ErbB receptor signaling directly controls oligodendrocyte progenitor cell transformation and spontaneous remyelination after spinal cord injury

We recently discovered a novel role for neuregulin-1 (Nrg1) signaling in mediating spontaneous regenerative processes and functional repair after spinal cord injury (SCI). We revealed that Nrg1 is the molecular signal responsible for spontaneous functional remyelination of dorsal column axons by peripheral nervous system (PNS)-like Schwann cells after SCI. Here, we investigate whether Nrg1/ErbB signaling controls the unusual transformation of centrally derived progenitor cells into these functional myelinating Schwann cells after SCI using a fate-mapping/lineage tracing approach. Specific ablation of Nrg1-ErbB receptors in central platelet-derived growth factor receptor alpha (PDGFRα)-derived lineage cells (using PDGFRαCreERT2/Tomato-red reporter mice crossed with ErbB3fl/fl/ErbB4fl/fl mice) led to a dramatic reduction in P0-positive remyelination in the dorsal columns following spinal contusion injury. Central myelination, assessed by Olig2 and proteolipid protein expression, was unchanged. Loss of ErbB signaling in PDGFRα lineage cells also significantly impacted the degree of spontaneous locomotor recovery after SCI, particularly in tests dependent on proprioception. These data have important implications, namely (a) cells from the PDGFRα-expressing progenitor lineage (which are presumably oligodendrocyte progenitor cells, OPCs) can differentiate into remyelinating PNS-like Schwann cells after traumatic SCI, (b) this process is controlled by ErbB tyrosine kinase signaling, and (c) this endogenous repair mechanism has significant consequences for functional recovery after SCI. Thus, ErbB tyrosine kinase receptor signaling directly controls the transformation of OPCs from the PDGFRα-expressing lineage into PNS-like functional remyelinating Schwann cells after SCI.     

The fate and function of oligodendrocyte progenitor cells after traumatic spinal cord injury

Oligodendrocyte progenitor cells (OPCs) are the most proliferative and dispersed population of progenitor cells in the adult central nervous system, which allows these cells to rapidly respond to damage. Oligodendrocytes and myelin are lost after traumatic spinal cord injury (SCI), compromising efficient conduction and, potentially, the long-term health of axons. In response, OPCs proliferate and then differentiate into new oligodendrocytes and Schwann cells to remyelinate axons. This culminates in highly efficient remyelination following experimental SCI in which nearly all intact demyelinated axons are remyelinated in rodent models. However, myelin regeneration comprises only one role of OPCs following SCI. OPCs contribute to scar formation after SCI and restrict the regeneration of injured axons. Moreover, OPCs alter their gene expression following demyelination, express cytokines and perpetuate the immune response. Here, we review the functional contribution of myelin regeneration and other recently uncovered roles of OPCs and their progeny to repair following SCI.     

Chronic alterations in the cellular composition of spinal cord white matter following contusion injury

Spinal cord injury (SCI) involves the loss of neurons and glia due to initial mechanical and secondary biochemical mechanisms. Treatment with the sodium channel blocker tetrodotoxin (TTX) reduces acute white matter pathology and increases both axon density and hindlimb function chronically at 6 weeks after injury. We investigated the cellular composition of residual white matter chronically to determine whether TTX also has a significant effect on the numbers and types of cells present. Rats received an incomplete thoracic contusion injury, in the presence or absence of TTX (0.15 nmole) injected focally, beginning at 15 min prior to injury. Six weeks later, cell density was significantly increased in the residual white matter of the dorsal, lateral, and ventral funiculi, both rostral and caudal to the injury site in both TTX-treated and injury control groups. Oligodendrocyte and astrocyte density was similar to normal but large numbers of cells expressing microglia/macrophage markers were present. Labeling with the progenitor markers nestin and NG2 showed that precursor cell density had also doubled or tripled as compared with uninjured controls. Some of these cells were also labeled for antigens that indicate their possible progression along an oligodendrocyte or astrocyte lineage. Our results support the hypothesis that the beneficial effect of TTX in SCI is related to its preservation of axons per se; no effect on chronic white matter cell composition was detected. They highlight the profound changes in cellular composition in preserved white matter chronically at 6 weeks after injury, including the accumulation of endogenous progenitor cells and the persistence of activated macrophages/microglia. The manipulation of these endogenous cells may be used in the future to enhance recovery after SCI.     

