confirming the results of tests recorded in the technically difficult environment of the operating room. In general, spinal cord stimulation recorded from the scalp or the spine was superior to peripheral nerve stimulation in yielding better defined responses. If the potential recorded from the muscle after stimulation of the spinal cord is indeed mediated through the motor pathway, this would be useful to assess motor function during surgery.