TGF-β/Smads信号通路与脊髓损伤后胶质瘢痕形成研究进展

脊髓损伤(spinal cord injury,SCI)是医学领域致死率、致残率最高的创伤之一,可直接引起神经轴突中断、神经元坏死、脊髓结构严重破坏导致严重功能缺陷。脊髓损伤后小胶质细胞激活、星形胶质细胞(astrocytes,AS)增殖和胶质瘢痕的形成等[1]是阻碍损伤脊髓轴突有效再生和生长的主要因素。国内外学者先后利用药物治疗、体外诱导及基因技术等     

星形胶质细胞在脊髓损伤中的研究进展

<正>近年来,脊髓损伤(spinal cord injury,SCI)的发病率在我国乃至世界呈明显上升趋势。脊髓损伤后的神经再生问题,一直是科研人员和临床工作者研究的一个重点和难点。脊髓损伤后,阻碍轴突再生的主要的因素是小胶质细胞激活、星形胶质细胞(astrocyte,AS)增殖活化和胶质瘢痕的形成[1]。而AS的增殖活化后并分泌多种细胞外基质共同形成神经胶质瘢痕是抑制脊髓损伤修复的最主要障碍。最近研究显示,星     

雪旺细胞与实验性脊髓损伤修复

脊髓损伤是神经科学领域致死率、致残率最高的创伤之一,可直接导致神经元坏死、神经轴突中断,脊髓结构严重破坏.而损伤后的微环境又有诸多不利于脊髓神经轴突再生的因素,如:多种神经营养因子的缺乏以及胶质疤痕和多种抑制再生的物质存在等.雪旺细胞是外周神经系统特有的胶质细胞,包绕轴突形成髓鞘,近年发现雪旺细胞不只在轴突周围形成髓鞘,它还有许多有利于调节损伤后轴突再生的独特功能.大量的动物实验已证明雪旺细胞移植和其他方法的联合应用能够克服这些因素达到促进轴突再生、髓鞘形成及功能恢复的目的,为脊髓损伤治疗带来了新的希望.     

脊髓损伤模型与再生实验研究（续）

二、脊髓损伤后轴突再生研究脊髓损伤的重点是损伤后的轴突再生Cajal、McCouch 和 Brown 以及 Lampert 和Creessman 等研究人员曾观察到哺乳动物 CNS,可以在损伤几天后出现轴突发芽,但轴突发芽最终死掉,认为是夭折性再生(abortive regeneration)。这种再生轴突缺乏继续生长和维持生存的现象在各     

中枢神经系统少突胶质细胞-髓鞘糖蛋白受体的作用及其研究进展

中枢神经系统（central nervous system，CNS）损伤后轴突再生困难，一直是临床神经疾病治疗的难点，长期以来，人们通过各种途径寻找其原因并试图攻克这一难关。近年来的研究表明，CNS受损后轴突不能再生可能有几方面的原因：成年神经元损伤后本身的再生能力下降；胶质瘢痕的形成；神经营养因子的缺乏及轴突再生抑制蛋白的作用等。其中关于轴突再生抑制蛋白的研究，成为神经再生研究的热点，     

神经组织移植与脊髓损伤的再生修复

脊髓损伤后神经再生与功能修复研究已进行了近百年。早期的研究大多限于观察轴突断端的生长。1928年,Cajal 等研究证实,在脊髓损伤的数天内,可见轴突有新芽(Sprouting)再生,但未见轴突的延长生长,新芽的最终结局仍是变性消失。此后,又有许多学者重复了有关脊髓损伤再生的研究,结论是:脊髓损伤后轴突再生能力极其有限,即使有一定程度的再生,也难以延长穿越损伤部位的瘢痕组织。这种中枢神经系统     

高压氧预处理对脊髓损伤后轴突再生影响实验研究

目的探讨高压氧预处理(HBO-PC)对成年大鼠脊髓损伤后轴突再生的影响,以探讨HBO-PC对脊髓损伤后的神经保护作用。方法成年雌性SD大鼠40只,体重250～300 g。随机分为HBO-PC组、非预处理组各20只。大鼠急性脊髓损伤模型制作,采用改良Allen’s法,分别于伤后8w行神经束路示踪(顺行和逆行),以脊髓损伤部位为中心,上下各2 cm取材,免疫组化染色及图像分析检测组织中示踪剂阳性纤维的表达。结果在胶质瘢痕的周围及上下两端,HBO-PC组示踪剂阳性纤维表达数量均明显高于非预处理组。结论 HBO-PC可诱导脊髓损伤后轴突再生,对神经损伤起到保护作用。     

