神经干细胞移植修复鼠脊髓损伤的实验研究

目的 :观察神经干细胞移植治疗对鼠脊髓损伤后神经结构修复和功能恢复的作用并探讨其作用机制。方法 :制备鼠T10 脊髓损伤模型 ,体外培养、诱导鼠神经干细胞 ,定量评价神经干细胞移植对脊髓损伤后神经结构修复和功能恢复的影响。结果 :与对照组相比 ,神经干细胞移植组明显的增强了GAP 43mRNA的表达 ,促进了脊髓ChAT阳性的运动神经元的再生、结构的修复和下肢运动功能的恢复。结论 :神经干细胞移植促进了脊髓损伤后神经结构的修复和功能的恢复 ,是急性脊髓损伤一种有效的治疗方案     

Cell Therapy for Spinal Cord Injury by Neural Stem/Progenitor Cells Derived from iPS/ES Cells

Reports of functional recovery from spinal cord injury after the transplantation of rat fetus-derived neural stem cells and embryonic stem cells has raised great expectations for the successful clinical use of stem cell transplantation therapy. However, the ethical issues involved in destroying human embryos or fertilized oocytes to obtain stem cells have been a major obstacle to developing clinically useful stem cell sources, and the transplantation of stem cells isolated from other human embryonic tissues has not yet been developed for use in clinical applications. Recently, induced pluripotent stem cells, which can serve as a source of cells for autologous transplantation, have been attracting a great deal of attention as a clinically viable alternative to stem cells obtained directly from tissues. In this review, we outline the neural induction of mouse embryonic stem cells and induced pluripotent stem cells, their therapeutic efficacy in spinal cord injury, and their safety in vivo.     

BDNF基因修饰神经干细胞移植治疗脊髓损伤的实验研究

目的研究BDNF基因修饰神经干细胞移植对脊髓损伤后神经细胞凋亡的影响。方法采用电控大鼠脊髓损伤打击装置制作大鼠脊髓损伤模型。120只SD大鼠随机分为4组:假手术组(Sham组),脊髓损伤组(SCI组),神经干细胞组(NSC组),BDNF基因修饰神经干细胞组(NSC-BDNF组)。通过免疫组化法检测大鼠脊髓BDNF、Bax、Bcl-2的表达,流式细胞仪检测大鼠脊髓细胞凋亡率。结果NSC-BDNF组中BDNF免疫阳性细胞光密度值较NSC、SCI组增加明显(P<0.05),表达时间及表达高峰延长,且Bcl-2的表达较其他组在各个时间点上均增高(P<0.05),而Bax的表达较其他组在各个时间点上均降低(P<0.05),凋亡率亦明显低于NSC和SCI组(P<0.01)。结论BDNF基因修饰神经干细胞移植可引起BDNF在损伤脊髓内有效表达,且明显的促进了脊髓损伤后Bcl-2的高表达,抑制了Bax的表达,从而降低了神经细胞的凋亡率。     

Stress-Resistant Neural Stem Cells Positively Influence Regional Energy Metabolism After Spinal Cord Injury in Mice

The importance of stem cells to ameliorate the devastating consequences of traumatic injuries in the adult mammalian central nervous system calls for improvements in the capacity of these cells to cope, in particular, with the host response to the injury. We have previously shown, however, that in the acutely traumatized spinal cord local energy metabolism led to decreased ATP levels after neural stem cell (NSC) transplantation. As this might counteract NSC-mediated regenerative processes, we investigated if NSC selected for increased oxidative stress resistance are better suited to preserve local energy content. For this purpose, we exposed wild-type (WT) NSC to hydrogen peroxide prior to transplantation. We demonstrate here that transplantation of WT-NSC into a complete spinal cord compression injury model even lowers the ATP content beyond the level detected in spinal cord injury–control animals. Compared to WT-NSC, stress-resistant (SR) NSC did not lead to a further decrease in ATP content. These differences between WT- and SR-NSC were observed 4 h after the lesion with subsequent transplantation. At 24 h after lesioning, these differences were no more as obvious. Thus, in contrast to native NSC, transplantation of NSC selected for oxidative stress resistance can positively influence local energy metabolism in the first hours after spinal cord compression. The functional relevance of this observation has to be tested in further experiments.     

