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1.
J Neurosci ; 27(42): 11201-13, 2007 Oct 17.
Article in English | MEDLINE | ID: mdl-17942715

ABSTRACT

Transforming growth factor beta1 (TGFbeta1) is a pleiotropic cytokine with potent neurotrophic and immunosuppressive properties that is upregulated after injury, but also expressed in the normal nervous system. In the current study, we examined the regulation of TGFbeta1 and the effects of TGFbeta1 deletion on cellular response in the uninjured adult brain and in the injured and regenerating facial motor nucleus. To avoid lethal autoimmune inflammation within 3 weeks after birth in TGFbeta1-deficient mice, this study was performed on a T- and B-cell-deficient RAG2-/- background. Compared with wild-type siblings, homozygous deletion of TGFbeta1 resulted in an extensive inflammatory response in otherwise uninjured brain parenchyma. Astrocytes increased in GFAP and CD44 immunoreactivity; microglia showed proliferative activity, expression of phagocytosis-associated markers [alphaXbeta2, B7.2, and MHC1 (major histocompatibility complex type 1)], and reduced branching. Ultrastructural analysis revealed focal blockade of axonal transport, perinodal damming of axonal organelles, focal demyelination, and myelin debris in granule-rich, phagocytic microglia. After facial axotomy, absence of TGFbeta1 led to a fourfold increase in neuronal cell death (52 vs 13%), decreased central axonal sprouting, and significant delay in functional recovery. It also interfered with the microglial response, resulting in a diminished expression of early activation markers [ICAM1 (intercellular adhesion molecule 1), alpha6beta1, and alphaMbeta2] and reduced proliferation. In line with axonal and glial findings in the otherwise uninjured CNS, absence of endogenous TGFbeta1 also caused an approximately 10% reduction in the number of normal motoneurons, pointing to an ongoing and potent trophic role of this anti-inflammatory cytokine in the normal as well as in the injured brain.


Subject(s)
Central Nervous System/metabolism , Central Nervous System/pathology , Inflammation Mediators/physiology , Transforming Growth Factor beta1/physiology , Age Factors , Animals , Brain/metabolism , Brain/pathology , Brain/physiology , Cell Survival/physiology , Central Nervous System/cytology , Inflammation/metabolism , Inflammation/pathology , Inflammation/prevention & control , Mice , Mice, Transgenic , Neurodegenerative Diseases/metabolism , Neurodegenerative Diseases/pathology , Neurodegenerative Diseases/prevention & control
2.
Proc Natl Acad Sci U S A ; 104(28): 11814-9, 2007 Jul 10.
Article in English | MEDLINE | ID: mdl-17606926

ABSTRACT

Changes in the molecular and cellular composition of the CNS after injury or disease result in the formation of an inhibitory environment that inhibits the regeneration of adult mammalian CNS neurons. Although a dramatic change in the CNS environment after traumatic injury or disease is hemorrhage because of vascular rupture or leakage of the blood-brain barrier, the potential role for blood proteins in repair processes remains unknown. Here we show that the blood protein fibrinogen is an inhibitor of neurite outgrowth that is massively deposited in the spinal cord after injury. We show that fibrinogen acts as a ligand for beta3 integrin and induces the transactivation of EGF receptor (EGFR) in neurons. Fibrinogen-mediated inhibition of neurite outgrowth is reversed by blocking either beta3 integrin or phoshorylation of EGFR. Inhibition of Src family kinases that mediate the cross-talk between integrin and growth factor receptors rescue the fibrinogen-induced phosphorylation of EGFR. These results identify fibrinogen as the first blood-derived inhibitor of neurite outgrowth and suggest fibrinogen-induced EGFR transactivation on neuronal cells as a molecular link between vascular and neuronal damage in the CNS after injury.


Subject(s)
ErbB Receptors/metabolism , Fibrinogen/physiology , Growth Inhibitors/physiology , Integrin beta3/physiology , Neural Inhibition/physiology , Neurites/metabolism , Animals , ErbB Receptors/physiology , Female , Fibrinogen/metabolism , Growth Inhibitors/metabolism , Humans , Integrin beta3/metabolism , Mice , Mice, Inbred C57BL , Neurites/physiology , Phosphorylation , Rats , Rats, Inbred F344 , Transcriptional Activation/physiology
3.
Exp Neurol ; 203(1): 8-21, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17014846