低能激光照射及嗅鞘细胞移植在脊髓损伤中的应用

脊髓损伤是一种严重危害人类健康的疾病，因其少突胶质细胞不能形成轴突迁移引导的通道，并分泌抑制因子，以及星形胶质细胞在损伤区快速反应性增生形成胶质瘢痕，抑制轴突的生长，使脊髓损伤的治疗成为目前医学领域的一个棘手问题。嗅鞘细胞具有良好的轴突再生和准确形成靶特异性突轴连接的能力；低能激光照射对神经系统及嗅鞘细胞的作用包括，保留甚至促进受损神经元的活性，减少瘢痕的形成，阻止神经元的退行性改变，又能通过干拢一些因素而增强嗅鞘细胞的活性。嗅鞘细胞移植及低能激光照射在脊髓损伤的修复中具有重要的作用。     

骨髓基质细胞移植治疗脊髓全横断损伤超微结构观察

目的观察骨髓基质细胞（MSCs）移植治疗脊髓全横断损伤（SCI）超微结构,探讨内源性细胞与再生轴突关系。方法通过全骨髓法培养、纯化MSCs,SCI9d后移植MSCs,通过免疫荧光组化观察细胞移植后损伤区轴突再生情况,免疫荧光双标、免疫电镜观察再生轴突与内源性细胞关系。结果移植8W后实验组脊髓损伤区可见大量神经微丝蛋白200（NF200）阳性纤维,对照组脊髓损伤区未见明显的NF200阳性纤维。免疫荧光双标结果显示损伤区NF200阳性纤维和2,3＇-环核苷酸磷酸而酯酶（CNP）阳性细胞之间存在密切的空间关系,免疫电镜显示CNP阳性细胞通过伸长丝状伪足形成再生轴突支架,内源性施万细胞参与再生轴突髓鞘形成。结论MSCs移植可促进损伤区轴突再生,宿主自身CNP阳性细胞和施万细胞参与损伤轴突的再生和髓鞘形成。     

移植人脐带间充质干细胞修复大鼠脊髓损伤

背景：已知人脐带间充质干细胞对脊髓损伤存在着潜在的治疗价值,然而,当前对移植人脐带间充质干细胞治疗脊髓损伤及机制方面研究很少。 目的：观察人脐带间充质干细胞对脊髓损伤大鼠的治疗效果。 方法：40只Wistar大鼠建立脊髓损伤模型,38只造模成功后随机摸球法分为3组：空白对照组：只接受单纯损伤,不做任何移植;DMEM移植组：损伤后1周予以5 μL DMEM局部移植;细胞移植组：损伤后1周予以5 μL准备好的人脐带间充质干细胞局部移植(细胞数1×106)。移植后对实验动物通过BBB评分、体感诱发电位与运动诱发电位观察后肢功能恢复情况。分别于损伤后2,4,6,8,10周随机于细胞移植组抽取大鼠2只,免疫组织化学染色观察人脐带间充质干细胞存活、迁移、分化,通过胶质纤维酸性蛋白阳性细胞染色比较各组损伤局部胶质瘢痕形成面积。 结果与结论：BBB评分损伤后4周细胞移植组高于其他两组(P < 0.05),损伤后12周细胞移植组与其他两组相比SEP、MEP潜伏期缩短、波幅值增高(P < 0.05)。免疫组织化学染色示人脐带间充质干细胞可向神经元、星形胶质细胞和少突胶质细胞分化,分化的少突胶质细胞并包绕轴突形成髓鞘。细胞移植组损伤局部胶质瘢痕面积均小于其他两组(P < 0.05),空白对照组、DMEM移植组间差异无显著性(P > 0.05)。提示未经体外诱导的人脐带间充质干细胞可于损伤大鼠脊髓体内向神经元、星形胶质细胞、少突胶质细胞分化,减小胶质瘢痕,并促进脊髓损伤大鼠神经功能的恢复。     