A cell population that strongly expresses the CB1 cannabinoid receptor in the ependyma of the rat spinal cord

The cells surrounding the central canal of the spinal cord are a source of stem/precursor cells that may give rise to neurons, astrocytes, or oligodendrocytes. However, they are a heterogeneous population that remains poorly understood. Here we describe a subpopulation characterized by their strong expression of the CB₁ cannabinoid receptor, oval/round soma, apical nucleus, a variable number of cilia (0, 1, or 2), and the presence of a single short and occasionally ramified basal process. These cells are mainly located in the lateral and dorsal central canal throughout the spinal cord. These CB cells are closely related to the basal lamina labyrinths or fractones derived from subependymal microglia. In addition, CB cells express some stem/precursor cell markers, including vimentin, nestin, Sox2, Sox9, and GLAST, but not others such as CD15 or GFAP. In addition, this cell population does not proliferate in the intact adult spinal cord, although up to 50% of these cells express the proliferation marker Ki67 in newly born rats or after a spinal cord contusion. The present findings contribute to our understanding of the spinal cord central canal structure and reveal the targets for endocannabinoids inside this neurogenic niche. J. Comp. Neurol. 521:233–251, 2013. © 2012 Wiley Periodicals, Inc.     

Interactions between intraspinal Schwann cells and the cellular constituents normally occurring in the spinal cord: an ultrastructural study in the irradiated rat

Relationships between intraspinal Schwann cells and neuroglia, particularly, astrocytes, were studied following X-irradiation of the spinal cord in 3-day-old rats. Initially, this exposure results in a depletion of the neuroglial population. By 10 days post-irradiation (P-I), gaps occur in the glia limitans, although the overlying basal lamina remains intact. Development of and myelination by intraspinal Schwann cells is well underway by 15 days P-I. These Schwann cell-occupied regions have a paucity of astrocyte processes, a finding which persists throughout the study (60 days P-I), and several types of Schwann cell-neuroglial interfaces are observed, including: (1) astrocyte separation of Schwann cells from oligodendrocyte-myelinated regions; (2) intermingling of Schwann cell-myelinated axons and oligodendrocyte-myelinated axons in the absence of astrocyte processes; and (3) ensheathment of unmyelinated axons by astrocyte processes which separate these axons from the Schwann cells. The gaps in the glia limitans widen as the P-I interval increases. At 45 and 60 days P-I, the basal lamina no longer forms a singular, continuous covering over the spinal cord surface, but follows instead a rather tortuous course over the disrupted glia limitans and the intraspinal Schwann cells. Although the mode of initial occurrence of Schwann cells within the spinal cord is not yet understood, the data indicate that the astrocyte population is involved in that process, as well as in limiting the further development of Schwann cells within the substance of the spinal cord.     

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.     

人胚胎雪旺细胞脊髓内移植治疗晚期脊髓损伤

目的 采用人胚雪旺细胞移植治疗晚期脊髓损伤,并探讨其疗效及安全性。方法 显微镜下切除脊髓损伤节段增厚的瘢痕组织,松解粘连,切开囊腔或空洞。取人胚胎背根神经节,培养成雪旺细胞并贴附于可吸收薇乔3-0紫色线及薇乔网的载体上,然后将其移植到脊髓损伤部位。本组共治疗53例,其中男42例,女11例,年龄2～58岁,伤后时间为4个月～19年。结果 雪旺细胞移植后2～8w时随访,按美国脊髓损伤学会（ASIA）脊髓损伤神经功能分类国际标准评价,53例患者的脊髓功能均有部分恢复,其中运动功能由术前（41.49±15.83）分提高到术后（44.62±15.39）分,轻触觉由（57.89±22.87）分提高到（63.94±23.67）分,针刺觉由（55.96±20.99）分提高到（59.68±20.57）分。患者术后无脊髓感染、功能损伤加重及死亡等并发症。术后复查MRI示脊髓无瘤样增生及空洞扩大。结论 人胚雪旺细胞移植治疗晚期脊髓损伤安全可行,能促进晚期脊髓损伤患者脊髓神经功能的部分恢复。     

