SD大鼠脊髓损伤后微环境的模拟实验

BACKGROUND: We built Sprague-Dawley rat models with mild, moderate, and severe spinal cord injuries to accord with the spinal cord injury types for basic empirical study, and consequently to further understand the microenvironmental change in Sprague-Dawley rats with spinal cord injury, and to provide help for clinical treatment. OBJECTIVE: To observe the changes in nerve function, pathological manifestation and motor sensory evoked potential in Allen’s models and Sprague-Dawley rats with complete spinal cord transection at different time points after spinal cord injury by simulating the microenviroment in Sprague-Dawley rats. METHODS: A total of 125 healthy adult female Sprague-Dawley rats were selected and randomly divided into group sham operation group, 100 gcf hit potential group (20 g×5 cm), 200 gcf hit potential (20 g×10 cm), 300 gcf hit potential group (20 g×15 cm), and spinal cord complete transection group with 25 rats in each group. At 1, 5, 7, 14 and 28 days after model establishment, the degree of spinal cord injury was identified by the BBB scores of motion function, motor evoked potential, and pathological section. RESULTS AND CONCLUSION: (1) Totally 24 Sprague-Dawley rats died in the experiment. The death rate and the rate of complications were highest in the spinal cord complete transection group. The BBB score of each group was decreased. The BBB scores in every group increased as time went on. There were significant differences between each surgery group and the sham operation group at corresponding time points (P < 0.05).  No significant difference was found between the 300 gcf hit potential group and the spinal cord complete transection group at corresponding time points (P > 0.05). (2) In each surgery group, the infiltration of inflammatory cells and obvious swelling of neurons were visible at 1 day after injury. Neural cells reduced with time prolonged. At 28 days after injury, a large number of astrocytes proliferated, scar and spinal cord cavity formed. Above symptoms were worse in the 300 gcf hit potential group and spinal cord complete transection group than in the 100 gcf and 200 gcf hit potential groups. (3) Significant differences in amplitude and latency were detectable between each surgery group and the sham operation group (P < 0.05). No significant difference in amplitude and latency was detected between the 300 gcf hit potential group and the spinal cord complete transection group at corresponding time points (P > 0.05). Results confirmed that hit potential of 20 g×5 cm, 20 g×10 cm and 20 g×15 cm can simulate the microenvironment of Sprague-Dawley rats with mild, moderate and severe spinal cord injury. The rate of complication was lower in modified Allen’s model of different hit potentials than in models of spinal cord complete transection, and was more accorded with basic research.       

炎症小体活化对脊髓损伤局部免疫微环境的影响

炎症小体是一种高分子量的多蛋白复合物,能够诱导细胞在炎性和应激的病理条件下死亡。脊髓损伤后,损伤局部微环境会出现多种功能不同的免疫细胞。自然条件下损伤脊髓局部微环境中的免疫细胞亚群是“阴阳冶失衡的,破坏性的亚群和细胞因子占主导地位,是造成脊髓损伤病理损伤的重要机制。在中枢神经系统的损伤过程中,炎症小体发挥极为核心的作用。虽然,炎症小体的活化及其导致的IL-1β和IL-18在脊髓损伤部位均有表达,但迄今未见它们对脊髓损伤局部免疫微环境影响的报道。根据目前的研究进展,我们认为炎症小体的活化可以影响脊髓损伤局部免疫微环境,这是一个有趣的课题,但相关的研究确鲜有报道。本综述主要对四种与中枢神经系统损伤相关的炎症小体在结构、功能等方面进行阐述,对炎症小体活化与脊髓损伤免疫微环境的关系进行探讨。     