ABSTRACT

Cellular and extracellular inhibitors are thought to restrict axon growth after chronic spinal cord injury (SCI), confronting the axon with a combination of chronic astrocytosis and extracellular matrix-associated inhibitors that collectively constitute the chronic "scar." To examine whether the chronically injured environment is strongly inhibitory to axonal regeneration, we grafted permissive autologous bone marrow stromal cells (MSCs) into mid-cervical SCI sites of adult rats, 6 weeks post-injury without resection of the "chronic scar." Additional subjects received MSCs genetically modified to express neurotrophin-3 (NT-3), providing a further local stimulus to axon growth. Anatomical analysis 3 months post-injury revealed extensive astrocytosis surrounding the lesion site, together with dense deposition of the inhibitory extracellular matrix molecule NG2. Despite this inhibitory environment, axons penetrated the lesion site through the chronic scar. Robust axonal regeneration occurred into chronic lesion cavities expressing NT-3. Notably, chronically regenerating axons preferentially associated with Schwann cell surfaces expressing both inhibitory NG2 substrates and the permissive substrates L1 and NCAM in the lesion site. Collectively, these findings indicate that inhibitory factors deposited at sites of chronic SCI do not create impenetrable boundaries and that inhibition can be balanced by local and diffusible signals to generate robust axonal growth even without resecting chronic scar tissue.


Subject(s)
Bone Marrow Transplantation/methods , Cicatrix/metabolism , Growth Cones/metabolism , Nerve Growth Factors/metabolism , Nerve Regeneration/physiology , Spinal Cord Injuries/therapy , Animals , Antigens/metabolism , Bone Marrow Transplantation/trends , Cells, Cultured , Chronic Disease/therapy , Cicatrix/physiopathology , Disease Models, Animal , Extracellular Matrix/metabolism , Female , Gliosis/metabolism , Gliosis/physiopathology , Gliosis/prevention & control , Growth Cones/ultrastructure , Growth Inhibitors/metabolism , Nerve Growth Factors/genetics , Neural Cell Adhesion Molecule L1/metabolism , Neural Cell Adhesion Molecules/metabolism , Neurotrophin 3/genetics , Neurotrophin 3/metabolism , Proteoglycans/metabolism , Rats , Rats, Inbred F344 , Schwann Cells/metabolism , Stromal Cells/metabolism , Stromal Cells/transplantation , Treatment Outcome
4.
J Neurosci ; 24(28): 6402-9, 2004 Jul 14.
Article in English | MEDLINE | ID: mdl-15254096

ABSTRACT

Previous attempts to promote regeneration after spinal cord injury have succeeded in stimulating axonal growth into or around lesion sites but rarely beyond them. We tested whether a combinatorial approach of stimulating the neuronal cell body with cAMP and the injured axon with neurotrophins would propel axonal growth into and beyond sites of spinal cord injury. A preconditioning stimulus to sensory neuronal cell bodies was delivered by injecting cAMP into the L4 dorsal root ganglion, and a postinjury stimulus to the injured axon was administered by injecting neurotrophin-3 (NT-3) within and beyond a cervical spinal cord lesion site grafted with autologous bone marrow stromal cells. One to 3 months later, long-projecting dorsal-column sensory axons regenerated into and beyond the lesion. Regeneration beyond the lesion did not occur after treatment with cAMP or NT-3 alone. Thus, clear axonal regeneration beyond spinal cord injury sites can be achieved by combinatorial approaches that stimulate both the neuronal soma and the axon, representing a major advance in strategies to enhance spinal cord repair.


Subject(s)
Axons/drug effects , Bucladesine/therapeutic use , Nerve Regeneration/drug effects , Neurotrophin 3/therapeutic use , Spinal Cord Injuries/drug therapy , Animals , Axons/physiology , Bone Marrow Transplantation , Bucladesine/administration & dosage , Combined Modality Therapy , Drug Evaluation , Drug Evaluation, Preclinical , Drug Therapy, Combination , Female , Ganglia, Spinal , Injections, Intralesional , Neurons, Afferent/drug effects , Neurons, Afferent/physiology , Neurotrophin 3/administration & dosage , Rats , Rats, Inbred F344 , Stromal Cells/transplantation , Transplantation, Autologous
5.
J Neurosci ; 23(28): 9276-88, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14561854