Epidural Spinal Cord Stimulation Promotes Motor Functional Recovery by Enhancing Oligodendrocyte Survival and Differentiation and by Protecting Myelin after Spinal Cord Injury in Rats

Epidural spinal cord stimulation (ESCS) markedly improves motor and sensory function after spinal cord injury (SCI), but the underlying mechanisms are unclear.Here, we investigated whether ESCS affects oligodendrocyte differentiation and its cellular and molecular mechanisms in rats with SCI. ESCS improved hindlimb motor function at 7 days, 14 days, 21 days, and 28 days after SCI.ESCS also significantly increased the myelinated area at 28days, and reduced the number of apoptotic cells in the spinal white matter at 7 days. SCI decreased the expression of 20,30-cyclic-nucleotide 30-phosphodiesterase (CNPase,an oligodendrocyte marker) at 7 days and that of myelin basic protein at 28 days. ESCS significantly upregulated these markers and increased the percentage of Sox2/CNPase/DAPI-positive cells (newly differentiated oligodendrocytes) at 7 days. Recombinant human bone morphogenetic protein 4 (rh BMP4) markedly downregulated these factors after ESCS. Furthermore, ESCS significantly decreased BMP4 and p-Smad1/5/9 expression after SCI,and rh BMP4 reduced this effect of ESCS. These findings indicate that ESCS enhances the survival and differentiation of oligodendrocytes, protects myelin, and promotes motor functional recovery by inhibiting the BMP4-Smad1/5/9 signaling pathway after SCI.     

Epigenetics of Neural Repair Following Spinal Cord Injury

Spinal cord injury results from an insult inflicted on the spinal cord that usually encompasses its 4 major functions (motor, sensory, autonomic, and reflex). The type of deficits resulting from spinal cord injury arise from primary insult, but their long-term severity is due to a multitude of pathophysiological processes during the secondary phase of injury. The failure of the mammalian spinal cord to regenerate and repair is often attributed to the very feature that makes the central nervous system special—it becomes so highly specialized to perform higher functions that it cannot effectively reactivate developmental programs to re-build novel circuitry to restore function after injury. Added to this is an extensive gliotic and immune response that is essential for clearance of cellular debris, but also lays down many obstacles that are detrimental to regeneration. Here, we discuss how the mature chromatin state of different central nervous system cells (neural, glial, and immune) may contribute to secondary pathophysiology, and how restoring silenced developmental gene expression by altering histone acetylation could stall secondary damage and contribute to novel approaches to stimulate endogenous repair.     

GFP^＋-大鼠胚胎脊髓源神经干细胞的培养、分化和Neuregulin-1的表达

目的:从GFP -大鼠胚胎脊髓分离和培养神经干细胞(NSC),观察NSC的分化功能和Neuregulin-1的表达。方法:从孕16d的GFP -大鼠胚胎脊髓中分离、培养神经干细胞,用免疫组织化学方法观察神经球的Neuregulin-1表达及鉴定分化的细胞类型。结果:从大鼠胚胎脊髓能分离、培养出NSC。神经球能表达Neuregin-1和Nestin,并能进一步分化为神经元、星形胶质细胞和少突胶质细胞。结论:从GFP -大鼠胚胎脊髓能分离和培养出NSC,该NSC具有分化为用于治疗中枢神经疾病的多种神经细胞的潜能。     

Myelin and Microsomes from Rhesus Monkey Spinal Cord: Isolation and Effects of Trauma

