【作者】 王熠钊；

【导师】 黄晓琳；

【作者基本信息】 华中科技大学 ， 康复医学与理疗学， 2009， 博士

【摘要】 第一部分硬膜外脊髓电刺激诱发正常大鼠脊髓反射的特点实验一硬膜外脊髓电刺激电压变化对正常大鼠脊髓反射的影响目的研究正常大鼠麻醉状态下S1脊髓节段不同电压硬膜外脊髓电刺激所诱发的脊髓反射，探讨硬膜外脊髓电刺激电压变化对正常大鼠脊髓反射的影响。方法选取成年雌性Sprague-Dawley大鼠10只，麻醉后手术将电极植入S1脊髓节段。分别予400mV、600mV、1200mV硬膜外脊髓电刺激，以同心圆针电极记录大鼠下肢半腱肌肌腹的肌电信号，观察所诱导脊髓反射的特点。结果能引起大鼠下肢肌肉反应的阈值为300mV。三种电压强度的硬膜外脊髓电刺激能诱导出两种潜伏期成分的脊髓反射，较低的400mV、600mV电压强度可诱发出长潜伏期成分，潜伏期分别为5.27±0.36 ms和5.19±0.67ms；较高的1200mV电压强度可诱发出短潜伏期成分，潜伏期为2.57±0.23ms。结论不同电压的硬膜外脊髓电刺激可诱导出不同来源的脊髓反射。长潜伏期成分可能是兴奋脊髓背根传入神经后引起的单突触反射；短潜伏期成分可能是直接兴奋脊髓内的运动神经元或运动纤维后向下传导引起的肌肉兴奋电反应。实验二硬膜外脊髓电刺激频率变化对正常大鼠脊髓反射的影响目的研究正常大鼠麻醉状态下S1脊髓节段不同频率硬膜外脊髓电刺激所诱发的脊髓反射，探讨硬膜外脊髓电刺激频率变化对正常大鼠脊髓反射的影响。方法选取成年雌性Sprague-Dawley大鼠10只，麻醉后手术将电极植入S1脊髓节段。予波宽200μs、电压强度1200mV，频率分别为50Hz、60Hz、80Hz、100Hz硬膜外脊髓电刺激，以同心圆针电极记录大鼠下肢半腱肌肌腹的肌电信号，观察所诱导脊髓反射的特点。结果以50Hz、60Hz、80Hz、100Hz的ESCS刺激大鼠S1脊髓时，可诱发出脊髓反射。4种高频刺激后期均出现了脊髓反射脱落后不规律出现，部分大鼠出现了高频刺激后期脊髓反射完全消失。50Hz频率的ESCS所诱发脊髓反射的潜伏期和波宽分别为4.46±1.07ms和7.33±1.00ms，与另外三组相比有统计学差异。结论四种高频ESCS所诱发的不规律出现的脊髓反射，可能是一种单突触反射。高频刺激时脊髓反射的不规律出现可能与高频刺激的抑制作用有关。实验三硬膜外脊髓电刺激应用于正常大鼠不同脊髓节段所诱发脊髓反射的特点目的观察ESCS分别刺激正常大鼠L2及S1脊髓节段时所诱发的脊髓反射，探讨硬膜外脊髓电刺激应用于正常大鼠不同脊髓节段所诱发脊髓反射的特点。方法以同心圆针电极记录大鼠下肢胫前肌的EMG信号。将不同电压强度(400mV，600mV，1200mV)所诱发脊髓反射的潜伏期与经颅磁刺激得到的运动诱发电位的潜伏期相比较。将高频ESCS(50Hz、60Hz、80Hz、100Hz)分别应用于大鼠L2及S1脊髓节段，观察高频ESCS对脊髓反射的作用。结果L2脊髓节段电刺激的电压强度的改变并未对其诱发的脊髓反射有明显作用，并且L2脊髓节段电刺激所诱发脊髓反射的潜伏期与运动诱发电位的潜伏期无统计学差异。但不同电压强度的S1脊髓节段电刺激所诱发脊髓反射则显示出了不同的潜伏期。结论S1节段较低电压的电刺激时能诱发出一个较长潜伏期的脊髓反射，这个脊髓反射可能是电刺激兴奋了脊髓背根所致；较高电压的电刺激时能诱发出一个较短潜伏期的脊髓反射，这个脊髓反射可能是电刺激直接兴奋了脊髓的运动神经元或是运动神经纤维所致。L2节段较高电压刺激时，并未观察到类似现象，提示这可能与两个节段的结构不同有关。研究中我们记录到脊髓反射的波宽较小，这可能是与我们使用的同心圆记录电极的记录面积更局限有关；提示我们，研究中采用不同的记录电极，脊髓反射的波形可能会有不同。我们应用高频硬膜外电刺激时并未诱发下肢节律性的运动，这可能是因为我们难以给予实验中的麻醉大鼠一定的下肢感觉反馈所致；这提示我们记录清醒大鼠的EMG信号是十分必要的。第二部分硬膜外脊髓电刺激结合跑台训练对脊髓损伤大鼠大脑运动皮质及脊髓灰质前角超微结构的影响目的ESCS联合跑台训练已被证明可以促进脊髓损伤患者及脊髓损伤大鼠步行功能的恢复。我们拟研究硬膜外脊髓电刺激结合跑台训练对脊髓损伤大鼠大脑运动皮质，以及刺激部位脊髓灰质前角超微结构的影响。探讨ESCS联合跑台训练促进脊髓损伤患者及脊髓损伤大鼠步行功能的机制。方法：12只成年雌性大鼠随机分为4组：(1)脊髓损伤组(spinal cord injury group，SI)，(2)脊髓损伤+ESCS组(spinal cordinjury plus ESCS group，SE SE)，(3)脊髓损伤+跑台训练组(treadmill training group，TT)，(4)脊髓损伤+跑台训练+ESCS组(treadmill training plus ESCS group，TE)。所有大鼠先接受不完全脊髓损伤手术造模。造模手术后4周，SE组和TE组大鼠再接受电极植入手术。电极覆盖范围为L2～S1。电极植入术后4周，SE组大鼠要接受运动阈下的ESCS刺激，第天30分钟。TT组大鼠接受5cm／s的跑台训练，每天30分钟。TE组大鼠则每天同时接受ESCS和跑台训练，训练参数同SE组和TT组。SI组大鼠则不接受ESCS或是跑台训练，作为对照组。以上4组的干预都进行4周。4周后按电镜标本制作方法取大鼠大脑下肢运动皮质及脊髓前角灰质，以观察其超微结构变化。结果：经过4周的干预后，TT组和TE组大鼠的空地神经行为学评分都增加到了18左右，但两组间无统计学差异。而SI组和SE组则未见明显的增加。TT、TE组的大脑运动皮质血管直径都显著地较SI组和SE组增大。但TT、TE组间的血管直径无统计学差异，SI、SE组间的血管直径无统计学差异。无论是否接受ESCS或是跑台干预、大脑运动皮质突触和神经元的形态都无明显改变；刺激部位脊髓灰质前角的突触和神经元形态无明显改变。结论：ESCS应用时，脊髓腰膨大特定部位的局限电刺激可能对其作用有重要意义。实验中我们观察到的大脑运动皮质血管直径的增大可能是由于相关蛋白表达的改变引起的血管增生，从而增加了运动皮质的血管储备。脊髓灰质前角内突触及神经元形态并未观察到有显著改变，可能是由于我们的取材部位为损伤节段以下。ESCS联合跑台训练促进下肢功能恢复可能与刺激部位脊髓及大脑运动皮质神经通路的改变无明显联系，但仍需进一步研究。更多还原

【Abstract】 PARTⅠSpinal cord reflexes induced by epidurat spinal cord stimulationin normal adult ratsEXP.ⅠEffects of epidural spinal cord stimulation voltage alteration on spinal cordreflexes in normal adult ratsObjective Effects of epidural spinal cord stimulation voltage alterationon spinal cord reflexes in normal adult rats were investigated, expecting to find out whereand how the spinal cord reflexes were generated. Methods Ten adult femaleSprague-Dawley rats were anaesthetized, following with electrode placed at S1 spinal cordsegment. 