脊髓损伤大鼠骨组织微环境中RANKL/OPG表达的改变

目的研究脊髓损伤(SCI)大鼠骨组织微环境中RANKL/OPG表达的改变,探讨SCI对骨代谢的影响。方法 20只雄性6wSD大鼠,分为SCI组和Sham组。饲养3w后收集右侧胫骨测定骨密度,左侧胫骨切取后立刻液氮冷冻保存,一部分用于骨组织提取RNA,半定量RT-PCR分析RANKL、OPG、Col1α1、OCN基因的表达变化;另一部分用于酶免疫分析(EIA)方法测量骨组织中RANKL和OPG的含量。结果 3w时SCI组胫骨的骨密度明显低于Sham组,骨密度的下降既发生在胫骨近端干骺端(-26.3%,P0.05),又发生在胫骨干(-21.2%,P0.05)。RT-PCR检测表明,SCI后3w大鼠胫骨近端骨组织微环境中RANKL的表达显著上调(+122.2%,P0.01),OPG(-22.1%,P0.05)和OCN(-25.3%,P0.05)的表达显著下调。RANKL和OPG的表达改变也得到了EIA的证实。结论 SCI时RANKL过表达及OCN和OPG表达下调,提示SCI时易发生骨质疏松。     

脊髓损伤后前炎症细胞因子的表达

脊髓损伤 (spinal cord injury,SCI)后其病理发生发展过程由两种损伤机制主导 ,即原发性和继发性损伤机制。对于SCI来说 ,原发性损伤是一个不可逆转的过程 ,而继发性损伤则是一个可逆的且可控制的过程 [1 ] 。因此 ,探究继发性损伤机制具有重要的现实意义。在继发性损伤的早期 ,存在一个重要的炎症反应过程 ,它能够触发其后发生的一系列针对受损脊髓的细胞和分子水平的反应 ;此外 ,炎症反应可以导致受损脊髓局部胶质细胞的疤痕形成 ,也有可能阻碍脊髓的再生。现已明确 ,SCI后介导炎症反应发生发展的是一类重要的细胞因子 (cytokine) ,它…     

脊髓损伤后神经修复过程中的细胞微环境北大核心

背景:以往的研究显示单一改变脊髓损伤区域某一基因表达或者某一细胞的状态,对脊髓损伤后功能恢复无显著影响,而大量证据表明调控脊髓损伤后紊乱的细胞微环境是神经功能恢复的关键因素。目的:对脊髓损伤前后细胞微环境的生物学特性,包括多种细胞之间的相互调控以及细胞外组分对损伤神经修复的作用和机制进行综述。方法:由第一作者检索PubMed及Web of Science数据库,英文检索词为“spinal cord injury,glial cell,neuron,immune cell,neural stem cell,extracellular matrix,cytokine,extracellular vesicle,regeneration”。文献检索的时间范围为2000年1月至2021年12月,最终筛选出64篇文献进行分析。结果与结论:①脊髓损伤后,在细胞微环境的细胞组分中,占比最高的胶质细胞间的相互作用,以及与神经元的相互调控作用最为关键。②在脊髓损伤后的细胞外组分中,利用生物相容性良好的水凝胶模仿天然细胞外基质,可有效模拟和重建损伤区域内的细胞微环境,促进轴突伸长。③在脊髓损伤后的细胞外调节因子中,促炎因子如肿瘤坏死因子α和白细胞介素1β等加剧了细胞微环境的炎症反应,应用受体抑制剂或阻断相关通路抑制上述促炎因子的表达是一种有效的治疗方法,同时在脊髓微环境中增加白细胞介素10等抗炎因子的表达,抑制损伤区域炎症发展的研究也陆续出现。④最近被重视起来的细胞外囊泡作为传递信息的载体在细胞微环境中也发挥了重要作用。⑤文章揭示了脊髓损伤后细胞微环境中的包括细胞组分和细胞外组分之间的多组相互调控关系,证实了细胞微环境中各组分之间所发挥的神经修复作用并不是孤立的。     

脊髓损伤后骨骼肌变化的研究进展

脊髓损伤后机体发生一系列的改变,大部分是由于直接损伤造成,另外神经活动的减少、骨骼肌萎缩、肌无力等也起一部分作用。脊髓损伤不仅引起损伤局部的病变,还涉及所支配骨骼肌的改变。脊髓损伤成功的恢复,要求其效应器未发生不可逆性退行性变,因此要进一步提高脊髓损伤后运动功能恢复的效果,必须深入研究骨骼肌因素。     

