Hepatocyte growth factor reduces astrocytic scar formation and promotes axonal growth beyond glial scars after spinal cord injury

The formation of glial scars impedes growth of regenerating axons after CNS injuries such as spinal cord injury (SCI). Hepatocyte growth factor (HGF), originally identified as a mitogen for hepatocytes, exerts pleiotropic functions in the nervous system. HGF has been implicated in peripheral wound healing via regulation of the transforming growth factor beta (TGFβ), which is also a potent inducer of glial scar formation in CNS. In the present study, we found that HGF completely blocked secretion of TGFβ1 and β2 from activated astrocytes in culture. HGF also prevented expression of specific chondroitin sulfate proteoglycan (CSPG) species. To determine whether HGF inhibits glial scar formation in an in vivo SCI model, HGF overexpressing mesenchymal stem cells (HGF-MSCs) were transplanted into hemisection spinal cord lesions at C4. Transplantation of HGF-MSCs markedly diminished TGFβ isoform levels and reduced the extent of astrocytic activation. In addition, HGF-MSCs also significantly decreased neurocan expression and glycosaminoglycan chain deposition around hemisection lesions. Furthermore, animals treated with HGF-MSCs showed increased axonal growth beyond glial scars and improvement in recovery of forepaw function. Our results indicate that anti-glial scar effects of HGF, together with its known neurotrophic functions, could be utilized to ameliorate functional deficits following SCI.     

A time course for the focal elevation of synthesis of basic fibroblast growth factor and one of its high-affinity receptors (flg) following a localized cortical brain injury.

Traumatic injury to the CNS initiates transient and unsuccessful regeneration of damaged neural pathways, accompanied by reactive gliosis, angiogenesis, and deposition of a dense fibrous glial/meningeal scar at the wound site. Basic fibroblast growth factor (basic FGF) is a CNS protein with potent effects on neurons, glia, fibroblasts, and vascular endothelial cells. Hybridization and immunocytochemical methods were used to examine temporal and spatial changes in distribution and levels of basic FGF protein and mRNA and also of its receptor mRNA (flg), following a defined wound to the cerebral cortex of adult rat brains. In the injured brain, a rapid, transient increase in basic FGF mRNA and protein is readily detectable within 7 d of surgery and thereafter declines in the tissues bordering the lesion. The increased expression is localized to multiple cell types including macrophages, neurons, astrocytes, and vascular endothelial cells. The changes in immunoreactive basic FGF parallel changes in the bioactivity of extracted heparin-binding proteins, which include basic FGF. Focal increases in flg mRNA appear 7 d after injury and subside by 14 d. The changes in local basic FGF synthesis, concentration, localization, and bioactivity suggest that this growth factor may contribute to the cascade of cellular events that occur in CNS wound repair.     

SC1, a brain extracellular matrix glycoprotein related to SPARC and follistatin, is expressed by rat cerebellar astrocytes following injury and during development

In the nervous system, extracellular matrix components are believed to influence cell shape, proliferation and migration during development and following injury. SC1 is a secreted glycoprotein expressed during neural development and in the adult brain. The molecule shows partial sequence homology to the anti-adhesive extracellular matrix molecule SPARC/osteonectin and to follistatin. We have made a surgical lesion in the adult rat cerebellum and examined changes in SC1 expression at 1 to 14 days after injury. Dual in situ hybridization/immunohistochemistry demonstrated that SC1 mRNA was induced in astrocytes surrounding the wound, reaching maximal levels at 10 days post-lesion. Immunohistochemistry revealed changes in the deposition of SC1 protein in radial fibres of Bergmann glia. SC1 protein was also detected at the border of the lesion, suggesting an association with the glial scar. Double immunohistochemistry with the astrocytic marker GFAP demonstrated that astrocytes also express SC1 during postnatal development.     

