Regenerative treatment in spinal cord injury

Spinal cord injury is a devastating, traumatic event, and experienced mainly among young people. Until the modern era, spinal cord injury was so rapidly fatal that no seriously injured persons would survive long enough for regeneration to occur. Treatment of spinal cord injury can be summarized as follows: prevent further cord injury, maintain blood flow, relieve spinal cord compression, and provide secure vertebral stabilization so as to allow mobilization and rehabilitation, none of which achieves functional recovery. Previous studies have focused on analyzing the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as the tissue damage and neural cell death associated with secondary injury. Now, there are hundreds of current experimental and clinical regenerative treatment studies. One of the most popular treatment method is cell transplantation in injured spinal cord. For this purpose bone marrow stromal cells, mononuclear stem cells, mesenchymal stem cells, embryonic stem cells, neural stem cells, and olfactory ensheathing cells can be used. As a result, cell transplantation has become a promising therapeutic option for spinal cord injury patients. In this paper we discuss the effectiveness of stem cell therapy in spinal cord injury.     

Umbilical Cord Blood-Derived Stromal Cells Regulate Megakaryocytic Proliferation and Migration Through SDF-1/PECAM-1 Pathway

We have previously reported that human umbilical cord blood-derived stromal cells (hUCBDSCs) are able to enhance the expansion of CFU-Meg in vitro, particularly promote the megakaryocytic lineage recovery, and effectively protect the survival of irradiated mice. In this study, we demonstrated that hUCBDSCs secreted SDF-1 to stimulate PECAM-1 expression in HEL cells (MK cell line), and consequently promoted the proliferation and migration of HEL cells. On the other hand, SDF-1 knock down in hUCBDSCs or PECAM-1 knock down in HEL cells diminished or abrogated the above effect. In addition, SDF-1/PECAM-1 probably activated PI3K/Akt and MAPK/ERK1/2 pathways. This report for the first time defines a SDF-1/PECAM-1 signaling pathway in the proliferation and migration of MKs, which provides supportive evidence for the clinical applications of hUCBDSCs in the treatment of megakaryocytic injury.     

自噬通过BDNF/TrkB信号通路对脊髓损伤的保护作用

脊髓型颈椎病(cervical spondylotic myelopathy, CSM)是临床常见病、多发病,由该病引起的脊髓受损而产生的脊髓功能障碍是其最常见的临床特征。在脊髓损伤中,多种因素能通过自噬相关信号通路而激活自噬,适度的自噬可以对脊髓损伤起到神经保护作用,而增加细胞存活率、恢复和增加自噬通量可以改善损伤后的脊髓功能的恢复;在CSM脊髓损伤中,脑源性神经营养因子(brain derived neurotrophic factor, BDNF)与其受体原肌球蛋白受体激酶B(tropomyosin receptor kinase B, TrkB)及其相关通路是参与保护神经细胞的重要途径之一,细胞自噬在这一过程中起到了重要的调节作用。本文通过阐释自噬与BDNF/TrkB信号通路在CSM过程中的作用及其对脊髓损伤的保护机制,以期对基础研究及临床研究提供理论支持。     

E-Selectin Mediated Adhesion and Migration of Endothelial Colony Forming Cells Is Enhanced by SDF-1α/CXCR4

Objective

Endothelial-colony forming cells (ECFCs) can be readily expanded from human umbilical cord blood and can facilitate repair of endothelial injury. E-selectin and SDF-1α are produced following endothelial injury and can regulate endothelial progenitor homing. Mechanisms of vascular repair specific to the mode of injury have not been well described in homogenous cell populations such as ECFCs and are needed for development of more effective vascular repair strategies.

Methods and Results

Lipopolysaccharide (LPS)-induced endotoxic injury to mature human umbilical vein endothelial cells (HUVEC) was compared with hypoxic and radiation injury. E-selectin expression in HUVEC cells is markedly increased (208-fold) following LPS-induced injury and facilitates increased ECFC adhesion and migration function in vitro. SDF-1α expression remains unchanged in LPS-treated HUVEC cells but increases more than 2 fold in fibroblasts undergoing similar endotoxic injury. SDF-1α induces expression of E-selectin ligands on ECFCs and facilitates greater E-selectin-mediated adhesion and migration of ECFCs in a CXCR4-dependent manner. Induction of E-selectin expression in HUVECs following hypoxic or radiation injury is negligible, however, while SDF-1α is increased markedly following hypoxia, highlighting injury-specific synergism between mediators of vascular repair.