Dispersions of rhesus monkey spinal cord and brain were separated into large particle (crude mitochondrial plus nuclear) and small particle (crude microsomal) fractions; myelin was isolated from each of these preparative fractions. In brain preparations, almost all myelin was found in the large particle fraction; in contrast, almost half the myelin from spinal cord preparations was found in the small particle fraction. In addition, much larger amounts of partially degraded myelin were found in the fraction floating on 0.32 M sucrose and in the cytosol fraction of the spinal cord preparation in comparison to those of the brain preparations. These results suggest that rhesus monkey spinal cord myelin is more fragile than brain myelin; upon dispersion of spinal cord, more small myelin vesicles (isolated from the crude microsomal fraction) and more 0.32 M sucrose floating fraction (partially degraded myelin) are formed. After trauma of the spinal cord, the proportion of small vesicle myelin was increased at the expense of large vesicle myelin, lending further support to the hypothesis that spinal cord myelin is more fragile than brain myelin. Although the lipid compositions were similar, spinal cord myelin had a lower protein content and a lower 2′,3′-cyclic nucleotide phosphodiesterase specific activity than did brain myelin. The lipid composition of microsomes from brain differed somewhat from that of spinal cord microsomes.     

Glial Cells Shape Pathology and Repair After Spinal Cord Injury

Glial cell types were classified less than 100 years ago by del Rio-Hortega. For instance, he correctly surmised that microglia in pathologic central nervous system (CNS) were “voracious monsters” that helped clean the tissue. Although these historical predictions were remarkably accurate, innovative technologies have revealed novel molecular, cellular, and dynamic physiologic aspects of CNS glia. In this review, we integrate recent findings regarding the roles of glia and glial interactions in healthy and injured spinal cord. The three major glial cell types are considered in healthy CNS and after spinal cord injury (SCI). Astrocytes, which in the healthy CNS regulate neurotransmitter and neurovascular dynamics, respond to SCI by becoming reactive and forming a glial scar that limits pathology and plasticity. Microglia, which in the healthy CNS scan for infection/damage, respond to SCI by promoting axon growth and remyelination—but also with hyperactivation and cytotoxic effects. Oligodendrocytes and their precursors, which in healthy tissue speed axon conduction and support axonal function, respond to SCI by differentiating and producing myelin, but are susceptible to death. Thus, post-SCI responses of each glial cell can simultaneously stimulate and stifle repair. Interestingly, potential therapies could also target interactions between these cells. Astrocyte–microglia cross-talk creates a feed-forward loop, so shifting the response of either cell could amplify repair. Astrocytes, microglia, and oligodendrocytes/precursors also influence post-SCI cell survival, differentiation, and remyelination, as well as axon sparing. Therefore, optimizing post-SCI responses of glial cells—and interactions between these CNS cells—could benefit neuroprotection, axon plasticity, and functional recovery.     

Axon Guidance Molecules and Neural Circuit Remodeling After Spinal Cord Injury

…once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. Santiago Ramón y Cajal

Cajal’s neurotropic theory postulates that the complexity of the nervous system arises from the collaboration of neurotropic signals from neuronal and non-neuronal cells and that once development has ended, a paucity of neurotropic signals means that the pathways of the central nervous system are “fixed, ended, immutable”. While the capacity for regeneration and plasticity of the central nervous system may not be quite as paltry as Cajal proposed, regeneration is severely limited in scope as there is no spontaneous regeneration of long-distance projections in mammals and therefore limited opportunity for functional recovery following spinal cord injury. It is not a far stretch from Cajal to hypothesize that reappropriation of the neurotropic programs of development may be an appropriate strategy for reconstitution of injured circuits. It has become clear, however, that a significant number of the molecular cues governing circuit development become re-active after injury and many assume roles that paradoxically obstruct the functional re-wiring of severed neural connections. Therefore, the problem to address is how individual neural circuits respond to specific molecular cues following injury, and what strategies will be necessary for instigating functional repair or remodeling of the injured spinal cord.     