ABSTRACT

Increased expression of certain extracellular matrix (ECM) molecules after CNS injury is believed to restrict axonal regeneration. The chondroitin sulfate proteoglycans (CSPGs) are one such class of ECM molecules that inhibit neurite outgrowth in vitro and are upregulated after CNS injury. We examined growth responses of several classes of axons to this inhibitory environment in the presence of a cellular fibroblast bridge in a spinal cord lesion site and after a growth factor stimulus at the lesion site (fibroblasts genetically modified to secrete NGF). Immunohistochemical analysis showed dense labeling of the CSPGs NG2, brevican, neurocan, versican, and phosphacan at the host-lesion interface after spinal cord injury (SCI). Furthermore, robust expression of NG2, and to a lesser extent versican, was also observed throughout grafts of control and NGF-secreting fibroblasts. Despite this inhibitory milieu, several axonal classes penetrated control fibroblast grafts, including dorsal column sensory, rubrospinal, and nociceptive axons. Axon growth was amplified more in the presence of NGF-secreting grafts. Confocal microscopy demonstrated that axon growth was, paradoxically, preferentially associated with NG2-rich substrates in both graft types. NG2 expression also increased after sciatic nerve injury, wherein axons successfully regenerate. Cellular sources of NG2 in SCI and peripheral nerve lesion sites included Schwann cells and endothelial cells. Notably, these same cellular sources in lesion sites produced the cell adhesion molecules L1 and laminin, and these molecules all colocalized. Thus, axons grow along substrates coexpressing both inhibitory and permissive molecules, suggesting that regeneration is successful when local permissive signals balance and exceed inhibitory signals.


Subject(s)
Axons/physiology , Chondroitin Sulfate Proteoglycans/metabolism , Nerve Regeneration/physiology , Spinal Cord Injuries/physiopathology , Animals , Antigens/biosynthesis , Axons/drug effects , Cell Adhesion Molecules/biosynthesis , Cell Division/physiology , Cells, Cultured , Chondroitin Sulfate Proteoglycans/biosynthesis , Disease Models, Animal , Endothelium, Vascular/cytology , Endothelium, Vascular/metabolism , Extracellular Matrix/metabolism , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/transplantation , Graft Survival , Laminin/metabolism , Lectins, C-Type , Nerve Growth Factor/biosynthesis , Nerve Growth Factor/genetics , Nerve Growth Factor/pharmacology , Proteoglycans/biosynthesis , Rats , Rats, Inbred F344 , Schwann Cells/cytology , Schwann Cells/metabolism , Sciatic Neuropathy/pathology , Sciatic Neuropathy/physiopathology , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord Injuries/pathology , Versicans
6.
Exp Neurol ; 182(2): 399-411, 2003 Aug.
Article in English | MEDLINE | ID: mdl-12895450

ABSTRACT

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: neurocan, brevican, phosphacan, and versican. After spinal cord injury, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury.


Subject(s)
Chondroitin Sulfate Proteoglycans/metabolism , Nerve Tissue Proteins/metabolism , Spinal Cord Injuries/metabolism , Animals , Astrocytes/metabolism , Astrocytes/pathology , Brevican , Chondroitin Sulfate Proteoglycans/genetics , Disease Models, Animal , Disease Progression , Female , Glial Fibrillary Acidic Protein/biosynthesis , Immunohistochemistry , In Situ Hybridization , Lectins, C-Type , Nerve Tissue Proteins/genetics , Neurocan , Rats , Rats, Inbred F344 , Receptor-Like Protein Tyrosine Phosphatases, Class 5 , Spinal Cord Injuries/pathology , Time Factors , Versicans
7.
J Neurosci Res ; 72(6): 726-33, 2003 Jun 15.
Article in English | MEDLINE | ID: mdl-12774313

ABSTRACT

Studies using mouse axotomised facial motoneuron model show a strong and highly selective entry of CD3+ lymphocytes into the affected nucleus, with a maximum at Day 14, which coincides with the peak of neuronal cell death, microglial phagocytosis, and increased synthesis of interleukin-1 beta (IL1beta), tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma). We explored the possible involvement of these cytokines during the main phase of lymphocyte recruitment into the axotomised facial motor nucleus 7-21 days after nerve cut using mice homozygously deficient for IL1 receptor type 1 (IL1R1-/-), TNF receptor type 1 (TNFR1-/-), type 2 (TNFR2-/-) and type 1 and 2 (TNFR1&2-/-), IFNgamma receptor type 1 (IFNgammaR1-/-), and the appropriate controls for the genetic background. Transgenic deletion of IL1R1 led to a 54% decrease and that of TNFR2 to a 44% reduction in the number of CD3+ T-cells in the axotomised facial motor nucleus, with a similar relative decrease at Day 7, 14, and 21. Deletion of TNFR1 or IFNgammaR1 had no significant effect. Deletion of both TNFR1 and 2 (TNFR1&2-/-) caused a somewhat stronger, 63% decrease than did TNFR2 deletion alone, but this could be due to an almost complete inhibition of neuronal cell death. No mutations seemed to inhibit aggregation of CD3+ T-cells around glial nodules consisting of Ca-ion binding adaptor protein-1 (IBA1)+ phagocytotic microglia and neuronal debris. Altogether, the current data show the importance of IL1R1 and TNFR2 as the key players during the main phase of lymphocyte recruitment to the damaged part of the central nervous system.


