糖尿病介导的周细胞损伤对脊髓损伤后血脊屏障破坏的作用

糖尿病是一种常见的慢性代谢异常性疾病,可通过血糖异常诱导体内内环境紊乱,引起一系列急性或慢性并发症。慢性高血糖可引起大血管和微血管病变,该过程由错综复杂的分子机制协同调控,例如炎症反应、细胞内应激作用、细胞焦亡和细胞铁死亡等。糖尿病可抑制脊髓损伤后血脊屏障修复,加重神经功能损伤,从而不利于运动功能恢复。周细胞是神经血管单元的重要组成部分,参与调控血管再生、毛细血管血流量以及血脊屏障渗透性。脊髓损伤后,血脊屏障遭到破坏,周细胞覆盖率显著降低,血管正常功能受到巨大影响。糖尿病不仅参与调控周细胞的收缩表型和信号传导,而且改变周细胞分泌基因组谱,影响周细胞正常功能。此外,有研究证实,糖尿病促进脊髓损伤后周细胞丢失。本综述系统阐述了糖尿病对血管系统中周细胞的调控作用,及其介导的周细胞损伤对脊髓损伤后血脊屏障修复影响的研究进展。     

Inosine Enhances Axon Sprouting and Motor Recovery after Spinal Cord Injury

Although corticospinal tract axons cannot regenerate long distances after spinal cord injury, they are able to sprout collateral branches rostral to an injury site that can help form compensatory circuits in cases of incomplete lesions. We show here that inosine enhances the formation of compensatory circuits after a dorsal hemisection of the thoracic spinal cord in mature rats and improves coordinated limb use. Inosine is a naturally occurring metabolite of adenosine that crosses the cell membrane and, in neurons, activates Mst3b, a protein kinase that is part of a signal transduction pathway that regulates axon outgrowth. Compared to saline-treated controls, rats with dorsal hemisections that were treated with inosine showed three times as many synaptic contacts between corticospinal tract collaterals and long propriospinal interneurons that project from the cervical cord to the lumbar level. Inosine-treated rats also showed stronger serotonergic reinnervation of the lumbar cord than saline-treated controls, and performed well above controls in both open-field testing and a horizontal ladder rung-walking test. Inosine was equally effective whether delivered intracranially or intravenously, and has been shown to be safe for other indications in humans. Thus, inosine might be a useful therapeutic for improving outcome after spinal cord injury.     

Plasminogen Activator Promotes Recovery Following Spinal Cord Injury

Plasminogen activators play an important role in synaptic plasticity associated with the crossed phrenic phenomenon (CPP) and recovery of respiratory function after spinal cord injury. A genetic approach using knockout mice lacking various genes in the plasminogen activator/plasmin system has shown that induction of urokinase plasminogen activator (uPA) is required during the first hour after a C2-hemisection for the acquisition of the CPP response. The uPA knockout mice do not show the structural remodeling of phrenic motor neuron synapses characteristic of the CPP response. As shown here uPA acts in a cell signaling manner via binding to its receptor uPAR rather than as a protease, since uPAR knockout mice or knock-in mice possessing a modified uPA that is unable to bind to uPAR both fail to generate a CPP and recover respiratory function. Microarray data and real-time PCR analysis of mRNAs induced in the phrenic motor nucleus after C2-hemisection in C57Bl/6 mice as compared to uPA knockout mice indicate a potential cell signaling cascade downstream possibly involving β-integrin and Src, and other pathways. Identification of these uPA-mediated signaling pathways may provide the opportunity to pharmacologically upregulate the synaptic plasticity necessary for recovery of phrenic motoneuron activity following cervical spinal cord injury.     

缺血后处理对兔脊髓缺血再灌注微循环损伤的影响

探讨缺血后处理对兔脊髓缺血再灌注微循环损伤的影响.成年新西兰大白兔24只随机分为假手术组(C组),缺血再灌注损伤组(IR组),缺血后处理组(P组).IR组和P组采用Zivin改进法制备脊髓缺血再灌注模型,P组在缺血30 min后行复灌1 min/缺血1 min相同处理3次.采用激光多普勒检测缺血前,缺血时及再灌注各时点血流量值,在再灌注24 h时取兔脊髓组织作HE染色观察病理形态学,比色法检测脊髓组织一氧化氮(Nitric oxide,NO)的含量,放免法检测内皮素-1(Endothelin-1,ET-1)及免疫组化法检测血红素氧合酶(Hemeoxygenase-1,HO-1)的表达.研究发现与缺血前基础值相比,再灌注10 min时IR组与P组血流量均有增高,在再灌注30、60、120 min,IR组血流量值有不同程度的降低;与IR组相比,P组血流量值在再灌注各时点均有不同程度的增高.与IR组相比,P组NO含量与HO-1表达均有增加,ET-1含量明显减少,NO/ET-1显著高于IR组(P<0.05或0.01),且P组脊髓病理学损伤轻于IR组.结果表明缺血后处理可减轻兔脊髓缺血再灌注微循环损伤,改善脊髓血流量,...     

