【作者】 陈安；

【导师】 罗学港；

【作者基本信息】 中南大学 ， 神经生物学， 2009， 博士

【摘要】 第一部分大鼠红核脊髓束横断模型的建立目的:建立简易可靠的红核脊髓束横断动物模型。方法:成年SD大鼠随机分为模型组和假手术组。根据正常大鼠脊柱颈段的解剖特征,将大鼠颈曲由下突位变为上突位,在手术显微镜下不咬椎板,直接切开C3、C4间的黄韧带,暴露脊髓,横断颈髓右外侧索;假手术组大鼠只行黄韧带切开。用Fast-Blue(FB)逆行示踪红核脊髓束,检测红核神经元标记情况;对脊髓损伤部位纵切片进行Nissl染色,观察损伤部位结构破坏情况;采用自发性直立探究行为学测试方法,评判前肢的运动功能。结果:假手术组左侧红核神经元被FB标记,而模型组左侧红核未被FB标记;模型组Nissl染色显示脊髓右半的外侧白质及部分灰质断裂,而右侧的后索及脊髓左侧半保留完整;假手术组大鼠双侧前肢使用率相同,模型组大鼠在探究活动中不使用伤侧前肢。结论:采用非咬椎板法暴露脊髓,可将右侧的RST完全切断,本方法具有可靠性和简便实用性,制备的红核脊髓束横断模型是研究脊髓损伤的良好模型。第二部分BYHWD对大鼠红核脊髓束横断后神经元的存活、再生及功能修复的影响目的:探讨补阳还五汤(BYHWD)对红核脊髓束横断后神经元的存活、再生及功能修复的影响。方法:SD大鼠随机分为3组:①正常组,不予处理;②实验组:C3-C4右侧红核脊髓束横断后灌服补阳还五汤(25.65g生药/kg);③对照组:C3-C4右侧红核脊髓束横断后灌服蒸馏水。正常大鼠C3右外侧索FG逆行示踪定位红核脊髓束神经元在红核的分布;各组动物存活8周后,Nissl染色计数红核神经元并计算其平均截面积;用BDA神经束路顺行示踪方法检测轴突再生情况;采用自发性直立探究行为学测试方法评判前肢运动功能的恢复。结果:FG标记的红核位于中脑上丘层面,中脑水管的腹外侧,全长约为1200μm;红核神经元的数目和平均截面积实验组分别减少了22%和35%,对照组分别减少了42%和64%;实验组的大鼠在损伤的尾侧端可见少量BDA标记的纤维,对照组损伤的尾侧端则未见BDA标记的纤维;实验组伤侧前肢使用率明显高于对照组。结论:BYHWD提高了轴突横断后红核神经元的存活率,对红核神经元的萎缩有抑制作用;BYHWD能促进大鼠红核脊髓束横断后轴突的再生及部分功能修复。第三部分BYHWD对红核脊髓束横断后红核神经元BDNF和cAMP表达的影响目的:探索BYHWD对RST横断后红核神经元的BDNF和cAMP表达的影响。方法:成年SD大鼠随机分为正常组、实验组和对照组。其中实验组C3-C4右侧红核脊髓束横断后灌服补阳还五汤(25.65g生药/kg);对照组C3-C4右侧红核脊髓束横断后灌服蒸馏水。动物分别存活3、7、14或21天后处死。采用免疫组织化学及放射免疫方法,检测BYHWD对RST横断后不同时间点红核神经元BDNF的表达和cAMP含量。结果:BDNF阳性细胞计数及相对灰度值检测结果显示,术后3d和7d实验组和对照组红核神经元BDNF表达上调,与正常组比差异有显著性(P＜0.05);随后,对照组BDNF表达下调,但实验组仍保持上调趋势。cAMP放射免疫测定结果显示,术后第3天,实验组和对照组红核内cAMP含量均降低,与正常组比差异有显著性(P＜0.05);实验组在术后第7天,cAMP含量有所升高,至14、21天cAMP含量高于正常组(P＜0.05);对照组在随后各时间点,cAMP含量都低于正常组(P＜0.05)。结论:BYHWD能上调RST横断后红核中BDNF和cAMP的表达。更多还原

【Abstract】 PARTⅠEstablishment of the rubrospinal tract transection animal modelObjective To provide a simple and reliable animal model of rubrospinal tract（RST） transection.Methods Adult SD rats were divided into model group and sham operation group.According to the natural anatomical characters of rat spinal cord,cervical curves of the rats were changed from inferior to superior,rats received yellow ligment and a right-sided lateral funiculus transection or yellow ligment transection at the level between the 3^rd and 4^th cervical spinal cord（C3 and C4） under the operating microscope. Fast-Blue（FB） retrograde tracing of RST,Nissl staining of the spinal injured site and behavior analysis were carried out to evaluate the effect of RST transection.Results There was no FB labeled rubrospinal neuron in the model group,whereas FB labeled neurons were detected in the sham operation group;It was showed by Nissl staining that in the model group the right lateral funiculus and small part of gray matter were disconnected after transection,while the right posterial funiculus and the left half of the spinal cord remained intact;The injured forelimb was not used during spontaneous vertical exploration in the model group,whereas normal used in the sham operation group.Conclusion These results indicate that RST could be completely transected between C3 and C4 without damaging the vertebral lamina.It is not only simple but also practicable and reliable for the RST transection model made in this method.The RST transection model is good for the study of spinal cord injury. PARTⅡEffect of BYHWD on survival,regeneration and functional recovery of neurons after rubrospinai tract transectionObjective To explore the effect of BYHWD on survival, regeneration and functional recovery of neurons after rubrospinal tract transection.Methods Adult SD rats were divided into experimental group, control group and normal control group.Rats received treatment of either BYHWD or DW via gastrogavage after a right lateral funiculus transection at the level between C3 and C4 in the experimental or control group.Location of rubrospinal neurons in the RN was determined by Flouro-gold（FG） retrograde tracing.Eight weeks after injury,viability and atrophy of RN neurons were determined by Nissl staining,axon regeneration was examined by biotinylated dextran amine（BDA） tracing techniques,and functional recovery was studied by a test of forelimb usage during spontaneous vertical exploration.Results The FG labeled RN was 1200μm long,located ventrolateral to the aquaeductus cerebra at the superior colliculus level.RN cell number and mean somal size were decreased 22%and 35% in the experimental group and 42%and 64%in the control group respectively.There was a small number of BDA-labeled axons in the caudal of injury site in the experimental group,whereas no caudal axonal regeneration was detected in the control group.Rats in the experimental group used the injured forelimbs more often than those in the control group.Conclusion These results indicate that administration of BYHWD following RST transection leads to enhancement of axotomized neuron survival,prevention of neuronal atrophy,promotion of transected axon regeneration and functional recovery. PARTⅢEffect of BYHWD on expression of BDNF and cAMP of neurons in red nucleus after rubrospinal tract transectionObjective To explore the effects of BYHWD on expression of BDNF and cAMP of RN neurons after RST transection.Methods Adult SD rats were divided into experimental group, control group and normal control group.Rats received treatment of either BYHWD or DW via gastrogavage after a right lateral funiculus transection at the level between C3 and C4 in the experimental or control group.After survival for 3,7,14 or 21 days,animals were sacrificed. Immunohistochemistry and radioimmunoassay were employed for detection of BDNF expression and cAMP concentration in RN neurons after RST transection.Results Showed by BDNF positive cell count and average gray value of BDNF positive products,BDNF expression was up-regulated both in the experimental and control group at the 3^rd and 7^th day after injury.Whereafter,BDNF expression in the control group was reduced, while in the experimental group,it remained the high level as the 7^th day’s. It was found by cAMP radioimmunoassay that cAMP concentration was decreased both in the experimental and control group at the 3^rd day after injury.And it was then increased from the 7^th day and remained a high level until to the 21^st day in the experimental group（compared with normal control,P＜0.05）,whereas decreased in the control group （compared with normal control,P＜0.05）.Conclusion These results indicate that administration of BYHWD following RST transection leads to Up-regulation of BDNF and cAMP expression in red nucleus.更多还原