Injury-induced Regulation of Ciliary Neurotrophic Factor mRNA in the Adult Rat Brain

Ciliary neurotrophic factor (CNTF) is a pleiotropic molecule that acts as a neurotrophic factor for a wide range of embryonic neurons as well as a differentiation factor for sympathetic neuroblasts and O2A progenitor cells in culture. CNTF messenger RNA (mRNA) is present at very low levels in the normal adult rat central nervous system (CNS), but is dramatically up-regulated after an aspiration lesion of dorsal hippocampus and overlying cortex, in the area coincident with glial scar. The increased level of CNTF mRNA in lesioned hippocampus is maximal by 3 days and is sustained for up to 20 days, the longest time point examined. In contrast, mRNA levels for brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) were slightly decreased during the same period. In situ hybridization experiments revealed that cells expressing CNTF mRNA were concentrated at the margin of the wound, and also present within the gelfoam which filled the lesion cavity. This distribution of CNTF-expressing cells corresponded very closely to that of cells expressing high levels of glial fibrillary acidic protein mRNA at the wound site. Paralleling the observed increase in CNTF mRNA, increased levels of CNTF-like neurotrophic activity were apparent in soluble extracts of the lesioned tissues. This neurotrophic activity for ciliary ganglion neurons was completely blocked by the addition of neutralizing antiserum against CNTF. Basic fibroblast growth factor, which has been shown by others to increase after a similar lesion paradigm (Frautschy et al., Brain Res. , 553 , 291–299, 1991), does not contribute appreciably to this trophic activity. We conclude that CNTF is markedly increased as a function of injury to the CNS and that its expression is most likely restricted to reactive astrocytes in the glial scar.     

Decorin Attenuates Gliotic Scar Formation in the Rat Cerebral Hemisphere

The transforming growth factor-betas (TGF-betas) are potent fibrogenic factors implicated in numerous CNS pathologies in which fibrosis and neural dysfunction are causally associated. In this study, we aimed to demonstrate significant inhibition of fibrogenesis, glial scarring, and inflammation in penetrating incisional wounds of the rat brain using the proteoglycan decorin, which effectively inhibits TGF-beta activity. Adult rats were assigned to two treatment groups each receiving 14 daily intraventricular injections of 10 microliter total volume of: (i) saline plus 0.3% autologous rat serum = 30 microgram protein); or (ii) saline plus 30 microgram recombinant human decorin. On day 0 of the experiment, a stereotactically defined unilateral incisional lesion was placed through the cerebral cortex into the lateral ventricle and, after 14 days, brains were processed for immunohistochemical analysis of the lesion site. Specific antibodies were used to visualize the deposition within the wound of matrix molecules and the extent and nature of reactive astrocytosis and inflammation. Quantitative and qualitative image analysis of the fibrous scar was performed in sections from a defined anatomical plane through the wound to detect the antifibrotic effects of decorin treatment. Treatment of wounds with decorin led to a marked attenuation of all aspects of CNS scarring including matrix deposition, formation of an accessory glial limiting membrane, and inflammation. Our findings suggest that decorin is potentially applicable to a number of human CNS fibrotic diseases to arrest the deposition of excessive extracellular matrix components and maintain and/or restore functional integrity.     

Gene transfer to the spinal cord neural scar with lentiviral vectors: predominant transgene expression in astrocytes but not in meningeal cells

Viral vector-mediated overexpression of neurotrophins in cells constituting the neural scar may represent a powerful approach to rendering scar tissue of a central nervous system (CNS) lesion permissive for neuronal regrowth. In this study a lentiviral vector encoding green fluorescent protein (LV-GFP) was injected in and around the neural scar 2 weeks after a dorsal column lesion in the rat spinal cord in order to analyze transduction characteristics of the neural scar after 4, 7, and 14 days. GFP expression was found at all points after injection and increased from 4 to 7 days, with no apparent difference observed between 7 and 14 days. The core of the lesion was virtually devoid of GFP signal despite direct vector injections in this area. The colocalization of GFP with specific cell markers (GFAP, vimentin, Raldh2, NeuN, OX-42, ED-1, and NG-2) indicated that the predominant cells transduced in the rim of the lesion were astrocytes, with neurons, microglia, oligodendrocyte precursors, and macrophages transduced to a lesser extent. None of the Raldh2-positive meningeal cells, present in the core of the scar, expressed GFP. In vitro meningeal cells were readily transduced, indicating that in vivo the formation of an extracellular matrix might prevent LV particles from transducing cells in the core of the scar. Because astrocytes are important cellular constituents of the glial scar after CNS injury, transduction of astrocytes with LV vectors encoding neurotrophic factors like BDNF or NT-3 may be used to enhance regeneration of severed axonal tracts through or along boundaries of a CNS lesion.     