Restoring Cellular Energetics Promotes Axonal Regeneration and Functional Recovery after Spinal Cord Injury

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Umbilical Cord Blood Stem Cell Mediated Downregulation of Fas Improves Functional Recovery of Rats after Spinal Cord Injury

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins α-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.     

Protective Effect of Mild Hypothermia on Spinal Cord Ischemia-Induced Delayed Paralysis and Spinal Cord Injury

Neurochemical Research - To explore the mechanism regarding the regulation of spinal cord ischemia (SCI) in rats by mild hypothermia. A SCI rat model was established through aorta occlusion, and in...     

Transplantation of D15A-Expressing Glial-Restricted-Precursor-Derived Astrocytes Improves Anatomical and Locomotor Recovery after Spinal Cord Injury

The transplantation of neural stem/progenitor cells is a promising therapeutic strategy for spinal cord injury (SCI). In this study, we tested whether combination of neurotrophic factors and transplantation of glial-restricted precursor (GRPs)-derived astrocytes (GDAs) could decrease the injury and promote functional recovery after SCI. We developed a protocol to quickly produce a sufficiently large, homogenous population of young astrocytes from GRPs, the earliest arising progenitor cell population restricted to the generation of glia. GDAs expressed the axonal regeneration promoting substrates, laminin and fibronectin, but not the inhibitory chondroitin sulfate proteoglycans (CSPGs). Importantly, GDAs or its conditioned medium promoted the neurite outgrowth of dorsal root ganglion neurons in vitro. GDAs were infected with retroviruses expressing EGFP or multi-neurotrophin D15A and transplanted into the contused adult thoracic spinal cord at 8 days post-injury. Eight weeks after transplantation, the grafted GDAs survived and integrated into the injured spinal cord. Grafted GDAs expressed GFAP, suggesting they remained astrocyte lineage in the injured spinal cord. But it did not express CSPG. Robust axonal regeneration along the grafted GDAs was observed. Furthermore, transplantation of D15A-GDAs significantly increased the spared white matter and decreased the injury size compared to other control groups. More importantly, transplantation of D15A-GDAs significantly improved the locomotion function recovery shown by BBB locomotion scores and Tredscan footprint analyses. However, this combinatorial strategy did not enhance the aberrant synaptic connectivity of pain afferents, nor did it exacerbate posttraumatic neuropathic pain. These results demonstrate that transplantation of D15A-expressing GDAs promotes anatomical and locomotion recovery after SCI, suggesting it may be an effective therapeutic approach for SCI.     

The Asparaginyl Endopeptidase Legumain Is Essential for Functional Recovery after Spinal Cord Injury in Adult Zebrafish

Unlike mammals, adult zebrafish are capable of regenerating severed axons and regaining locomotor function after spinal cord injury. A key factor for this regenerative capacity is the innate ability of neurons to re-express growth-associated genes and regrow their axons after injury in a permissive environment. By microarray analysis, we have previously shown that the expression of legumain (also known as asparaginyl endopeptidase) is upregulated after complete transection of the spinal cord. In situ hybridization showed upregulation of legumain expression in neurons of regenerative nuclei during the phase of axon regrowth/sprouting after spinal cord injury. Upregulation of Legumain protein expression was confirmed by immunohistochemistry. Interestingly, upregulation of legumain expression was also observed in macrophages/microglia and neurons in the spinal cord caudal to the lesion site after injury. The role of legumain in locomotor function after spinal cord injury was tested by reducing Legumain expression by application of anti-sense morpholino oligonucleotides. Using two independent anti-sense morpholinos, locomotor recovery and axonal regrowth were impaired when compared with a standard control morpholino. We conclude that upregulation of legumain expression after spinal cord injury in the adult zebrafish is an essential component of the capacity of injured neurons to regrow their axons. Another feature contributing to functional recovery implicates upregulation of legumain expression in the spinal cord caudal to the injury site. In conclusion, we established for the first time a function for an unusual protease, the asparaginyl endopeptidase, in the nervous system. This study is also the first to demonstrate the importance of legumain for repair of an injured adult central nervous system of a spontaneously regenerating vertebrate and is expected to yield insights into its potential in nervous system regeneration in mammals.     