臂丛损伤后脊髓前角运动神经元超微结构改变

目的探讨大鼠臂丛损伤导致脊髓前角运动神经元死亡的机制。方法成年雄性SD大鼠24只,其中对照组6只,损伤组18只。建立3种臂丛损伤模型:右C7前根撕脱(A组);右C7前根撕脱+同侧C5～T1后根离断(B组);右C7前根撕脱+右C5与C6之间脊髓半横断(C组)。术后14d取C7节段脊髓,采用尼氏染色方法和透射电镜技术,观察脊髓前角运动神经元的存活率及其超微结构改变。结果术后2周A组脊髓前角运动神经元的存活率最高,B组居中,C组最低。3个臂丛损伤组C7前角均可见凋亡特征性改变:运动神经元内核染色质聚集靠边,核固缩、碎裂、核膜皱褶并内陷,并有染色质团块形成的凋亡小体。细胞体积缩小,胞浆内细胞器密集,线粒体轻度肿胀,核周粗面内质网减少,游离核糖体增多,胞浆内可见较多的空泡。神经元胞体周围的有髓神经纤维和无髓神经纤维呈轻度肿胀,髓鞘的板层结构消失。结论臂丛损伤诱导脊髓运动神经元死亡途径中存在凋亡和坏死两种机制,运动神经元可形成凋亡小体。     

Hippo 信号通路与炎症的研究进展

Hippo信号通路是由一系列发生级联反应激酶构成的,调控细胞增殖和凋亡的一条关键信号转导通路,该信号通路最先于果蝇体内发现,且进化上高度保守。在哺乳动物中,早期研究主要集中于Hippo信号在肿瘤、心血管系统、免疫系统等疾病的调控,随着对Hippo信号通路的研究不断深入,近年来研究发现,Hippo与某些炎症疾病相关,特别是以Hippo通路为信号传导的中心,与某些炎症因子的信号通路存在交互作用,进而参与炎症的发生、发展过程。本文就Hippo信号通路与免疫细胞分化发育、Toll样受体信号传导通路、TGF-β/Smad信号通路,以及与G蛋白偶联受体之间的相互作用来阐述Hippo信号通路对炎症的调控机制,为研究Hippo信号通路调控炎症的机理提供思路。     

脊髓损伤后白藜芦醇保护作用的研究进展

正白藜芦(resveratrol,Res)是一种化学名为3,5,4’-三羟基戊二烯的多酚类化合物,广泛地存在于花生、凤梨、朝槐、决明、桑子、大黄、首乌、金雀根、葡萄、虎杖、藜芦等植物中,尤以虎杖含量最高~[1]。白藜芦醇分子式为C14H12O3,相对分子质量228.25,本质是一种植物抗毒素,是葡萄属植物在外来病菌侵入、紫外线照射等不利因素影响下产生的~[2]。于1940年首次从毛叶黎芦的根部获得,其存在的形式主要有四种:顺、反式白藜芦醇及与葡萄糖结合形成的顺、反式白藜芦     

基因治疗脊髓损伤研究进展

脊髓损伤在全世界都有较高的发病率,迄今为止,临床上脊髓损伤后功能恢复仍非常困难.现在认为中枢神经系统再生困难主要是因为损伤处微环境不适合轴突再生,其中损伤处缺乏神经营养因子类物质被认为是最主要的原因之一.近年来,人们将神经营养因子基因导入损伤处对脊髓损伤进行基因治疗取得了一定的研究进展,本文对基因治疗脊髓损伤的目的基因、病毒载体和靶细胞等进行了综述.     

Impaired crossed facilitation of the corticospinal pathway after cervical spinal cord injury

