【作者】 王飞；

【导师】 朱庆三；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 本研究首先采用经背侧入路腹主动脉阻断术建立的脊髓缺血再灌注损伤模型(SCIIR),通过控制缺血时间模拟不同程度的脊髓缺血性损伤(SCII)对其稳定性、重复性和一致性进行了客观评价并观察了SCII后胶质瘢痕形成的一般规律。而后采用缺血60分钟SCIIR模型探讨蛛网膜下腔置管注射Noggin在脊髓缺血性损伤后神经功能恢复和胶质瘢痕形成过程中的治疗作用。本研究实验一将大鼠随机分为假手术组、缺血30、60、90分钟损伤组；实验二大鼠随机分组为假手术组、损伤对照组、Noggin治疗组。依据BBB评分标准对大鼠伤后6周内后肢运动功能变化进行观测,并行电生理检测,同时利用病理学、免疫组化、蛋白印迹等技术对大鼠SCII后4周内脊髓胶质瘢痕形成、病理组织学改变进行观察与评价。结果表明：不同缺血时间的SCIIR模型能够模拟不同程度SCII后神经功能恢复和胶质瘢痕形成过程,可用于脊髓损伤与修复的研究；缺血60min组大鼠运动功能恢复曲线稳定而规律,能够用来对SCII后治疗措施或药物进行疗效评价；Noggin的早期应用可以保护神经组织和轴突,减少了损伤范围,防止其在脊髓损伤后3-4周发生严重退行性变；Noggin可以通过调控星形胶质细胞的分化,抑制SCII后胶质瘢痕形成；Noggin可以促进SCII后神经功能的恢复,但对于轴突再生没有直接促进作用。更多还原

【Abstract】 Experimental Study on Glial Scar Formation and Intrathecal Injection of Noggin to Promote Functional Recovery after Spinal Cord Ischemic InjuryGlial scar is a formidable mechanical barrier, and extracellular matrix components such as CSPGs is an important chemical barrier to axonal regeneration, in borne and abroad most of the studies on glial scar in central nervous system focused on brain injury, spinal cord injury research on glial scar formation has mild,and mostly on moderate damage and sharp injury, research on spinal ischemic injury in this area were not reported yet. The basic pathological changes of spinal cord injury, including decreased blood flow in injury site, prolonged ischemia.reperfusion induces the release of free radicals, spinal cord ischemia induces hypoxia, ischemia and hypoxia secondary increase the pathological free radical reactions, and further aggravate spinal cord ischemic necrosis, giving rise to a vicious circle. In addition, clinically SCI is not complete cross-section in the anatomy and not serious, but the symptoms after decompression is aggravated, and later develope into the "secondary paralysis", the cause may be spinal cord ischemia-reperfusion injury. Therefore, the study on glial reaction and recovery of function after spinal ischemic injury is very practical. Bone morphogenetic protein (BMP) is a component of TGF-βfamily, which played an important role s and regulation of cell proliferation and differentiation in central nervous system development proces. BMP is considered to nerve growth inhibitor, BMP-4 and BMP-7 used in the cultivation of astrocytes can increase the expression of CSPGs, many studies found that inhibitor BMPs-Noggin can effectively promote axon growth, and contribute to recovery of neurological function after central nervous system ischemic.In the first part of this study, the stability, repeatability and consistency are evaluated during the scar formation and recovery of nerve function in rat spinal cord ischemia-reperfusion injury (SCIIR) model objectively and observed the scar formation after SCII; rats were randomly divided into sham-operated group and three ischemic groups which were treated for 30,60,90-minute ischemia correspondingly.Behavioral recovery was assessed in an open field environment for 4 weeks after the injury by using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Both SEP and MEP were recorded for 4 weeks.The morphological changes, tissue integrity, and scar formation were evaluated until 4 weeks after SCI using histological, immunohistochemical and western blot analyses. Results:1.The longer the ischemic time,the more severe the pathological damage,scar formation and expression of glial fibriallary acidic protein (GFAP) of spinal cord tissue after SCII for 4 weeks. The gray matter of the cord was severely damaged and the damage was the most severe in the 90 min ischemia group, second severe in the 60 min ischemia group, third severe in the30 min ischemia group and least severe in the control. The degree of evoked potential latency and amplitude changes in ischemia group at the same time point was I30> I60> 190. Conclusion:Different ischemic time SCIIR model can simulate varying degrees of SCII.Not only for spinal cord injury and repair studies, SCIIR model also can be used to evaluate the efficacy of different drugs; Especially,SCIIR model with ischemia for 60 minutes has the most high stability repeatability and consistency during the scar formation and recovery of nerve function;In the second part, the effects of noggin-a soluble BMP antagonist is investigated which was administered via intrathecally inject (IT)on the scar formation and repair of spinal cord tissues in rat SCII model, rats were randomly divided into three groups with 8 animals in each group:Ⅰsham operation group received operation but no spinal cord ischemia;Ⅱcontrol group received 60min ischemia and intrathecally injected (IT)0.01M PBS 10μl qd for 2 weeks after reperfusion;Ⅲtreatment group received IT noggin 5 ul+0.01 M PBS 5μl qd for 2 weeks. Behavioral recovery was assessed in an open field environment for 4 weeks after the injury by using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. The morphological changes, tissue integrity, and scar formation were evaluated until 4 weeks after SCI using histological, immunohistochemical and western blot analyses. Results:The administration of noggin via IT reduced the cavity size and glial scar formation at 3-4 weeks after SCII. Noggin also reduced the expression of core proteins of chondroitin sulfate proteoglycans (CSPGs) such as neurocan and phosphacan but not NG2. In particular, an intensive expression of GFAP and neurocan found around the cavity at 4 weeks was obviously suppressed by noggin. Immunostianing for neurofilament (NF) and Luxol fast blue (LFB) showed that noggin preserved well the axonal arrangement after SCII. The expression of GAP-43, a marker of regenerating axons, also apparently increased in grey matter by noggin.Conclusion:Early applications of Noggin can inhibit apoptosis, protect nervous tissue and axons, reduce the scope of damage and prevent serious degeneration of spinal cord in 3-4 weeks; Noggin can repress GFAP expression after SCII; noggin can reduce lesion volume and enhanced structural integrity after SCII; Noggin can contribute to recovery of neurological function on SCII, but be not the direct cause for axon regeneration.更多还原