Induced expression of polysialic acid in the spinal cord promotes regeneration of sensory axons

After spinal cord injury axonal regeneration is prevented by glial scar formation. In this study we examined whether induced expression of polysialic acid (PSA) in the lesion site would render the glial scar permissive to axonal regeneration after dorsal column transection. PSA was induced by lentiviral vector-mediated expression of polysialyltransferase (LV/PST). PSA expression increased astrocyte infiltration and permitted the penetration of regenerating axons across the caudal border of the lesion and into the lesion cavity. In LV/PST-injected animals with a peripheral nerve-conditioning lesion, 20 times more axons grew into the lesion cavity than those LV/GFP-injected plus conditioning lesion, and some axons grew across the cavity and extended to the rostral cord, while in LV/GFP group most ascending axons terminated at the caudal border of the lesion. Our result suggests that induced expression of PSA can provide a favorable environment for axonal regeneration.     

Long-term changes in the molecular composition of the glial scar and progressive increase of serotoninergic fibre sprouting after hemisection of the mouse spinal cord

The scarring process occurring after adult central nervous system injury and the subsequent increase in the expression of certain extracellular matrix molecules are known to contribute to the failure of axon regeneration. This study provides an immunohistochemical analysis of temporal changes (8 days to 1 year) in the cellular and molecular response of the Swiss mouse spinal cord to a dorsal hemisection and its correlation with the axonal growth properties of a descending pathway, the serotoninergic axons. In this lesion model, no cavity forms at the centre of the lesion. Instead, a dense fibronectin-positive tissue matrix occupies the centre of the lesion, surrounded by a glial scar mainly constituted by reactive astrocytes. NG2 proteoglycan and tenascin-C, potential axon growth inhibitors, are constantly associated with the central region. In the glial scar, tenascin-C is never observed and the expression of chondroitin sulphate proteoglycans (revealed with CS-56 and anti-NG2 antibodies) highly increases in the week following injury to progressively return to their control level. In parallel, there is an increasing expression of the polysialilated neural cell adhesion molecule by reactive astrocytes. These molecular changes are correlated with a sprouting process of serotoninergic axons in the glial scar, except in a small area in contact with the central region. All these observations suggest that while a part of the glial scar progressively becomes permissive to axon regeneration after mouse spinal cord injury, the border of the glial scar, in contact with the fibronectin-positive tissue matrix, is the real barrier to prevent axon regeneration.     

Neutralization of ciliary neurotrophic factor reduces astrocyte production from transplanted neural stem cells and promotes regeneration of corticospinal tract fibers in spinal cord injury

Transplantation of neural stem cells (NSC) into lesioned spinal cord offers the potential to increase regeneration by replacing lost neurons or oligodendrocytes. The majority of transplanted NSC, however, typically differentiate into astrocytes that may exacerbate glial scar formation. Here we show that blocking of ciliary neurotrophic factor (CNTF) with anti-CNTF antibodies after NSC transplant into spinal cord injury (SCI) resulted in a reduction of glial scar formation by 8 weeks. Treated animals had a wider distribution of transplanted NSC compared with the control animals. The NSC around the lesion coexpressed either nestin or markers for neurons, oligodendrocytes, or astrocytes. Approximately 20% fewer glial fibrillary acidic protein-positive/bromodeoxyuridine (BrdU)-positive cells were seen at 2, 4, and 8 weeks postgrafting, compared with the control animals. Furthermore, more CNPase(+)/BrdU(+) cells were detected in the treated group at 4 and 8 weeks. These CNPase(+) or Rip(+) mature oligodendrocytes were seen in close proximity to host corticospinal tract (CST) and 5HT(+) serotonergic axon. We also demonstrate that the number of regenerated CST fibers both at the lesion and at caudal sites in treated animals was significantly greater than that in the control animals at 8 weeks. We suggest that the blocking of CNTF at the beginning of SCI provides a more favorable environment for the differentiation of transplanted NSC and the regeneration of host axons.     

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well-established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.     