Conclusion

E-selectin mediates adhesion and migration of ECFCs following endotoxic endothelial injury. SDF-1α augments E-selectin mediated ECFC adhesion and migration in a CXCR4-dependent manner.     

Upregulation of miR-199a-5p Protects Spinal Cord Against Ischemia/Reperfusion-Induced Injury via Downregulation of ECE1 in Rat

Ischemia–reperfusion (I/R)-induced spinal cord injury can cause apoptotic damage and subsequently act as a blood–spinal cord barrier damage. MicroRNAs (miRNAs) contributed to the process of I/R injury by regulating their target mRNAs. miR-199a-5p is involved in brain and heart I/R injury; however, its function in the spinal cord is not yet completely clarified. In this study, we investigated the role of miR-199a-5p on spinal cord I/R via the endothelin-converting enzyme 1, especially the apoptosis pathway. In the current study, the rat spinal cord I/R injury model was established, and the Basso Beattie Bresnahan scoring, Evans blue staining, HE staining, and TUNEL assay were used to assess the I/R-induced spinal cord injury. The differentially expressed miRNAs were screened using microarray. miR-199a-5p was selected by unsupervised hierarchical clustering analysis. The dual-luciferase reporter assay was used for detecting the regulatory effects of miR-199a-5p on ECE1. In addition, neuron expression was detected by immunostaining assay, while the expressions of p-ERK, ERK, p-JNK, JNK, caspase-9, Bcl-2, and ECE1 were evaluated by Western blot. The results indicated the successful establishment of the I/R-induced spinal cord injury model; the I/R induced the damage to the lower limb motor. Furthermore, 18 differentially expressed miRNAs were detected in the I/R group compared to the sham group, and miR-199a-5p protected the rat spinal cord injury after I/R. Moreover, miR-199a-5p negatively regulated ECE1, and silencing the ECE1 gene also protected the rat spinal cord injury after I/R. miR-199a-5p or silencing of ECE1 also regulated the expressions of caspase-9, Bcl-2, p-JNK, p-ERK, and ECE1 in rat spinal cord injury after I/R. Therefore, we demonstrated that miR-199a-5p might protect the spinal cord against I/R-induced injury by negatively regulating the ECE1, which could aid in developing new therapeutic strategies for I/R-induced spinal cord injury.     

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.     

Leukotriene B4 Release and Polymorphonuclear Cell Infiltration in Spinal Cord Injury

Activation of arachidonic acid occurs after spinal cord injury. Leukotriene B4 is a lipoxygenase metabolite of arachidonic acid. In a rat model of experimental spinal cord injury, we found that the leukotriene B4 content was less than the sensitivity of our assay (8 pg/mg of protein) in non-traumatized spinal cord. Leukotriene B4 was detectable in traumatized cord (mean +/- SE, 25 +/- 5 pg/mg of protein; n = 3). Release of leukotriene B4 from spinal cord slices into the incubation medium was also noted after trauma (9 +/- 1 pg/mg of protein; n = 12) and was enhanced by exposure of traumatized spinal cord slices to the calcium ionophore A23187 (375 +/- 43 pg/mg of protein; n = 12). The amount of leukotriene B4 released corresponded to the extent of post-traumatic polymorphonuclear cell infiltration determined by a myeloperoxidase assay. Results from this study suggest that the source of leukotriene B4 in spinal cord injury is infiltrating polymorphonuclear cells.     