Up-regulation of Connexin43 in the glial scar following photothrombotic ischemic injury

Several types of CNS injury and various diseases are associated with the development of a glial scar. Astrocytes are major components of the glial scar. They are interconnected by gap junctions, with connexin43 (Cx43) being the most prominent channel protein. We applied a model of focal cerebral ischemia to study the spatio-temporal expression of glial fibrillary acidic protein, as well as of Cx43 mRNA and protein in gliotic tissue up to 60 days after injury. Reactive astrocytes enveloping the lesion up-regulated their Cx43 mRNA and protein. A band of reactive astrocytes filling in the lesion exhibited elevated Cx43 and showed a high degree of proliferation. Because of these findings, we hypothesize a role for Cx43 in glial scar formation, specifically in the proliferation of astrocytes.     

Connective tissue growth factor is expressed by a subset of reactive astrocytes in human cerebral infarction

Connective tissue growth factor (CTGF), a transforming growth factor (TGF)-beta1 downstream mediator, is a secreted cell matrix-inducing peptide involved in both tissue regeneration mechanisms, such as wound repair, and also in aberrant deposition of extracellular matrix. The present study reports CTGF expression by cells associated with matrix deposition and glial scar formation in human cerebral infarction. CTGF was localized by immunohistochemistry in 17 brains of patients after focal infarction and in three neuropathologically normal control brains. CTGF expression was selectively localized to the cytoplasm of stellate reactive astrocytes. Compared to peripheral areas and brain controls without neuropathological findings, the total number CTGF+ astrocytes was significantly higher (P < 0.0001) in border zones adjacent to the core, corresponding to the penumbra. These numbers were significantly increased at day 1 and day 3 and remained persistently elevated up to several months post-infarction (P < 0.0001). The restricted expression and accumulation of CTGF+ reactive astrocytes adds convincing evidence for CTGF participation in the gliotic astrocyte CNS injury response involved in glial scar formation. CTGF can be considered a sensitive marker of early human astrocyte activation and a possible target for pharmacological intervention of aberrant matrix deposition.     

Positioned to inhibit: netrin-1 and netrin receptor expression after spinal cord injury

Netrin-1 regulates axon extension during embryonic development and is expressed by neurons and myelinating oligodendrocytes in the adult CNS. To investigate the potential role of netrin-1 after spinal cord injury, we examined the expression of netrin-1 and netrin receptors after sagittal myelotomy in adult rats. This lesion targets spinal commissural projections, which respond to netrin-1 during development. Netrin-1 mRNA and protein levels were dramatically reduced at the site of injury and reduced expression persisted for at least 7 months. Neither netrin-1 protein nor mRNA was associated with the glial scar, but netrin-1 was expressed by neurons and oligodendrocytes immediately adjacent to the lesion. The post-injury distribution detected is similar to that reported for myelin-associated inhibitors of axon regeneration, such as Nogo, and is distinct from the distribution of inhibitors associated with a glial scar. DCC and UNC-5 homologue (UNC5H) expression also was reduced after injury. Although UNC5H levels recovered, DCC expression at the site of injury remained approximately 50% of pre-injury values at 7 months. Increased UNC5H immunoreactivity was associated with fibers in the superficial layers of the dorsal horn and in fibers located in white matter adjacent to the lesion. The dominant expression of UNC5H on axons and neurons in the spinal cord after injury and the persistent expression of netrin-1 by oligodendrocytes surrounding the lesion are consistent with the hypothesis that netrin-1 is a myelin-associated inhibitor of axonal regeneration after spinal cord injury.     

N-cadherin at the glial scar in the rat.