Subject(s)
Brain Stem/pathology , Cell Movement/physiology , Cytokines/physiology , Lymphocytes/pathology , Animals , Axotomy , Brain Stem/metabolism , Cell Communication/physiology , Cytokines/deficiency , Cytokines/genetics , Facial Nerve/metabolism , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Microglia/pathology , Phagocytosis/physiology , Receptors, Cytokine/deficiency , Receptors, Cytokine/genetics , Receptors, Cytokine/physiology
8.
Exp Neurol ; 181(1): 47-56, 2003 May.
Article in English | MEDLINE | ID: mdl-12710933

ABSTRACT

Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examined whether delivery of neurotrophic factors after chronic spinal cord injury would also promote axonal growth and influence functional outcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblasts genetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later (6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to the lesion site and showed a modest but significant 1.5-point improvement in locomotor scores (P < 0.05) on the BBB scale, compared to control-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improves functional outcomes compared to non-growth-factor-treated animals.


Subject(s)
Axons/drug effects , Genetic Therapy/methods , Neurotrophin 3/pharmacology , Spinal Cord Injuries/therapy , Animals , Axons/physiology , Cells, Cultured , Chronic Disease , Disease Models, Animal , Fibroblasts/metabolism , Fibroblasts/transplantation , Gene Transfer Techniques , Genes, Reporter , Graft Survival , Humans , Motor Activity , Neurotrophin 3/biosynthesis , Neurotrophin 3/genetics , Pyramidal Tracts/drug effects , Pyramidal Tracts/injuries , Pyramidal Tracts/pathology , Rats , Treatment Outcome
9.
J Comp Neurol ; 449(1): 88-101, 2002 Jul 15.
Article in English | MEDLINE | ID: mdl-12115695

ABSTRACT

Little is known about molecular and cellular responses to spinal cord injury in primates. In this study, the normal milieu of the primate spinal cord was disturbed by multiple needle penetrations and cell injections in the mid-thoracic spinal cord; subsequent effects on local axons and expression of extracellular matrix (ECM) molecules were examined, together with effects of cellular delivery of nerve growth factor (NGF) to the injured region. Four adult rhesus monkeys each received injections of two grafts of autologous fibroblasts genetically modified to secrete human NGF, and, in control injection sites, two separate grafts of autologous fibroblasts transduced to express the reporter gene, beta-galactosidase. Three months later, Schwann cells extensively infiltrated the region of localized injury and penetrated both NGF and control fibroblast grafts. Marked upregulation of several ECM molecules occurred, including chondroitin and heparan sulfate proteoglycans and type IV collagen, in or adjacent to all injection sites. Schwann cells were an apparent source of some ECM expression. Spinal cord sensory axons and putative coerulospinal axons extended into both graft types, but they penetrated NGF grafts to a significantly greater extent. Many of these axons expressed the cell adhesion molecule L1. Thus, extensive cellular and molecular changes occur at sites of localized primate spinal cord injury and grafting, attributable in part to migrating Schwann cells, and are accompanied by spontaneous axonal plasticity. These molecular and cellular events closely resemble those observed in the rodent spinal cord after injury. Furthermore, as in rodent studies, cellular delivery of a trophic factor significantly augments axonal plasticity in the primate spinal cord.


Subject(s)
Axons/physiology , Nerve Growth Factor/administration & dosage , Spinal Cord Injuries , Transplants , Animals , Axons/drug effects , Cell Division/drug effects , Cell Division/physiology , Drug Delivery Systems/methods , Drug Delivery Systems/statistics & numerical data , Extracellular Matrix/drug effects , Extracellular Matrix/metabolism , Fibroblasts/drug effects , Fibroblasts/physiology , Humans , Macaca mulatta , Male , Nerve Growth Factor/metabolism , Neuronal Plasticity/drug effects , Neuronal Plasticity/physiology , Schwann Cells/drug effects , Schwann Cells/physiology , Spinal Cord Injuries/drug therapy , Spinal Cord Injuries/metabolism , Spinal Cord Injuries/pathology , Transplants/statistics & numerical data
10.
J Neurosci ; 22(11): 4611-24, 2002 Jun 01.
Article in English | MEDLINE | ID: mdl-12040068