Hydralazine inhibits compression and acrolein-mediated injuries in ex vivo spinal cord

We have previously shown that acrolein, a lipid peroxidation byproduct, is significantly increased following spinal cord injury in vivo , and that exposure to neuronal cells results in oxidative stress, mitochondrial dysfunction, increased membrane permeability, impaired axonal conductivity, and eventually cell death. Acrolein thus may be a key player in the pathogenesis of spinal cord injury, where lipid peroxidation is known to be involved. The current study demonstrates that the acrolein scavenger hydralazine protects against not only acrolein-mediated injury, but also compression in guinea pig spinal cord ex vivo . Specifically, hydralazine (500 μmol/L to 1 mmol/L) can significantly alleviate acrolein (100–500 μmol/L)-induced superoxide production, glutathione depletion, mitochondrial dysfunction, loss of membrane integrity, and reduced compound action potential conduction. Additionally, 500 μmol/L hydralazine significantly attenuated compression-mediated membrane disruptions at 2 and 3 h following injury. This was consistent with our findings that acrolein-lys adducts were increased following compression injury ex vivo , an effect that was prevented by hydralazine treatment. These findings provide further evidence for the role of acrolein in spinal cord injury, and suggest that acrolein-scavenging drugs such as hydralazine may represent a novel therapy to effectively reduce oxidative stress in disorders such as spinal cord injury and neurodegenerative diseases, where oxidative stress is known to play a role.     

Migration of human umbilical cord blood mesenchymal stem cells mediated by stromal cell-derived factor-1/CXCR4 axis via Akt, ERK, and p38 signal transduction pathways

Human mesenchymal stem cells (hMSCs) have been used for cell-based therapies in degenerative disease and as vehicles for delivering therapeutic genes to sites of injury and tumors. Recently, umbilical cord blood (UCB) was identified as a source for MSCs, and human UCB-derived MSCs (hUCB-MSCs) can serve as an alternative source of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, migration signaling pathways required for homing and recruitment of hUCB-MSCs are not fully understood. Stromal cell-derived factor-1 (SDF-1), a ligand for the CXCR4 chemokine receptor, plays a pivotal role in mobilization and homing of stem cells and modulates different biological responses in various stem cells. In this study, expression of CXCR4 in hUCB-MSCs was studied by western blot analysis and the functional role of SDF-1 was assessed. SDF-1 induced the migration of hUCB-MSCs in a dose-dependent manner. The induced migration was inhibited by the CXCR4-specific peptide antagonist (AMD3100) and by inhibitors of phosphoinositide 3-kinase (LY294002), mitogen-activated protein kinase/extracellular signal related kinase (PD98059) and p38MAPK inhibitor (SB203580). hUCB-MSCs treated with SDF-1 displayed increased phosphorylation of Akt, ERK and p38, which were inhibited by AMD3100. Small-interfering RNA-mediated knock-down of Akt, ERK and p38 blocked SDF-1 induced hUCB-MSC migration. In addition, SDF-1-induced actin polymerization was also blocked by these inhibitors. Taken together, these results demonstrate that Akt, ERK and p38 signal transduction pathways may be involved in SDF-1-mediated migration of hUCB-MSCs.     

Upregulation of vascular endothelial growth factor receptors Flt-1 and Flk-1 following acute spinal cord contusion in rats.

To investigate the possible role of vascular endothelial growth factor (VEGF) in the injured spinal cord, we analyzed the distribution and time course of the two tyrosine kinase receptors for VEGF, Flt-1 and Flk-1, in the rat spinal cord following contusion injury using a weight-drop impactor. The semi-quantitative RT-PCR analysis of Flt-1 and Flk-1 in the spinal cord showed slight upregulation of these receptors following spinal cord injury. Although mRNAs for Flt-1 and Flk-1 were constitutively expressed in neurons, vascular endothelial cells, and some astrocytes in laminectomy control rats, their upregulation was induced in association with microglia/macrophages and reactive astrocytes in the vicinity of the lesion within 1 day in rats with a contusion injury and persisted for at least 14 days. The spatiotemporal expression of Flt-1 in the contused spinal cord mirrored that of Flk-1 expression. In the early phase of spinal cord injury, upregulation of Flt-1 and Flk-1 mRNA occurred in microglia/macrophages that infiltrated the lesion. In addition, the expression of both receptors increased progressively in reactive astrocytes within the vicinity of the lesion, predominately in the white matter, and almost all reactive astrocytes coexpressed Flt-1 or Flk-1 and nestin. These results suggest that VEGF may be involved in the inflammatory response and the astroglial reaction to contusion injuries of the spinal cord via specific VEGF receptors.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.     