Rolipram联合少突胶质前体细胞移植治疗大鼠脊髓损伤

背景：脊髓损伤轴突难以再生涉及到多方面原因,综合运用多种治疗方法应该更有效。 目的：应用Rolipram联合少突胶质前体细胞移植治疗大鼠脊髓损伤,观察轴突再生和再髓鞘化的情况。 方法：Wistar大鼠利用ALLEN法打击形成脊髓损伤模型。Rolipram治疗组和联合治疗组背部皮下埋置ALZET渗透性微泵内装Rolipram 200 μL,每小时释放0.5 μL,2 mg/(kg•d),可持续14 d,1周后局部注入0.012 5 μmol 的双丁酸环腺苷酸;细胞移植组和联合治疗组移植少突胶质前体细胞,脊髓损伤组注入同等量的生理盐水,术后每周测定BBB运动评分,4周取材,冰冻切片,苏木精-伊红染色,免疫荧光染色。 结果与结论：伤后4周Rolipram治疗组和联合治疗组BBB运动评分高于脊髓损伤组和细胞移植组;免疫荧光显示Rolipram治疗组和联合治疗组损伤局部NF200表达旺盛;细胞移植组和联合治疗组少突胶质前体细胞存活并表达髓鞘碱性蛋白,后者存活数量更多,并且有髓纤维增多。说明应用Rolipram提高环磷腺苷水平促进了大鼠脊髓损伤功能的恢复,联合少突胶质前体细胞移植可产生协同促进作用。     

Chemokine antagonist infusion promotes axonal sparing after spinal cord contusion injury in rat

Spinal cord injury produced by mechanical contusion causes the onset of acute and chronic degradative events. These include blood brain barrier disruption, edema, demyelination, axonal damage and neuronal cell death. Posttraumatic inflammation after spinal cord injury has been implicated in the secondary injury that ultimately leads to neurologic dysfunction. Studies after spinal cord contusion have shown expression of several chemokines early after injury and suggested a role for them in the ordered recruitment of inflammatory cells at the lesion site (McTigue et al. [1998] J. Neurosci. Res. 53:368-376; Lee et al., [2000] Neurochem Int). We have demonstrated previously that infusion of the broad-spectrum chemokine receptor antagonist (vMIPII) in the contused spinal cord initially attenuates leukocyte infiltration, suppresses' gliotic reaction and reduces neuronal damage after injury. These changes are accompanied by increased expression of bcl-2, the endogenous apoptosis inhibitor, and reduced neuronal apoptosis (Ghirnikar et al. [2000] J. Neurosci. Res. 59:63-73). We demonstrate that 2 and 4 weeks of vMIPII infusion in the contusion-injured spinal cord also results in decreased hematogenous infiltration and is accompanied by reduced axonal degeneration in the gray matter. Luxol fast blue and MBP immunoreactivity indicated reduced myelin breakdown in the dorsal and ventral funiculi. Increased neuronal survival in the ventral horns of vMIPII infused cords was seen along with increased bcl-2 staining in them. Immunohistochemical identification of fiber phenotypes showed increased presence of calcitonin gene related peptide, choline acetyl transferase and tyrosine hydroxylase positive fibers as well as increased GAP43 staining in treated cords. These results suggest that sustained reduction in posttraumatic cellular infiltration is beneficial for tissue survival. A preliminary report of this study has been published (Eng et al. [2000] J. Neurochem. 74(Suppl):S67B). In contrast to vMIPII, infusion of MCP-1 (9-76), a N-terminal analog of the MCP-1 chemokine showed only a modest reduction in cellular infiltration at 14 and 21 dpi without significant tissue survival after spinal cord contusion injury. Comparing data on tissue survival obtained with vMIPII and MCP-1 (9-76) further validate the importance of the use of broad-spectrum antagonists in the treatment of spinal cord injury. Controlling the inflammatory reaction and providing a growth permissive environment would enhance regeneration and ultimately lead to neurological recovery after spinal cord injury. J. Neurosci. Res. 64:582-589, 2001. Published 2001 Wiley-Liss, Inc.     