Following injury to the central nervous system (CNS), astrocytes become reactive and in many cases form a glial scar. Very little is known about the adhesive interactions between astrocytes at the glial scar, even though reactive gliosis and scar formation are a central issue in CNS wound healing. In the present study, we examine the role of cadherin in the process of scar formation using immunohistochemistry and immunoblot methods. When a stab wound was made in the cerebral cortex of the rat, cadherins were consistently upregulated by the reactive astrocytes at the glial scar. Our immunoblot analysis demonstrates that the increase in cadherin immunoreactivity was due to a threefold upregulation of a single protein with a molecular weight of 135 kDa. The size (135 kDa) and location of the immunoreactive protein at regions of cell-cell contact in cultured astrocytes indicates that the immunoreactive protein is N-cadherin. These data are the first to demonstrate that N-cadherin plays a prominent role in the response of astrocytes to injury, including the formation and maintenance of the glial scar.     

Expression of protein zero is increased in lesioned axon pathways in the central nervous system of adult zebrafish

The immunoglobulin superfamily molecule protein zero (P0) is important for myelin formation and may also play a role in adult axon regeneration, since it promotes neurite outgrowth in vitro. Moreover, it is expressed in the regenerating central nervous system (CNS) of fish, but not in the nonregenerating CNS of mammals. We identified a P0 homolog in zebrafish. Cell type-specific expression of P0 begins in the ventromedial hindbrain and the optic chiasm at 3-5 days of development. Later (at 4 weeks) expression has spread throughout the optic system and spinal cord. This is consistent with a role for P0 in CNS myelination during development. In the adult CNS, glial cells constitutively express P0 mRNA. After an optic nerve crush, expression is increased within 2 days in the entire optic pathway. Expression peaks at 1 to 2 months and remains elevated for at least 6 months postlesion. After enucleation, P0 mRNA expression is also upregulated but fails to reach the high levels observed in crush-lesioned animals at 4 weeks postlesion. Spinal cord transection leads to increased expression of P0 mRNA in the spinal cord caudal to the lesion site. The glial upregulation of P0 mRNA expression after a lesion of the adult zebrafish CNS suggests roles for P0 in promoting axon regeneration and remyelination after injury.     

Spinal cord injury induces differential expression of the profibrotic semaphorin 7A in the developing and mature glial scar

Semaphorin 7A (Sema7A) is involved in the formation of the central nervous system during development by operating axon guidance and neuronal migration. We investigated the expression of the TGFβ‐inducible Sema7A following spinal cord injury (SCI). After SCI, Sema7A⁺ cells accumulated specifically in lesion areas resulting in significantly enhanced Sema7A expression at the injury site (P < 0.0001). During the first days lesional Sema7A expression was confined to neurons, ballooned neurite fibers/retraction bulbs, and endothelial cells. At day 7, we observed Sema7A expression by components of the glial scar, such as reactive astrocytes and pronounced extracellular Sema7A deposition. In the direct perilesional rim, Sema7A⁺ astrocytes coexpressed the activation‐associated intermediate filament vimentin. In the injured spinal cord, numbers of Sema7A⁺ cells reached maximum levels at day 14. The restricted accumulation of Sema7A⁺ reactive astrocytes and Sema7A deposition in fibronectin⁺ extracellular matrix territories suggests a participation of the fibrostimulatory Sema7A in the developing and maturating scar following SCI. In addition, Sema7A appears to be marker a for astrocyte activation. © 2010 Wiley‐Liss, Inc.     

NG2 proteoglycan-expressing cells of the adult rat brain: possible involvement in the formation of glial scar astrocytes following stab wound

Stab wound lesion to the adult central nervous system induces strong proliferative response that is followed by the formation of a dense astroglial scar. In order to determine the origin of those astrocytes composing the glial scar, the cell proliferation marker bromodeoxyuridine (BrdU) was administered to lesioned rats that were fixed 3 h or 6 days later. At 3 h after the BrdU administration, labeled nuclei were frequently associated with either NG2(+) cells or microglia/macrophages, but rarely with astrocytes expressing glial fibrillary acidic protein (GFAP). Six days later, by contrast, numerous BrdU-labeled nuclei were associated with astrocytes located along the lesion borders. After the injection of a viral vector of the green fluorescent protein (GFP) into the lesional cavity, GFP was preferentially detected within NG2- or GFAP-labeled cells when lesioned animals were fixed 1 or 6 days after the injections, respectively. The combined detection of glial markers within cells present in the lesioned area indicated that, although they rarely express GFAP, the marker of mature astrocytes, NG2(+) cells located along the lesion borders frequently express nestin and vimentin, i.e., two markers of immature astrocytes. Lastly, chronic treatment of lesioned rats with dexamethasone was found to inhibit the proliferation of NG2(+) cells present within the lesioned area and to subsequently alter the formation of a dense astroglial scar. Taken together, these data strongly suggest that following a surgical lesion, at least a portion of the astrocytes that constitute the glial scar are issued from resident NG2(+) cells.     

Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury

We have examined the injury response of astrocytes in the immature hamster brain in this study, focusing on alterations in the expression of glial fibrillary acidic protein (GFAP) and vimentin. In the adult CNS these two type III intermediate filament (IF) proteins have been shown to undergo robust increases in expression in response to axonal injury. Since injury to the immature CNS reportedly elicits less glial scar formation than adult brain injury, we examined the possibility that immature astrocytes respond differently than adult astrocytes to CNS injury with respect to IF gene expression. In situ hybridization using a ³⁵S-labeled cDNA GFAP probe was done on brainstem sections obtained 2,7 and 14 days after unilateral transection of the corticospinal tract in P8 hamster pups. The results indicated that substantial increases in GFAP mRNA were associated with the degenerating portion of the corticospinal tract by 2 days after axotomy and that the levels remained elevated for at least 14 days. Double-label immunofluorescence studies of this material suggested that GFAP as well as vimentin protein levels were also increased in many astrocytes in and around the degenerating corticospinal tract 2–14 days after axotomy. Most of the reactive astocytes in the degenerating regions exhibited increases in GFAP and vimentin immunostaining but some vimentin-negative GFAP-positive reactive astrocytes were also observed, particularly in regions surrounding the actual degenerative zones. The results from these experiments revealed that immature astrocytes have the potential for altering their normal developmental program of GFAP and vimentin expression after injury and mount a response that is qualitatively similar to that of astrocytes after CNS injury in the adult animal.     

Proliferation of a subpopulation of reactive astrocytes following needle-insertion lesion in rat

It is well known that traumatic injuries of the CNS induce a gliotic reaction, characterized by the presence of reactive astrocytes. Reactive astrocytes exhibit enhanced expression of the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), hypertrophy, and thickened processes. Recently, we have demonstrated that injuries of the CNS induce a re-expression of an embryonic intermediate filament-associated protein, IFAP-70/280 kDa. Based on IFAP-70/280 kDa immunolabeling, we have shown that reactive astrocytes, activated by stab-wound injury, can be divided into two major groups: 1. persistent IFAP+/GFAP+ cells which are close to the wound in the area of glial scar, and 2. transient IFAP-/GFAP+ cells which are farther from the wound. In this study, we use BrdU incorporation to examine proliferation in these two groups of reactive astrocytes induced by stab injury of the rat cerebrum. Triple/double-label immunofluorescence microscopy was performed using antibodies to IFAP-70/280 kDa, GFAP, and BrdU. The results showed that BrdU+ reactive astrocytes (GFAP+) were always IFAB-70/280 kDa+ as well. However, not all IFAP+ reactive astrocytes are BrdU+. BrdU+ signal was not observed in any IFAP- reactive astrocytes. At five days post-lesion, IFAP+ reactive astrocytes were increasing in the area of the wound (0-50 micrograms from the wound edge), but had reached a peak in the proximal area (50-800 micrograms away from the wound edge). At eight days post-lesion, IFAP+ reactive astrocytes achieved the highest percentage in the wound area. At the same time, BrdU-containing reactive astrocytes occupied an area closer to the wound. By 20 days post-lesion, following the formation of the gliotic scar at the stab-wound, a few IFAP+/GFAP+ cells still persisted. BrdU-containing reactive astrocytes were only observed in the scar. These results indicate that many IFAP+ reactive astrocytes close to the wound, in contrast to the IFAP- ones farther from the wound, appear to regain their proliferative potential to increase in number and participate in the formation of the gliotic scar.     