In uninjured humans, it is well established that voluntary contraction of muscles on one side of the body can facilitate transmission in the contralateral corticospinal pathway. This crossed facilitatory effect may favor interlimb coordination and motor performance. Whether this aspect of corticospinal function is preserved after chronic spinal cord injury (SCI) is unknown. Here, using transcranial magnetic stimulation, we show in patients with chronic cervical SCI (C(5)-C(8)) that the size of motor evoked potentials (MEPs) in a resting intrinsic hand muscle remained unchanged during increasing levels of voluntary contraction with a contralateral distal or proximal arm muscle. In contrast, MEP size in a resting hand muscle was increased during the same motor tasks in healthy control subjects. The magnitude of voluntary electromyography was negatively correlated with MEP size after chronic cervical SCI and positively correlated in healthy control subjects. To examine the mechanisms contributing to MEP crossed facilitation we examined short-interval intracortical inhibition (SICI), interhemispheric inhibition (IHI), and motoneuronal behavior by testing F waves and cervicomedullary MEPs (CMEPs). During strong voluntary contractions SICI was unchanged after cervical SCI and decreased in healthy control subjects compared with rest. F-wave amplitude and persistence and CMEP size remained unchanged after cervical SCI and increased in healthy control subjects compared with rest. In addition, during strong voluntary contractions IHI was unchanged in cervical SCI compared with rest. Our results indicate that GABAergic intracortical circuits, interhemispheric glutamatergic projections between motor cortices, and excitability of index finger motoneurons are neural mechanisms underlying, at least in part, the lack of crossed corticospinal facilitation observed after SCI. Our data point to the spinal motoneurons as a critical site for modulating corticospinal transmission after chronic cervical SCI.     

Targeting IKK/NF-κB pathway reduces infiltration of inflammatory cells and apoptosis after spinal cord injury in rats

IKK/NF-κB pathway is a principal player in the regulation of inflammation and apoptosis in spinal cord injury (SCI). We have previously shown that BMS-345541, a selective inhibitor of IKK, could inhibit NF-κB activation and ameliorate the motor function of hind limbs in rats after SCI. However its neuroprotective mechanism in SCI is still unclear. In this study, we investigated the neuroprotective effects of BMS-345541 and possible mechanisms by which BMS-345541 acts against inflammatory cells infiltration and apoptosis in rat SCI models. SCI induced neutrophils infiltration when examined by myeloperoxidase activity, which was prevented by BMS-345541 through inhibiting the expression of ICAM-1. BMS-345541 inhibited activation of caspase-3, and attenuated apoptotic cell death in spinal cord by modulating Bcl-2 and Bax expression following SCI. Our study indicates that BMS-345541 may play an important role on the anti-inflammatory and anti-apoptotic effects with experimental SCI. Our results imply the IKK inhibitors may be useful in the therapy of spinal cord injury.     

Acute and chronic changes in aquaporin 4 expression after spinal cord injury

The effect of spinal cord injury (SCI) on the expression levels and distribution of water channel aquaporin 4 (AQP4) has not been studied. We have found AQP4 in gray and white matter astrocytes in both uninjured and injured rat spinal cords. AQP4 was detected in astrocytic processes that were tightly surrounding neurons and blood vessels, but more robustly in glia limitans externa and interna, which were forming an interface between spinal cord parenchyma and cerebrospinal fluid (CSF). Such spatial distribution of AQP4 suggests a critical role that astrocytes expressing AQP4 play in the transport of water from blood/CSF to spinal cord parenchyma and vice versa. SCI induced biphasic changes in astrocytic AQP4 levels, including its early down-regulation and subsequent persistent up-regulation. However, changes in AQP4 expression did not correlate well with the onset and magnitude of astrocytic activation, when measured as changes in GFAP expression levels. It appears that reactive astrocytes began expressing increased levels of AQP4 after migrating to the wound area (thoracic region) two weeks after SCI, and AQP4 remained significantly elevated for months after SCI. We also showed that increased levels of AQP4 spread away from the lesion site to cervical and lumbar segments, but only in chronically injured spinal cords. Although overall AQP4 expression levels increased in chronically-injured spinal cords, AQP4 immunolabeling in astrocytic processes forming glia limitans externa was decreased, which may indicate impaired water transport through glia limitans externa. Finally, we also showed that SCI-induced changes in AQP4 protein levels correlate, both temporally and spatially, with persistent increases in water content in acutely and chronically injured spinal cords. Although correlative, this finding suggests a possible link between AQP4 and impaired water transport/edema/syringomyelia in contused spinal cords.     