缺血性脑损伤与神经干细胞的再生

脑缺血可在成年哺乳动物脑内诱发神经再生,这可能有潜在的治疗价值。本文阐述了脑缺血后神经干细胞的增殖、迁移、分化和整合情况及血管微生态的重要作用,分析了侧脑室下区、齿状回和其他部位的神经再生情况以及老年脑神经再生的特点。     

蛛网膜下腔移植自体激活许旺细胞治疗大鼠脊髓损伤***

背景：许旺细胞能够分泌多种神经营养因子,促进脊髓损伤功能的恢复。但异体许旺细胞移植可引发自身免疫反应,且在移植方式上,局部移植无法避免二次损伤,静脉移植虽可以透过血脊髓屏障到达损伤局部,但不能达到有效的治疗浓度。 目的：探讨经蛛网膜下腔移植自体激活许旺细胞对脊髓损伤大鼠功能恢复的影响。 方法：66只大鼠均建立脊髓损伤模型,造模后随机分为3组,自体激活许旺细胞组通过结扎单侧隐神经从而激活许旺细胞,自体未激活许旺细胞组、模型对照组仅在相同部位手术但不结扎神经。切除各组手术远端1 cm神经,采用组织块法进行许旺细胞的体外分离培养及纯化。1周后,自体激活许旺细胞组、自体未激活许旺细胞组分别通过蛛网膜下腔注入经Hoechst33342标记的对应许旺细胞悬液,模型对照组仅注入等量DMEM。对脊髓损伤后肢体功能的恢复进行BBB运动功能评分及脚印分析,通过苏木精-伊红染色和GFAP染色从组织学角度评价脊髓损伤恢复情况。 结果与结论：从术后第4周开始,自体激活许旺细胞组BBB后肢功能评分明显优于另两组(P < 0.05)。移植后2周,可见迁移至大鼠脊髓损伤局部的许旺细胞。与自体未激活许旺细胞组比较,移植后5周自体激活许旺细胞组的前后足中心距离、后肢第3足趾外旋角度均显著减小(P < 0.05),移植后13周损伤区胶质瘢痕面积明显减小(P < 0.05),损伤区空洞面积明显减小(P < 0.05)。证实经蛛网膜下腔移植自体激活许旺细胞可以促进脊髓损伤的恢复。     

Dihydroprogesterone Increases the Gene Expression of Myelin Basic Protein in Spinal Cord of Diabetic Rats

Alterations in myelin membranes, as well as in the expression of myelin proteins have been reported in experimental models of diabetes. Data here reported show for the first time that the mRNA levels of two isoforms of myelin basic protein (MBP), 18.5 and 21.5 kDa, are decreased in the spinal cord of streptozotocin-treated rats and that treatment with a neuroactive steroid, such as progesterone (P), may counteract this effect. Interestingly, metabolism of progesterone into dihydroprogesterone (DHP) by the enzyme 5α-reductase seems to exert an important role in such an effect. As here demonstrated, 5α-reductase mRNA and DHP levels are reduced by diabetes in spinal cord, but treatment with P, is able to counteract these effects. Moreover, treatment with DHP is able to mimic the effect of P on MBP gene expression. Thus, the effects of P here observed are due to its enzymatic conversion into DHP. Because DHP, like P, interacts with P receptor (PR), the present results may suggest the importance to analyze the effects of PR modulators as tools of therapeutic strategies for diabetic complications occurring in nervous system.     

碱性成纤维生长因子和表皮生长因子对脊髓神经干细胞增殖与分化的影响

目的观察碱性成纤维生长因子(bFGF)和表皮生长因子(EGF)对胚胎脊髓神经干细胞(NSC)增殖与分化的影响。方法从14 d胚胎大鼠的脊髓组织中分离培养脊髓NSC,并随机分为3组:EGF组、bFGF组和bFGF+EGF组。通过光镜观察不同时间点各组脊髓NSC克隆细胞团数量及直径大小,并采用免疫荧光染色检测各组脊髓NSC向神经元和星形胶质细胞分化的情况。结果①EGF组培养1、3、7 d后NSC克隆细胞团数量和直径均少于bFGF和bFGF+EGF组,差异有统计学意义(P0.05)。而bFGF+EGF组仅在培养1 d时克隆细胞团数量多于bFGF组,在培养3、7 d时差异无统计学意义。②EGF组分化细胞中神经元比例显著少于bFGF和bFGF+EGF组,星形胶质细胞数量明显大于bFGF和bFGF+EGF组,差异有统计学意义(P0.05)。而bFGF和bFGF+EGF组组间差异无统计学意义。结论 EGF对脊髓NSC克隆形成有一定作用,而bFGF能较好地促进克隆细胞团的形成及生长,两者联合应用在培养早期可显著促进克隆细胞团形成。EGF可诱导脊髓NSC更多分化为星形胶质细胞,而bFGF则可促进脊髓NSC向神经元分化。     