Distant microglial and astroglial activation secondary to experimental spinal cord lesion

This paper analysed whether glial responses following a spinal cord lesion is restricted to a scar formation close to the wound or they might be also related to widespread paracrine trophic events in the entire cord. Spinal cord hemitransection was performed in adult rats at the thoracic level. Seven days and three months later the spinal cords were removed and submitted to immunohistochemistry of glial fibrillary acidic protein (GFAP) and OX42, markers for astrocytes and microglia, as well as of basic fibroblast growth factor (bFGF), an astroglial neurotrophic factor. Computer assisted image analysis was employed in the quantification of the immunoreactivity changes. At the lesion site an increased number of GFAP positive astrocytes and OX42 positive phagocytic cells characterized a dense scar formation by seven days, which was further augmented after three months. Morphometric analysis of the area and microdensitometric analysis of the intensity of the GFAP and OX42 immunoreactivities showed reactive astrocytes and microglia in the entire spinal cord white and gray matters 7 days and 3 months after surgery. Double immunofluorescence demonstrated increased bFGF immunostaining in reactive astrocytes. The results indicated that glial reaction close to an injury site of the spinal cord is related to wounding and repair events. Although gliosis constitutes a barrier to axonal regeneration, glial activation far from the lesion may contribute to neuronal trophism and plasticity in the lesioned spinal cord favoring neuronal maintenance and fiber outgrowth.     

Expression of TGFbeta2 but not TGFbeta1 correlates with the deposition of scar tissue in the lesioned spinal cord

Transforming growth factor-betas (TGFbetas) are implicated in fibrotic pathologies. TGFbeta1 and -beta2 expression is increased around the glial/fibrotic scar in the injured brain. Moreover, local injection of TGFbeta antagonists into cerebral wounds reduces glial scarring. Here, we monitored expression of TGFbeta1 and -beta2 mRNA and protein in the spinal cord after transection of the dorsal funiculi. Levels of TGFbeta1 mRNA were most elevated over the acute inflammatory phase, while TGFbeta2 mRNA levels were raised locally about the wound, particularly in astrocytes and neovascular endothelial cells, over the subacute period of scarring. TGFbeta protein production also increased after injury. Both TGFbeta1 and TGFbeta2 were found in hematogenous inflammatory cells, while TGFbeta1 was also neuron-associated, and high levels of TGFbeta2 were localized to multiple cell types in the wound, including reactive astrocytes, during the period of glial/collagen scar formation. The cellular localization and temporal pattern of expression of TGFbeta after spinal cord injury suggest that TGFbeta1 modulates the inflammatory and neuronal responses, while TGFbeta2 regulates glial/collagen scarring.     

Lesion-induced changes in the expression of ciliary neurotrophic factor and its receptor in rat optic nerve

There is evidence that ciliary neurotrophic factor (CNTF) is involved in reactive changes following lesions of the nervous system. To investigate, whether differences in the regulation of CNTF and CNTF receptor α (CNTFRα) contribute to the differences in PNS and CNS responses to injury, we have studied their expression on the mRNA and protein level in the rat optic nerve following a crush lesion to compare them with the situation in peripheral nerve. Seven days after the lesion, CNTF mRNA and protein levels were markedly decreased at the lesion site, concommitant with the disappearance of GFAP- and CNTF-immunopositive astrocytes. CNTF levels in proximal and distal parts were less affected. This was in contrast to the situation in the PNS, where CNTF was downregulated at and distal to the lesion site. Different from other CNS regions, optic nerve astrocytes expressed CNTFRα mRNA under normal conditions. Following lesion, CNTFRα was reduced substantially only in distal and proximal parts of the optic nerve but continued to be expressed at high levels at the lesion site, suggesting that GFAP-negative, CNTF-responsive cells are present there. Our results suggest that differences in lesion-induced changes in the optic and sciatic nerve reflect differences in the response to injury of astrocytes and Schwann cells. In the light of the known actions of CNTF in inducing astrogliosis, the expression pattern observed in the optic nerve indicates that CNTF and CNTFRα are involved in glial scar formation in the lesion area. GLIA 23:239–248, 1998. © 1998 Wiley-Liss, Inc.