ABSTRACT

Keratan sulfate proteoglycans (KSPGs) are extracellular matrix molecules that appear to establish boundaries for axonal growth in the developing brain and spinal cord. In vitro studies confirm that KSPGs define inhibitory boundaries to extending neurites. The aim of the current study was to investigate whether KSPGs are expressed after spinal cord injury (SCI) and thereby might act as potential inhibitors of axonal growth. Adult Fischer 344 rats were subjected to spinal cord lesions, and the temporal and spatial expression of KSPGs was examined using the 5D4 monoclonal anti-KSPG antibody. In the intact spinal cord, a subpopulation of microglia expressed 5D4-KSPG throughout the white and gray matter. Within 24 hr of injury, 5D4-KSPG immunoreactivity substantially increased and appeared on cellular profiles in close proximity to the spinal cord lesion site, peaking 3 d after injury. Double immunolabeling revealed that 5D4-KSPG expression arose from multiple cell types at the lesion site, including reactive microglia, macrophages, and oligodendrocyte progenitors. Astrocytes were not identified as a source of 5D4-KSPG. The robust and extensive production of 5D4-KSPG at sites of SCI precedes the expression of other putatively inhibitory proteoglycan molecules such as chondroitin sulfate proteoglycans. This is the first demonstration that KSPGs are expressed after SCI in a temporal and spatial relationship that could exert an early and important role in modulating axonal growth after SCI.


Subject(s)
Chondroitin Sulfate Proteoglycans/biosynthesis , Keratan Sulfate/biosynthesis , Macrophages/metabolism , Microglia/metabolism , Spinal Cord Injuries/metabolism , Stem Cells/metabolism , Animals , Antibodies, Monoclonal , Antibody Specificity , Disease Models, Animal , Disease Progression , Female , Immunohistochemistry , Lumican , Macrophages/pathology , Microglia/pathology , Oligodendroglia/metabolism , Oligodendroglia/pathology , Rats , Rats, Inbred F344 , Spinal Cord/cytology , Spinal Cord/metabolism , Spinal Cord/pathology , Spinal Cord Injuries/pathology , Stem Cells/pathology
11.
J Neurosci ; 22(7): 2792-803, 2002 Apr 01.
Article in English | MEDLINE | ID: mdl-11923444

ABSTRACT

Several extracellular matrix (ECM) molecules have been identified as potent inhibitors of neurite outgrowth in vitro and are believed to limit axonal growth after CNS injury. Recent studies have shown that different members of the chondroitin sulfate proteoglycan (CSPG) class of putatively inhibitory ECM molecules are expressed after a number of CNS injuries. The purpose of this study was to evaluate the relative amounts of individual CSPGs expressed after spinal cord injury (SCI) and identify their cells of origin. Evaluation of total soluble CSPGs 2 weeks after dorsal column lesion in the rat demonstrated that NG2 is highly upregulated and is a major CSPG species. Immunocytochemical analysis further demonstrated that NG2 expression is upregulated within 24 hr of injury, peaks at 1 week, and remains elevated for at least an additional 7 weeks. NG2 expression results from a multicellular response to injury, including both reactive macrophages and oligodendrocyte progenitors; astrocytes were not identified as a major source of NG2. Immunocytochemical analysis of other CSPG family members 7 d after injury showed moderate upregulation of versican, brevican, and neurocan, and downregulation of phosphacan. Axonal tracing experiments demonstrated dense NG2 labeling adjacent to the forward processes of transected corticospinal tract axons in a spatial profile that could restrict axonal growth. Thus, NG2 is a major component of this putatively inhibitory class of ECM molecules expressed at sites of SCI and may restrict axonal regeneration.


Subject(s)
Antigens/metabolism , Chondroitin Sulfate Proteoglycans/metabolism , Macrophages/metabolism , Oligodendroglia , Proteoglycans/metabolism , Spinal Cord Injuries/metabolism , Stem Cells/metabolism , Animals , Astrocytes/pathology , Axons/metabolism , Axons/pathology , Disease Progression , Female , Immunoblotting , Immunohistochemistry , Macrophages/pathology , Microglia/pathology , Oligodendroglia/cytology , Rats , Rats, Inbred F344 , Silver Staining , Spinal Cord Injuries/pathology , Stem Cells/pathology
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