400mV, 600mV, 1200mV epidural spinal cord stimulation were appliedrespectively, while EMG signal was recorded with concentric needle electrodes atsemitendinosus of the rats, to observe the characteristics of spinal cord reflexes. ResultsThe threshold for generating a hind limb muscle respond is 300mV. Three kinds ofepidural spinal cord stimulation could induce 2 kinds of spinal cord reflexes. Lowerstimulation voltages, including 400mV, 600mV, had induced the short latency spinal cordreflexes, which were 5.27±0.36 ms and 5.19±0.67ms respectively. The higher 1200mV stimulation voltage had induced the long latency spinal cord reflexes, which were 2.57±0.23ms. Conclusion Different voltages of epidural spinal cord stimulation could inducedifferent spinal cord reflexes generated differently. The long latency reflexes might bemonosynaptic responses mediated by dorsal root excitement, while the short latencyreflexes might be sarcous electric activity mediated by direct excitement of motor neuron ormotor fiber.EXP.ⅡEffects of epidural spinal cord stimulation frequency alteration on spinalcord reflexes in normal adult ratsObjective Effects of epidural spinal cord stimulation frequencyalteration on spinal cord reflexes in normal adult rats were investigated, expecting to findout where and how the spinal cord reflexes were generated. Methods Ten adult femaleSprague-Dawley rats were anaesthetized, following with electrode placed at S1 spinal cordsegment. 50Hz, 60Hz, 80Hz, 100Hz epidural spinal cord stimulations were appliedrespectively, while EMG signal was recorded with concentric needle electrodes atsemitendinosus of the rats, to observe the characteristic of spinal cord reflexes. ResultsSpinal cord reflexes could be generated by 50Hz, 60Hz, 80Hz, 100Hz epidural spinalcord stimulations, while the late stage of high frequency stimulations could induce spinalcord reflexes lost, and appeared irregularly. Some of the rats appeared with spinal cordvanishing totally at the late stage of the stimulations. Latency and duration of spinal cordreflexes induced by 50Hz epidural spinal cord stimulations, which were 4.46±1.07ms and7.33±1.00ms respectively, statistically differed from the ones initiated by 60Hz, 80Hz,100HzESCS. Conclusion Spinal cord reflexes induced by high frequency epiduralspinal cord stimulation might be some kind of monosynaptic responses. Irregularly appearances of spinal cord reflexes induced by high frequency stimulation might due to theinhibitory effect of the high frequency stimulation.EXP.ⅢEpidural stimulation on different spinal segments induced spinal cordreflexes recorded using a concentric needle electrode in normal adult ratsObjective Spinal cord reflexes induced by epidural spinal cordstimulation of L2/S1 spinal cord segments in normal adult rats were recorded, expecting tofind out how the spinal cord reflexes were generated. Methods EMG signals weredetected using a concentric needle electrode inserted into the tibia muscle. Reflexlatencies induced by different stimulation voltages (400 mV, 600 mV, 1200 mV) werecompared with magnetic transcranial stimulation-induced motion evoked potentials. Theeffects upon the spinal cord reflex under epidural spinal cord stimulation with variousfrequencies (50 Hz, 60 Hz, 80 Hz, and 100 Hz) were also investigated. ResultsElectrical stimulation of L2 segment with different voltages did not induce statisticallysignificant changes in the latency of spinal cord reflexes. However, different voltageelectrical stimulation of S1 segment induced different reflexes. Conclusion Low voltageelectrical stimulation of S1 segment induced reflexes with longer