Mice lacking L1 cell adhesion molecule have deficits in locomotion and exhibit enhanced corticospinal tract sprouting following mild contusion injury to the spinal cord

L1 is a member of the immunoglobulin superfamily of cell adhesion molecules that is associated with axonal growth, including formation of the corticospinal tract (CST). The present study describes the effects of L1 deletion on hindlimb function in locomotion, and examines the role of L1 in recovery and remodeling after contusive spinal cord injury (SCI) in mice. Uninjured adult L1 knockout (Y/-) mice had impaired performance on locomotor tests compared with their wild-type littermates (Y/+). Anterograde tracing demonstrated that CST axons project to thoracic, but not lumbar, levels of the spinal cord of Y/- mice, and revealed a diversion of these fibers from their position in the base of the dorsal columns. Retrograde tracing also revealed reduced numbers of descending projections from paraventricular hypothalamus and red nuclei to the lumbar spinal cord in Y/- mice. SCI at the mid-thoracic level produced a lesion encompassing the center of the spinal cord, including the site of the dorsal CST and surrounding gray matter (GM). The injury caused lasting deficits in fine aspects of locomotion. There was no effect of genotype on final lesion size or the growth of axons into the lesion area. However, injured Y/- mice demonstrated a robust expansion of CST projections throughout the GM of the cervical and thoracic spinal cord rostral to the lesion compared with Y/+ littermates. Thus, L1 is important for the development of multiple spinal projections and also contributes to the restriction of CST sprouting rostral to the site of a SCI in adults.     

Effect of glial cells on remyelination after spinal cord injury

Remyelination plays a key role in functional recovery of axons after spinal cord injury. Glial cells are the most abundant cells in the central nervous system. When spinal cord injury occurs, many glial cells at the lesion site are immediately activated, and different cells differentially affect inflammatory reactions after injury. In this review, we aim to discuss the core role of oligodendrocyte precursor cells and crosstalk with the rest of glia and their subcategories in the remyelination process. Activated astrocytes influence prolif-eration, differentiation, and maturation of oligodendrocyte precursor cells, while activated microglia alter remyelination by regulating the inflammatory reaction after spinal cord injury. Understanding the interac-tion between oligodendrocyte precursor cells and the rest of glia is necessary when designing a therapeutic plan of remyelination after spinal cord injury.     

Prominent oligodendrocyte genesis along the border of spinal contusion lesions

Oligodendrocyte (OL) loss and axon demyelination occur after spinal cord injury (SCI). OLs may be replaced, however, by proliferating NG2+ progenitor cells. Indeed, new OLs have been noted in ventral white matter after SCI. Since tissue adjacent to lesion cavities is exposed to different mediators compared with outlying spared tissue, the authors used a rat SCI model to compare NG2 cell proliferation and OL genesis adjacent to lesion cavities with that in spared tissue closer to meninges. NG2 cells proliferated throughout the first week postinjury and accumulated along lesion borders, especially within gray matter. By 3 days postinjury (dpi), new OLs were detected throughout the cross-sections; between 4 and 7 dpi, however, oligogenesis was restricted to lesion borders. New OLs derived from cells proliferating during 1-7 dpi increased dramatically by 14 dpi; most were located along lesion borders and in spared gray matter. Oligogenesis continued along lesion borders during the second week postinjury. Overall OL numbers were reduced at 3 dpi in spared tissue, but rebounded to normal levels by 14 dpi. Surprisingly, lesion borders maintained normal OL numbers at 3 dpi, which then rose to exceed preinjury levels at 7 and 14 dpi. These results indicate that oligogenesis is protracted after SCI and leads to increased OL numbers. Most new OLs are formed in regions of greatest NG2 cell proliferation. Thus, the adult spinal cord spontaneously develops a dynamic gliogenic zone along lesion borders.     