Restoring walking after spinal cord injury

One of the most obvious deficits following a spinal cord injury is the difficulty in walking, forcing many patients to use wheelchairs for locomotion. Over the past decade considerable effort has been directed at promoting the recovery of walking and to find effective treatments for spinal cord injury. Advances in our knowledge of the neuronal control of walking have led to the development of a promising rehabilitative strategy in patients with partial spinal cord injury, namely treadmill training with partial weight support. The current focus is on developing more efficient training protocols and automating the training to reduce the physical demand for the therapists. Mechanisms underlying training-induced improvements in walking have been revealed to some extent in animal studies. Another strategy for improving the walking in spinal cord injured patients is the use of functional electric stimulation of nerves and muscles to assist stepping movements. This field has advanced significantly over the past decade as a result of developments in computer technology and the miniaturization of electronics. Finally, basic research on animals with damaged spinal cords has focused on enhancing walking and other motor functions by promoting growth and regeneration of damaged axons. Numerous important findings have been reported yielding optimism that techniques for repairing the injured spinal cord will be developed in the near future. However, at present no strategy involving direct treatment of the injured spinal cord has been established for routine use in spinal cord injured patients. It now seems likely that any successful protocol in humans will require a combination of a treatment to promote re-establishing functional connections to neuronal networks in the spinal cord and specialized rehabilitation training to shape the motor patterns generated by these networks for specific behavioral tasks.     

干细胞移植治疗脊髓损伤的研究进展

背景：干细胞移植作为治疗脊髓损伤最具前景的方法,已经在大量的动物实验和临床试验中得到证实。 目的：综述干细胞移植治疗脊髓损伤的相关研究进展。 方法：应用计算机检索2006-01/2010-12中国知网、Medline数据库相关文章,中文检索词“干细胞,脊髓损伤,细胞移植”,英文检索词“stem cells,spinal cord injury,cells transplantation”,共检索到文献494篇,最终纳入符合标准的文献24篇。 结果与结论：研究发现,移植的干细胞可以在脊髓内迁移、分化为神经元并分泌神经营养物质,促进神经组织的修复,改善神经功能。胚胎干细胞最早用于治疗脊髓损伤,但潜在的致瘤性等成为其临床应用的障碍;神经干细胞理论上是治疗脊髓损伤的首选干细胞,由于分离纯化技术要求严格,费用昂贵等使其在研究中进展缓慢;骨髓间充质干细胞来源丰富、取材方便,可行自体移植,避开了伦理学和移植后排斥等问题,目前被认为是一种理想的自体干细胞移植来源。随着细胞联合移植、基因修饰及组织工程支架移植治疗脊髓损伤的研究不断取得进展,许旺细胞、嗅鞘细胞的应用范围和治疗效果也得到提升。     

大鼠脊髓压迫性损伤解压后pEGFR的表达变化

目的　探讨大鼠脊髓压迫性损伤（compressed spinal cord injury,CSCI）解压后表皮生长因子受体（phosphorylated epidermal growth factor receptor,pEGFR）、pAkt1的表达变化及其与神经功能、有髓神经纤维数量变化的相关性,为CSCI解压后治疗策略的制定和药物的研发提供实验基础。 方法　采用自行设计的方法制作SD大鼠CSCI模型,造模成功后解压。运用BBB（Basso Beattie Bresnahan）评分观察动物解压后神经功能的恢复情况;通过Luxol fast blue（LFB）染色检测解压后1、7、14、21 d有髓神经纤维数量变化;运用免疫荧光双标（Double-labeling immunoflurescence）、免疫印迹（western blotting,WB）检测pEGFR、 pAkt1的表达变化。 结果　CSCI解压后,BBB评分和有髓神经纤维数量随时间延长而逐渐增加;与此同时,pEGFR、pAkt1表达亦上调且与BBB评分、有髓神经纤维数量增加趋势一致。 结论　CSCI解压后,神经功能有一定改善、有髓神经纤维数量有一定增加,这些变化与pEGFR表达上调有关,提示EGFR的活化参与了CSCI解压后的内源性修复。     