latencies, which may bedue to excitation of dorsal root neurons. High voltage electrical stimulation of S1segments induced short latency reflexes, which may be due to direct excitation of motorneurons or nerve fibers. We recorded wave shapes that were different to those describedin previous studies, which may be related to the more localized recording area of theconcentric needle electrode used in this study. This highlights the need for furtherresearch using different recording electrodes. We were unable to induce rhythmichindlimb movement with high frequency electrical stimulation, possibly due to suppressedsensory feedback in anesthetized rats. Therefore, further studies that record EMG signals in waking rats are necessary.PARTⅡEffects of epidural spinal cord stimulation combined withtreadmill training on stimulated spinal cord and cerebral motor cortexultrastructure after moderate spinal cord injury in ratsObjective Epidural spinal cord stimulation (ESCS) combined withtreadmill training has been proven to help spinal cord injury patients and rats regainwalking ability. We plan to investigate how this procedure affects the ultrastructure of thestimulated spinal cord and cerebral motor cortex after moderate spinal cord injury in rats.Methods Twelve adult female Sprague-Dawley rats were randomly distributed intofour groups: (1) spinal cord injury group (SI), (2) spinal cord injury plus ESCS group (SE),(3) spinal cord injury plus treadmill training group (TT), and (4) spinal cord injury plustreadmill training and ESCS group (TE). All rats received a moderate spinal cord injurysurgery. Four weeks after the surgery, SE and TE rats received an electrode implantationprocedure, with the electrode field covering spinal cord segments L2～S1. Four weeks afterelectrode implantation, the SE rats received subthreshold ESCS for 30 minutes per day. TTrats received 5cm/s treadmill training for 30 minutes per day. TE rats received ESCS whilecarrying out treadmill training, with parameters equal to those of the SE and TT rats. SI ratsreceived no intervention, thus functioning as a control group. All procedures in these fourgroups lasted four weeks. After four weeks intervention, tissues of stimulated spinal cordand cerebral motor cortex were cut off following standard protocols for electronmicroscopy. Results After four weeks of intervention, TT and TE animals improvedtheir open field locomotion score to 18 (there were no significant differences between thetwo groups). In contrast, no significant improvement was observed in groups SI and SE. The diameters of cortical blood vessels were significantly larger in the TT and TE groupswhen compared to the SI and SE groups. There were no significant differences between theTT and TE groups or between the SI and SE groups. Synapses and neurons of thestimulated spinal cord and cerebral motor cortex were similar regardless of whether ratsunderwent ESCS combined with treadmill training or not. Conclusion Localizedstimulation on the lumbar enlargement may play an important role in ESCS’s effectiveness.Treadmill trained rats showed widened motor cortex blood vessels, which could be due tothe expression of angiogenesis-related proteins leading to an increase in the size of thecapillary reserve. ESCS and treadmill training might not contribute to changes in thestimulated spinal cord and cortical pathways underlying the recovery of walking ability,which still needs more investigation.更多还原