Embryonic stem cell-derived L1 overexpressing neural aggregates enhance recovery after spinal cord injury in mice

An obstacle to early stem cell transplantation into the acutely injured spinal cord is poor survival of transplanted cells. Transplantation of embryonic stem cells as substrate adherent embryonic stem cell-derived neural aggregates (SENAs) consisting mainly of neurons and radial glial cells has been shown to enhance survival of grafted cells in the injured mouse brain. In the attempt to promote the beneficial function of these SENAs, murine embryonic stem cells constitutively overexpressing the neural cell adhesion molecule L1 which favors axonal growth and survival of grafted and imperiled cells in the inhibitory environment of the adult mammalian central nervous system were differentiated into SENAs and transplanted into the spinal cord three days after compression lesion. Mice transplanted with L1 overexpressing SENAs showed improved locomotor function when compared to mice injected with wild-type SENAs. L1 overexpressing SENAs showed an increased number of surviving cells, enhanced neuronal differentiation and reduced glial differentiation after transplantation when compared to SENAs not engineered to overexpress L1. Furthermore, L1 overexpressing SENAs rescued imperiled host motoneurons and parvalbumin-positive interneurons and increased numbers of catecholaminergic nerve fibers distal to the lesion. In addition to encouraging the use of embryonic stem cells for early therapy after spinal cord injury L1 overexpression in the microenvironment of the lesioned spinal cord is a novel finding in its functions that would make it more attractive for pre-clinical studies in spinal cord regeneration and most likely other diseases of the nervous system.     

Glial and axonal regeneration following spinal cord injury

Spinal cord injury (SCI) has been regarded clinically as an irreversible damage caused by tissue contusion due to a blunt external force. Past research had focused on the analysis of the pathogenesis of secondary injury that extends from the injury epicenter to the periphery, as well as tissue damage and neural cell death associated with secondary injury. Recent studies, however, have proven that neural stem (progenitor) cells are also present in the brain and spinal cord of adult mammals including humans. Analyses using spinal cord injury models have also demonstrated active dynamics of cells expressing several stem cell markers, and methods aiming at functional reconstruction by promoting the potential self-regeneration capacity of the spinal cord are being explored. Furthermore, reconstruction of the neural circuit requires not only replenishment or regeneration of neural cells but also regeneration of axons. Analysis of the tissue microenvironment after spinal cord injury and research aiming to remove axonal regeneration inhibitors have also made progress. SCI is one of the simplest central nervous injuries, but its pathogenesis is associated with diverse factors, and further studies are required to elucidate these complex interactions in order to achieve spinal cord regeneration and functional reconstruction.Key words: glia, regeneration, spinal cord, injury, axon     

Cell surface peptidase CD26/dipeptidylpeptidase IV regulates CXCL12/stromal cell-derived factor-1 alpha-mediated chemotaxis of human cord blood CD34+ progenitor cells

CD26/dipeptidylpeptidase IV (DPPIV) is a membrane-bound extracellular peptidase that cleaves dipeptides from the N terminus of polypeptide chains. The N terminus of chemokines is known to interact with the extracellular portion of chemokine receptors, and removal of these amino acids in many instances results in significant changes in functional activity. CD26/DPPIV has the ability to cleave the chemokine CXCL12/stromal cell-derived factor 1alpha (SDF-1alpha) at its position two proline. CXCL12/SDF-1alpha induces migration of hemopoietic stem and progenitor cells, and it is thought that CXCL12 plays a crucial role in homing/mobilization of these cells to/from the bone marrow. We found that CD26/DPPIV is expressed by a subpopulation of CD34(+) hemopoietic cells isolated from cord blood and that these cells have DPPIV activity. The involvement of CD26/DPPIV in CD34(+) hemopoietic stem and progenitor cell migration has not been previously examined. Functional studies show that the N-terminal-truncated CXCL12/SDF-1alpha lacks the ability to induce the migration of CD34(+) cord blood cells and acts to inhibit normal CXCL12/SDF-1alpha-induced migration. Finally, inhibiting the endogenous CD26/DPPIV activity on CD34(+) cells enhances the migratory response of these cells to CXCL12/SDF-1alpha. This process of CXCL12/SDF-1alpha cleavage by CD26/DPPIV on a subpopulation of CD34(+) cells may represent a novel regulatory mechanism in hemopoietic stem and progenitor cells for the migration, homing, and mobilization of these cells. Inhibition of the CD26/DPPIV peptidase activity may therefore represent an innovative approach to increasing homing and engraftment during cord blood transplantation.     