Roles of ES Cell-Derived Gliogenic Neural Stem/Progenitor Cells in Functional Recovery after Spinal Cord Injury

Transplantation of neural stem/progenitor cells (NS/PCs) following the sub-acute phase of spinal cord injury (SCI) has been shown to promote functional recovery in rodent models. However, the types of cells most effective for treating SCI have not been clarified. Taking advantage of our recently established neurosphere-based culture system of ES cell-derived NS/PCs, in which primary neurospheres (PNS) and passaged secondary neurospheres (SNS) exhibit neurogenic and gliogenic potentials, respectively, here we examined the distinct effects of transplanting neurogenic and gliogenic NS/PCs on the functional recovery of a mouse model of SCI. ES cell-derived PNS and SNS transplanted 9 days after contusive injury at the Th10 level exhibited neurogenic and gliogenic differentiation tendencies, respectively, similar to those seen in vitro. Interestingly, transplantation of the gliogenic SNS, but not the neurogenic PNS, promoted axonal growth, remyelination, and angiogenesis, and resulted in significant locomotor functional recovery after SCI. These findings suggest that gliogenic NS/PCs are effective for promoting the recovery from SCI, and provide essential insight into the mechanisms through which cellular transplantation leads to functional improvement after SCI.     

促凋亡基因Bax在大鼠脊髓损伤后的表达及意义

目的:研究促凋亡基因Bax表达与脊髓损伤(spinal cord injury,SCI)程度的关系.方法:Wistar大鼠36只,随机分成3组,为正常对照组、轻(中)度损伤组和重度损伤组.大鼠在脊髓损伤后14天处死,HE和Nissel染色观察脊髓组织形态结构和病理学变化,免疫组织化学S-P法检测脊髓中Bax表达情况.结果:Bax蛋白在大鼠脊髓损伤前后表达阳性率分别为5.6%和58.3%,有显著性差异(P＜0.05);轻(中)度脊髓损伤和重度脊髓损伤中的Bax的阳性率分别为18.5%和59.3%,有显著性差异(P＜0.05).结论:Bax基因表达与大鼠脊髓损伤有密切关系,且随着损伤程度加重Bax表达也增强.     

Relationship between Spinal Cord Volume and Spinal Cord Injury due to Spinal Shortening

Vertebral column resection is associated with a risk of spinal cord injury. In the present study, using a goat model, we aimed to investigate the relationship between changes in spinal cord volume and spinal cord injury due to spinal shortening, and to quantify the spinal cord volume per 1-mm height in order to clarify a safe limit for shortening. Vertebral column resection was performed at T10 in 10 goats. The spinal cord was shortened until the somatosensory-evoked potential was decreased by 50% from the baseline amplitude or delayed by 10% relative to the baseline peak latency. A wake-up test was performed, and the goats were observed for two days postoperatively. Magnetic resonance imaging was used to measure the spinal cord volume, T10 height, disc height, osteotomy segment height, and spinal segment height pre- and postoperatively. Two of the 10 goats were excluded, and hence, only data from eight goats were analyzed. The somatosensory-evoked potential of these eight goats demonstrated meaningful changes. With regard to neurologic function, five and three goats were classified as Tarlov grades 5 and 4 at two days postoperatively. The mean shortening distance was 23.6 ± 1.51 mm, which correlated with the d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment (r = 0.95, p < 0.001) and with the height of the T10 body (r = 0.79, p = 0.02). The mean d-value (post-pre) of the spinal cord volume per 1-mm height of the osteotomy segment was 142.87 ± 0.59 mm³ (range, 142.19–143.67 mm³). The limit for shortening was approximately 106% of the vertebral height. The mean volumes of the osteotomy and spinal segments did not significantly change after surgery (t = 0.310, p = 0.765 and t = 1.241, p = 0.255, respectively). Thus, our results indicate that the safe limit for shortening can be calculated using the change in spinal cord volume per 1-mm height.     

Arylsulfatase B Improves Locomotor Function after Mouse Spinal Cord Injury

Bacterial chondroitinase ABC (ChaseABC) has been used to remove the inhibitory chondroitin sulfate chains from chondroitin sulfate proteoglycans to improve regeneration after rodent spinal cord injury. We hypothesized that the mammalian enzyme arylsulfatase B (ARSB) would also enhance recovery after mouse spinal cord injury. Application of the mammalian enzyme would be an attractive alternative to ChaseABC because of its more robust chemical stability and reduced immunogenicity. A one-time injection of human ARSB into injured mouse spinal cord eliminated immunoreactivity for chondroitin sulfates within five days, and up to 9 weeks after injury. After a moderate spinal cord injury, we observed improvements of locomotor recovery assessed by the Basso Mouse Scale (BMS) in ARSB treated mice, compared to the buffer-treated control group, at 6 weeks after injection. After a severe spinal cord injury, mice injected with equivalent units of ARSB or ChaseABC improved similarly and both groups achieved significantly more locomotor recovery than the buffer-treated control mice. Serotonin and tyrosine hydroxylase immunoreactive axons were more extensively present in mouse spinal cords treated with ARSB and ChaseABC, and the immunoreactive axons penetrated further beyond the injury site in ARSB or ChaseABC treated mice than in control mice. These results indicate that mammalian ARSB improves functional recovery after CNS injury. The structural/molecular mechanisms underlying the observed functional improvement remain to be elucidated.