Serotonergic Facilitation of Forelimb Functional Recovery in Rats with Cervical Spinal Cord Injury

Serotonergic agents can improve the recovery of motor ability after a spinal cord injury. Herein, we compare the effects of buspirone, a 5-HT_1A receptor partial agonist, to fluoxetine, a selective serotonin reuptake inhibitor, on forelimb motor function recovery after a C4 bilateral dorsal funiculi crush in adult female rats. After injury, single pellet reaching performance and forelimb muscle activity decreased in all rats. From 1 to 6 weeks after injury, rats were tested on these tasks with and without buspirone (1–2 mg/kg) or fluoxetine (1–5 mg/kg). Reaching and grasping success rates of buspirone-treated rats improved rapidly within 2 weeks after injury and plateaued over the next 4 weeks of testing. Electromyography (EMG) from selected muscles in the dominant forelimb showed that buspirone-treated animals used new reaching strategies to achieve success after the injury. However, forelimb performance dramatically decreased within 2 weeks of buspirone withdrawal. In contrast, fluoxetine treatment resulted in a more progressive rate of improvement in forelimb performance over 8 weeks after injury. Neither buspirone nor fluoxetine significantly improved quadrupedal locomotion on the horizontal ladder test. The improved accuracy of reaching and grasping, patterns of muscle activity, and increased excitability of spinal motor–evoked potentials after buspirone administration reflect extensive reorganization of connectivity within and between supraspinal and spinal sensory-motor netxcopy works. Thus, both serotonergic drugs, buspirone and fluoxetine, neuromodulated these networks to physiological states that enabled markedly improved forelimb function after cervical spinal cord injury.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-020-00974-8.Key Words: Serotonin, Spinal cord injury, Buspirone, Fluoxetine, Forelimb     

Neuroprotective Effects of Interleukin-10 Following Excitotoxic Spinal Cord Injury

Intraspinal injection of quisqualic acid (QUIS) produces excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic spinal cord injury (SCI). Inflammatory responses appear to be a major component of the secondary neuronal injury initiated by SCI and play a role in the pathogenesis of QUIS-induced injury. IL-10 is a potent antiinflammatory cytokine that has been shown to reduce inflammation and improve functional outcome in human and animal models of inflammatory diseases. We propose the administration of IL-10 following excitotoxic SCI will attenuate the inflammatory response, thus resulting in increased neuronal survival. Female, Sprague-Dawley rats were given intraspinal injections of QUIS followed by either intraspinal (5 ng, n = 8) or systemic injections (5 microgram n = 14) of IL-10. Survival times were varied (2-3 days) in order to produce a range of injury states and inflammatory involvement. When administered intraspinally, IL-10 significantly exacerbated the QUIS damage (P < 0.05), resulting in an 11.2% increase in lesion volume. When given systemically, IL-10 significantly decreased lesion volume by 18.1% in the more advanced injury (P < 0.05), but did not effect the more acute injury. These divergent effects were attributed to the modest inflammatory response in the short-term injury compared to the more robust inflammatory response in the more chronic injury. In conclusion, reducing the inflammatory response to SCI by systemic administration of IL-10 resulted in a significant reduction in neuronal damage, suggesting that targeting injury-induced inflammation may be an effective treatment strategy for acute SCI.     

Reversion of Injured Adult Neurons to an Embryonic State by Grafts of Neural Progenitor Cells After Spinal Cord Injury

Spinal cord injury(SCI)is a severe condition characterized by neurological dysfunction,dependence,respiratory failure,psychological morbidities,and an increased mortality rate[1].Globally,its incidence ranges from 250,000 to 500,000 annually,resulting in tremendous psychological and financial burdens[1].