脊髓损伤后的康复实践结果

背景：脊髓损伤患者的康复结果与患者的损伤程度、治疗方法、康复时间及后期治疗等多因素有关。跨学科、全面、专业的脊髓损伤康复单元能为脊髓损伤患者提供更好的恢复。目的：综合评价脊髓损伤康复单元干预或其组合干预的效果。方法：检索2003至2014年Springer及PubMed数据库,检索词：spinal cord injury,rehabilitation practice,outcomes。根据纳入排除标准,阅读标题和摘要进行初筛,选用44篇英文文献进行分析。结果与结论：许多研究采用基于实践证据的方法,识别多种康复实践方法,把信息与结果联系以评价康复干预的效果。研究显示创伤及非创伤性混合样本中的目标实现与年龄没有差异,大多数康复结果很少有性别差异。最初入院到专科脊髓损伤中心的绝大多数脊髓损伤患者的并发症通常最低,患者尽早入住到跨学科、全面、专业的脊髓损伤单位比延迟入住缩短住院总时间。脊髓损伤患者接受正规、全面的门诊医护随访,在健康感知、独立性、抑郁症没有显著差异,但特定继发情况出现频率显著减少、程度减低。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

急性不完全脊髓损伤模型大鼠相关炎症因子的表达

背景：随着现代交通和工矿事业的发展,脊髓损伤成为临床常见的多发病,严重的影响了人类身心健康和生活质量。 目的：检测在大鼠急性脊髓损伤模型中磷酸化信号转导与转录激活因子3（p-STAT3）介导的IL-1β、IL-6、IL-17等炎症因子的表达变化情况,探讨IL-1β、IL-6、IL-17等炎症因子在急性脊髓损伤后炎症反应过程中的作用。 方法：雄性成年SD大鼠75只,随机分为对照组和脊髓损伤1h、6h、24h和72h四个亚组（n=15只）。采用改良Allen氏法建立脊髓损伤模型,对照组仅行椎板全切除术。在建模之后相应的时间点获取损伤段脊髓、脾脏组织,免疫组织化学染色法检测IL-6和IL-17在损伤段脊髓组织中的分布及表达情况,蛋白免疫印迹法检测p-STAT3在损伤段脊髓组织中的表达变化,采用RT-PCR法检测IL-1βmRNA、IL-6mRNA和IL-17AmRNA在脾脏组织中的表达情况。 结果与结论：脊髓损伤后p-STAT3、IL-1β、IL-6和IL-17的表达明显高于对照组（P＜0.05）。损伤后炎症因子表达量立即升高,IL-1β和IL-6在损伤后6h小时到达高峰,随后开始下降;p-STAT3和IL-17在损伤后24h到达高峰,随后开始下降,至损伤后72h表达量仍高于对照组。损伤早期p-STAT3介导的促炎因子IL-1β和IL-6表达增加,可能导致损伤区域炎症级联放大,诱导继发性脊髓损伤;促炎因子IL-17的异常升高,可能在继发性损伤炎症反应中起着重要作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

大鼠脊髓发育及损伤后NIDD mRNA表达的实验研究

目的：探讨在大鼠发育过程中及急性脊髓损伤后脊髓组织中NIDD （nNOS-interacting DHHC domain-containing protein with dendritic mRNA）mRNA的表达变化及意义。方法：采用改良Allen＇s打击法，咬除T8-10椎板后，造成大鼠脊髓损伤模型，致伤量为10×10g·cm；借助实时荧光定量PCR、原位杂交与免疫荧光结合的方法，定量、定位研究发育过程中及脊髓损伤后早期大鼠脊髓组织中NIDD mRNA与nNOS mRNA表达的时间和空间分布特征。结果：大鼠发育过程中，胚胎16d的大鼠脊髓中可见NIDD mRNA的高表达，在生后1d，与nNOS共表达于尚未分化成熟的前角，在白质也见NIDD的阳性信号。成年后呈低表达；nNOS mRNA于生后1～3d出现表达高峰；脊髓损伤后NIDD mRNA表达明显增多，在8h到达高峰，分布于脊髓前角、中间带、中央管周围及后角nNOS阳性的神经元，7d恢复至正常水平；nNOS mRNA在损伤后8h达到高峰，1d降低至正常水平。而且，在脊髓损伤后NIDD mRNA与nNOS mRNA二者表达呈正相关。结论：胎鼠脊髓中，NIDD高表达于nNOS阳性细胞，提示其在脊髓组织的发育成熟过程中的作用可能与nNOS相关。脊髓损伤后脊髓组织中NIDD与nNOS表达增多，提示在脊髓损伤的病理过程中，NIDD可能通过调节nNOS的细胞亚定位及活性而发挥一定的生物学作用。