myelin-basic-protein and Spinal-Cord-Injuries

Neurodegenerative diseases (NDD) are disorders characterized by the progressive loss of neurons affecting motor, sensory, and/or cognitive functions. The incidence of these diseases is increasing and has a great impact due to their high morbidity and mortality. Unfortunately, current therapeutic strategies only temporarily improve the patients' quality of life but are insufficient for completely alleviating the symptoms. An interaction between the immune system and the central nervous system (CNS) is widely associated with neuronal damage in NDD. Usually, immune cell infiltration has been identified with inflammation and is considered harmful to the injured CNS. However, the immune system has a crucial role in the protection and regeneration of the injured CNS. Nowadays, there is a consensus that deregulation of immune homeostasis may represent one of the key initial steps in NDD. Dr. Michal Schwartz originally conceived the concept of "protective autoimmunity" (PA) as a well-controlled peripheral inflammatory reaction after injury, essential for neuroprotection and regeneration. Several studies suggested that immunizing with a weaker version of the neural self-antigen would generate PA without degenerative autoimmunity. The development of CNS-related peptides with immunomodulatory neuroprotective effect led to important research to evaluate their use in chronic and acute NDD. In this review, we refer to the role of PA and the potential applications of active immunization as a therapeutic option for NDD treatment. In particular, we focus on the experimental and clinical promissory findings for CNS-related peptides with beneficial immunomodulatory effects.

Topics: Alzheimer Disease; Amyotrophic Lateral Sclerosis; Animals; Autoantigens; Autoimmunity; Glatiramer Acetate; Humans; Immunization, Passive; Immunologic Factors; Immunomodulation; Myelin Basic Protein; Nerve Regeneration; Neurodegenerative Diseases; Neuroprotection; Parkinson Disease; Peptide Fragments; Peptides; Proteostasis Deficiencies; Spinal Cord Injuries; Stroke

In addition to its traditional role in reproduction, progesterone (PROG) has demonstrated neuroprotective and promyelinating effects in lesions of the peripheral and central nervous systems, including the spinal cord. The latter is a target of PROG, as nuclear receptors, as well as membrane receptors, are expressed by neurons and/or glial cells. When spinal cord injury (SCI) is produced at the thoracic level, several genes become sensitive to PROG in the region caudal to the lesion site. Although the cellular machinery implicated in PROG neuroprotection is only emerging, neurotrophins, their receptors, and signaling cascades might be part of the molecules involved in this process. In rats with SCI, a 3-d course of PROG treatment increased the mRNA of brain-derived neurotrophic factor (BDNF) and BDNF immunoreactivity in perikaryon and processes of motoneurons, whereas chromatolysis was strongly prevented. The increased expression of BDNF correlated with increased immunoreactivity for the BDNF receptor TrkB and for phosphorylated cAMP-responsive element binding in motoneurons. In the same SCI model, PROG restored myelination, according to measurements of myelin basic protein (MBP) and mRNA levels, and further increased the density of NG2+-positive oligodendrocyte progenitors. These cells might be involved in remyelination of the lesioned spinal cord. Interestingly, similarities in the regulation of molecular parameters and some cellular events attributed to PROG and BDNF (i.e., choline acetyltransferase, Na,K-ATPase, MBP, chromatolysis) suggest that BDNF and PROG might share intracellular pathways. Furthermore, PROG-induced BDNF might regulate, in a paracrine or autocrine fashion, the function of neurons and glial cells and prevent the generation of damage.

Topics: Animals; Brain-Derived Neurotrophic Factor; Cyclic AMP Response Element-Binding Protein; Myelin Basic Protein; Myelin Sheath; Neurons; Neuroprotective Agents; Oligodendroglia; Progesterone; Receptors, Progesterone; Spinal Cord; Spinal Cord Injuries; Stem Cells

Autoimmunity is usually considered only as a cause of disease; nevertheless, human T-cell repertoires are filled naturally with autoimmune lymphocytes. Here, we review evidence that autoimmune T cells can help heal damaged tissues, indicating that natural autoimmunity could also be a cause of health.

Topics: Animals; Autoantigens; Autoimmunity; Central Nervous System; Chemotaxis, Leukocyte; Contusions; Encephalomyelitis, Autoimmune, Experimental; Humans; Injections, Intraperitoneal; Lymphocyte Activation; Models, Immunological; Myelin Basic Protein; Myelin Sheath; Neovascularization, Physiologic; Nerve Crush; Nerve Degeneration; Optic Nerve Injuries; Regeneration; Spinal Cord Injuries; T-Lymphocyte Subsets; Wound Healing

Spinal cord injury (SCI) can cause a variety of cells apoptosis, neurodegeneration, and eventually permanent paralysis. This study aimed to examine whether transplanting human umbilical cord mesenchymal stem cells (hucMSCs) can promote locomotor function recovery, reduce apoptosis and inhibit demyelination in SCI models.. Rats were allocated into Sham group (spinal cord exposure only), SCI + PBS group (spinal cord impact plus phosphate-buffered saline (PBS) injections), SCI + hucMSCs group (spinal cord impact plus hucMSCs injections) groups. Behavioral tests, Basso-Beattie-Bresnahan locomotion scores (BBB scores), were carried out at 0, 3, 7, 14, 21, 28 days after SCI surgery. Hematoxylin-eosin staining observed spinal cord morphology. Nissl staining detected the number of nissl bodies. Myelin basic protein (MBP) and oligodendrocyte (CNPase) were examed by immunohistochemical staining. The apoptosis of oligodendrocyte and neurons were detected by immunofluorescence.. The 28-day behavioral test showed that the BBB score of rats in the SCI + hucMSCs group increased significantly, comparing to the SCI + PBS group. The numbers of nissl bodies and myelin sheath in the damaged area of SCI + hucMSCs group were also significantly increased compared to the SCI + PBS group. HucMSCs transplanting decreased the expression of protein level of Caspase-3 and Bax and increased the Bcl-2, MBP and CNPase, rescued the apoptosis of neurons and the oligodendrocyte.. These results showed that hucMSCs can improve motor function, tissue repairing and reducing apoptosis in SCI rats.

Topics: Animals; Apoptosis; Blood-Brain Barrier; Demyelinating Diseases; Female; Gene Expression Regulation; Humans; Locomotion; Male; Mesenchymal Stem Cell Transplantation; Myelin Basic Protein; Neurons; Oligodendroglia; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries

Historically, the membrane attack complex, composed of complement components C5b-9, has been connected to lytic cell death and implicated in secondary injury after a CNS insult. However, studies to date have utilized either non-littermate control rat models, or mouse models that lack significant C5b-9 activity. To investigate what role C5b-9 plays in spinal cord injury and recovery, we generated littermate PVG C6 wildtype and deficient rats and tested functional and histological recovery after moderate contusion injury using the Infinite Horizon Impactor. We compare the effect of C6 deficiency on recovery of locomotor function and histological injury parameters in PVG rats under two conditions: (1) animals maintained as separate C6 WT and C6-D homozygous colonies; and (2) establishment of a heterozygous colony to generate C6 WT and C6-D littermate controls. The results suggest that maintenance of separate homozygous colonies is inadequate for testing the effect of C6 deficiency on locomotor and histological recovery after SCI, and highlight the importance of using littermate controls in studies involving genetic manipulation of the complement cascade.

Topics: Animals; Behavior, Animal; Complement C6; Complement Membrane Attack Complex; Disease Models, Animal; Female; Genotype; Glial Fibrillary Acidic Protein; Gray Matter; Hereditary Complement Deficiency Diseases; Heterozygote; Locomotion; Male; Myelin Basic Protein; Rats, Mutant Strains; Selective Breeding; Spinal Cord Injuries; Thoracic Vertebrae; White Matter

Apoptosis plays an important role in various diseases, including spinal cord injury (SCI). Hyperbaric oxygen (HBO) and erythropoietin (EPO) promote the recovery from SCI, but the relationship between apoptosis and the combination therapeutic effect is not completely clear.. The purpose of this study was to investigate the effects of HBO and EPO on SCI and the mechanisms that underlie their therapeutic benefits.. The study was designed to explore the effects of HBO and EPO on SCI through a randomized controlled trial.. Sixty young developing female Sprague-Dawley rats were randomly divided into groups of 12 rats receiving sham, SCI, HBO, EPO, or HBO plus EPO. The SCI model was modified with the Allen method to better control consistency. HBO was performed for 1 hour per day for a total of 21 days, and EPO was given once per week for a total of 3 weeks. Both methods were performed 2 hours after SCI. Locomotor function was evaluated with the 21-point Basso-Beattie-Bresnahan Locomotor Rating Scale, an inclined-plane test, and a footprint analysis. All genes were detected by Western blotting and immunohistochemistry. The level of cell apoptosis was determined by Hoechst staining.. The results showed that HBO and EPO promoted the recovery of locomotor function in the hind limbs of rats by inhibiting the apoptosis of neurons. During this period, the expression of B-cell lymphoma/leukemia 2 protein (Bcl-2) increased significantly, whereas the expression of Bcl-2-associated X protein (Bax) and cleaved caspase 3 decreased significantly, indicating the inhibition of apoptosis. Meanwhile, the expression of G protein-coupled receptor 17 decreased, and that of myelin basic protein increased, suggesting that there may be a potential connection between demyelination and neuronal apoptosis.. The limitations of the study include deviations in the preparation of SCI models; lack of reverse validation of molecular mechanisms; absence of in vitro cell experiments; and only one time point after SCI was studied.. HBO and EPO treatments are beneficial for SCI, especially when the 2 therapies are combined.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase 3; Combined Modality Therapy; Drug Administration Schedule; Erythropoietin; Female; Hyperbaric Oxygenation; Locomotion; Myelin Basic Protein; Neurons; Proto-Oncogene Proteins c-bcl-2; Random Allocation; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Recovery of Function; Spinal Cord Injuries

To investigate the recovery effect of exosomes derived from micro ribonucleic acid (miR)-133b-modified adipose-derived stem cells (ADSCs) on neurological function after spinal cord injury (SCI) and its mechanism.. The SCI model of rats was used and divided into the following 5 groups: sham-operation group, 4 d SCI group, phosphate-buffered saline (PBS) group, miR-control group and miR-133b group. At 96 h after operation, rats were euthanatized, and spinal tissues were removed. Next, the level of miR-133b was detected via reverse transcription-polymerase chain reaction (RT-PCR), and the expression of RhoA protein was measured via Western blotting. Moreover, expressions of proteins associated with the axon regeneration pathway, including phosphorylated-cAMP-response element binding protein (p-CREB), CREB, phosphorylated-signal transducer and activator of transcription 3 (p-STAT3) and STAT3, along with expressions of neurofilament (NF), growth associated protein 43 (GAP43), glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP), were tested by Western blotting and immunofluorescence staining.. The miR-133b mimics significantly upregulated the expression of miR-133b in adipose-derived stem cells (ADSCs), compared to blank group (p<0.05). The expression of miR-133b was significantly decreased in 4 d SCI group compared with that in sham-operation group (p<0.001). The RhoA expression was statistically increased in 4 d SCI group compared with that in sham-operation group (p<0.001), and it was partially impaired by using miR-133b compared with that in 4 d SCI group (p<0.001). Expressions of NF, GAP43, GFAP and MBP were remarkably higher in 4 d SCI group than those in sham-operation group (p<0.01), and they were also significantly increased in miR-133b group than those in 4 d SCI group (p<0.01). Besides, our data showed a significant increase of p-CREB/CREB, p-STAT3/STAT3, NF, GAP-43, GFAP and MBP in miR-133b group compared to those in 4 d SCI group, with statistical reduction of RhoA (p<0.05).. We showed that exosomes derived from miR-133b-modified ADSCs can significantly promote the recovery of neurological function of SCI animals through affecting the signaling pathway related to axon regeneration and expressions of NF, GAP-43, GFAP and MBP.

Topics: Adipose Tissue; Animals; Axons; Disease Models, Animal; Exosomes; GAP-43 Protein; Glial Fibrillary Acidic Protein; Humans; Male; Mesenchymal Stem Cells; MicroRNAs; Myelin Basic Protein; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Recovery of Function; Signal Transduction; Spinal Cord Injuries; Up-Regulation

Accumulating evidence indicates that a suitable scaffold designed for the spinal cord injury (SCI) was needed to enhance the survival of transplanted Bone mesenchymal stem cells (BMSCs) and to promote nerve regeneration. The current study was aimed to evaluate the effect of the porous silk fibroin scaffold (PSFSs) seeded with BMSCS on nerve regeneration, myelination and functional recovery after SCI. We previously demonstrated that the PSFSs could bridge defected nerve with nerve fibers when applied to the transected spinal cord. And we found that BMSCs were adhered to the scaffold closely and have good biological compatibility with PSFSs. PSFSs seeded with BMSCs exhibited significant improvement in complete transverse thoracic SCI rat models. Flow cytometric assay also indicated that BMSCs grew well and adhered closely to the surface of the scaffold. The Basso-Beattie-Bresnehan (BBB) scores at each time point showed that the hindlimb motor function of each transplant group was also significantly restored. Meanwhile, growth associated protein 43 (GAP-43)marker of damaged axons regeneration and myelin basic protein (MBP) marker of maintaining the myelin structural and functional integrity, all markedly increased in PSFSs seeded with BMSCs models. Together, these results demonstrated that transplantation of PSFSs seeded with BMSCS could promote the nerve regeneration, myelination and functional recovery after SCI.

Topics: Animals; Fibroins; GAP-43 Protein; Mesenchymal Stem Cell Transplantation; Microscopy, Electron, Scanning; Motor Activity; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Porosity; Rats, Sprague-Dawley; Spinal Cord Injuries; Tissue Scaffolds

Human natural killer-1 (HNK-1) cell antigen is a glycan epitope involved in several neural events, such as neuritogenesis, myelination, synaptic plasticity and regeneration of the nervous system after injury. We have recently identified the small organic compound ursolic acid (UA) as a HNK-1 mimetic with the aim to test its therapeutic potential in the central nervous system. UA, a plant-derived pentacyclic triterpenoid, is well known for its multiple biological functions, including neuroprotective, antioxidant and anti-inflammatory activities. In the present study, we evaluated its functions in a mouse model of spinal cord injury (SCI) and explored the molecular mechanisms underlying its positive effects. Oral administration of UA to mice 1 h after SCI and thereafter once daily for 6 weeks enhanced the regaining of motor functions and axonal regrowth, and decreased astrogliosis. UA administration decreased levels of proinflammatory markers, including interleukin-6 and tumor necrosis factor-α, in the injured spinal cord at the acute phase of inflammation and activated the mitogen-activated protein kinase and phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin pathways in the injured spinal cord. Taken together, these results suggest that UA may be a candidate for treatment of nervous system injuries.

Topics: Animals; Axons; CD57 Antigens; Cytokines; Drug Evaluation, Preclinical; Female; Intermediate Filaments; Mice, Inbred C57BL; Motor Activity; Myelin Basic Protein; Oligosaccharides; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Spinal Cord Injuries; TOR Serine-Threonine Kinases; Triterpenes; Ursolic Acid

Neural cell adhesion molecule L1 contributes to nervous system development and maintenance by promoting neuronal survival, neuritogenesis, axonal regrowth/sprouting, myelination, and synapse formation and plasticity. L1 also enhances recovery after spinal cord injury and ameliorates neurodegenerative processes in experimental rodent models. Aiming for clinical translation of L1 into therapy we screened for and functionally characterized in vitro the small organic molecule phenelzine, which mimics characteristic L1 functions.. The present study was designed to evaluate the potential of this compound in vivo in a mouse model of spinal cord injury.. In mice, intraperitoneal injection of phenelzine immediately after severe thoracic compression, and thereafter once daily for 6 weeks, improved hind limb function, reduced astrogliosis and promoted axonal regrowth/sprouting at 4 and 5 weeks after spinal cord injury compared to vehicle control-treated mice. Phenelzine application upregulated L1 expression in the spinal cord and stimulated the cognate L1-mediated intracellular signaling cascades in the spinal cord tissue. Phenelzine-treated mice showed decreased levels of pro-inflammatory cytokines, such as interleukin-1β, interleukin-6, and tumor necrosis factor-α in the injured spinal cord during the acute phase of inflammation.. This study provides new insights into the role of phenelzine in L1-mediated neural functions and modulation of inflammation. The combined results raise hopes that phenelzine may develop into a therapeutic agent for nervous system injuries.

Topics: Analysis of Variance; Animals; Biogenic Amines; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Gene Expression Regulation; Locomotion; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Monoamine Oxidase Inhibitors; Myelin Basic Protein; Neural Cell Adhesion Molecule L1; Phenelzine; Recovery of Function; Spinal Cord Injuries; Time Factors; TOR Serine-Threonine Kinases

Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals' neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.

Topics: Animals; Axons; Biocompatible Materials; Feasibility Studies; Female; Green Fluorescent Proteins; Mice; Mice, Transgenic; Myelin Basic Protein; Nerve Regeneration; Polyglactin 910; Spinal Cord Injuries; Spinal Nerves; Tissue Scaffolds

Neurotoxic A1 astrocytes are induced by inflammation after spinal cord injury (SCI), and the inflammation-related Nuclear Factor Kappa B (NFκB) pathway may be related to A1-astrocyte activation. Mesenchymal stem cell (MSC) transplantation is a promising therapy for SCI, where transplanted MSCs exhibit anti-inflammatory effects by downregulating proinflammatory factors, such as Tumor Necrosis Factor (TNF)-α and NFκB. MSC-exosomes (MSC-exo) reportedly mimic the beneficial effects of MSCs. Therefore, in this study, we investigated whether MSCs and MSC-exo exert inhibitory effects on A1 astrocytes and are beneficial for recovery after SCI.. The effects of MSC and MSC-exo on SCIinduced A1 astrocytes, and the potential mechanisms were investigated in vitro and in vivo using immunofluorescence and western blot. In addition, we assessed the histopathology, levels of proinflammatory cytokines and locomotor function to verify the effects of MSC and MSC-exo on SCI rats.. MSC or MSC-exo co-culture reduced the proportion of SCIinduced A1 astrocytes. Intravenously-injected MSC or MSC-exo after SCI significantly reduced the proportion of A1 astrocytes, the percentage of p65 positive nuclei in astrocytes, and the percentage of TUNEL-positive cells in the ventral horn. Additionally, we observed decreased lesion area and expression of TNFα, Interleukin (IL)-1α and IL-1β, elevated expression of Myelin Basic Protein (MBP), Synaptophysin (Syn) and Neuronal Nuclei (NeuN), and improved Basso, Beattie & Bresnahan (BBB) scores and inclined-plane-test angle. In vitro assay showed that MSC and MSC-exo reduced SCI-induced A1 astrocytes, probably via inhibiting the nuclear translocation of the NFκB p65.. MSC and MSC-exo reduce SCI-induced A1 astrocytes, probably via inhibiting nuclear translocation of NFκB p65, and exert antiinflammatory and neuroprotective effects following SCI, with the therapeutic effect of MSCexo comparable with that of MSCs when applied intravenously.

Topics: Animals; Astrocytes; Cells, Cultured; Cytokines; Down-Regulation; Exosomes; Fluorescent Dyes; I-kappa B Kinase; Locomotion; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Microscopy, Fluorescence; Myelin Basic Protein; Phosphorylation; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Transcription Factor RelA

Tacrine is a small organic compound that was discovered to mimic the functions of the neural cell adhesion molecule L1 by promoting the cognate functions of L1 in vitro, such as neuronal survival, neuronal migration, neurite outgrowth, and myelination. Based on studies indicating that L1 enhances functional recovery in different central and peripheral nervous system disease paradigms of rodents, it deemed interesting to investigate the beneficial role of tacrine in the attractive zebrafish animal model, by evaluating functional recovery after spinal cord injury. To this aim, larval and adult zebrafish were exposed to tacrine treatment after spinal cord injury and monitored for locomotor recovery and axonal regrowth. Tacrine promoted the rapid recovery of locomotor activities in both larval and adult zebrafish, enhanced regrowth of severed axons and myelination, and reduced astrogliosis in the spinal cords. Tacrine treatment upregulated the expression of L1.1 (a homolog of the mammalian recognition molecule L1) and enhanced the L1.1-mediated intracellular signaling cascades in the injured spinal cords. These observations lead to the hope that, in combination with other therapeutic approaches, this old drug may become a useful reagent to ameliorate the deficits resulting from acute and chronic injuries of the mammalian nervous system.

Topics: Animals; Creatine Kinase; Gliosis; Larva; Lasers; Locomotion; Myelin Basic Protein; Neural Cell Adhesion Molecule L1; Phosphatidylinositol 3-Kinases; Phosphorylation; Photons; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Recovery of Function; RNA, Messenger; Signal Transduction; Small Molecule Libraries; Spinal Cord; Spinal Cord Injuries; Tacrine; Time Factors; Tumor Suppressor Protein p53; Zebrafish

Olig2 is one of the most critical factors during CNS development, which belongs to b-HLH transcription factor family. Previous reports have shown that Olig2 regulates the remyelination processes in CNS demyelination diseases models. However, the role of Olig2 in contusion spinal cord injury (SCI) and the possible therapeutic effects remain obscure. This study aims to investigate the effects of overexpression Olig2 by lentivirus on adult spinal cord injury rats.. Injection of Lenti-Olig2 significantly increased the number of oligodendrocytes lineage cells and enhanced myelination after SCI. More importantly, the introduction of Olig2 greatly improved hindlimb locomotor performances. Other oligodendrocyte-related transcription factors, which were downregulated or upregulated after injury, were reversed by Olig2 induction.. Our findings provided the evidence that overexpression Olig2 promotes myelination and locomotor recovery of contusion SCI, which gives us more understanding of Olig2 on spinal cord injury treatment.

Topics: Animals; Disease Models, Animal; Evoked Potentials, Motor; Evoked Potentials, Somatosensory; Exploratory Behavior; Female; Gangliosides; Gene Expression Regulation; Hindlimb; Ki-67 Antigen; Lentivirus; Locomotion; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries

Neural progenitor cells (NPCs) grafted to sites of spinal cord injury (SCI) extend numerous axons over long distances and form new synaptic connections with host neurons. In the present study we examined the myelination of axons emerging from NPC grafts. Rat embryonic day 14 (E14) multipotent NPCs constitutively expressing GFP were grafted into adult C5 spinal cord hemisection lesions; 3months later we examined graft-derived axonal diameter and myelination using transmission electron microscopy. 104 graft-derived axons were characterized. Axon diameter ranged from 0.15 to 1.70μm, and 24% of graft-derived axons were myelinated by host oligodendrocytes caudal to the lesion. The average diameter of myelinated axons (0.72±0.3μm) was significantly larger than that of non-myelinated axons (0.61±0.2μm, p<0.05). Notably, the G-ratio of myelinated graft-derived axons (0.77±0.01) was virtually identical to that of the normal, intact spinal cord described in published reports. These findings indicate that axons emerging from early stage neural grafts into the injured spinal cord recapitulate both the small/medium size range and myelin thickness of intact spinal cord axons.

Topics: Adenomatous Polyposis Coli Protein; Animals; Axons; Cells, Cultured; Embryo, Mammalian; Female; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Intercellular Signaling Peptides and Proteins; Microscopy, Electron, Scanning Transmission; Myelin Basic Protein; Myelin Sheath; Neural Stem Cells; Rats; Rats, Inbred F344; Rats, Transgenic; Spinal Cord Injuries

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein-coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss-of-function were accompanied by increased numbers of Olig2- and CC1-positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.

Topics: Animals; Animals, Newborn; Autophagy-Related Proteins; Brain-Derived Neurotrophic Factor; Cyclic AMP; Gene Expression Regulation, Developmental; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Mice; Mice, Transgenic; Myelin Basic Protein; Myelin Proteolipid Protein; Myelin Sheath; Nogo Proteins; Oligodendroglia; Receptor, PAR-2; Spinal Cord; Spinal Cord Injuries; Stem Cells

Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub-ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory-related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid-cervical transplantation of SVZ-derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non-GFP rats. Whole body plethysmography conducted at 4 and 8wks post-transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P=0.003) or hypercapnic respiratory challenge (P=0.019). Phrenic nerve output was assessed at 8wks post-transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P<0.001). Stereological analyses at 8wks post-injury indicated survival of ~50% of transplanted NPCs with ~90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P<0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ-derived NPCs can improve respiratory recovery following high cervical SCI.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Animals, Newborn; CD11b Antigen; Cervical Vertebrae; Disease Models, Animal; Female; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Hypoxia; Lateral Ventricles; Male; Myelin Basic Protein; Neural Stem Cells; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Recovery of Function; Respiration Disorders; Spinal Cord Injuries

The adult mammalian forebrain comprises two distinct populations of neural stem cells (NSCs): rare, Oct4 positive, primitive NSCs (pNSCs) and definitive NSC (dNSC) which are more abundant and express GFAP. The pNSCs are upstream of the dNSCs in the neural stem cell lineage. Herein we show that pNSC and dNSC populations can also be isolated from the developing and adult spinal cord. Spinal cord derived pNSCs are similarly rare, Oct4 expressing cells that are responsive to leukemia inhibitory factor and dNSCs are 4-5X more abundant and express GFAP. We demonstrate that myelin basic protein (MBP) is inhibitory to both pNSC and dNSC derived colony formation. Similar to what is seen in the adult forebrain following injury, spinal cord injury results in a significant increase in the size of the dNSC and pNSC pools. Hence, both primitive and definitive neural stem cells can be isolated from along the embryonic and adult neuraxis in vivo and their behavior is regulated by MBP and injury. Stem Cells 2017;35:485-496.

Topics: Aging; Animals; Cell Proliferation; Cell Separation; Colony-Forming Units Assay; Forkhead Transcription Factors; Glial Fibrillary Acidic Protein; Mice, Mutant Strains; Mice, Transgenic; Myelin Basic Protein; Myelin Sheath; Nestin; Neural Stem Cells; Octamer Transcription Factor-3; Spheroids, Cellular; Spinal Cord; Spinal Cord Injuries

Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction attributable to both mechanical damage and secondary biochemical and physiological responses. Upregulation of cell cycle proteins occurs in both neurons and glia after SCI and may contribute to these changes. Increased cell cycle protein is associated with neuronal and oligodendroglial apoptosis, reactive astrogliosis, glial scar formation, and microglial activation. Here, using lentiviral vectors (LV), we induced the expression of the cyclin-dependent kinase (CDK) inhibitor p27

Topics: Animals; Apoptosis; Astrocytes; Caspase 3; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p27; Enzyme Activation; Genetic Vectors; Inflammation Mediators; Lentivirus; Locomotion; Male; Microglia; Myelin Basic Protein; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Transgenes

Oligodendrocyte progenitor cell (OPC) differentiation is an important therapeutic target to promote remyelination in multiple sclerosis (MS). We previously reported hyperphosphorylated and aggregated microtubule-associated protein tau in MS lesions, suggesting its involvement in axonal degeneration. However, the influence of pathological tau-induced axonal damage on the potential for remyelination is unknown. Therefore, we investigated OPC differentiation in human P301S tau (P301S-htau) transgenic mice, both in vitro and in vivo following focal demyelination. In 2-month-old P301S-htau mice, which show hyperphosphorylated tau in neurons, we found atrophic axons in the spinal cord in the absence of prominent axonal degeneration. These signs of early axonal damage were associated with microgliosis and an upregulation of IL-1β and TNFα. Following in vivo focal white matter demyelination we found that OPCs differentiated more efficiently in P301S-htau mice than wild type (Wt) mice. We also found an increased level of myelin basic protein within the lesions, which however did not translate into increased remyelination due to higher susceptibility of P301S-htau axons to demyelination-induced degeneration compared to Wt axons. In vitro experiments confirmed higher differentiation capacity of OPCs from P301S-htau mice compared with Wt mice-derived OPCs. Because the OPCs from P301S-htau mice do not ectopically express the transgene, and when isolated from newborn mice behave like Wt mice-derived OPCs, we infer that their enhanced differentiation capacity must have been acquired through microenvironmental priming. Our data suggest the intriguing concept that damaged axons may signal to OPCs and promote their differentiation in the attempt at rescue by remyelination.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; CD11b Antigen; Cell Death; Cell Differentiation; Cell Movement; Cell Proliferation; Demyelinating Diseases; Disease Models, Animal; Gene Expression Regulation; Humans; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mutation; Myelin Basic Protein; Nerve Tissue Proteins; Neurons; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Spinal Cord Injuries; Stem Cells; tau Proteins

This study aims to investigate the therapeutic effects and mechanisms of x-ray treatment on rats following spinal cord injury (SCI).. Forty-six female Sprague-Dawley rats were subjected to spinal cord injury using the modified Allen weight-drop method. The animals were randomly divided into six groups. Two of the animal groups were irradiated with 10 Gy at the lesion site; another two groups were irradiated with 20 Gy; and the last two groups without irradiation were regarded as the sham group. One of the each of two animal groups was euthanized at different time points at 4 and 12 weeks, respectively, after irradiation. Spinal cord calluses were assessed using kinology and electrophysiology and histology methods.. In all of the groups, the neurofilament (NF) counts at 14 weeks were found to be higher than that at 6 weeks after SCI. Both 10-Gy irradiated and 20-Gy irradiated groups were higher than those of the sham group at each time point (P < 0.05). The myelin basic protein (MBP) count decreased at 14 weeks after SCI in the irradiated groups (P < 0.05) but increased at 14 weeks in the sham group (P < 0.05). Furthermore, the MBP count of the irradiated groups was lower than that of the sham group at 14 weeks (P < 0.05). The glial fibrillary acidic protein (GFAP) and Nogo-A counts at 14 weeks were higher than those at 6 weeks in all the groups (P < 0.05), and there was no statistical significance with kinology and electrophysiology tests in all groups.. A self-repair mechanism exists after spinal cord injury, which lasts at least 14 weeks. X-ray therapy promotes the regeneration of the spinal cord system after injury.

Topics: Animals; Disease Models, Animal; Dose-Response Relationship, Radiation; Female; Glial Fibrillary Acidic Protein; Intermediate Filaments; Myelin Basic Protein; Nerve Regeneration; Nogo Proteins; Radiotherapy Dosage; Rats, Sprague-Dawley; Spinal Cord Injuries

Myelin basic protein (MBP) activated T cells (MBP-T) play an important role in the damage and repair process of the central nervous system (CNS). However, whether these cells play a beneficial or detrimental role is still a matter of debate. Although some studies showed that MBP-T cells are mainly helper T (Th) cells, their subtypes are still not very clear. One possible explanation for MBP-T immunization leading to conflicting results may be the different subtypes of T cells are responsible for distinct effects. In this study, the Th1 and Th2 type MBP-T cells (MBP-Th1 and -Th2) were polarized in vitro, and their effects on the local immune microenvironment and tissue repair of spinal cord injury (SCI) after adoptive immunization were investigated. In MBP-Th1 cell transferred rats, the high levels of pro-inflammatory cells (Th1 cells and M1 macrophages) and cytokines (IFN-γ, TNF-α, -β, IL-1β) were detected in the injured spinal cord; however, the anti-inflammatory cells (Th2 cells, regulatory T cells, and M2 macrophages) and cytokines (IL-4, -10, and -13) were found in MBP-Th2 cell transferred animals. MBP-Th2 cell transfer resulted in decreased lesion volume, increased myelination of axons, and preservation of neurons. This was accompanied by significant locomotor improvement. These results indicate that MBP-Th2 adoptive transfer has beneficial effects on the injured spinal cord, in which the increased number of Th2 cells may alter the local microenvironment from one primarily populated by Th1 and M1 cells to another dominated by Th2, Treg, and M2 cells and is conducive for SCI repair.

Topics: Adoptive Transfer; Analysis of Variance; Animals; Cytokines; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Macrophages; Motor Activity; Motor Disorders; Myelin Basic Protein; Nitric Oxide Synthase Type II; Psychomotor Performance; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells

Spinal cord injury (SCI) is a debilitating event with multiple mechanisms of degeneration leading to life-long paralysis. Biomaterial strategies, including bridges that span the injury and provide a pathway to reconnect severed regions of the spinal cord, can promote partial restoration of motor function following SCI. Axon growth through the bridge is essential to characterizing regeneration, as recovery can occur via other mechanisms such as plasticity. Quantitative analysis of axons by manual counting of histological sections can be slow, which can limit the number of bridge designs evaluated. In this study, we report a semi-automated process to resolve axon numbers in histological sections, which allows for efficient analysis of large data sets.. Axon numbers were estimated in SCI cross-sections from animals implanted with poly(lactide co-glycolide) (PLG) bridges with multiple channels for guiding axons. Immunofluorescence images of histological sections were filtered using a Hessian-based approach prior to threshold detection to improve the signal-to-noise ratio and filter out background staining associated with PLG polymer.. Semi-automated counting successfully recapitulated average axon densities and myelination in a blinded PLG bridge implantation study.. Axon counts obtained with the semi-automated technique correlated well with manual axon counts from blinded independent observers across sections with a wide range of total axons.. This semi-automated detection of Hessian-filtered axons provides an accurate and significantly faster alternative to manual counting of axons for quantitative analysis of regeneration following SCI.

Topics: Analysis of Variance; Animals; Axons; Biocompatible Materials; Electronic Data Processing; Female; Lactic Acid; Mice; Mice, Inbred C57BL; Microscopy, Electron; Myelin Basic Protein; Nerve Regeneration; Neurofilament Proteins; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Spinal Cord Injuries

Emerging evidence indicates that microglia activation plays an important role in spinal cord injury (SCI) caused by trauma. Studies have found that inhibiting the Rho/Rho-associated protein kinase (ROCK) signaling pathway can reduce inflammatory cytokine production by microglia. In this study, Western blotting was conducted to detect ROCK2 expression after the SCI; the ROCK Activity Assay kit was used for assay of ROCK pathway activity; microglia morphology was examined using the CD11b antibody; electron microscopy was used to detect microglia phagocytosis; TUNEL was used to detect tissue cell apoptosis; myelin staining was performed using an antibody against myelin basic protein (MBP); behavioral outcomes were evaluated according to the methods of Basso, Beattie, and Bresnahan (BBB). We observed an increase in ROCK activity and microglial activation after SCI. The microglia became larger and rounder and contained myelin-like substances. Furthermore, treatment with fasudil inhibited neuronal cells apoptosis, alleviated demyelination and the formation of cavities, and improved motor recovery. The experimental evidence reveals that the ROCK inhibitor fasudil can regulate microglial activation, promote cell phagocytosis, and improve the SCI microenvironment to promote SCI repair. Thus, fasudil may be useful for the treatment of SCI.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Apoptosis; Male; Microglia; Myelin Basic Protein; Myelin Sheath; Phagocytosis; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; rho-Associated Kinases; Spinal Cord Injuries

The cytokine, interleukin (IL)-25, is thought to be critically involved in inducing a type 2 immune response which may contribute to regeneration after central nervous system (CNS) trauma. We investigated whether applying recombinant IL-25, locally or systemically, in a mouse model of spinal cord injury (SCI) improves functional and histological recovery.. Repeated systemic administration of IL-25 did not influence functional recovery following SCI. In contrast, a single local administration of IL-25 significantly worsened locomotor outcome, which was evident from a decreased Basso mouse scale (BMS) score compared with phosphate-buffered saline (PBS)-treated controls. This was accompanied by a significant increase in lesion size, demyelination, and T helper cell infiltration.. These data show for the first time that IL-25 is either ineffective when applied systemically or detrimental to spinal cord recovery when applied locally. Our findings question the potential neuroprotective role of IL-25 following CNS trauma.

Topics: Animals; Astrocytes; Calcium-Binding Proteins; CD4 Antigens; Disease Models, Animal; Female; Glial Fibrillary Acidic Protein; Interleukins; Locomotion; Macrophages; Mice; Mice, Inbred BALB C; Microfilament Proteins; Myelin Basic Protein; Recovery of Function; Spinal Cord Injuries; T-Lymphocytes; Time Factors

After spinal cord (SC)-injury, a non-modulated immune response contributes to the damage of neural tissue. Protective autoimmunity (PA) is a T cell mediated, neuroprotective response induced after SC-injury. Immunization with neural-derived peptides (INDP), such as A91, has shown to promote-in vitro-the production of neurotrophic factors. However, the production of these molecules has not been studied at the site of injury.. In order to evaluate these issues, we performed four experiments in adult female Sprague-Dawley rats. In the first one, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) concentrations were evaluated at the site of lesion 21 days after SC-injury. BDNF and NT-3 were significantly increased in INDP-treated animals. In the second experiment, proliferation of anti-A91 T cells was assessed at chronic stages of injury. In this case, we found a significant proliferation of these cells in animals subjected to SC-injury + INDP. In the third experiment, we explored the amount of BDNF and NT3 at the site of injury in the chronic phase of rats subjected to either SC-contusion (SCC; moderate or severe) or SC-transection (SCT; complete or incomplete). The animals were treated with INDP immediately after injury. Rats subjected to moderate contusion or incomplete SCT showed significantly higher levels of BDNF and NT-3 as compared to PBS-immunized ones. In rats with severe SCC and complete SCT, BDNF and NT-3 concentrations were barely detected. Finally, in the fourth experiment we assessed motor function recovery in INDP-treated rats with moderate SC-injury. Rats immunized with A91 showed a significantly higher motor recovery from the first week and up to 4 months after SC-injury.. The results of this study suggest that PA boosted by immunization with A91 after moderate SC-injury can exert its benefits even at chronic stages, as shown by long-term production of BDNF and NT-3 and a substantial improvement in motor recovery.

Topics: Animals; Autoimmunity; Brain-Derived Neurotrophic Factor; Chronic Disease; Disease Models, Animal; Female; Motor Activity; Myelin Basic Protein; Neurotrophin 3; Peptide Fragments; Random Allocation; Rats, Sprague-Dawley; Recovery of Function; Severity of Illness Index; Spinal Cord; Spinal Cord Injuries; Time Factors; Vaccination

The pathophysiology of spinal cord injury (SCI) has a classically bad prognosis. It has been demonstrated that human umbilical cord blood stem cells (hUCBSCs) and Melissa officinalis (MO) are useful for the prevention of neurological disease.. Thirty-six adult male rats were randomly divided into intact, sham, control (SCI), MO, hUCBSC, and MO-hUCBSC groups. Intraperitoneal injection of MO (150 mg/kg) was commenced 24 hr post-SCI and continued once a day for 14 days. Intraspinal grafting of hUCBSCs was commenced immediately in the next day. The motor and sensory functions of all animals were evaluated once a week after the commencement of SCI. Electromyography (EMG) was performed in the last day in order to measure the recruitment index. Immunohistochemistry, reverse transcription-polymerase chain reaction, and transmission electron microscopy evaluations were performed to determine the level of astrogliosis and myelination.. The results revealed that motor function (MO-hUCBSC: 15 ± 0.3, SCI: 8.2 ± 0.37, p < .001), sensory function (MO-hUCBSC: 3.57 ± 0.19, SCI: 6.38 ± 0.23, p < .001), and EMG recruitment index (MO-hUCBSC: 3.71 ± 0.18, SCI: 1.6 ± 0.1, p < .001) were significantly improved in the MO-hUCBSC group compared with SCI group. Mean cavity area (MO-hUCBSC: 0.03 ± 0.03, SCI: 0.07 ± 0.004, p < .001) was reduced and loss of lower motor neurons (MO-hUCBSC: 7.6 ± 0.43, SCI: 3 ± 0.12, p < .001) and astrogliosis density (MO-hUCBSC: 3.1 ± 0.15, SCI: 6.25 ± 1.42, p < 0.001) in the ventral horn of spinal cord were prevented in MO-hUCBSC group compared with SCI group.. The results revealed that the combination of MO and hUCBSCs in comparison with the control group has neuroprotective effects in SCI.

Topics: Animals; Antigens, CD; Bromodeoxyuridine; Cord Blood Stem Cell Transplantation; Disease Models, Animal; Electromyography; Evoked Potentials, Motor; Glial Fibrillary Acidic Protein; Humans; Male; Melissa; Myelin Basic Protein; Neurologic Examination; Neuroprotective Agents; Phosphopyruvate Hydratase; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Time Factors

Electroacupuncture (EA) has been used worldwide to treat demyelinating diseases, but its therapeutic mechanism is poorly understood. In this study, a custom-designed model of compressed spinal cord injury (CSCI) was used to induce demyelination. Zusanli (ST36) and Taixi (KI3) acupoints of adult rats were stimulated by EA to demonstrate its protective effect. At 14 days after EA, both locomotor skills and ultrastructural features of myelin sheath were significantly improved. Phenotypes of proliferating cells were identified by double immunolabeling of 5-ethynyl-2'-deoxyuridine with antibodies to cell markers: NG2 [oligodendrocyte precursor cell (OPC) marker], 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) (oligodendrocyte marker), and glial fibrillary acidic protein (GFAP) (astrocyte marker). EA enhanced the proliferation of OPCs and CNPase, as well as the differentiation of OPCs by promoting Olig2 (the basic helix-loop-helix protein) and attenuating Id2 (the inhibitor of DNA binding 2). EA could also improve myelin basic protein (MBP) and protect existing oligodendrocytes from apoptosis by inhibiting caspase-12 (a representative of endoplasmic reticulum stress) and cytochrome c (an apoptotic factor and hallmark of mitochondria). Therefore, our results indicate that the protective effect of EA on neural myelin sheaths is mediated via promotion of oligodendrocyte proliferation and inhibition of oligodendrocyte death after CSCI.

Topics: Animals; Astrocytes; Cell Death; Cell Differentiation; Demyelinating Diseases; Electroacupuncture; Glial Fibrillary Acidic Protein; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Oligodendroglia; Rats, Sprague-Dawley; Spinal Cord Injuries; Stem Cells

Vaccination of spinal cord injury (SCI) mice with myelin basic protein-derived peptide (A91) pulsed dendritic cells (DC) to enhance brain-derived neurotrophic factor and neurotrophin-3 (NT-3) expression in injured spinal cord.. To investigate the effect of A91-pulsed DC (A91-DC) on expression of neurotrophic factor in injured spinal cord.. SCI leads to progressive secondary tissue degeneration, and no satisfactory treatment is currently available. Accumulating evidence indicates that administration of neurotrophic factors to injured spinal cord is partially successful at promoting nerve tissue repair. However, most of strategy can cause secondary injury and limiting their wide clinical application.. Proliferation of T cells and the capability of CD4 T cells to secret neurotrophic factors were first measured in vitro to demonstrate the stimulus action of the A91-DC. In SCI mice model, enzyme-linked immunosorbent assay and immunofluorescence was employed to investigate the brain-derived neurotrophic factor and NT-3 expression in injured spinal cord. Furthermore, the neuroprotective effect of A91-DC in injured spinal cord was examined through histology measurement.. In this study, we demonstrated that A91-DC promoted the capability of T cells to secret neurotrophic factors and in the subacute phase of SCI. Moreover, vaccination with A91-DC enhanced the expression level of brain-derived neurotrophic factor and NT-3 and exerted neuroprotective effect in injured spinal cord.. The findings of study demonstrate that the therapeutic strategy of vaccination A91-DC is a potential minimally invasive approach that could provide strong neurotrophic factor support after SCI.

Topics: Animals; Brain-Derived Neurotrophic Factor; Dendritic Cells; Disease Models, Animal; Mice; Mice, Inbred BALB C; Myelin Basic Protein; Neuroprotective Agents; Neurotrophin 3; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Treatment Outcome; Vaccines

Positively charged oligo[poly(ethylene glycol) fumarate] (OPF+) scaffolds loaded with Schwann cells bridge spinal cord injury (SCI) lesions and support axonal regeneration in rat. The regeneration achieved is not sufficient for inducing functional recovery. Attempts to increase regeneration would benefit from understanding the effects of the scaffold and transplanted cells on lesion environment. We conducted morphometric and stereological analysis of lesions in rats implanted with OPF+ scaffolds with or without loaded Schwann cells 1, 2, 3, 4, and 8 weeks after thoracic spinal cord transection. No differences were found in collagen scarring, cyst formation, astrocyte reactivity, myelin debris, or chondroitin sulfate proteoglycan (CSPG) accumulation. However, when scaffold-implanted animals were compared with animals with transection injuries only, these barriers to regeneration were significantly reduced, accompanied by increased activated macrophages/microglia. This distinctive and regeneration permissive tissue reaction to scaffold implantation was independent of Schwann cell transplantation. Although the tissue reaction was beneficial in the short term, we observed a chronic fibrotic host response, resulting in scaffolds surrounded by collagen at 8 weeks. This study demonstrates that an appropriate biomaterial scaffold improves the environment for regeneration. Future targeting of the host fibrotic response may allow increased axonal regeneration and functional recovery.

Topics: Animals; Astrocytes; Calcium-Binding Proteins; Female; Fumarates; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Macrophages; Microfilament Proteins; Microglia; Myelin Basic Protein; Phenotype; Polyethylene Glycols; Prosthesis Implantation; Proteoglycans; Rats, Sprague-Dawley; Schwann Cells; Spinal Cord Injuries; Time Factors; Tissue Scaffolds

Oscillating field stimulation (OFS) has been used in attempts to treat spinal cord injury (SCI) and has been shown to improve remyelination after SCI in rats. However, some controversies regarding the effects of OFS have been presented in previous papers. Oligodendrocytes (OLs) are the main cell for remyelination and are derived from the differentiation of oligodendrocyte precursor cells (OPCs). To date, it has been unclear whether the differentiation of OPCs can be regulated by OFS. The goal of this study was to determine if OFS can improve the differentiation of OPCs and promote the recovery of neurological function after SCI in rats. Immature and mature OLs were observed in spinal cord slices through immunofluorescence staining. Levels of adenosine triphosphate (ATP) and the cytokine leukemia inhibitory factor (LIF) were detected by enzyme-linked immunosorbent assay (ELISA). Basso-Beattie-Bresnahan (BBB) scores and transcranial magnetic motor-evoked potentials (tcMMEPs) were used to evaluate the locomotor outcomes of rats after SCI. Our results showed a significant improvement in the differentiation of OPCs and the content of ATP and LIF in the injured spinal cord in the OFS group. Furthermore, BBB scores and tcMMEPs were significantly improved in the rats stimulated by OFS. These findings suggest that OFS can improve the differentiation of OPCs and promote the recovery of neurological function following SCI in rats.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Blood-Brain Barrier; Cell Differentiation; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Evoked Potentials, Motor; Female; Leukemia Inhibitory Factor; Motor Activity; Myelin Basic Protein; Nervous System Diseases; Oligodendroglia; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Stem Cells; Time Factors; Transcranial Direct Current Stimulation

Oligodendrocyte Progenitor Cells (OPCs) reside in the central nervous system (CNS) and are responsible for remyelinating axons after a spinal cord injury (SCI). However, the remyelination process is incomplete and abnormal due to the inability of OPCs to fully differentiate at the site of injury. In this study a newly developed injectable chitosan sponge crosslinked using guanosine 5'-diphosphate (GDP) was used to enhance OPC survival, attachment and differentiation. This purine-based biomaterial is the first of its kind and its inception was based on the growing body of literature concerning the role of purinergic signalling in the CNS. GDP-crosslinked chitosan sponges are rapidly-gelling and can be easily administered in situ using an injection system based on a double-lumen design. The chitosan sponges prompted OPC differentiation even in the presence of mitogens. Moreover, neurotrophin-3 (NT-3) was successfully entrapped in the sponges and a sustained release for up to 30 days was achieved. OPCs were shown to differentiate into mature oligodendrocytes that express myelin basic protein (MBP) when cultured on sponges containing NT-3. These findings, along with the suitable physicochemical and biological properties, make these sponges conducive to use as viable therapeutic agents for enhancing remyelination post-SCI.

Topics: Animals; Cell Differentiation; Chitosan; Drug Delivery Systems; Guanosine Diphosphate; Myelin Basic Protein; Nerve Regeneration; Oligodendroglia; Porifera; Purines; Spinal Cord Injuries; Stem Cells

Current treatments to restore neurological deficits caused by axonal disconnection following central nervous system (CNS) injury are extremely limited. Protein phosphatase 2A (PP2A), one of the main serine-threonine phosphatases in mammalian cells, dephosphorylates collapsin response mediator protein-2 (CRMP2) in the developing CNS. In our study, we found that the major CNS inhibiting substrates, including chondroitin sulfate proteoglycans (CSPGs) and myelin associated glycoproteins (MAG), activated epidermal growth factor receptor (EGFR), but inactivated PP2A and downstream CRMP2. Both EGFR inactivation and PP2A activation promoted axon elongation in vitro in the presence of inhibitory substrates. EGFR blockage by AG1478 selectively attenuated the inactive form of PP2A in pY307 phosphorylation, thus increasing PP2A activity. EGFR activation by EGF attenuated PP2A activity, whereas mutation of Y307 to phenylalanine abolished the effect. Furthermore, PP2A activity was down-regulated immediately after spinal cord injury (SCI) in rats. Chronic application of d-erythro-sphingosine (DES), the PP2A agonist, to spinal cord-lesioned rats enhanced the activity of this phosphatase and dephosphorylated CRMP2 around the lesion. PP2A activation induced significant axon sprouting in the lesioned spinal cord and promoted function recovery after SCI. These findings suggest that PP2A works downstream of EGFR and dephosphorylates CRMP2 after CNS injury. Therefore, therapies targeting PP2A may be effective following SCI.

Topics: Animals; Axons; Biotin; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Dextrans; Disease Models, Animal; Enzyme Inhibitors; ErbB Receptors; Male; Mutation; Myelin Basic Protein; Neurons; Phosphorylation; Protein Phosphatase 2; Quinazolines; Rats; Rats, Sprague-Dawley; Recovery of Function; Sphingosine; Spinal Cord Injuries; Tyrphostins

Thymosin β4 (Tβ4) has many physiological functions that are highly relevant to spinal cord injury (SCI), including neuronal survival, anti-inflammation, wound repair promotion, and angiogenesis. The present study investigated the therapeutic value of Tβ4 in SCI, with a focus on its neuroprotective, anti-inflammatory, and vasculoprotective properties. Tβ4 or a saline control was administered by intraperitoneal injection 30 min, 3 days, or 5 days after SCI with mild compression in rat. Locomotor recovery was tested with the Basso-Beattie-Bresnahan scale and a footprint analysis. All behavioral assessments were markedly improved with Tβ4 treatment. Histological examination at 7 days post injury showed that the numbers of surviving neurons and oligodendrocytes were significantly increased in Tβ4-treated animals compared to saline-treated controls. Levels of myelin basic protein, a marker of mature oligodendrocytes, in Tβ4-treated rats were 57.8% greater than those in saline-treated controls. The expression of ED1, a marker of activated microglia/macrophages, was reduced by 36.9% in the Tβ4-treated group compared to that of the saline-treated group. Tβ4 treatment after SCI was also associated with a significant decrease in pro-inflammatory cytokine gene expression and a significant increase in the mRNA levels of IL-10 compared to the control. Moreover, the size of lesion cavity delineated by astrocyte scar in the injured spinal cord was markedly reduced in Tβ4-treated animals compared to saline-treated controls. Given the known safety of Tβ4 in clinical trials and its beneficial effects on SCI recovery, the results of this study suggested that Tβ4 is a good candidate for SCI treatment in humans.

Topics: Animals; Cell Survival; Cytokines; Disease Models, Animal; Interleukin-10; Macrophages; Male; Microglia; Myelin Basic Protein; Neuroimmunomodulation; Neurons; Neuroprotective Agents; Oligodendroglia; Random Allocation; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Thymosin; Time Factors

To investigate the effects of chondroitinase ABC (ChABC) on axonal myelination and glial scar after spinal cord injury (SCI) in rats.. Seventy-two adult male Sprague Dawley rats were randomly assigned into ChABC treatment group (group A), saline treatment group (group B), and sham operation group (group C), 24 rats in each group. In groups and B, the SCI model was established with modified Allen's method and then the rats of groups A and B were administrated by subarachnoid injection of 6 microL ChABC (1 U/mL) and saline respectively at 1 hour after injury and every day for 1 week; the rats of group C served as control, which canal was opened without damage to spinal cord. At 1, 7, 14, and 28 days after operation, the locomotor functions were evaluated according to the Basso-Beattie-Bresnahan (BBB) score scale; and the spinal cord samples were harvested for HE staining, Nissl staining, and immunohistochemistry analysis to detect the change of myelin basic protein (MBP), growth associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP) of the injured spinal cord.. At different time points, the BBB score of group C was significantly higher than those of groups A and B (P < 0.05), and the BBB score of group was significantly better than that of group B at 14 and 28 days after operation (P < 0.05). HE staining and Nissl staining showed that the morphous and the neuron number of the remainant injured spinal cord in group A were better than those in group B. The integral absorbance (IA) values of MBP and GAP-43 and the positive area of GFAP after SCI in groups A and B were significantly higher than those in group C at different time points (P < 0.05), and the IA values of MBP and GAP-43 were significantly higher in group A than those in group B at 7, 14, and 28 days after operation (P < 0.05), but the positive area of GFAP was significantly smaller in group A than that in group B (P < 0.05).. The ChABC can effectively improve the microenvironment of the injured spinal cord of rats, enhance the expressions of MBP and GAP-43, and inhibit the expression of GFAP, which promotes the axonal regeneration and myelination, attenuate glial scar formation, and promote the recovery of nerve function.

Topics: Animals; Astrocytes; Axons; Chondroitin ABC Lyase; Cicatrix; Disease Models, Animal; GAP-43 Protein; Glial Fibrillary Acidic Protein; Immunohistochemistry; Male; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries

The endoplasmic reticulum (ER) stress response (ERSR) is activated to maintain protein homeostasis or induce apoptosis in the ER in response to distinct cellular insults including hypoxia, inflammation, and oxidative damage. Recently, we showed ERSR activation in a mouse model of a contusive spinal cord injury (SCI) and an improved hindlimb locomotor function following SCI when the pro-apoptotic arm of ERSR was genetically inhibited. The objective of the current study was to explore if the pharmacological enhancement of the homeostatic arm of the ERSR pathway can improve the functional outcome after SCI. Salubrinal enhances the homeostatic arm of the ERSR by increasing phosphorylation of eIF2α. Salubrinal significantly enhanced the levels of phosphorylated eIF2α protein and modulated the downstream ERSR effectors assessed at the lesion epicenter 6h post-SCI. Hindlimb locomotion showed significant improvement in animals treated with salubrinal. Treadmill-based-gait assessment showed a significant increase in maximum speed of coordinated walking and a decrease in rear stance time and stride length in salubrinal-treated animals. This improved functional recovery corresponded with increased white matter sparing and decreased oligodendrocyte apoptosis. In addition, salubrinal protected cultured mouse oligodendrocyte progenitor cells against the ER stress-inducing toxin tunicamycin. These data suggest that boosting the homeostatic arm of the ERSR reduces oligodendrocyte loss after traumatic SCI and support the contention that pharmacological targeting of the ERSR after CNS trauma is a therapeutically viable approach.

Topics: Activating Transcription Factor 4; Animals; Animals, Newborn; Cerebral Cortex; Cinnamates; Disease Models, Animal; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gait Disorders, Neurologic; Gene Expression Regulation; Glutamate-Ammonia Ligase; Heat-Shock Proteins; Homeostasis; Locomotion; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Nerve Fibers, Myelinated; Oligodendroglia; Phosphorylation; Protein Phosphatase 1; Recovery of Function; Spinal Cord Injuries; Thiourea; Tunicamycin

The dysmyelinated axons of shiverer mice exhibit impaired conduction characteristics, similar to early postnatal axons before myelination, whereas the patterns of neuronal activity and connectivity are relatively comparable with those of wild-type myelinated axons. This unique dysmyelination pattern is exploited in the present study to determine the role of compact myelin in the loss and recovery of function following traumatic spinal cord injury (SCI). We applied in vivo diffusion tensor imaging (DTI) and post-mortem immunohistochemistry analysis to examine changes in myelin and axonal integrity, and evaluated these changes in concert with the analysis of locomotor function from 1 to 4 weeks following a mid-thoracic contusion injury in homozygous shiverer and heterozygous littermate mice. The DTI biomarkers, axial and radial diffusivities, are noninvasive indicators of axon and myelin integrity in response to SCI of both myelinated and dysmyelinated spinal cord. We show that myelin is critical for normal hind limb function in open field locomotion. However, when the functional outcome is limited during chronic SCI, the extent of recovery is associated with residual axonal integrity and independent of the extent of intact myelin at the lesion epicenter.

Topics: Animals; Anisotropy; Axons; Diffusion Tensor Imaging; Female; Heterozygote; Mice; Mice, Neurologic Mutants; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neurofilament Proteins; Recovery of Function; Spinal Cord Injuries; Staining and Labeling; Time Factors

Nogo receptor 1 (NgR1) mediates the inhibitory effects of several myelin-associated inhibitors (MAIs) on axonal regeneration in the central nervous system. A truncated soluble NgR1 (sNgR) has been reported to act as a decoy receptor to block the actions of MAIs. In this study, we fused the sNgR to nerve growth factor (NGF) and used NGF as a carrier to deliver sNgR to the intercellular space to neutralize MAIs. NGF in NGF-sNgR remained biologically active and induced sprouting of calcitonin gene related peptide containing axons when expressed in the spinal cord using a lentiviral vector (LV). Secreted NGF-sNgR promoted neurite outgrowth of dissociated dorsal root ganglion neurons on myelin protein substrate. In a rat dorsal column transection model, regenerating sensory axons were found to grow into the lesion cavity in animals injected with LV/NGF-sNgR, while in animals injected with LV/GFP or LV/NGF-GFP few sensory axons entered the lesion cavity. The results indicate that NGF-sNgR fusion protein can reduce the inhibition of MAIs and facilitate sensory axon regeneration. The fusion constructs may be modified to target other molecules to promote axonal regeneration and the concept may also be adapted to develop gene therapy strategies to treat other disorders.

Topics: Animals; Axons; Calcitonin Gene-Related Peptide; Cell Differentiation; Disease Models, Animal; Gene Expression Regulation; Lentivirus; Male; Myelin Basic Protein; Myelin Proteins; Nerve Growth Factor; Nerve Regeneration; Neurites; Nogo Proteins; PC12 Cells; Rats; Rats, Wistar; Recombinant Fusion Proteins; Serotonin; Spinal Cord Injuries

Kallikrein 6 (KLK6) is a secreted serine protease preferentially expressed by oligodendroglia in CNS white matter. Elevated levels of KLK6 occur in actively demyelinating multiple sclerosis (MS) lesions and in cases of spinal cord injury (SCI), stroke, and glioblastoma. Taken with recent evidence establishing KLK6 as a CNS-endogenous activator of protease-activated receptors (PARs), we hypothesized that KLK6 activates a subset of PARs to regulate oligodendrocyte physiology and potentially pathophysiology. Here, primary oligodendrocyte cultures derived from wild type or PAR1-deficient mice and the murine oligodendrocyte cell line, Oli-neu, were used to demonstrate that Klk6 (rodent form) mediates loss of oligodendrocyte processes and impedes morphological differentiation of oligodendrocyte progenitor cells (OPCs) in a PAR1-dependent fashion. Comparable gliopathy was also elicited by the canonical PAR1 agonist, thrombin, as well as PAR1-activating peptides (PAR1-APs). Klk6 also exacerbated ATP-mediated oligodendrogliopathy in vitro, pointing to a potential role in augmenting excitotoxicity. In addition, Klk6 suppressed the expression of proteolipid protein (PLP) RNA in cultured oligodendrocytes by a mechanism involving PAR1-mediated Erk1/2 signaling. Microinjection of PAR1 agonists, including Klk6 or PAR1-APs, into the dorsal column white matter of PAR1(+/+) but not PAR1(-/-) mice promoted vacuolating myelopathy and a loss of immunoreactivity for myelin basic protein (MBP) and CC-1(+) oligodendrocytes. These results demonstrate a functional role for Klk6-PAR1 signaling in oligodendroglial pathophysiology and suggest that antagonists of PAR1 or its protease agonists may represent new modalities to moderate demyelination and to promote myelin regeneration in cases of CNS white matter injury or disease.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Animals, Newborn; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Gene Expression Regulation; Humans; Kallikreins; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Myelin Proteolipid Protein; Oligodendroglia; Receptor, PAR-1; Receptor, PAR-2; Signal Transduction; Spinal Cord Injuries; Thrombin

To study the role and effect of Schwann cells (SCs) remyelination in contused spinal cord.. Green fluorescence protein expressing-SCs were transplanted into the epicenter, rostral and caudal tissues of the injury site at 1 week after the spinal cords were contused. At 6 weeks, the spinal cords were removed for cryosections, semithin sections and ultrathin sections, and then immunocytochemical staining of myelin basic protein (MBP), P0 protein (P0) and S100 protein (S100) was carried out on the cryosections. Qualitative and semiquantitative analyses were performed on the cryosections and semithin sections. Ultrastructure of myelinated fibers was observed on the ultrathin sections under electron microscope.. Transplanted SCs and myelinated fibers immunocytochemically labeled by MBP, P0 as well as S100 distributed in whole injured area. The quantity of myelinated fibers labeled by the three myelin proteins showed no statistical difference, however, which was significantly larger than that of controls. On the semithin sections, the experimental group demonstrated more myelinated fibers in the injured area than the controls, but the fibers had smaller diameter and thinner myelin sheath under electron microscope.. SCs can promote regeneration of injured nerve fibers and enhance remyelination, which may be histological basis of SCs-mediated functional repair of injured spinal cords.

Topics: Animals; Immunohistochemistry; Microscopy, Electron; Myelin Basic Protein; Myelin P0 Protein; Nerve Regeneration; Rats; Rats, Sprague-Dawley; S100 Proteins; Schwann Cells; Spinal Cord Injuries

Iatrogenic injury to the spinal cord (SC) is not an uncommon complication of spinal surgery. In an attempt to establish a preventive therapy for anticipated SC injury, we tested the effect of a single dose (SD) vaccine vs. the addition of a booster dose (BD) of a neural-derived peptide (A91) prior to SC contusion. Immunization with A91 immediately after SC injury has demonstrated to induce significant tissue protection and motor recovery. After injury, only the BD vaccination schedule had a neuroprotective effect. It was capable of improving neurological recovery that was always significantly higher than the one observed in rats with SD immunization or those only treated with PBS. Toward the end of study, animals treated with an A91 BD presented a BBB score of 9.75±0.17 (mean±standard deviation) while rats treated with SD or PBS had a score of 6.6±0.7 and 5.6±0.6 respectively. In the next step we attempted to corroborate the neuroprotective effect induced by A91 immunization. For this purpose, we assessed the survival of rubrospinal neurons (RSNs) and ventral horn neurons (VHNs) sixty days after SC injury. BD vaccination induced a significant survival of both RSNs and VHNs after injury. Finally, the failure or success of this therapy (SD or BD respectively) was associated with a lower (SD) or higher (BD) A91-specific T cell proliferation. Prophylactic neuroprotection with an initial and subsequent booster dose of A91 may improve recovery after SC injury sustained during invasive spinal surgery procedures.

Topics: Animals; Cell Proliferation; Cell Survival; Female; Immunization, Secondary; Myelin Basic Protein; Neurons; Neuroprotective Agents; Peptides; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; T-Lymphocytes; Vaccines

Spinal cord injury (SCI) represents a devastating central nervous system disease that still lacks sufficient therapies. Here, dogs are increasingly recognized as a preclinical animal model for the development of future therapies. The aim of this study was a detailed characterization of axonopathy in canine intervertebral disc disease, which produces a mixed contusive and compressive injury and functions as a spontaneous translational animal model for human SCI. The results revealed an early occurrence of ultrastructurally distinct axonal swelling. Immunohistochemically, enhanced axonal expression of β-amyloid precursor protein, non-phosphorylated neurofilament (n-NF) and growth-associated protein-43 was detected in the epicenter during acute canine SCI. Indicative of a progressive axonopathy, these changes showed a cranial and caudally accentuated spatial progression in the subacute disease phase. In canine spinal cord slice cultures, immunoreactivity of axons was confined to n-NF. Real-time quantitative polymerase chain reaction of naturally traumatized tissue and slice cultures revealed a temporally distinct dysregulation of the matrix metalloproteinases (MMP)-2 and MMP-9 with a dominating expression of the latter. Contrasting to early axonopathy, diminished myelin basic protein immunoreactivity and phagocytosis were delayed. The results present a basis for assessing new therapies in the canine animal model for translational research that might allow partial extrapolation to human SCI.

Topics: Animals; Axons; Disease Models, Animal; Dogs; Female; GAP-43 Protein; Gene Expression Regulation; Hypoxanthine Phosphoribosyltransferase; Intervertebral Disc Degeneration; Intervertebral Disc Displacement; Macrophages; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Microglia; Microscopy, Electron, Transmission; Microscopy, Immunoelectron; Myelin Basic Protein; Neurofilament Proteins; Organ Culture Techniques; Peptide Elongation Factor 1; Phagocytes; RNA, Messenger; Spinal Cord; Spinal Cord Injuries; Statistics, Nonparametric

Immunisation with neural-derived peptides is a promising strategy in models of spinal cord (SC) injury. Recent studies have also demonstrated that the addition of glutathione monoethyl ester (GHSE) to this strategy further improves motor recovery, tissue protection and neuronal survival after SC injury. As it is realistic to envision that this combination therapy could be tested in clinical trials, the therapeutic window should be experimentally explored before implementing its use in SC-injured human beings. For this purpose, 50 rats (10 per group) were subjected to a moderate SC contusion. The combined therapy was initiated at 10 min., 24, 72 or 120 hr after injury. Motor recovery and the survival of rubrospinal (RS) and ventral horn (VH) neurones were evaluated 60 days after injury. Results showed a significant motor improvement even if the combined therapy was initiated up to 72 hr after injury. BBB scores were as follows: 10 min.: 10.5 ± 0.7, 24 hr: 10.7 ± 0.5, 72 hr: 11.0 ± 1.3 and PBS: 6.7 ± 1 (mean ± S.D.). Initiation of combined therapy 120 hr after injury had no beneficial effect on motor recovery. Survival of RS and VH neurones was significantly higher in animals treated during the first 72 hr than those treated only with PBS. In this case again, animals treated with combined therapy 120 hr after injury did not present significant survival of neurones. Treatment with this combined strategy has a clinically feasible therapeutic window. This therapy provides enough time to transport and diagnose the patient and allows the concomitant use of other neuroprotective therapies.

Topics: Animals; Anterior Horn Cells; Cell Survival; Disease Models, Animal; Drug Administration Schedule; Drug Therapy, Combination; Female; Glutathione; Motor Activity; Myelin Basic Protein; Neurons; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Time Factors; Time-to-Treatment

Manipulation of various components of the endoplasmic reticulum (ER) stress response (ERSR) has led to functional recovery in diabetes, cancer, and several neurodegenerative diseases, indicating its use as a potential therapeutic intervention. One of the downstream pro-apoptotic transcription factors activated by the ERSR is CCAAT enhancer binding protein (C/EBP) homologous protein (CHOP). Recently, we showed significant recovery in hindlimb locomotion function after moderate contusive spinal cord injury (SCI) in mice null for CHOP. However, more than 40% of human SCI are complete. Thus the present study examined the potential therapeutic modulation of CHOP in a more severe SCI injury. Contused wild-type spinal cords showed a rapid activation of PERK, ATF6, and IRE-1, the three arms of the ERSR signaling pathway, specifically at the injury epicenter. Confocal images of phosphorylated EIF2α, GRP78, CHOP, ATF4, and GADD34 localized the activation of the ERSR in neurons and oligodendrocytes at the injury epicenter. To directly determine the role of CHOP, wild-type and CHOP-null mice with severe contusive SCI were analyzed for improvement in hindlimb locomotion. Despite the loss of CHOP, the other effectors in the ERSR pathway were significantly increased beyond that observed previously with moderate injury. Concomitantly, Basso Mouse Scale (BMS) scores and white matter sparing between the wild-type and CHOP-null mice revealed no significant differences. Given the complex pathophysiology of severe SCI, ablation of CHOP alone is not sufficient to rescue functional deficits. These data raise the caution that injury severity may be a key variable in attempting to translate preclinical therapies to clinical practice.

Topics: Animals; Apoptosis; Behavior, Animal; Blotting, Western; Claudins; Contusions; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Female; Heat-Shock Proteins; Immunohistochemistry; Locomotion; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Nerve Tissue Proteins; Protein Phosphatase 1; Real-Time Polymerase Chain Reaction; RNA; RNA, Messenger; Spinal Cord; Spinal Cord Injuries; Transcription Factor CHOP

Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by axonal/neuronal damage that may be caused by defective remyelination. Current therapies aim to slow the rate of degeneration, however there are no treatment options that can stop or reverse the myelin sheath damage. Bone marrow mesenchymal stem cells (MSCs) are a potential candidate for the cell implantation-targeted therapeutic strategies, but the pro-remyelination effects of MSCs when directly injected into a demyelinated cord lesion have been questioned. Neurotrophin-3 (NT-3) has been shown to serve a crucial role in the proliferation, differentiation and maturation of oligodendrocyte lineages. Here, we showed that implantation of NT-3 gene-modified MSCs via a recombinant adenoviral vector (Adv) into a region of ethidium bromide (EB)-induced demyelination in the spinal cord resulted in significant improvement of locomotor function and restoration of electrophysiological properties in rats. The morphological basis of this recovery was evidenced by robust myelin basic protein (MBP) expression and the extensive remyelination. AdvNT-3-MSC implants promote the endogenous remyelinating cells to participate directly in myelination, which was confirmed under light and electron microscopy. Our study suggested that genetically modified MSCs could be a potential therapeutic avenue for improving the efficacy of stem cell treatment for neurodegenerative diseases such as MS.

Topics: Animals; Demyelinating Diseases; Female; Genetic Vectors; Mesenchymal Stem Cell Transplantation; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neurotrophin 3; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Thoracic Vertebrae

Immunization with neurally derived peptides (INDP) boosts the action of an autoreactive immune response that has been shown to induce neuroprotection in several neurodegenerative diseases, especially after spinal cord (SC) injury. This strategy provides an environment that promotes neuronal survival and tissue preservation. The mechanisms by which this autoreactive response exerts its protective effects is not totally understood at the moment. A recent study showed that INDP reduces lipid peroxidation. Lipid peroxidation is a neurodegenerative phenomenon caused by the increased production of reactive nitrogen species such as nitric oxide (NO). It is possible that INDP could be interfering with NO production. To test this hypothesis, we examined the effect of INDP on the amount of NO produced by glial cells when cocultured with autoreactive T cells. We also evaluated the amount of NO and the expression of the inducible form of nitric oxide synthase (iNOS) at the injury site of SC-injured animals. The neural-derived peptides A91 and Cop-1 were used to immunize mice and rats with SC injury. In vitro studies showed that INDP significantly reduces the production of NO by glial cells. This observation was substantiated by in vivo experiments demonstrating that INDP decreases the amount of NO and iNOS gene expression at the site of injury. The present study provides substantial evidence on the inhibitory effect of INDP on NO production, helpingour understanding of the mechanisms through which protective autoimmunity promotes neuroprotection.

Topics: Animals; Cell Proliferation; Glatiramer Acetate; Immunization; Mice; Mice, Inbred BALB C; Myelin Basic Protein; Nitric Oxide; Nitric Oxide Synthase Type II; Peptides; Rats; Rats, Inbred F344; Spinal Cord; Spinal Cord Injuries; T-Lymphocytes

Previous studies have shown the existence of either cellular or humoral MBP-reactive elements up to 5 years after spinal cord injury (SCI), but not the presence of both after 10 years.. Twelve SCI patients, with more than 10 years of evolution, and 18 healthy blood donors were studied. Lymphocyte proliferation (colorimetric-BrdU ELISA assay) and antibody titers against MBP (ELISA Human IgG MBP-specific assay) were assessed.. SCI patients presented a significant T-cell proliferation against MBP (lymphocyte proliferation index: 3.7 ± 1.5, mean ± SD) compared to control individuals (0.7 ± 0.3; P < 0.001). Humoral response analysis yielded a significant difference (P < 0.0001) between the antibody titers of controls and SCI patients. A significant correlation between cellular and humoral responses was observed. Finally, patients with an ASIA B presented the highest immune responses.. This work demonstrates, for the first time, the existence of both cellular and humoral responses against MBP in the chronic stages (>10 years) of injury.

Topics: Adult; Antibodies; Case-Control Studies; Cell Proliferation; Chronic Disease; Female; Humans; Immunity, Humoral; Longitudinal Studies; Male; Middle Aged; Myelin Basic Protein; Paraplegia; Spinal Cord Injuries; T-Lymphocytes; Time Factors

Neural stem cell (NSC) transplantation is a major focus of current research for treatment of spinal cord injury (SCI). However, it is very important to promote the survival and differentiation of NSCs into myelinating oligodendrocytes (OLs). In this study, myelin basic protein-activated T (MBP-T) cells were passively immunized to improve the SCI microenvironment. Olig2-overexpressing NSCs were infected with a lentivirus carrying the enhanced green fluorescent protein (GFP) reporter gene to generate Olig2-GFP-NSCs that were transplanted into the injured site to differentiate into OLs. Transferred MBP-T cells infiltrated the injured spinal cord, produced neurotrophic factors, and induced the differentiation of resident microglia and/or infiltrating blood monocytes into an "alternatively activated" anti-inflammatory macrophage phenotype by producing interleukin-13. As a result, the survival of transplanted NSCs increased fivefold in MBP-T cell-transferred rats compared with that of the vehicle-treated control. In addition, the differentiation of MBP-positive OLs increased 12-fold in Olig2-GFP-NSC-transplanted rats compared with that of GFP-NSC-transplanted controls. In the MBP-T cell and Olig2-GFP-NSC combined group, the number of OL-remyelinated axons significantly increased compared with those of all other groups. However, a significant decrease in spinal cord lesion volume and an increase in spared myelin and behavioral recovery were observed in Olig2-NSC- and NSC-transplanted MBP-T cell groups. Collectively, these results suggest that MBP-T cell adoptive immunotherapy combined with NSC transplantation has a synergistic effect on histological and behavioral improvement after traumatic SCI. Although Olig2 overexpression enhances OL differentiation and myelination, the effect on functional recovery may be surpassed by MBP-T cells.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cells, Cultured; Female; Gene Expression Regulation; Lymphocyte Activation; Myelin Basic Protein; Nerve Tissue Proteins; Neural Stem Cells; Oligodendrocyte Transcription Factor 2; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Stem Cell Transplantation; T-Lymphocytes

The previous studies suggested that some subpopulations of T lymphocytes against central nervous system (CNS) antigens, such as myelin basic protein (MBP), are neuroprotective. But there were few reports about the effect of these T cells on axon regeneration. In this study, the neonatally thymectomied (Tx) adult rats which contain few T lymphocytes were subjected to spinal cord hemisection and then passively immunized with MBP-activated T cells (MBP-T). The regeneration and dieback of transected axons of cortico-spinal tract (CST) were detected by biotin dextran amine (BDA) tracing. The behavioral assessments were performed using the Basso, Beattie, and Bresnahan locomotor rating scale. We found that passive transferring of MBP-T could attenuate axonal dieback. However, no significant axon regeneration and behavioral differences were observed among the normal, Tx and sham-Tx (sTx) rats with or without MBP-T passive immunization. These results indicate that passive transferring of MBP-T cells can attenuate axonal dieback and promote neuroprotection following spinal cord injury (SCI), but may not promote axon regeneration.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antigens, CD; Biotin; Cell Proliferation; Cytokines; Dextrans; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Female; Functional Laterality; Immunization, Passive; Locomotion; Myelin Basic Protein; Nerve Regeneration; Pyramidal Tracts; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; T-Lymphocytes; Thymectomy

MicroRNAs have been shown to effectively regulate gene expression at the translational level. Recently, we identified novel microRNAs that were upregulated in a mouse model of spinal cord injury. Among those, we have focused on microRNA 486, which directly represses NeuroD6 expression through a conserved sequence in its untranslated region. We correlated the overexpression of microRNA 486 in motor neurons with a poor outcome due to progressive neurodegeneration and a pathophysiology that is mediated by reactive oxygen species. The expression of microRNA 486 was induced by reactive oxygen species that were produced by inflammatory factors, and reactive oxygen species were accumulated in response to the knockdown of NeuroD6, which enhances the downregulation of glutathione peroxidase 3 and thioredoxin-like 1 after traumatic spinal cord injury. NeuroD6 directly bound to regulatory regions of thioredoxin-like 1 and glutathione peroxidase 3 in motor neurons and activated their expression, which promoted reactive oxygen species scavenging. Moreover, knocking down microRNA 486 induced the expression of NeuroD6, which effectively ameliorated the spinal cord injury and allowed the mice to recover motor function. The infusion of exogenic NeuroD6 in spinal cord injury lesions effectively blocked apoptosis by reactivating thioredoxin-like 1 and glutathione peroxidase 3, which was accompanied by a recovery of motor function. Collectively, these findings have identified a novel microRNA in spinal cord injury lesions called microRNA 486, demonstrating a new role for NeuroD6 in neuroprotection, and suggest a potential therapeutic target for spinal cord injuries.

Topics: Adenosine Triphosphate; Analysis of Variance; Animals; Basic Helix-Loop-Helix Transcription Factors; Caspase 3; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Mice; Mice, Inbred ICR; MicroRNAs; Motor Activity; Motor Neurons; Myelin Basic Protein; Nerve Degeneration; Neural Stem Cells; Neurofilament Proteins; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; Oligodeoxyribonucleotides, Antisense; Peroxidase; Reactive Oxygen Species; Recovery of Function; RNA, Small Interfering; Spinal Cord Injuries; Time Factors

Microglia and macrophages (MG/MΦ) have a diverse range of functions depending on unique cytokine stimuli, and contribute to neural cell death, repair, and remodeling during central nervous system diseases. While IL-1 has been shown to exacerbate inflammation, it has also been recognized to enhance neuroregeneration. We determined the activating phenotype of MG/MΦ and the impact of IL-1 in an in vivo spinal cord injury (SCI) model of IL-1 knock-out (KO) mice. Moreover, we demonstrated the contribution of IL-1 to both the classical and alternative activation of MG in vitro using an adult MG primary culture.. SCI was induced by transection of the spinal cord between the T9 and T10 vertebra in wild-type and IL-1 KO mice. Locomotor activity was monitored and lesion size was determined for 14 days. TNFα and Ym1 levels were monitored to determine the MG/MΦ activating phenotype. Primary cultures of MG were produced from adult mice, and were exposed to IFNγ or IL-4 with and without IL-1β. Moreover, cultures were exposed to IL-4 and/or IL-13 in the presence and absence of IL-1β.. The locomotor activity and lesion area of IL-1 KO mice improved significantly after SCI compared with wild-type mice. TNFα production was significantly suppressed in IL-1 KO mice. Also, Ym1, an alternative activating MG/MΦ marker, did not increase in IL-1 KO mice, suggesting that IL-1 contributes to both the classical and alternative activation of MG/MΦ. We treated primary MG cultures with IFNγ or IL-4 in the presence and absence of IL-1β. Increased nitric oxide and TNFα was present in the culture media and increased inducible NO synthase was detected in cell suspensions following co-treatment with IFNγ and IL-1β. Expression of the alternative activation markers Ym1 and arginase-1 was increased after exposure to IL-4 and further increased after co-treatment with IL-4 and IL-1β. The phenotype was not observed after exposure of cells to IL-13.. We demonstrate here in in vivo experiments that IL-1 suppressed SCI in a process mediated by the reduction of inflammatory responses. Moreover, we suggest that IL-1 participates in both the classical and alternative activation of MG in in vivo and in vitro systems.

Topics: Animals; Arginase; CD11b Antigen; Cells, Cultured; Central Nervous System; Cytokines; Disease Models, Animal; Doxorubicin; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Interleukin-1; Interleukin-1alpha; Interleukin-1beta; Macrophages; Mice; Mice, Inbred C57BL; Mice, Knockout; Microglia; Microtubule-Associated Proteins; Motor Activity; Myelin Basic Protein; Nitric Oxide; Spinal Cord Injuries

Spinal cord injury (SCI) induces an immune response during which microglia, the resident immunocompetent cells of the central nervous system, become activated and migrate to the site of damage. Depending on their state of activation, microglia secrete neurotoxic or neurotrophic factors that influence the surrounding environment and have a detrimental or restorative effect following SCI, including causing or protecting bystander damage to nearby undamaged tissue. Subsequent infiltration of macrophages contributes to the SCI outcome. We show here that suppressing microglia/macrophage activation using the tripeptide macrophage/microglia inhibitory factor (MIF/TKP) reduced secondary injury around the lesion epicenter in the murine dorsal hemisection model of SCI; it decreased the hypertrophic change of astrocytes and caused an increase in the number of axons present within the lesion epicenter. Moreover, timely inhibition of microglial/macrophage activation prevented demyelination and axonal dieback by modulating oligodendrocyte survival and oligodendrocyte precursor maturation. Microglia/macrophages located within or proximal to the lesion produced neurotoxic factors, such as tumor necrosis factor alpha (TNF-α). These results suggest that microglia/macrophages within the epicenter at early time points post injury are neurotoxic, contributing to demyelination and axonal degeneration and that MIF/TKP could be used in combination with other therapies to promote functional recovery.

Topics: Animals; Animals, Newborn; Antigens; Autophagy-Related Proteins; Axons; Bromodeoxyuridine; Calcium-Binding Proteins; Cell Proliferation; Cells, Cultured; Cholera Toxin; Chondroitin Sulfate Proteoglycans; Coculture Techniques; Cytokines; Disease Models, Animal; Drug Administration Schedule; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Glial Fibrillary Acidic Protein; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Ki-67 Antigen; Lipopolysaccharides; Macrophage Activation; Mice; Mice, Inbred C57BL; Microfilament Proteins; Microglia; Microscopy, Electron, Transmission; Myelin Basic Protein; Neuroprotective Agents; Oligodendroglia; Oligopeptides; Peptide Fragments; Proteoglycans; Spinal Cord Injuries; Time Factors

The present study assessed the ability of a combined immunomodulatory treatment using (1) selective depletion of peripheral macrophages with liposomal-encapsulated clodronate, and (2) rolipram, a type 4 phosphodiesterase (PDE4) inhibitor, to promote neuroprotection and improve locomotor recovery following experimental contusion SCI. We demonstrate that delivery of either liposomal clodronate or rolipram alone promotes neuroprotection, enhances myelinated tissue sparing, and improves hindlimb functional recovery. Combined treatment with liposomal clodronate and rolipram produced the greatest improvement in locomotor recovery (inter-limb coordination, paw placement, and toe clearance), at 4 weeks post-injury (2.9 points). Retrograde tracing revealed substantial axonal sparing and/or sprouting from several brainstem motor nuclei, and hindlimb motor cortex. The combined treatment with these two drugs promoted the greatest amount of axonal sparing (3- to 4-fold increase compared to controls). Histological assessments revealed that combined treatment with clodronate/ rolipram resulted in a significant reduction in lesion volume (51%) and lesion area at the injury epicenter (45%), and significantly increased the extent of myelinated tissue sparing. Immunohistochemical studies showed a qualitative reduction in the accumulation of ED-1(+) macrophages within the injured spinal cord 5 weeks after injury. Our results demonstrate robust neuroprotection and improved hindlimb locomotor function using a combined immunomodulatory treatment strategy consisting of liposomal clodronate and rolipram. The present data suggest that clinical trials with acute delivery of combination immunomodulatory therapies may be warranted. This article is part of a Special Issue entitled "Interaction between repair, disease, & inflammation."

Topics: Analysis of Variance; Animals; Axons; Brain Stem; Clodronic Acid; Contusions; Disease Models, Animal; Drug Therapy, Combination; Ectodysplasins; Exploratory Behavior; Female; Glial Fibrillary Acidic Protein; Hindlimb; Immunologic Factors; Liposomes; Locomotion; Macrophages; Motor Cortex; Myelin Basic Protein; Myelin Sheath; Neural Pathways; Neurofilament Proteins; Neuroprotective Agents; Phosphodiesterase 4 Inhibitors; Psychomotor Performance; Rats; Recovery of Function; Rolipram; Spinal Cord Injuries; Stilbamidines; Vimentin

Activation of the unfolded protein response (UPR) is involved in the pathogenesis of numerous CNS myelin abnormalities; yet, its direct role in traumatic spinal cord injury (SCI)-induced demyelination is not known. The UPR is an evolutionarily conserved cell defense mechanism initiated to restore endoplasmic reticulum homeostasis in response to various cellular stresses including infection, trauma, and oxidative damage. However, if uncompensated, the UPR triggers apoptotic cell death. We demonstrate that the three signaling branches of UPR including the PERK, ATF6, and IRE1α are rapidly initiated in a mouse model of contusive SCI specifically at the injury epicenter. Immunohistochemical analyses of the various UPR markers revealed that in neurons, the UPR appeared at 6 and 24-h post-SCI. In contrast, in oligodendrocytes and astroglia, UPR persisted at least for up to 3 days post-SCI. The UPR-associated proapoptotic transcriptional regulator CHOP was among the UPR markers upregulated in neurons and oligodendrocytes, but not in astrocytes, of traumatized mouse spinal cords. To directly analyze its role in SCI, WT and CHOP null mice received a moderate T9 contusive injury. Deletion of CHOP led to an overall attenuation of the UPR after contusive SCI. Furthermore, analyses of hindlimb locomotion demonstrated a significant functional recovery that correlated with an increase in white-matter sparing, transcript levels of myelin basic protein, and Claudin 11 and decreased oligodendrocyte apoptosis in CHOP null mice in contrast to WT animals. Thus, our study provides evidence that the UPR contributes to oligodendrocyte loss after traumatic SCI.

Topics: Activating Transcription Factor 4; Analysis of Variance; Animals; Apoptosis; Basic Helix-Loop-Helix Transcription Factors; Caspase 3; Disease Models, Animal; DNA-Binding Proteins; Endoplasmic Reticulum Stress; Eukaryotic Initiation Factor-2; Female; Locomotion; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Nerve Fibers, Myelinated; Nerve Tissue Proteins; Oligodendrocyte Transcription Factor 2; Oligodendroglia; Recovery of Function; Regulatory Factor X Transcription Factors; RNA, Messenger; Spinal Cord Injuries; Time Factors; Transcription Factor CHOP; Transcription Factors; Unfolded Protein Response; Up-Regulation

Macrophages exert divergent effects in the injured CNS, causing either neurotoxicity or regeneration. The mechanisms regulating these divergent functions are not understood but can be attributed to the recruitment of distinct macrophage subsets and the activation of specific intracellular signaling pathways. Here, we show that impaired signaling via the chemokine receptor CX3CR1 promotes recovery after traumatic spinal cord injury (SCI) in mice. Deficient CX3CR1 signaling in intraspinal microglia and monocyte-derived macrophages (MDMs) attenuates their ability to synthesize and release inflammatory cytokines and oxidative metabolites. Also, impaired CX3CR1 signaling abrogates the recruitment or maturation of MDMs with presumed neurotoxic effects after SCI. Indeed, in wild-type mice, Ly6C(lo)/iNOS(+)/MHCII(+)/CD11c(-) MDMs dominate the lesion site, whereas CCR2(+)/Ly6C(hi)/MHCII(-)/CD11c(+) monocytes predominate in the injured spinal cord of CX3CR1-deficient mice. Replacement of wild-type MDMs with those unable to signal via CX3CR1 resulted in anatomical and functional improvements after SCI. Thus, blockade of CX3CR1 signaling represents a selective anti-inflammatory therapy that is able to promote neuroprotection, in part by reducing inflammatory signaling in microglia and MDMs and recruitment of a novel monocyte subset.

Topics: Analysis of Variance; Animals; Antigens, Ly; CD11 Antigens; Cells, Cultured; Chemokine CXCL1; CX3C Chemokine Receptor 1; Disease Models, Animal; Flow Cytometry; Gene Expression Regulation; Green Fluorescent Proteins; Macrophages; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Activity; Myelin Basic Protein; Nitric Oxide; Nitric Oxide Synthase Type II; Receptors, Chemokine; Recovery of Function; Signal Transduction; Spinal Cord Injuries

To investigate neurological effects of transplanting bone marrow-derived mesenchymal stem cells (BMSCs) transfected with the basic fibroblast growth factor (bFGF) gene in spinal cord-injured rats.. Ninety-six male adult Sprague-Dawley rats were randomized into four groups: (1) pcDNA3.1-bFGF group; (2) pcDNA3.1 group; (3) BMSCs group; and (4) vehicle control (DMEM) group. After the rat model of acute spinal cord injury (SCI) was established, 1×10(6) BMSCs or cells transfected with pcDNA3.1-bFGF or pcDNA3.1 were injected into rats of groups 1-3. At days 1, 7, 14, and 21 after injection, the Basso-Beattie-Bresnahan (BBB) locomotor rating scale was used to evaluate recovery of motor function. Expression changes of bFGF, myelin basic protein (MBP), and NF200 were examined by immunohistochemistry.. The BBB score of DMEM group was significantly lower than those of groups 1-3 (P<0.05), but the score of pcDNA3.1-bFGF group was significantly higher than that of BMSCs group or pcDNA3.1 group at day 14 or 21 after injection (P<0.01). The number of bFGF-positive neurons in rats of pcDNA3.1-bFGF group was significantly higher than those of groups 1-3 at any time point (P<0.05). The optical density values of NF200-positive neurons and MBP-positive MBP axons in rats of pcDNA3.1-bFGF group were significantly higher than those of groups 1-3 at day 7 or 14 after injection (P<0.05).. bFGF gene-modified BMSCs not only effectively promoted axonal outgrowth but also enhanced recovery of neurological function after SCI in rats, and may be a good candidate to evaluate gene therapy of SCI in man.

Topics: Animals; Bone Marrow Cells; Disease Models, Animal; Fibroblast Growth Factor 2; Locomotion; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Myelin Basic Protein; Nerve Regeneration; Neurofilament Proteins; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries; Transfection

Previous reports of functional recovery from spinal cord injury (SCI) in rodents and monkeys after the delayed transplantation of neural stem/progenitor cells (NS/PCs) have raised hopes that stem cell therapy could be used to treat SCI in humans. More research is needed, however, to understand the mechanism of functional recovery. Oligodendrocytes derived from grafted NS/PCs remyelinate spared axons in the injured spinal cord. Here, we studied the extent of this remyelination's contribution to functional recovery following contusive SCI in mice. To isolate the effect of remyelination from other possible regenerative benefits of the grafted cells, NS/PCs obtained from myelin-deficient shiverer mutant mice (shi-NS/PCs) were used in this work alongside wild-type NS/PCs (wt-NS/PCs). shi-NS/PCs behaved like wt-NS/PCs in vitro and in vivo, with the exception of their myelinating potential. shi-NS/PC-derived oligodendrocytes did not express myelin basic protein in vitro and formed much thinner myelin sheaths in vivo compared with wt-NS/PC-derived oligodendrocytes. The transplantation of shi-NS/PCs promoted some locomotor and electrophysiological functional recovery but significantly less than that afforded by wt-NS/PCs. These findings establish the biological importance of remyelination by graft-derived cells for functional recovery after the transplantation of NS/PCs into the injured spinal cord.

Topics: Animals; Axons; Cell Differentiation; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Electrophysiology; Embryonic Stem Cells; Female; Lentivirus; Luminescent Measurements; Mice; Mice, Inbred C57BL; Mice, SCID; Myelin Basic Protein; Myelin Sheath; Neural Stem Cells; Oligodendroglia; Recovery of Function; Spinal Cord Injuries; Spinal Cord Regeneration; Stem Cell Transplantation; Thoracic Vertebrae

Bone morphogenetic proteins (BMPs) play a critical role in regulating cell fate determination during central nervous system (CNS) development. In light of recent findings that BMP-2/4/7 expressions are upregulated after spinal cord injury, we hypothesized that the BMP signaling pathway is important in regulating cellular composition in the injured spinal cord. We found that BMP expressions were upregulated in neural stem cells (NSCs), neurons, oligodendrocytes and microglia/macrophages. Increased expression levels of pSmad1/5/8 (downstream molecules of BMP) were detected in neurons, NSCs, astrocytes, oligodendrocytes and oligodendroglial progenitor cells (OPCs). Active astrocytes which form the astroglial scar were probably derived from NSCs, OPCs and resident astrocytes. Since quiescent NSCs in the normal adult spinal cord will proliferate and differentiate actively into neural cells after traumatic injury, we proposed that BMPs can regulate cellular components by controlling NSC differentiation. Neurosphere culture from adult mouse spinal cord showed that BMP-4 promoted astrocyte differentiation from NSCs while suppressing production of neurons and oligodendrocytes. Conversely, inhibition of BMP-4 by Noggin notably decreased the ratio of astrocyte to neuron numbers. However, intrathecal administration of Noggin in the injured spinal cord failed to attenuate glial fibrillar acidic protein (GFAP) expression even though it effectively reduced pSmad expression. Noggin treatment did not block phosphorylation of Stat3 and the induction of GFAP in the injured spinal cord, suggesting that in addition to the BMP/Smad pathway, the JAK/STAT pathway may also be involved in the regulation of GFAP expression after spinal cord injury.

Topics: Animals; Astrocytes; Bone Morphogenetic Proteins; Bromodeoxyuridine; Carrier Proteins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Disease Models, Animal; Female; Gene Expression Regulation; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Nerve Tissue Proteins; Neurons; Nuclear Proteins; Oligodendroglia; RNA, Messenger; Signal Transduction; Spinal Cord Injuries; Stem Cells; Time Factors

Lipid peroxidation (LP) is one of the most harmful mechanisms developed after spinal cord (SC) injury. Several strategies have been explored in order to control this phenomenon. Protective autoimmunity is a physiological process based on the modulation of inflammatory cells that can be boosted by immunizing with neural-derived peptides, such as A91. Since inflammatory cells are among the main contributors to lipid peroxidation, we hypothesized that protective autoimmunity could reduce LP after SC injury. In order to test this hypothesis, we designed two experiments in SC contused rats. First, animals were immunized with a neural-derived peptide seven days before injury. With the aim of inducing the functional elimination of CNS-specific T cells, for the second experiment, animals were tolerized against SC-protein extract and thereafter subjected to a SC injury. The lipid-soluble fluorescent products were used as an index of lipid peroxidation and were assessed after injury. Immunization with neural-derived peptides reduced lipid peroxidation after SC injury. Functional elimination of CNS-specific T cells avoided the beneficial effect induced by protective autoimmunity. The present study demonstrates the beneficial effect of immunizing with neural-derived peptides on lipid peroxidation inhibition; besides this, it also provides evidence on the neuroprotective mechanisms exerted by protective autoimmunity.

Topics: Animals; Autoimmunity; Immunization; Lipid Peroxidation; Myelin Basic Protein; Neuropeptides; Ovalbumin; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; T-Lymphocytes

The effect of Nano PGE(1) (nanoparticles containing prostaglandin E(1)) on spinal cord injury (SCI) was investigated in rat model. Nano PGE(1) significantly and dose-dependently promoted the recovery from SCI-induced motor dysfunction, and the potency of Nano PGE(1) was comparable with successive treatment of Lipo PGE(1), and was superior to single treatment of Lipo PGE(1). Distribution study revealed that Nano PGE(1) sustained longer in the blood. In the injured spinal cord, gradual accumulation and longer retention were observed. Lipo PGE(1) was also accumulated with time, but over 10 fold less. It should be noted that over 80 fold more of PGE(1) were detected in Nano PGE(1)-treated injured spinal cord as compared with that in normal ones. Nano PGE(1)-treated injured spinal cord had less lesion cavity with increased MBP expression. Also, HGF production significantly increased as compared with that of SCI control. These findings could lead to the conclusion that Nano PGE(1) had the therapeutic potential for SCI, which might be partly ascribed by the efficient distribution of Nano PGE(1) to the injured spinal cord. The sustained release of PGE(1) would have increased HGF production, and both would have promoted cell survival and endogenous repair.

Topics: Alprostadil; Animals; Chemistry, Pharmaceutical; Delayed-Action Preparations; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Compounding; Female; Hepatocyte Growth Factor; Hindlimb; Motor Activity; Motor Neurons; Muscle, Skeletal; Myelin Basic Protein; Nanoparticles; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Transcription Factors

Most experimental models of spinal cord injury (SCI) in rodents induce damage in the thoracic cord and subsequently examine hindlimb function as an indicator of recovery. In these models, functional recovery is most attributable to white-matter preservation and is less influenced by grey-matter sparing. In contrast, most clinical cases of SCI occur at the lower cervical levels, a region in which both grey-matter and white-matter sparing contribute to functional motor recovery. Thus experimental cervical SCI models are beginning to be developed and used to assess protective and pharmacological interventions following SCI. The objective of this study was to characterize a model of graded cervical hemicontusion SCI with regard to several histological and behavioral outcome measures, including novel forelimb behavioral tasks. Using a commercially available rodent spinal cord impactor, adult male rats received hemicontusion SCI at vertebral level C5 at 100, 200, or 300 kdyn force, to produce mild, moderate, or severe injury severities. Tests of skilled and unskilled forelimb and locomotor function were employed to assess functional recovery, and spinal cord tissue was collected to assess lesion severity. Deficits in skilled and unskilled forelimb function and locomotion relating to injury severity were observed, as well as decreases in neuronal numbers, white-matter area, and white-matter gliosis. Significant correlations were observed between behavioral and histological data. Taken together, these data suggest that the forelimb functional and locomotor assessments employed here are sensitive enough to measure functional changes, and that this hemicontusion model can be used to evaluate potential protective and regenerative therapeutic strategies.

Topics: Animals; Benzoxazines; Cell Count; Contusions; Disease Models, Animal; Fluorescence; Forelimb; Functional Laterality; Glial Fibrillary Acidic Protein; Gliosis; Health; Immunohistochemistry; Locomotion; Male; Motor Skills; Myelin Basic Protein; Neuroglia; Neurons; Oxazines; Psychomotor Performance; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries

Randomized, double-blinded animal experiment for neural functional recovery from spinal cord injury (SCI) through vaccination with immature dendritic cells (DCs) pulsed with homogenate protein of spinal cord (hpDCs) in mice.. To study the effect of hpDCs in the recovery from SCI in mice.. Immature DCs pulsed with homogenate protein of spinal cord, myelin basic protein (MBP) or phosphate-buffer solution (PBS) were injected into spinal cord-injured mice locally or peritoneally. The functional recovery of spinal cord (open-field locomotor rating scale of Basso, Beattie and Bresnahan, BBB score) was measured weekly. The areas of injured region and cyst as well as the thickness of the glial scar were measured and the expressions of glial fibrillary acidic protein, neurofilament and nestin were detected to confirm the BBB scores.. Eighty-four days after injection, the BBB score of the hpDCs group (peritoneally injected mice) reached 18.2+/-1.1, significantly higher than that the scores of the mbpDCs and control groups (16.3+/-2.1 and 10.0+/-2.0, respectively). The areas of injured region and cyst as well as the thickness of the glial scar of the hpDCs group were less than that of the control group. Meanwhile, the expression of nestin lasts up to 56 days after injection in the hpDCs group, while it disappeared in the mbpDCs and PBS groups.. Implanting DCs pulsed with homogenate protein of spinal cord, but not mbpDCs or PBS alone, locally or peritoneally, have a significant effect on functional recovery and neural preservation from SCI.

Topics: Animals; Autoimmunity; Dendritic Cells; Disease Models, Animal; Glial Fibrillary Acidic Protein; Immunohistochemistry; Injections, Intraperitoneal; Injections, Spinal; Intermediate Filament Proteins; Mice; Mice, Inbred BALB C; Motor Activity; Myelin Basic Protein; Nerve Fibers, Myelinated; Nerve Regeneration; Nerve Tissue Proteins; Nestin; Neurofilament Proteins; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Treatment Outcome; Vaccination

Previous works have reported that the transplantation of neural stem cells (NSCs) may improve functional recovery after spinal cord injury (SCI), but these results have been mainly obtained in rat models. In the present work, the authors sought to determine whether the transplantation of human NSCs improves functional outcome in a canine SCI model and whether transplanted NSCs survive and differentiate.. Human NSCs (HB1. F3 clone) were used in this work. Lateral hemisection at the L2 level was performed in dogs and either (1) Matrigel (200 microl) alone as a growth-promoting matrix or (2) Matrigel seeded with human NSCs (10(7) cells/200 microl) were transplanted into hemisected gaps. Using a canine hind limb locomotor scale, functional outcomes were assessed over 12 weeks. Immunofluorescence staining was performed to examine cell survival, differentiation and axonal regeneration.. Compared with dogs treated with Matrigel alone, dogs treated with Matrigel + human NSCs showed significantly better functional recovery (10.3 +/- 0.7 versus 15.6 +/- 0.7, respectively, at 12 weeks; p<0.05). Human nuclei-positive cells were found mainly near hemisected areas in dogs treated with Matrigel + NSCs. In addition, colocalization of human nuclei and neuronal nuclei or myelin basic protein was clearly observed. Moreover, the Matrigel + NSC group showed more ascending sensory axon regeneration.. The transplantation of human NSCs has beneficial effects on functional recovery after SCI, and these NSCs were found to differentiate into mature neurons and/or oligodendrocytes. These results provide baseline data for future clinical applications.

Topics: Animals; Antigens, Nuclear; Biomarkers; Calcitonin Gene-Related Peptide; Cell Differentiation; Cell Nucleus; Cells, Cultured; Collagen; Disability Evaluation; Disease Models, Animal; Dogs; Drug Combinations; Glial Fibrillary Acidic Protein; Graft Survival; Growth Cones; Humans; Laminin; Motor Activity; Myelin Basic Protein; Nerve Regeneration; Nerve Tissue Proteins; Neurogenesis; Proteoglycans; Recovery of Function; Spinal Cord Injuries; Stem Cell Transplantation; Stem Cells; Treatment Outcome

Traumatic spinal cord injury (SCI) causes severe and permanent functional deficits due to the primary mechanical insult followed by secondary tissue degeneration. The cascade of secondary degenerative events constitutes a range of therapeutic targets which, if successfully treated, could significantly ameliorate functional loss after traumatic SCI. During the early hours after injury, potent pro-inflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) are synthesized and released, playing key roles in secondary tissue degeneration. In the present investigation, the ability of rolipram and thalidomide (FDA approved drugs) to reduce secondary tissue degeneration and improve motor function was assessed in an experimental model of spinal cord contusion injury. The combined acute single intraperitoneal administration of both drugs attenuated TNF-alpha and IL-1beta production and improved white matter sparing at the lesion epicenter. This was accompanied by a significant (2.6 point) improvement in the BBB locomotor score by 6 weeks. There is, at present, no widely accepted intervention strategy that is appropriate for the early treatment of human SCI. The present data suggest that clinical trials for the acute combined application of rolipram and thalidomide may be warranted. The use of such "established drugs" could facilitate the early initiation of trials.

Topics: Animals; Disease Models, Animal; Disease Progression; Drug Therapy, Combination; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Immunosuppressive Agents; Interleukin-1beta; Locomotion; Male; Myelin Basic Protein; Nerve Regeneration; Neurofilament Proteins; Phosphodiesterase Inhibitors; Psychomotor Performance; Rats; Rats, Inbred Lew; Recovery of Function; Rolipram; Spinal Cord Injuries; Thalidomide; Time Factors; Tumor Necrosis Factor-alpha

Progenitors that express NG2-proteoglycan are the predominant self-renewing cells within the CNS. NG2 progenitors replenish oligodendrocyte populations within the intact stem cell niche, and cycling NG2 cells are among the first cells to react to CNS insults. We investigated the role of NG2 progenitors after spinal cord injury and how bone morphogen protein signals remodel the progressive postinjury (PI) niche. Progeny labeled by an NG2-specific reporter virus undergo a coordinated shift in differentiation profile. NG2 progeny born 24 h PI produce scar-forming astrocytes and transient populations of novel phagocytic astrocytes shown to contain denatured myelin within cathepsin-D-labeled endosomes, but NG2 progenitors born 7 d PI differentiate into oligodendrocytes and express myelin on processes that wrap axons. Analysis of spinal cord mRNA shows a temporal shift in the niche transcriptome of ligands that affect PI remodeling and direct progenitor differentiation. We conclude that NG2 progeny are diverse lineages that obey progressive cues after trauma to replenish the injured niche.

Topics: Analysis of Variance; Animals; Animals, Newborn; Antigens; Apoptosis; Caspase 3; Cell Differentiation; Disease Models, Animal; Glial Fibrillary Acidic Protein; Green Fluorescent Proteins; Mice; Myelin Basic Protein; Nerve Growth Factors; Proteoglycans; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Signal Transduction; Spinal Cord Injuries; Stem Cell Niche; Stem Cell Transplantation; Stem Cells; Time Factors; Transfection

Progenitor cells isolated from adult brain tissue are important tools for experimental studies of remyelination. Cells harvested from neurogenic regions in the adult brain such as the subependymal zone have demonstrated remyelination potential. Multipotent cells from the progenitor fraction have been isolated from the adult olfactory bulb (OB) but their potential to remyelinate has not been studied.. We used the buoyant density gradient centrifugation method to isolate the progenitor fraction and harvest self-renewing multipotent neural cells grown in monolayers from the adult green-fluorescent protein (GFP) transgenic rat OB. OB tissue was mechanically and chemically dissociated and the resultant cell suspension fractionated on a Percoll gradient. The progenitor fraction was isolated and these cells were plated in growth media with serum for 24 hrs. Cells were then propagated in N2 supplemented serum-free media containing b-FGF. Cells at passage 4 (P4) were introduced into a demyelinated spinal cord lesion. The GFP(+) cells survived and integrated into the lesion, and extensive remyelination was observed in plastic sections. Immunohistochemistry revealed GFP(+) cells in the spinal cord to be glial fibrillary acidic protein (GFAP), neuronal nuclei (NeuN), and neurofilament negative. The GFP(+) cells were found among primarily P0(+) myelin profiles, although some myelin basic protein (MBP) profiles were present. Immuno-electron microscopy for GFP revealed GFP(+) cell bodies adjacent to and surrounding peripheral-type myelin rings.. We report that neural cells from the progenitor fraction of the adult rat OB grown in monolayers can be expanded for several passages in culture and that upon transplantation into a demyelinated spinal cord lesion provide extensive remyelination without ectopic neuronal differentiation.

Topics: Animals; Axons; Brain; Cell Differentiation; Green Fluorescent Proteins; Microscopy, Immunoelectron; Myelin Basic Protein; Myelin Sheath; Neuroglia; Olfactory Bulb; Rats; Rats, Transgenic; Spinal Cord; Spinal Cord Injuries; Stem Cells

This study was designed to investigate functional recovery after the transplantation of mesenchymal stem cells (MSCs) or neurally differentiated MSCs (NMSCs) derived from bone marrow in a rat model of spinal cord injury (SCI). Sprague-Dawley rats were subjected to incomplete SCI using an NYU impactor to create a free drop contusion at the T9 level. The SCI rats were then classified into three groups; MSCs, NMSCs, and phosphate-buffered saline (PBS)-treated groups. The cells or PBS were administrated 1 week after SCI. Basso-Beattie-Bresnahan (BBB) locomotor rating scores were measured at 1-week intervals for 9 weeks. Somatosensory evoked potentials (SSEPs) and motor evoked potentials (MEPs) were also recorded 8 weeks after transplantation. While transplantation of MSCs led to a clear tendency of motor recovery, NMSC-treated rats had significantly improved BBB scores and showed significantly shortened initial latency, N1 latency, and P1 latency of the SSEPs compared to PBS controls. In addition, 5-bromo-2-deoxyuridine (BrdU)-prelabeled MSCs costained for BrdU and glial fibrillary acidic protein (GFAP) or myelin basic protein (MBP) were found rostrally and caudally 5 mm each from the epicenter of the necrotic cavity 4 weeks after transplantation. These results suggest that neurally differentiated cells might be an effective therapeutic source for functional recovery after SCI.

Topics: Animals; Bone Marrow Cells; Bromodeoxyuridine; Cell Differentiation; Disease Models, Animal; Female; Glial Fibrillary Acidic Protein; Immunohistochemistry; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Motor Activity; Myelin Basic Protein; Nervous System; Rats; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord Injuries

ABSTRACT Diffusion tensor imaging (DTI) and quantitative T(2) magnetic resonance imaging (MRI) were used to characterize ex vivo the white matter damage at 3 and 8 weeks following dorsal column transection (DC Tx) injury at the cervical level C5 of rat spinal cords. Luxol Fast Blue (LFB) and myelin basic protein (MBP) staining was used to assess myelin damage, and neurofilament-H in combination with neuron specific beta-III-tubulin (NF/Tub) staining was used to assess axonal damage. Average values of myelin water fraction (MWF), fractional anisotropy (FA), longitudinal diffusivity (D(long)), transverse diffusivity (D(trans)), and average diffusivity (D(ave)) were calculated in the fasciculus gracilis, fasciculus cuneatus, and the dorsal corticospinal tract (CST) 5 mm cranial, as well as 5 and 10 mm caudal to injury and correlated with histology. These tracts were selected as these contain bundles of parallel ascending and descending axons in very circumscribed areas with little intermingling of other axonal populations. Axonal and myelin degeneration occur cranial to injury in the funiculus gracilis and caudal to injury in the CST. Both MWF and D(trans) showed significant correlation with LFB staining at 3 weeks (0.64 and -0.49, respectively) and 8 weeks post-injury (0.88 and -0.71, respectively). Both D(long) and FA correlated significantly with NF/Tub staining at 3 weeks post-injury (0.78 and 0.64, respectively), while only D(long) displayed significant correlation 8 weeks post-injury (0.58 and 0.33, respectively). This study demonstrates that quantitative MRI can accurately characterize white matter damage in DC Tx model of injury in rat spinal cord.

Topics: Animals; Anisotropy; Biomarkers; Body Water; Diffusion; Diffusion Magnetic Resonance Imaging; Disease Models, Animal; Disease Progression; Immunohistochemistry; Male; Myelin Basic Protein; Nerve Fibers, Myelinated; Neural Pathways; Neurofilament Proteins; Predictive Value of Tests; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Injuries; Staining and Labeling; Time Factors; Trauma Severity Indices; Tubulin; Wallerian Degeneration

The central nervous system (CNS) is considered to be an immune-privileged site. For a long time, autoimmunity-induced inflammation has been viewed as an important mediator of secondary damage in the CNS following injury. However, other studies also suggest that autoimmunity is protective and beneficial. To investigate whether protective autoimmunity is present following spinal cord injury (SCI), we employed neonatally thymectomized (Tx) rats which contain few T lymphocytes in their peripheral blood, and passively immunized them with T lymphocytes activated by myelin basic protein (MBP) or spinal cord homogenate (SCH). Here we report that, among Tx, sham-Tx (sTx) and normal rats that received a contusive SCI, no significant histological and behavioral differences were found, suggesting that the endogenous T lymphocytes had no significant influence on the pathogenesis of secondary SCI. In rats passively immunized with MBP- or SCH-activated T cells (MBP-T or SCH-T, respectively), similar numbers of CD4(+) T cells were found to infiltrate into the injured spinal cords. However, only the MBP-T immunization showed neuroprotection, evidenced by the reduction of post-traumatic neuronal losses and improvement of functional recovery. These results collectively suggest that not all T lymphocytes against CNS antigens are neuroprotective and that a subpopulation of them, such as those of MBP-T cells, could be beneficial for SCI repair.

Topics: Analysis of Variance; Animals; Autoimmunity; Cell Survival; Cytoprotection; Flow Cytometry; Fluorescent Antibody Technique; Immunization, Passive; Inflammation; Lymphocyte Activation; Microscopy, Electron; Motor Activity; Motor Neurons; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Recovery of Function; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord; Spinal Cord Injuries; Staining and Labeling; T-Lymphocytes; Thoracic Vertebrae; Thymectomy; Time Factors

Umbilical cord blood (UCB) is known to have stem/progenitor cells. We earlier showed that novel progenitors could be isolated from cryopreserved human UCB with high efficiency. The multipotent progenitor cells were induced to differentiate into neural-lineage cells under the appropriate condition. In this study, we confirmed these neurally induced progenitor cells (NPCs), containing higher quantities of nerve growth factor, promoted functional recovery in rats with spinal cord injury (SCI). Sprague-Dawley rats with SCI achieved a modest improvement in locomotor rating scale until 10 weeks after transplantation of the NPCs. SCI rats treated with NPCs also showed somatosensory-evoked potentials were recovered, and grafted cells especially exhibited oligodendrocytic phenotype around the necrotic cavity. These findings suggest that UCB-NPCs might be a therapeutic resource to repair damaged spinal cords.

Topics: Animals; Behavior, Animal; Cell Differentiation; Cells, Cultured; Cord Blood Stem Cell Transplantation; Electrophysiology; Enzyme-Linked Immunosorbent Assay; Evoked Potentials, Somatosensory; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Male; Motor Activity; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Recovery of Function; Reverse Transcriptase Polymerase Chain Reaction; Spinal Cord Injuries; Stem Cells

Injuries to the cauda equina/conus medullaris portion of the spinal cord can result in motor, sensory, and autonomic dysfunction, and neuropathic pain. In rats, unilateral avulsion of the motor efferents from the lumbosacral spinal cord results in at-level allodynia, along with a corresponding glial and inflammatory response in the dorsal horn of the spinal cord segments immediately rostral to the lesion. Here, we investigated the fate of intramedullary primary sensory projections following a motor efferent lesion. The lumbosacral (L6 and S1) ventral roots were unilaterally avulsed from the rat spinal cord (VRA; n=9). A second experimental group had the avulsed roots acutely reimplanted into the lateral funiculus (Imp; n=5), as this neural repair strategy is neuroprotective, and promotes the functional reinnervation of peripheral targets. A laminectomy-only group served as controls (Lam; n=7). At 8 weeks post-lesion, immunohistochemical examination showed a 42% reduction (P<0.001) in the number of RT97-positive axons in the ascending tracts of the dorsal funiculus of the L4-5 spinal segment in VRA rats. Evidence for degenerating myelin was also present. Reimplantation of the avulsed roots ameliorated axon and myelin degeneration. Axons in the descending dorsal corticospinal tract were unaffected in all groups, suggesting a specificity of this lesion for spinal primary sensory afferents. These results show for the first time that a lesion restricted to motor roots can induce the degeneration of intramedullary sensory afferents. Importantly, reimplantation of the lesioned motor roots ameliorated sensory axon degeneration. These data further support the therapeutic potential for reimplantation of avulsed ventral roots following trauma to the cauda equina/conus medullaris.

Topics: Animals; Calcium-Binding Proteins; Cauda Equina; Disease Models, Animal; Female; Functional Laterality; Glial Fibrillary Acidic Protein; Indoles; Microfilament Proteins; Myelin Basic Protein; Nerve Degeneration; Nerve Regeneration; Neurofilament Proteins; Rats; Rats, Sprague-Dawley; Recovery of Function; Replantation; Spinal Cord Injuries; Spinal Nerve Roots

Methylprednisolone (MP) is used to treat a variety of neurological disorders involving white matter injury, including multiple sclerosis, acute disseminated encephalomyelitis, and spinal cord injury (SCI). Although its mechanism of action has been attributed to anti-inflammatory or antioxidant properties, we examined the possibility that MP may have direct neuroprotective activities. Neurons and oligodendrocytes treated with AMPA or staurosporine died within 24 h after treatment. MP attenuated oligodendrocyte death in a dose-dependent manner; however, neurons were not rescued by the same doses of MP. This protective effect was reversed by the glucocorticoid receptor (GR) antagonist (11, 17)-11-[4-(dimethylamino)phenyl]-17-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (RU486) and small interfering RNA directed against GR, suggesting a receptor-dependent mechanism. MP reversed AMPA-induced decreases in the expression of anti-apoptotic Bcl-x(L), caspase-3 activation, and DNA laddering, suggesting anti-apoptotic activity in oligodendrocytes. To examine whether MP demonstrated this selective protection in vivo, neuronal and oligodendrocyte survival was assessed in rats subjected to spinal cord injury (SCI); groups of rats were treated with or without MP in the presence or absence of RU486. Eight days after SCI, MP significantly increased oligodendrocytes (CC-1-immunoreactive cells) after SCI, but neuronal (neuronal-specific nuclear protein-immunoreactive cells) number remained unchanged; RU486 reversed this protective effect. MP also inhibited SCI-induced decreases in Bcl-x(L) and caspase-3 activation. Consistent with these findings, the volume of demyelination, assessed by Luxol fast blue staining, was attenuated by MP and reversed by RU486. These results suggest that MP selectively inhibits oligodendrocyte but not neuronal cell death via a receptor-mediated action and may be a mechanism for its limited protective effect after SCI.

Topics: Analysis of Variance; Animals; Benzothiadiazines; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; DNA Fragmentation; Dose-Response Relationship, Drug; Embryo, Mammalian; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Female; L-Lactate Dehydrogenase; Methylprednisolone; Myelin Basic Protein; Neurons; Neuroprotective Agents; Oligodendroglia; Phosphopyruvate Hydratase; Rats; Rats, Long-Evans; RNA, Small Interfering; Spinal Cord Injuries

Three independent experiments in a rat model of contusive spinal cord (SC) injury were performed. Two studied the alterations induced by SC injury on some immunological aspects of the T-cell response. The third one evaluated the motor recovery of rats with low-thoracic injuries.. To examine the effect of level, intensity and phase of SC injury on T-cell proliferation and T-cell-dependent antibody response.. Neuroimmunology Department, UIMEN, IMSS-CAMINA Research Center.. Lymphocyte proliferation and hemagglutination assays were performed. Animals were injured either moderately or severely at T1 or T12 SC segments. Analysis of peripheral T-cell proliferation in response to mitogens and to myelin basic protein (MBP), as well as of antibody production against a T-dependent antigen, was performed at acute, subacute and chronic phases.. A significant decrease of both response to mitogens and antibody production was found especially during the acute phase and in animals with severe and high (T1)-level injury. Animals with low (T12) and moderate contusions recovered to control levels at the chronic phase. An autoimmune reaction against MBP was observed only in animals with severe contusion at low level.. The intensity, level and phase of SC injury differentially alter the function of T cells. These results will allow a better interpretation of studies directed to elucidate the role of T lymphocytes in various processes developed after SC injury.

Topics: Animals; Antigen-Antibody Reactions; Cell Proliferation; Central Nervous System; Concanavalin A; Erythrocytes; Female; Hemagglutination; Locomotion; Myelin Basic Protein; Rats; Rats, Inbred F344; Sheep; Spinal Cord Injuries; Spleen; Stimulation, Chemical; T-Lymphocytes

We hypothesized that heme oxygenase (HO)-1, the inducible form of HO, represents an important defense against early oxidative injury in the traumatized spinal cord by stabilizing the blood-spinal cord barrier and limiting the infiltration of leukocytes. To test this hypothesis, we first examined the immunoexpression of HO-1 and compared barrier permeability and leukocyte infiltration in spinal cord-injured HO-1-deficient (+/-) and wild-type (WT, +/+) mice. Heme oxygenase was expressed in both endothelial cells and glia of the injured cord. Barrier disruption to luciferase and infiltration of neutrophils were significantly greater in the HO-1+/- than WT mice at 24 h postinjury (P

Topics: Aldehydes; Animals; Blood Vessels; Blotting, Western; Brain; Enzyme Induction; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Immunohistochemistry; Inflammation; Male; Malondialdehyde; Mice; Mice, Inbred C57BL; Mice, Knockout; Myelin Basic Protein; Neutrophil Infiltration; Oxidative Stress; Spinal Cord; Spinal Cord Injuries

This study characterized the Infinite Horizon (IH) Impactor for use in mouse models of contusion spinal cord injury (SCI), and investigated the feasibility and reliability of using magnetic resonance imaging (MRI) as a method to accurately measure lesion volume after mouse contusion SCI. Eight-week-old female C57Bl/6 mice received a mild (30 kilodyne), moderate (50 kilodyne), or severe (70 kilodyne) contusion injury at the T9 vertebral level. Uninjured control mice received a T9 laminectomy only. Functional recovery was assessed using the Basso, Beattie, Bresnahan (BBB) and Basso Mouse Scale (BMS) open-field locomotor rating scales. Next, 4% paraformaldehyde-perfused spinal cords were collected between the T6 and T12 spinal roots, and stored in phosphate-buffered saline (PBS) at 4 degrees C until MRI analysis. MRI lesion volumes were determined using T1-weighted images on a 7-Tesla MRI. Histology was performed on 20-microm polyester wax-embedded sections processed from the same spinal cords for stereological determination of fibronectin lesion volume and myelin basic protein spared white matter volume. Area of spared white matter at the epicenter was also analyzed. The results demonstrated that the IH Impactor produced precise, graded contusion SCI in mice. Lesion volumes were positively correlated with force of impact, and negatively correlated with spared white matter and functional recovery. Additionally, similar lesion volumes were detected using fibronectin staining and MRI analysis, although MRI may be more sensitive for milder injuries. These results give researchers more options in how to analyze spinal cord injuries in animal models.

Topics: Animals; Behavior, Animal; Brain; Contusions; Female; Fibronectins; Image Processing, Computer-Assisted; Locomotion; Magnetic Resonance Imaging; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Plastic Embedding; Reproducibility of Results; Spinal Cord Injuries

The inflammatory response has been characterized in the lumbosacral segments (L4-S1) of rats after spinal transection at T8. Immune cells were identified immunohistochemically using antibodies to complement type 3 receptor, CD11b (OX-42), the macrophage lysosomal antigen, CD68 (ED1), major histocompatibility complex class II (MHC II), and CD163 (ED2), a marker of perivascular cells. One week after cord transection, OX-42+ microglial density had nearly doubled. In the white matter, microglia became enlarged, often with retracted processes. In contrast, microglia in the grey matter remained ramified although nearly half of those lying medially contained clusters of ED1+ granules. After 8 weeks, ED1+ (+/-MHC II) macrophages were prominent in regions of Wallerian degeneration extending from dorsolateral to ventral funiculi. Microglial density remained raised in grey matter, particularly in the ventral horns of L4/5. Ramified microglia expressing MHC II+ (+/-ED1) extended from deep in the dorsal columns and around the central canal to the ventral columns. More ED2+ (+/-MHC II) perivascular and meningeal cells were recruited and expressed ED1. Thus, distinct from their conversion into macrophages in the white matter, the activation of ramified microglia after degeneration in the grey matter involves expression of ED1 without morphologic transformation.

Topics: Animals; CD11b Antigen; Cell Count; Ectodysplasins; Female; Histocompatibility Antigens Class II; Lumbosacral Region; Microglia; Myelin Basic Protein; Nerve Tissue; Rats; Rats, Wistar; Spinal Cord Injuries; Thoracic Vertebrae; Time Factors

It is now widely accepted that progesterone (PROG) brings neuroprotection in lesions of the peripheral and central nervous system. Spinal cord trauma leads to neuronal degeneration, astrogliosis, demyelination, and proliferation of oligodendrocyte-precursor cells (OPCs). In this work, we studied the effects of PROG on myelin-related parameters in rats with complete spinal cord transection (TRX). To this end, sham-operated controls and rats with TRX at thoracic level T10 received vehicle or PROG (4 mg/kg/day) during 3 days. Three variables were measured in the lumbar L4 region below the lesion: (1) expression of myelin basic protein (MBP) at the protein and mRNA levels; (2) density of NG2-immunopositive cells as markers for OPCs; and (3) number of cells immunopositive for RIP, an antibody staining mature oligodendrocytes. TRX decreased MBP immunostaining in the corticospinal tract (CST) and dorsal ascending tract (DAT) but not the ventral funiculus (VF). NG2+ cells, which were detected in low number in controls, increased after TRX in the gray and white matter. RIP-positive cell number, however, remained unchanged. PROG treatment of rats with TRX enhanced the expression of MBP protein and mRNA in CST and DAT, but not VF and highly stimulated the number of cells showing NG2 immunostaining over untreated lesioned rats. Instead, density of RIP positive cells was similar in the PROG-treated and untreated lesioned groups. We propose that PROG effects on expression of MBP and the number of NG2 immunopositive cells may contribute to neuroprotection, as they go in parallel with previous results showing enhanced biochemical and morphological parameters of motoneurons of animals with TRX receiving PROG treatment.

Topics: Animals; Antigens; Cell Count; Male; Myelin Basic Protein; Oligodendroglia; Progesterone; Proteoglycans; Rats; Rats, Sprague-Dawley; RNA, Messenger; Spinal Cord Injuries; Stem Cells

As a consequence of secondary pathophysiological mechanisms elicited after spinal cord injury (SCI), oligodendrocytes die by waves of apoptosis. This ultimately results in demyelination of intact axons leading to a loss of their conducting properties. Preservation of as few as 5% to 10% of myelinated axons in individual tracts can confer locomotor recovery. Thus, strategies aimed at rescuing mature oligodendrocytes ensheathing viable axons are likely to be of therapeutic significance. We report that leukemia inhibitory factor (LIF) can prevent oligodendrocyte apoptosis, notably contralateral to the spinal cord lesion, through the induction of the JAK/STAT and Akt signaling pathways as well as by potentiating the expression of the antiapoptotic molecule, cIAP2. Reduced oligodendrocyte apoptosis after SCI with LIF administration resulted in a substantial decrease in demyelination shown by the preservation of lamellated myelin surrounding viable axons and deposition of the degraded myelin basic protein. The data suggest that LIF signals survival in oligodendrocytes after SCI, prevents the secondary wave of demyelination, and thereby reduces inhibitory myelin deposits.

Topics: Animals; Axotomy; Baculoviral IAP Repeat-Containing 3 Protein; Cell Death; Demyelinating Diseases; Disease Models, Animal; Female; Gene Expression; Immunoprecipitation; In Situ Nick-End Labeling; Inhibitor of Apoptosis Proteins; Interleukin-6; Leukemia Inhibitory Factor; Leukemia Inhibitory Factor Receptor alpha Subunit; Mice; Mice, Inbred C57BL; Myelin Basic Protein; Oligodendroglia; Receptors, Cytokine; Receptors, OSM-LIF; Severity of Illness Index; Spinal Cord Injuries; STAT Transcription Factors; Ubiquitin-Protein Ligases

The natural history of post-traumatic demyelination and myelin repair in the human spinal cord is largely unknown and has remained a matter of speculation. A wealth of experimental studies indicate that mild to moderate contusive injuries to the mammalian spinal cord evolve into a cavity with a preserved rim of white matter in which a population of segmentally demyelinated axons persists. It is believed that such injured axons have abnormal conduction properties. Theoretically, such axons might show improved function if myelin repair occurred. Schwann cells can remyelinate axons affected by multiple sclerosis, but little evidence exists that such repair can occur spontaneously following traumatic human SCI. Therefore, it is important to determine if chronic demyelination is present following human spinal cord injury. There are no previous reports that have conclusively demonstrated demyelination in the human spinal cord following traumatic spinal cord injury using immunohistochemical techniques. Immunohistochemical methods were used to study the distribution of peripheral and central myelin proteins as well as axonal neurofilament at the injury epicenter in 13 postmortem chronically injured human spinal cords 1-22 years following injury. Of these seven could be assessed by our methods. We found that some axonal demyelination can be detected even a decade following human SCI and indirect evidence that invading Schwann cells contributed to restoration of myelin sheaths around some spinal axons.

Topics: Adult; Aged; Aged, 80 and over; Demyelinating Diseases; Diagnostic Imaging; Female; Glial Fibrillary Acidic Protein; Humans; Immunohistochemistry; Male; Middle Aged; Myelin Basic Protein; Myelin Proteolipid Protein; Neurofilament Proteins; Postmortem Changes; Schwann Cells; Spinal Cord Injuries; Survival Rate; Time Factors

Inhibition of the small GTPase ras homology protein (Rho) or its downstream target, the Rho-associated kinase (ROCK), has been shown to promote axon regeneration and to improve functional recovery following spinal cord injury (SCI) in the adult rat. Here, we have analyzed the expression of RhoA and RhoB following spinal cord injury in order to assess whether Rho is a possible target for late pharmacological intervention. In control spinal cords, RhoA(+) cells were almost absent, whereas RhoB was localized to some ependymal cells, a few microglia, and some dissociated neurons. In injured spinal cords, RhoA(+) and RhoB(+)cells accumulated at perilesional areas and in the developing necrotic core early after injury at day 1. After reaching their maximum levels (RhoA at day 3; RhoB at day 1), RhoA(+) and RhoB(+) cell numbers remained significantly elevated until day 28. In areas remote from the lesion (> or =0.75 mm), a more discrete accumulation of RhoA(+) and RhoB(+) cells was observed, primarily in areas of ongoing Wallerian degeneration. RhoA and RhoB were predominantly expressed by polymorphonuclear granulocytes, ED1(+) microglia/macrophages, oligodendrocytes, some neurons, and swollen axons/neurites. Furthermore, expression was located to lesional, reactive astrocytes and fibroblastoid cells confined to areas of scar formation. Our experiments have determined that most RhoA(+) and RhoB(+) cells (>70%) are of mononuclear origin. The persistent presence of lesional RhoA(+) and RhoB(+) axon/neurite fibers over a period of 4 weeks after injury suggests that Rho inhibition is a putative therapeutic concept also for delayed intervention after SCI.

Topics: Amyloid beta-Protein Precursor; Animals; Brain; Cell Count; Cyclooxygenase 2; Ectodysplasins; Gene Expression Regulation; Glial Fibrillary Acidic Protein; Immunohistochemistry; Lymphocytes; Male; Membrane Proteins; Myelin Basic Protein; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Lew; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; Spinal Cord Injuries; Time Factors

Injuries to the central nervous system (CNS) trigger an inflammatory reaction with potentially devastating consequences. In this report we compared the characteristics of the inflammatory response on spinal cord injury (SCI) caused by a stab wound between the T7 and T9 vertebrae and spontaneous experimental autoimmune encephalomyelitis (EAE). SCI and EAE were compared in two types of myelin basic protein Ac1-11-specific T-cell receptor transgenic mice: T/R+ mice harbor regulatory T cells, and T/R- mice lack regulatory T cells. Our results show that 8 days after SCI, T/R- mice developed a strong T-cell infiltrate in the spinal cord, with remarkable down-modulation of CD4 expression that was accompanied by a local increase in Mac-3+ and F4/80+ reactivity and diffuse local and distal astrogliosis. In contrast, T/R+ mice exhibited a modest increase in CD4+ cells localized to the site of injury, without CD4 down-modulation; focal astrogliosis was restricted to the site of the lesion, although Mac-3+ and F4/80+ cells were also present. Similarly to T/R- mice that underwent SCI, T cells displaying down-modulated CD4 expression were found in the CNS of older T/R- mice afflicted by spontaneous EAE. Overall, our results suggest that common mechanisms regulate T-cell accumulation in CNS lesions of different causes, such as mechanic lesion or autoimmune-mediated damage.

Topics: Animals; CD4 Antigens; Encephalomyelitis, Autoimmune, Experimental; Flow Cytometry; Immunohistochemistry; Lymphocyte Activation; Mice; Mice, Transgenic; Myelin Basic Protein; Spinal Cord Injuries; T-Lymphocytes

The intraspinal cues that orchestrate T-cell migration and activation after spinal contusion injury were characterized using B10.PL (wild-type) and transgenic (Tg) mice with a T-cell repertoire biased toward recognition of myelin basic protein (MBP). Previously, we showed that these strains exhibit distinct anatomical and behavioral phenotypes. In Tg mice, MBP-reactive T-cells are activated by spinal cord injury (SCI), causing more severe axonal injury, demyelination, and functional impairment than is found in non-Tg wild-type mice (B10.PL). Conversely, despite a robust SCI-induced T-cell response in B10.PL mice, no overt T-cell-mediated pathology was evident. Here, we show that chronic intraspinal T-cell accumulation in B10.PL and Tg mice is associated with a dramatic and sustained increase in CXCL10/IP-10 and CCL5/RANTES mRNA expression. However, in Tg mice, chemokine mRNA were enhanced 2- to 17-fold higher than in B10.PL mice and were associated with accelerated intraspinal T-cell influx and enhanced CNS macrophage activation throughout the spinal cord. These data suggest common molecular pathways for initiating T-cell responses after SCI in mice; however, if T-cell reactions are biased against MBP, molecular and cellular determinants of neuroinflammation are magnified in parallel with exacerbation of neuropathology and functional impairment.

Topics: Animals; Chemokine CCL5; Chemokine CXCL10; Chemokines; Chemokines, CC; Chemokines, CXC; Chemotaxis, Leukocyte; Contusions; Genes, T-Cell Receptor beta; Growth Substances; Insulin-Like Growth Factor I; Interleukin 1 Receptor Antagonist Protein; Lymphocyte Activation; Lymphokines; Macrophages; Mice; Mice, Transgenic; Microglia; Myelin Basic Protein; Myelitis; RNA, Messenger; Sialoglycoproteins; Spinal Cord Injuries; T-Cell Antigen Receptor Specificity; T-Lymphocyte Subsets; Transforming Growth Factor beta; Transforming Growth Factor beta1

Spinal cords of adult cats were transected and subsequently reconnected with the biocompatible porous poly (N-[2-hydroxypropyl] methacrylamide) hydrogel, NeuroGel. Tissue repair was examined at various time points from 6-21 months post reconstructive surgery. We examined two typical phenomena, astrogliosis and scar formation, in spines reconstructed with the gel and compared them to those from transected non-reconstructed spines. Confocal examination with double immunostaining for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) showed that the interface formed between the hydrogel and the spine stumps did prevent scar formation and only a moderate gliosis was observed. The gel implant provided an adequate environment for growth of myelinated fibers and we saw angiogenesis within the gel. Electron microscopy showed that regenerating axons were myelinated by Schwann cells rather than oligodendrocytes. Moreover, the presence of the gel implant lead to a considerable reduction in damage to distal caudal portions of the spine as assessed by the presence of more intact myelinated fibers and a reduction of myelin degradation. Neurologic assessments of hindlimb movement at various times confirmed that spinal cord reconstruction was not only structural but also functional. We conclude that NeuroGel lead to functional recovery by providing a favorable substrate for regeneration of transected spinal cord, reducing glial scar formation and allowing angiogenesis.

Topics: Animals; Biocompatible Materials; Cats; Cicatrix; Female; Glial Fibrillary Acidic Protein; Gliosis; Hindlimb; Hydrogels; Methacrylates; Microscopy, Electron; Motor Activity; Myelin Basic Protein; Myelin Sheath; Neovascularization, Physiologic; Nerve Fibers, Myelinated; Nerve Regeneration; Recovery of Function; Schwann Cells; Spinal Cord Injuries; Treatment Outcome

Myelin-reactive T-cells are activated by traumatic spinal cord injury (SCI) in rodents and humans. Despite the historical association of these cells with experimental and clinical neuropathology, recent data suggest a neuroprotective role for myelin-reactive T-cells. Because of the biological and therapeutic implications of these findings, we attempted to reproduce the original neuroprotective vaccine protocols in a model of rat SCI. Specifically, MBP-reactive T-cell function was enhanced in SCI rats via passive or active immunization. Locomotor function was assessed using a standardized locomotor rating scale (Basso-Beattie-Bresnahan scale) and was correlated with myelin and axon sparing. The functional and anatomical integrity of the rubrospinal pathway also was analyzed using the inclined plane test and anatomical tract tracing. MBP-immunized rats exhibited varying degrees of functional impairment, exacerbated lesion pathology, greater rubrospinal neuron loss, increased intraspinal T-cell accumulation, and enhanced macrophage activation relative to SCI control groups. These data are consistent with the conventional view of myelin-reactive T-cells as pathological effector cells.

Topics: Animals; Disease Models, Animal; Disease Progression; Female; Immunization, Passive; Motor Activity; Myelin Basic Protein; Myelin Sheath; Myelitis; Rats; Rats, Inbred Lew; Recovery of Function; Spinal Cord; Spinal Cord Injuries; T-Lymphocytes; Vaccination

Immune system activity has traditionally been considered harmful for recovery after spinal cord injury (SCI). Recent evidence suggests, however, that immune activity--and specifically autoimmune activity--is evoked by the insult, is beneficial if properly regulated and is amenable to boosting. Thus, for example, vaccination with an altered peptide ligand derived from myelin basic protein reduces the progressive degeneration of neurons that escaped the initial insult, thereby promoting recovery after SCI. As the steroid drug methylprednisolone (MP) is currently the only treatment available for patients with SCI, our purpose in the present study was to examine the mutual compatibility of the two treatments within the post-traumatic therapeutic window. We show, using rats of two different strains, that if MP is injected concomitantly with the therapeutic vaccination, the beneficial effect of the vaccination is diminished. However, if MP is given immediately after the insult and the vaccination 48 h later, MP does not detract from the beneficial effect of the vaccination. These results demonstrate that the therapeutic window after SCI can accommodate immediate administration of MP plus a delayed therapeutic vaccination.

Topics: Analysis of Variance; Animals; Behavior, Animal; Cell Count; Cell Division; Cells, Cultured; Dextrans; Ectodysplasins; Encephalomyelitis, Autoimmune, Experimental; Female; Immunohistochemistry; Immunotherapy, Active; Laminectomy; Membrane Proteins; Methylprednisolone; Motor Activity; Mycobacterium; Myelin Basic Protein; Neuroprotective Agents; Peptide Fragments; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Rhodamines; Species Specificity; Spinal Cord Injuries; T-Lymphocytes; Time Factors; Tryptophan

Injury-induced self-destructive processes cause significant functional loss after incomplete spinal cord injury (SCI). Cellular elements of both the innate (macrophage) and the adaptive (T-cell) immune response can, if properly activated and controlled, promote post-traumatic regrowth and protection after SCI. Dendritic cells (DCs) trigger activation of effector and regulatory T-cells, providing a link between the functions of the innate and the adaptive immune systems. They also initiate and control the body's response to pathogenic agents and regulate immune responses to both foreign and self-antigens. Here we show that post-injury injection of bone marrow-derived DCs pulsed with encephalitogenic or nonencephalitogenic peptides derived from myelin basic protein, when administered (either systemically or locally by injection into the lesion site) up to 12 d after the injury, led to significant and pronounced recovery from severe incomplete SCI. No significant protection was seen in DC recipients deprived of mature T-cells. Flow cytometry, RT-PCR, and proliferation assays indicated that the DCs prepared and used here were mature and immunogenic. Taken together, the results suggest that the DC-mediated neuroprotection was achieved via the induction of a systemic T-cell-dependent immune response. Better preservation of neural tissue and diminished formation of cysts and scar tissue accompanied the improved functional recovery in DC-treated rats. The use of antigen-specific DCs may represent an effective way to obtain, via transient induction of an autoimmune response, the maximal benefit of immune-mediated repair and maintenance as well as protection against self-destructive compounds.

Topics: Animals; Autoimmunity; Cells, Cultured; Dendritic Cells; Disease Models, Animal; Encephalomyelitis, Autoimmune, Experimental; Magnetic Resonance Imaging; Motor Activity; Myelin Basic Protein; Nerve Regeneration; Peptide Fragments; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Treatment Outcome; Vaccination

Spinal cord injury (SCI) is associated with progressive neurodegeneration and dysfunction. Multiple cellular and molecular mechanisms are involved in this pathogenesis. In particular, the activation of proteases following trauma can cause apoptosis in the spinal cord. Calpain, a calcium-dependent cysteine protease, plays a major role in apoptosis following trauma. We identified apoptosis and decrease in transcription of the genes for proteolipid protein (PLP) and myelin basic protein (MBP) in five 1-cm long spinal cord segments (S1, distant rostral; S2, near rostral; S3, lesion; S4, near caudal; and S5, distant caudal) 24 h after induction of SCI (40 g.cm force) in rats by weight-drop method. Sham rats underwent laminectomy and did not receive injury. Internucleosomal DNA fragmentation occurred prominently in the lesion (S3), moderately in near segments (S2 and S4), and slightly in distant segments (S1 and S5) of injured rats, indicating the occurrence of apoptosis in the lesion and penumbra. Levels of transcription of PLP and MBP were reduced highly in the lesion and moderately in near segments, suggesting that apoptotic loss of cells impaired biosynthesis of two important structural components of myelin. Immediate administration of the calpain inhibitor E-64-d (1 mg/kg) to injured rats prevented apoptosis and restored transcription of these genes, indicating the therapeutic efficacy of calpain inhibitor for treatment of SCI.

Topics: Animals; Apoptosis; Calpain; DNA Fragmentation; Electrophoresis, Agar Gel; Leucine; Myelin Basic Protein; Myelin Proteolipid Protein; Nucleosomes; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Spinal Cord Injuries; Transcription, Genetic

Functional loss after spinal cord injury (SCI) is caused, in part, by demyelination of axons surviving the trauma. Administration of guanosine (8 mg/kg/day, i.p.) for 7 consecutive days, starting 5 weeks after moderate SCI in rats, improved locomotor function and spinal cord remyelination. Myelinogenesis was associated with an increase in the number of mature oligodendrocytes detected in guanosine-treated spinal cord sections in comparison with controls. These data indicate that guanosine-induced remyelination resulted, at least in part, from activation of endogenous oligodendrocyte lineage cells. These findings may have significant implications for chronic demyelinating diseases.

Topics: Animals; Chronic Disease; Female; Guanosine; Motor Activity; Myelin Basic Protein; Myelin Sheath; Nerve Fibers, Myelinated; Rats; Rats, Wistar; Recovery of Function; Spinal Cord Injuries

Alterations of the blood-spinal cord barrier (BSCB) following spinal cord injury (SCI) and leakage of serum proteins induce vasogenic edema and cell damage. The possibility that two members of the neurotrophin family, BDNF or IGF-1 induce neuroprotection by attenuating the BSCB permeability following trauma was examined in a rat model. Repeated topical application of BDNF or IGF-1 (0.1 1microg, 0.5 microg or 1 microg in 10 microl) onto the spinal cord 30 min before SCI or 2, 5, 10 or 30 min thereafter significantly attenuated BSCB permeability to Evans blue and iodine. In the neurotrophin treated rats. edema formation, degradation of MBP, and myelin vesiculation were much less frequent compared to the untreated traumatised rats. The protective effect of BDNF and IGF-1 was most pronounced at the high dose (1 microg in 10 microl) given either 30 min before or within 10 min after SCI. The observations suggest that early intervention with neurotrophins in high doses following trauma (within 10 min) attenuates disturbances of the fluid microenvironment of the spinal cord. This indicates that BSCB opening plays an important role in SCI induced myelin vesiculation and cord pathology.

Topics: Animals; Axons; Brain-Derived Neurotrophic Factor; Capillary Permeability; Diffuse Axonal Injury; Edema; Endothelium, Vascular; Insulin-Like Growth Factor I; Male; Microcirculation; Myelin Basic Protein; Rats; Rats, Sprague-Dawley; Spinal Cord; Spinal Cord Diseases; Spinal Cord Injuries

To investigate the protective effect of pSVPoMcat (myelin basic protein microgene) modifying Schwann cell on injured spinal neurons.. A model of rat spinal cord injured by hemisection was used. One hundred and twenty healthy SD rats of both sexes weighing 250-300 g were divided into three groups: Group A (n=40, treated with implantation of pSVPoMcat modifying Schwann cell), Group B (n= 40, treated with implantation of Schwann cell only) and Group C (n=400, treated with sham operation as the control). One week after operation the rat functional recovery was observed dynamically by using combined behavioral score (CBS) and cortical somatasensory evoked potentials, the spinal cord sections were stained by Nissl, acid phosphatase enzyme histochemistry and cell apoptosis was examined by methye green, terminal deoxynucleotidyl and the dUTP Nick end labeling technique. Quantitative analysis was done by computer image analysis system.. In Group A the injured neurons recovered well morphologically. The imaging analysis showed a result of Group A

Topics: Acid Phosphatase; Animals; Apoptosis; Cell Transplantation; Disease Models, Animal; Evoked Potentials, Somatosensory; Female; Gene Transfer Techniques; Male; Methyl Green; Myelin Basic Protein; Nerve Regeneration; Rats; Rosaniline Dyes; Schwann Cells; Spinal Cord Injuries

The ability of rats or mice to withstand the consequences of injury to myelinated axons in the CNS was previously shown to depend on the ability to manifest a T cell-mediated protective immune response, which is amenable to boosting by myelin-specific T cells. Here we show that this ability, assessed by retinal ganglion cell survival after optic nerve injury or locomotor activity after spinal cord contusion, is decreased if the animals were immunized as neonates with myelin proteins (resulting in their nonresponsiveness as adults to myelin proteins) or injected with naturally occurring regulatory CD4(+)CD25(+) T cells immediately after the injury, and is improved by elimination of these regulatory T cells. In nude BALBc mice replenished with a splenocyte population lacking CD4(+)CD25(+) regulatory T cells, significantly more neurons survived after optic nerve injury than in nude mice replenished with a complete splenocyte population or in matched wild-type controls. In contrast, neuronal survival in wild-type BALBc mice injected with CD4(+)CD25(+) regulatory T cells immediately after injury was significantly worse than in noninjected controls. These findings suggest that the ability to cope with the sequelae of a CNS insult is affected unfavorably by nonresponsiveness to myelin self-antigens and favorably by conditions allowing rapid expression of an autoimmune response. The regulatory T cells might represent an evolutionary compromise between the need to avoid autoimmune diseases and the need for autoimmunity on alert for the purpose of tissue maintenance.

Topics: Adoptive Transfer; Animals; Autoimmunity; Axons; CD4-Positive T-Lymphocytes; Cell Separation; Cell Survival; Central Nervous System; Disease Susceptibility; Female; Gamma Rays; Guinea Pigs; Immunization; Lymphocyte Depletion; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Myelin Basic Protein; Nerve Crush; Nerve Regeneration; Neurons; Optic Nerve Injuries; Radiation Chimera; Radiation Injuries, Experimental; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Receptors, Interleukin-2; Retinal Ganglion Cells; Spinal Cord Injuries; T-Lymphocyte Subsets; Thymectomy

Neurotrophins enhance the survival of cells in the nervous system under both physiological and pathological conditions, such as those caused by disease or trauma. We recently demonstrated that expression of brain-derived neurotrophic factor (BDNF) was up-regulated in neurons and glia after compression-induced spinal cord injury (SCI). We show here the effects of BDNF on the oligodendrocyte survival and functional recovery after SCI. The effects of intrathecally administered BDNF on both Cu/Zn superoxide dismutase (CuZnSOD) and myelin basic protein (MBP) expression were examined using rats that had received compression-induced spinal cord injury. CuZnSOD expression in the spinal cord was down-regulated within 24 h of compression-induced injury and then recovered. Continuous infusion of BDNF inhibited the acute down-regulation of CuZnSOD expression. In situ hybridization showed that CuZnSOD was expressed in both neurons and glia. Although MBP expression was greatly reduced after injury, BDNF administration promoted the recovery of MBP expression nearly to a control level after 2 wk. Furthermore, BDNF administration also prompted behavioral recovery. These results suggest BDNF's usefulness in human clinical applications. The attenuation of CuZnSOD down-regulation may be related to a protective effect of BDNF and the promotion of MBP up-regulation may be related to a long-lasting restorative effect.

Topics: Animals; Blotting, Northern; Brain-Derived Neurotrophic Factor; Cell Survival; Free Radical Scavengers; Humans; Immunohistochemistry; In Situ Hybridization; Male; Motor Activity; Myelin Basic Protein; Oligodendroglia; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Superoxide Dismutase

Lymphocytes respond to myelin proteins after spinal cord injury (SCI) and may contribute to post-traumatic secondary degeneration. However, there is increasing evidence that autoreactive T-lymphocytes may also convey neuroprotection and promote functional recovery after CNS injury. To clarify the role of myelin autoreactive lymphocytes after SCI, we performed contusion injuries in the thoracic spinal cord of transgenic (Tg) mice in which >95% of all CD4+ T-lymphocytes are reactive with myelin basic protein (MBP). We observed significantly impaired recovery of locomotor and reflex function in Tg mice compared with non-Tg (nTg) littermates. Measures of functional impairment in Tg mice correlated with significantly less white matter at the injury site, and morphometric comparisons of injured Tg and nTg spinal cords revealed increased rostrocaudal lesion expansion (i.e., secondary degeneration) in Tg mice. Rostrocaudal to the impact site in SCI-nTg mice, demyelination was restricted to the dorsal funiculus, i.e., axons undergoing Wallerian degeneration. The remaining white matter appeared normal. In contrast, lymphocytes were colocalized with regions of demyelination and axon loss throughout the white matter of SCI-Tg mice. Impaired neurological function and exacerbated neuropathology in SCI-Tg mice were associated with increased intraspinal production of proinflammatory cytokine mRNA; neurotrophin mRNA was not elevated. These data suggest that endogenous MBP-reactive lymphocytes, activated by traumatic SCI, can contribute to tissue injury and impair functional recovery. Any neuroprotection afforded by myelin-reactive T-cells is likely to be an indirect effect mediated by other non-CNS-reactive lymphocytes. Similar to the Tg mice in this study, a subset of humans that are genetically predisposed to autoimmune diseases of the CNS may be adversely affected by vaccine therapies designed to boost autoreactive lymphocyte responses after CNS trauma. Consequently, the safe implementation of such therapies requires that future studies define the mechanisms that control T-cell function within the injured CNS.

Topics: Animals; Autoimmunity; Axons; Behavior, Animal; CD4-Positive T-Lymphocytes; Cytokines; