【作者】 麻彬；

【导师】 贾连顺；

【作者基本信息】 第二军医大学 ， 外科学， 2009， 博士

【摘要】 研究背景:随着医学的进步,人们对疾病认识程度的加深,马尾神经压迫损害(Cauda equina compression,CEC)及由此导致的马尾神经综合征(Cauda equina syndrome, CES)愈来愈引起学者们的重视。据统计,普通人群中CES的发病率为1:100,000到1:33,000之间,患有下腰痛的人群中CES发病率为万分之4,而在患有腰椎间盘突出的患者中,CES发病率更是达到了1%～10%。我国近30%的腰椎疾患的患者存在不同程度的CEC症状。马尾神经压迫损害及其继发性病理生理反应可直接导致组织器官神经功能损伤及功能障碍,导致下肢疼痛、运动感觉功能、大小便功能及性功能障碍,且其治疗困难,致残率非常高,给患者本人造成了极大的痛苦,也给家庭和社会带来沉重的负担。目前对CEC的研究相对较少而且缓慢,预后难以预料,主要是该病的病理生理机制非常复杂,对此认识还不够全面、深入。以往研究显示,马尾神经压迫损害致神经元的凋亡是造成马尾神经压迫临床症状的重要原因,但究竟是哪些基因参与了马尾神经压迫损害导致的神经元的凋亡尚不清楚。国内外近年研究显示,马尾神经压迫损伤后,其局部病理改变如不能得到及时改善,则形成恶性循环,由马尾神经的局部损害到广泛性病理损害,可产生顺行性、逆行性变性及传递性神经元变性,以至于产生跨细胞的中枢神经元变性,而致使其结构和功能难以修复,造成永久性的功能损害。在此实验基础上,本课题拟通过建立实验性大鼠马尾神经压迫模型,就马尾神经压迫损害的基础进行初步探讨。第一部分:马尾神经压迫动物模型的建立及马尾神经压迫电生理的实验研究目的:对SD大鼠腰椎区域相关组织解剖行初步观测,建立合适的马尾神经压迫损害动物模型,并在此基础上进行实验性大鼠急性马尾神经压迫后行为学、神经电生理及组织学研究。方法:首先对SD大鼠腰椎区域相关组织解剖做了初步观测,得到SD大鼠腰椎区域相关解剖数据,根据测量数据制作改良的实验性大鼠急性马尾神经压迫动物模型。之后将实验动物随机分为以下4组:CON组:空白对照组(n=8);SHAM组:假手术组(n=8);CCC组:经典马尾神经压迫组(n=8);MCC组:改良马尾神经压迫组(n=9)。手术建立改良的实验性大鼠急性马尾神经压迫动物模型:显露L4～L5的椎板,扩大L4/5椎间隙,切除L4/5间隙的黄韧带,向尾端塞入10.0×1.0×(1.1-1.3)mm硅胶条1根。并与经典实验性大鼠急性马尾神经压迫动物模型进行比较。术后第1、3、7、14、28天对此模型进行了行为学评价,包括:BBB评分、电子von Frey机械测痛法测定大鼠缩足潜伏期、热板实验、甩尾实验、步行分析。结合HE染色及尼氏染色,光镜下对实验性大鼠急性马尾神经压迫模型进行组织病理学评价。在此基础上,使用诱发电位记录仪,进行了脊髓诱发电位(SCEP)N1波潜伏期及运动神经传导速度(MNCV)的动态测定,了解实验性大鼠急性马尾神经压迫后电生理的动态变化。结果:模型制作大鼠死亡率低。步行分析强迫运动评价显示,模型组大鼠术后出现明显神经源性间歇性跛行,并且持续稳定,CON组与SHAM相比无明显差异;CCC组及MCC组相比无明显差异。BBB评分显示,与对照组相比,模型组术后静态运动功能减退不明显,非强迫运动功能术后恢复较早;相关感觉行为指标评价出现了明显感觉障碍,主要表现为痛觉及热觉减退,以术后早期明显,随着时间延长,感觉功能逐渐恢复,但未恢复到术前水平。通过对压迫DRG(DRG)、马尾神经(CE)及脊髓、圆锥的大体观、组织病理学的动态观察,可见神经根压迫后其有髓神经纤维数目减少和组织结构的改变,如神经膜细胞胞质肿胀和细胞水肿、尼氏体减少、华勒变性、神经元轴突脱髓鞘变等。电生理研究发现,马尾神经受压后SCEP发生的变化明显,潜伏期明显延长,至术后14天仍不能完全恢复;MNCV的下降恢复较快,术后7天即恢复到术前水平。结论:本部分实验结果说明,改良的实验性大鼠急性马尾神经压迫模型可以复制出神经源性间歇性跛行,且更接近于椎管狭窄对马尾神经的压迫,为临床马尾神经压迫导致的疼痛及麻木治疗的研究建立了可供参考的动物模型。行为学评价说明,马尾神经压迫性损伤后,出现感觉功能障碍,且随着时间延长,神经功能有所恢复,但由于压迫因素未解除,神经功能不能恢复到术前水平。电生理实验研究说明马尾神经感觉支对机械压迫的反应比较敏感,而运动支相对则较容易恢复。第二部分:马尾神经压迫后DRG、马尾神经与相应脊髓神经细胞凋亡及PUMA的表达目的:研究马尾神经压迫后DRG、马尾神经与相应脊髓神经细胞有无凋亡的发生;了解马尾神经压迫后是否导致PUMA表达的变化以及表达数量随时间的变化,为手术时机的选择提供依据,以及为手术的疗效提供评价标准。方法:将50只SD大鼠随机分为下列几组:A组:空白对照组(n=10);B组:马尾神经压迫1d组(n=8);C组:马尾神经压迫3d组(n=8);D组:马尾神经压迫7d组(n=8);E组:马尾神经压迫14d组(n=8);F组:马尾神经压迫28d组(n=8)。用免疫组织化学的方法检测DRG、马尾神经与相应脊髓中PUMA的表达。具体如下:4%的多聚甲醛灌注固定大鼠,取出标本,经石蜡包埋后切片,厚度在4μm。二甲苯脱蜡后,经梯度乙醇至水和TBS冲洗,用3%过氧化氢封闭内源性过氧化酶约5分钟,加入0.01M柠檬酸缓冲液(pH6.0),煮沸20分钟,再用5-10%山羊血清室温封闭20分钟。一抗孵育1h小时和二抗孵育30分钟后,用DAB显色处理,最后经TBS冲洗,苏木素复染和二甲苯透明后,用中性树胶封片。用末端转移酶介导的dUTP缺口末端标记(Tunel)法检测DRG、马尾神经与相应脊髓中细胞凋亡:4μm石蜡切片常规脱蜡至水,3%过氧化氢封闭,蛋白酶K处理后置入Tunel混合液,37℃,1h,Streptavidin-HRP 30min后,0.04% DAB+0.03%H2O2显色10min,苏木素衬染和二甲苯透明后,常规树脂封片。采用实时定量PCR技术检测DRG、马尾神经与相应脊髓中PUMA基因mRNA的表达水平:TRIzol法抽提细胞总RNA,RNA的鉴定及定量,逆转录至cDNA,引物设计与合成,BLAST确认引物特异性,测定各样品的Ct值,以β-actin浓度为参照对待测样品的Ct值进行校正。结果:免疫组化结果示对照组未见明显PUMA阳性深染区。当大鼠马尾神经压迫后,相应脊髓神经元胞体可见大量阳性深染区,主要集中在腹角及背角。Tunel染色示大鼠马尾神经压迫术后,在相应脊髓节段发现神经元凋亡,以脊髓前背角明显,与对照组相比,P<0.05;而各组DRG及马尾神经凋亡细胞少见,与对照组比较,P>0.05。术前DRG、马尾神经与脊髓中PUMA基因mRNA的表达水平,组间比较,P>0.05;术后相应脊髓中PUMA基因mRNA的表达水平随时间出现动态变化,术后第3天出现显著的PUMA增量调节,结果与术前相比,P<0.05;DRG、马尾神经中PUMA基因mRNA的表达水平变化与术前相比无明显变化,P>0.05。结论:大鼠马尾神经压迫术后,可导致逆行性脊髓、背根节及马尾神经出现神经元凋亡。马尾神经受压后可在相应脊髓部位出现显著的PUMA增量调节。第三部分:PUMA信号通路在马尾神经压迫损害致逆行性脊髓损害的作用目的:在蛋白水平检测大鼠马尾神经压迫逆行性导致相应脊髓神经元凋亡作用中的凋亡相关蛋白的表达,探讨PUMA这一凋亡通路中的重要因子在马尾神经压迫中的作用,以及PUMA、p53、SirT2间的相互作用机制。方法:用免疫组织化学的方法检测DRG、马尾神经与脊髓中p53、SirT2的表达。具体方法同上一部分。用Western-Blot方法检测脊髓中PUMA、p53、SirT2的表达:取100mg组织加入0.2ml裂解液进行匀浆提取蛋白,将所有蛋白样品调至等浓度后,加入上样缓冲液进行电泳,经转膜、封闭和孵育一抗、二抗后鉴定。结果:p53免疫组织化学染色结果示:正常对照组少见明显阳性深染区。当大鼠马尾神经压迫后,相应脊髓神经元胞体可见大量阳性深染区。SirT2免疫组织化学染色结果示:A组在脊髓前背角可见阳性深染区。马尾神经受压迫后,脊髓内神经元胞体阳性深染区减少,颜色减淡,术后各组间差别不明显。马尾神经及DRG压迫前后未发现明显阳性表达。Western blot结果示,马尾神经压迫损伤后第3天,在相应脊髓组织内检测到显著的PUMA增量调节,同时出现p53的高表达;SirT2在相应的时间点出现表达量减少。结论:大鼠马尾神经压迫损伤可以导致逆行性脊髓神经元出现凋亡,PUMA是其中的关键基因。PUMA在马尾神经压迫病理损害中的作用机制可能是通过p53通路,从而促进细胞凋亡。SirT2可能的作用是负向调节p53,从而抑制PUMA的表达。更多还原

【Abstract】 IntroductionAlong with the progress of medical knowledge and the deepening understanding of disease extent, more and more attention of Cauda Equina Compression (CEC) is paid to by scholars. Cauda equina syndrome (CES), a rare neurological disorder, is a combination of signs and symptoms resulting from lesion of the nerves in the CE. Typical manifestations can be associated variably with the disorders characterized by low back pain, unilateral or usually bilateral sciatica, bilateral weakness of the lower extremities, saddle or perianal hypoesthesia or anesthesia, sexual impotence, together with rectal and bladder sphincter dysfunction. The incidence of CES is variable, depending on the etiology of the syndrome. The prevalence among the general population has been estimated between 1:100,000 and 1:33,000. The most common cause of CES is herniation of a lumbar intervertebral disc. It is reported by approximately 1% to 10% of patients with herniated lumbar disks. The prevalence among patients with low back pain is approximately four in 10000. About nearly 30% of the lumbar spine disease in patients with different levels of existence CEC symptoms in our country. CEC has created the enormous pain for the patient, also gives the family and the society brings the serious burden. CEC researches are relatively few and slow now, and the prognosis seems poor. The mean reason for this is that the pathology physiology mechanism of CEC is very complex, and there is no comprehensive knowledge regarding it. Previous studies have shown that, the apoptosis of neuron caused by CEC is an important reason for the clinical symptoms. The domestic and foreign recent research suggests that it would forms the vicious circle after the CEC if the partial pathology change cannot have the prompt improvement. Local damage would cause extensive pathological damage, including anterograde damage, retrograde damage, transneuronal neuronal degeneration, and even transcellular central neuron degeneration. Thus the structure and function are hard to be repaired and lead to nonvolatile functional lesion. On this experimental basis, we plan to establish the animal model of CEC and do some preliminary study on this topic.SECTION ONE:Establishment and evaluation of the animal model of cauda equina compression and the nerve electrophysiological researchObjective: To do preliminary observation on lumbar region anatomy of the SD rat and relevant organization, and to establish the CEC animal models. To research on the neural physiological and behavioral, histologic and nerve electrophysiological changes based on this animal model.Methods: We observed and measured the nomal anatomy of SD rats’lumbar region and relevant organization, and made certain the cross sectional area of lumbar spinal canal in SD rat. On the basis of above, we established a modified CEC animal model to analyze the effects of the pressure on the behavioral, histologic and nerve electrophysiological changes in rats. Eight-to-nine-week-old male SD rats were randomly divided into four groups: control group(CON),n=8; sham group(SHAM),n=8; classic cauda equina compression group (CCC),n=8; modified cauda equina compression group (MCC),n=9. The method to establish the modified CEC animal model: reveal the vertebral plate of L4 and L5, and excise the yellow ligament of L4/5. Insert a silicon rubber (10.0×1.0×1.1 mm to10.0×1.0×1.3 mm) to the caudal end. After induction of spinal stenosis walking function was measured using a treadmill apparatus and sensory functions were tested by measuring thermal and tactile withdrawal threshold (von Frey filaments) and BBB score for the period of 28 days after stenosis, which is day 1, 3, 7, 14, and 28. Using HE and Nissl’s staining, we have taken a histopathology evaluation. Then, We measured the spinal cord evoked potential (SCEP) and motor nerve conduction velocity (MNCV) by using evoked potential recorder to investigate the nerve electrophysiological changes in CEC model.Results: There is a low death rate in all experimental groups. The modified CEC animal model could mimic the situation of walking of human being which is neurological intermittent claudication. After the surgical compression, a significant running dysfunction, as evidenced by shortening of running distance, was measured as soon as 24 h after stenosis. This effect persisted for 28 days after surgery. There is no statistical difference between CCC and MCC. Similarly, a significant thermal and algaesthesia hyposensitivity was measured for a period of 28 days. BBB score decreased after the CEC, but recovered soon. There is no statistical difference between CCC and MCC in BBB score. HE and Nissl’s stain reveal that the number of medullated nerve fibers and tigroid body decrease, Wallerian’s degeneration and demyelinate of neuron axon. SCEP increased significantly and couldn’t recover till 14 days after surgery; while MNCV recovered at the 7th day after surgery.Conclusions: The modified CEC animal model could mimic the situation of walking of human being which is neurological intermittent claudication and is an easy and effective method to make CEC animal model. Behavior evaluation of this model suggests that sensory function and motor dysfunction could happen after CEC. Results of nerve electrophysiological experiment reveal that, the sensory brunch of CE is much more sensitive than locomotive brunch.SECTION TWO:Apoptosis of cellula nervosa and expression of PUMA in DRG, cauda equina and corresponding spinal cord after cauda equina compressionObjective: To learn if cauda equina compression could result in apoptosis in DRG, cauda equina and corresponding spinal cord after cauda equina compression, and to detect the expression of PUMA and find the change pattern with time.Methods: Fifty eight-to-nine-week-old male SD rats were randomly divided into six groups: group A: control group (n=10); group B: one day after CEC (n=8); group C: three days after CEC (n=8); group D: seven days after CEC (n=8); group E: 14 days after CEC (n=8); group F: 28 days after CEC (n=8). The expressions of PUMA in DRG, cauda equina and corresponding spinal cord were detected by immunohistochemical methods described as follows: 10% formalin-fixed pancreatic tissue and paraffin-embedded pancreatic tissue were cut into sections with thickness of 4μm and placed onto glass-slides. Following deparaffinization in xylene, the slides were rehydrated and washed in Tris Buffered Saline (TBS). The endogenous peroxidase activity was quenched by 5 min incubation in a mixture of 3% hydrogen peroxide solution and 100% methanol. After being boiled in citrate buffer pH 6.0 for 20 mins, sections were sealed and left at room temperature for 20 min with 10% nonimmune goat serum in Tris-buffered saline solution (pH 7.5). After that, they were incubated with primary antibody at room temperature for 1 h, then with secondary antibody at room temperature for 30min. After washing with TBS, slides were kept in diaminobenzidine for 5-10 min and counterstained with Mayers hematoxylin.The expressions of PUMA in DRG, cauda equina and corresponding spinal cord were detected by TdT-mediated X-dUTP nick end labeling (Tunel) methods described as follows: paraffin-embedded pancreatic tissue were cut into sections with thickness of 4μm and placed onto glass-slides. Following deparaffinization in xylene, the slides were rehydrated and washed in TBS. The endogenous peroxidase activity was quenched by 5 min incubation in a mixture of 3% hydrogen peroxide solution and protease K. After Tunel mixed liquor for one hour and Streptavidin-HRP for 30min. After washing with TBS, slides were kept in diaminobenzidine for 5-10 min and counterstained with Mayers hematoxylin.Detect the mRNA expression level of PUMA in DRG, cauda equina and corresponding spinal cord by use Real Time PCR.Results: Immunohistochemistry stain of p53 suggests that there are few positive cell in control group. Positive cell increased after the compression of CE could be found. We can find positive expression of Tunel in corresponding spinal cord tissue. The expression in spinal cord is significantly increased than control group (P<0.05). The expression of PUMA among DRG, cauda equina and corresponding spinal cord has no statistical difference (P>0.05). The expression of PUMA increased significantly on the third day after the compression (P<0.05), while there is no statistical difference of PUMA expression in DRG or CE after the surgery versus control group (P>0.05).Conclusions: cauda equina compression could lead to retrogressional spinal cord lesion. The expression of PUMA increased significantly after the compression.SECTION THREE: Preliminary study of the Basic Research on the nosogenesis of spinal cord after cauda equina compressionObjective: To detect the expression of apoptosis-associated protein in spinal cord after CEC in protein level; to approach the effect of PUMA in the process of apoptosis; to research the mechanism of interaction of PUMA, p53 and SirT2 in the pathogenesy of CEC.Methods: Detect the expressions of PUMA in corresponding spinal cord by using immunohistochemical methods described in section one.The expression of protein PUMA, p53 and SirT2 in spinal cord was detected by Western-Blot. Spinal cord tissue (100 mg) was homogenized in Lysis Buffer containing 50mM Tris·Cl (pH7.5), 150mM NaCl, 1% NP-40, 0.5% sodium desoxycholate (w/v), 0.1% SDS (w/v),10 mM mercaptoethanol,10mg/ml PMSF,5μg/ml Pepstatin,and 5μg/ml leupeptin (pH7.4). Electrophoresis of homogenated aliquots of protein was carried out using polyacrylamid gels. Separated proteins were then electrotransferred to the nitrocellulose membranes. These membranes were incubated in primary antibody for 24 h at 4°C and then in the secondary peroxidase-conjugated antibody for 2h. Immunoreactive protein bands were visualized using an enhanced chemiluminescence reaction kit.Results: Immunohistochemistry stain of p53 suggests that there are few positive cells in control group. We can see that the positive cell increased after the compression of CE. Immunohistochemistry stain of SirT2 suggests that there are positive cell in control group. After the compression of CE, the anachromasis area decreased and lighted. The result of western blot suggests that at the day 3 after CEC, the expression of PUMA and p53 increased significantly and that of SirT2 decreased at the corresponding time.Conclusions: Retrogressive apoptosis could happen after the compression of cauda equina. PUMA is the key gene in this course. The mechanism of action is depended the p53. p53 is involved in puma up-regulation and promote the apoptosis, while SirT2 may down-regulate the expression of PUMA.更多还原