Accumulation of the inhibitory receptor EphA4 may prevent regeneration of corticospinal tract axons following lesion

Abstract We have examined the expression of Eph receptors and their ephrin ligands in adult rat spinal cord before and after lesion. Neurons in adult motor cortex express EphA4 mRNA, but the protein is undetectable in uninjured corticospinal tract. In contrast, after dorsal column hemisection EphA4 protein accumulates in proximal axon stumps. One of the ligands for EphA4, ephrinB2, is normally present in the grey matter flanking the corticospinal tract but after injury is markedly up-regulated in astrocytes in the glial scar. The result is that, after a lesion, corticospinal tract axons bear high levels of EphA4 and are surrounded to front and sides by a continuous basket of cognate inhibitory ephrin ligand. We suggest that a combination of EphA4 accumulation in the injured axons and up-regulation of ephrinB2 in the surrounding astrocytes leads to retraction of corticospinal axons and inhibition of their regeneration in the weeks after a spinal lesion.     

Transforming growth factor α transforms astrocytes to a growth-supportive phenotype after spinal cord injury

Astrocytes are both detrimental and beneficial for repair and recovery after spinal cord injury (SCI). These dynamic cells are primary contributors to the growth-inhibitory glial scar, yet they are also neuroprotective and can form growth-supportive bridges on which axons traverse. We have shown that intrathecal administration of transforming growth factor α (TGFα) to the contused mouse spinal cord can enhance astrocyte infiltration and axonal growth within the injury site, but the mechanisms of these effects are not well understood. The present studies demonstrate that the epidermal growth factor receptor (EGFR) is upregulated primarily by astrocytes and glial progenitors early after SCI. TGFα directly activates the EGFR on these cells in vitro, inducing their proliferation, migration, and transformation to a phenotype that supports robust neurite outgrowth. Overexpression of TGFα in vivo by intraparenchymal adeno-associated virus injection adjacent to the injury site enhances cell proliferation, alters astrocyte distribution, and facilitates increased axonal penetration at the rostral lesion border. To determine whether endogenous EGFR activation is required after injury, SCI was also performed on Velvet (C57BL/6J-Egfr(Vel)/J) mice, a mutant strain with defective EGFR activity. The affected mice exhibited malformed glial borders, larger lesions, and impaired recovery of function, indicating that intrinsic EGFR activation is necessary for neuroprotection and normal glial scar formation after SCI. By further stimulating precursor proliferation and modifying glial activation to promote a growth-permissive environment, controlled stimulation of EGFR at the lesion border may be considered in the context of future strategies to enhance endogenous cellular repair after injury.     

Modification of the regenerative response of dorsal column axons by olfactory ensheathing cells or peripheral axotomy in adult rat

The regeneration of sciatic-dorsal column (DC) axons following DC crush injury and treatment with olfactory ensheathing cells (OECs) and/or sciatic axotomy ("conditioning lesion") was evaluated. Sciatic-DC axons were examined with a transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and evaluated at chronic time points, 2-26 weeks post-lesion. With DC injury alone (n = 7), sciatic-DC axons were localized to the caudal border of the lesion terminating in reactive end bulbs with no indication of growth into the lesion. In contrast, treatment with either a heterogeneous population of OECs (equal numbers of p75- and fibronectin-positive OECs) (n = 9) or an enriched population of OECs (75% p75-positive OECs) (n = 6) injected either directly into the lesion or 1-mm rostral and caudal to the injury, stimulated DC axon growth into the lesion. A similar regenerative response was observed with a conditioning lesion either concurrent to (n = 4) or 1 week before (n = 4) the DC injury. In either of the latter two paradigms, some DC axons grew across the injury, but no axons grew into the rostral intact spinal cord. Upon combining OEC treatment with the conditioning lesion (n = 21), the result was additive, increasing DC axon growth beyond the rostral border of the lesion in best cases. Additional factors that may limit DC regeneration were tested including formation of the glial scar (immunoreactivity to glial fibrillary acidic protein in astrocytes and to chondroitin sulfate proteoglycans), which remained similar between treated and untreated groups.     

Matrix metalloproteases: degradation of the inhibitory environment of the transected optic nerve and the scar by regenerating axons

After injury to the central nervous system, a glial/collagen scar forms at the lesion site, which is thought to act as a physicochemical barrier to regenerating axons. We have shown that scar formation in the transected optic nerve (ON) is attenuated when robust growth of axons is stimulated. Matrix metalloproteases (MMP), modulated by tissue inhibitors of MMP (TIMP), degrade a wide variety of extracellular matrix components (ECM) and may be activated by growing axons to remodel the ECM to allow regeneration through the inhibitory environment of the glial or collagen scar. Here, we investigate whether MMP levels are modulated in a nonregenerating (scarring) versus a regenerating (nonscarring) model of ON injury in vivo. Western blotting and immunohistochemistry revealed that MMP-1, -2, and -9 levels were higher and TIMP-1 and TIMP-2 levels were lower in regenerating compared to nonregenerating ON and retinae. In situ zymography demonstrated significantly greater MMP-related gelatinase activity in the regenerating model, mainly colocalized to astrocytes in the proximal ON stump and around the lesion site. These results suggest that activation of MMP and coincident down-regulation of TIMP may act to attenuate the inhibitory scarring in the regenerating ON, thus transforming the ON into a noninhibitory pathway for axon regrowth.     

Neuropeptide Y-producing neurons of the arcuate nucleus regenerate axons after surgical deafferentation of the mediobasal hypothalamus

Dorsolateral and ventomedial surgical deafferentation of the hypothalamus were used to study the capacity of different types of neuropeptide Y-containing axons afferent to the dorsal hypothalamus to regenerate through surgical lesions. The kinetics of the postlesional responses of transected neuropeptide Y-axons was studied on 30–40 μm thick vibratome sections, either (i) by light or electron microscopy after peroxidase immunostaining for neuropeptide Y or (ii) by confocal microscopy after double fluorescence immunostaining for neuropeptide Y and for glial fibrillary acidic protein. The dorsolateral cut was found to sever 2 main pathways containing neuropeptide Y axons located, respectively, below the bed nucleus of the stria terminalis and in the perifornical region. In both regions transected fibers were found to abut onto the surgical lesion, but even 45 days after the lesion, they were very rarely observed to penetrate into the astroglial scar formed along the lesion. The ventromedial cut was found to sever numerous neuropeptide axons that originate in the underlying arcuate nucleus. Seven to 15 days after the lesion neuropeptide Y fibers located below this type of cut presented a dramatic increase in both their numerical density and their immunostaining intensity. With increasing post-surgery times, an increased number of neuropeptide Y fibers was observed to penetrate and to cross the lesional scar formed by densely packed astrocytic processes. Electron microscope observations further demonstrated that 45 days after the lesion, numerous neuropeptide Y-immunoreactive axonal profiles were included in the scar matrix, which appeared to be mainly composed of closely interdigitating astrocytic processes containing dense bundles of filaments. These data indicate that, in contrast to other neuropeptide Y neurons innervating the dorsal hypothalamus, neuropeptide Y neurons of the arcuate nucleus regenerate axons through the astroglial scar produced by a surgical lesion placed in the ventromedial hypothalamus. © 1993 Wiley-Liss, Inc.     

Degeneration and sprouting of identified descending supraspinal axons after contusive spinal cord injury in the rat

Contusive spinal cord injury (SCI) results in the formation of a chronic lesion cavity surrounded by a rim of spared fibers. Tissue bridges containing axons extend from the spared rim into the cavity dividing it into chambers. Whether descending axons can grow into these trabeculae or whether fibers within the trabeculae are spared fibers remains unclear. The purposes of the present study were (1) to describe the initial axonal response to contusion injury in an identified axonal population, (2) to determine whether and when sprouts grow in the face of the expanding contusion cavity, and (3) in the long term, to see whether any of these sprouts might contribute to the axonal bundles that have been seen within the chronic contusion lesion cavity. The design of the experiment also allowed us to further characterize the development of the lesion cavity after injury. The corticospinal tract (CST) underwent extensive dieback after contusive SCI, with retraction bulbs present from 1 day to 8 months postinjury. CST sprouting occurred between 3 weeks and 3 months, with penetration of CST axons into the lesion matrix occurring over an even longer time course. Collateralization and penetration of reticulospinal fibers were observed at 3 months and were more extensive at later time points. This suggests that these two descending systems show a delayed regenerative response and do extend axons into the lesion cavity and that the endogenous repair can continue for a very long time after SCI.