LPA receptor expression in the central nervous system in health and following injury

Lysophosphatidic acid (LPA) is released from platelets following injury and also plays a role in neural development but little is known about its effects in the adult central nervous system (CNS). We have examined the expression of LPA receptors 1-3 (LPA_1–3) in intact mouse spinal cord and cortical tissues and following injury. In intact and injured tissues, LPA₁ was expressed by ependymal cells in the central canal of the spinal cord and was upregulated in reactive astrocytes following spinal cord injury. LPA₂ showed low expression in intact CNS tissue, on grey matter astrocytes in spinal cord and in ependymal cells lining the lateral ventricle. Following injury, its expression was upregulated on astrocytes in both cortex and spinal cord. LPA₃ showed low expression in intact CNS tissue, viz. on cortical neurons and motor neurons in the spinal cord, and was upregulated on neurons in both regions after injury. Therefore, LPA_1–3 are differentially expressed in the CNS and their expression is upregulated in response to injury. LPA release following CNS injury may have different consequences for each cell type because of this differential expression in the adult nervous system.     

脊髓缺血损伤合并脓毒症后脊髓ZnT1表达的研究

目的：研究脊髓缺血损伤合并脓毒血症后大鼠脊髓的病理改变及脊髓组织中锌转运体1（zinc transporter1,ZnT1）的表达规律。方法：将32只wistar大鼠随机分为假手术组（s组,n=8）、腹主动脉阻断组（I／R组,n=8）、内毒素组（LPS组,n=8）和腹主动脉阻断＋内毒素组（I／R＋LPS组,n=8）。用HE染色的方法检测脊髓组织病理损害,用免疫组织化学的方法检测脊髓组织中ZnTl的表达规律。结果：1．病理结果改变：除S组外,I／R组、LPS组、UR＋LPS组三组大鼠HE染色切片中均可见脊髓组织损伤,各组脊髓损伤的严重程度有以下规律：S组〈I/R组〈LPS组〈I／R＋LPS组。2．免疫组化结果：脊髓损伤组ZnT1的表达较假手术组均增加（P〈0．05）。结论：1．脊髓缺血损伤合并内毒素攻击可导致严重的脊髓损伤。2．腹主动脉阻断合并内毒素攻击所致脊髓损伤早期脊髓组织中ZnT1表达上调,可能通过调节脊髓损伤早期脊髓组织中锌稳态平衡进而在脊髓损伤后脊髓神经元的病理生理活动中发挥重要作用,这一实验结果可为寻找早期脊髓损伤预防措施提供新的思路。     

Spinal cord injury reveals multilineage differentiation of ependymal cells

Spinal cord injury often results in permanent functional impairment. Neural stem cells present in the adult spinal cord can be expanded in vitro and improve recovery when transplanted to the injured spinal cord, demonstrating the presence of cells that can promote regeneration but that normally fail to do so efficiently. Using genetic fate mapping, we show that close to all in vitro neural stem cell potential in the adult spinal cord resides within the population of ependymal cells lining the central canal. These cells are recruited by spinal cord injury and produce not only scar-forming glial cells, but also, to a lesser degree, oligodendrocytes. Modulating the fate of ependymal progeny after spinal cord injury may offer an alternative to cell transplantation for cell replacement therapies in spinal cord injury.