New vascular tissue rapidly replaces neural parenchyma and vessels destroyed by a contusion injury to the rat spinal cord.

Blood vessels identified by laminin staining were studied in uninjured spinal cord and at 2, 4, 7, and 14 days following a moderate contusion (weight drop) injury. At 2 days after injury most blood vessels had been destroyed in the lesion epicenter; neurons and astrocytes were also absent, and few ED1+ cells were seen infiltrating the lesion center. By 4 days, laminin associated with vessel staining was increased and ED1+ cells appeared to be more numerous in the lesion. By 7 days after injury, the new vessels formed a continuous cordon oriented longitudinally through the lesion center. ED1+ cells were abundant at this time point and were found in the same area as the newly formed vessels. Astrocyte migration from the margins of the lesion into the new cordon was apparent. By 14 days, a decrease in the number of vessels in the lesion center was observed; in contrast, astrocytes were more prominent in those areas. In addition to providing a blood supply to the lesion site, protecting the demise of the newly formed vascular bridge might provide an early scaffold to hasten axonal regeneration across the injury site.     

NG2 expression in rats with acute T10 spinal cord injury

Rat models of T10 spinal cord injury were established with a clamp method.NG2 expression was detected with immunohistochemical staining and western blot.Ten days after spinal cord injury,the number of NG2-positive cells in the damaged areas and NG2 absorbance were both significantly increased.The findings indicate that acute T10 spinal cord injury in rats can lead to upregulation of NG2 protein expression in damaged areas.     

Astrogliosis limits the integration of peripheral nerve grafts into the spinal cord

Segments of peripheral nerve were grafted into the site of a spinal cord transection in mice. To determine the relationship of spinal cord astrocytes to graft derived Schwann cells, graft sites were examined with immunohistochemical as well as conventional histological techniques. Myelin derived from Schwann cells, as identified by immunoreactivity to antibodies against its major protein PO, was strictly confined to the graft. Astrocytes and astrocytic filaments, identified by immunoreactivity to antibodies against glial fibrillary acidic protein (GFAP), predominated at the graft-spinal cord interface, bordering the most central penetration of Schwann cell myelin. Occasional GFAP-positive astrocytes were observed within the graft. It appears that astrocytes limit the penetration of Schwann cells from peripheral nerve grafts into the spinal cord.     

Single-chain variable fragment antibodies against the neural adhesion molecule CHL1 (close homolog of L1) enhance neurite outgrowth

The neural cell adhesion molecule CHL1 (close homolog of L1) plays important roles in neurite outgrowth and neuronal survival in vitro. Reproducible and functionally active CHL1 antibodies are critical for a better understanding of the functional properties of CHL1 in vitro and in vivo. We have isolated human single-chain variable fragment (scFv) antibodies against mouse CHL1 from a human synthetic phage display library. To improve the binding activity of such antibodies, a clone (C12) was selected for affinity maturation by combined random mutagenesis of the V(H) gene and site-directed cassette mutagenesis to introduce random mutations in the complementarity determining region 3 (CDR3) of the V(L) gene. From the mutant phage display library, we selected a clone (6C2) that gave the strongest signal as determined by ELISA. The dissociation constant of 6C2 (Kd 2.28 x 10(-8) M) was increased approximately 85-fold compared with the wild-type clone C12 (Kd 1.93 x 10(-6) M). 6C2 detected CHL1 by Western blot analysis in mouse brain homogenates and detected CHL1 in CHL1-transfected cells by immunofluorescence. Furthermore, the wild-type and affinity-matured antibodies promoted neurite outgrowth of hippocampal and cerebellar neurons in vitro. Our results suggest that the affinity-matured CHL1 scFv antibody will serve a range of applications in vitro and in vivo.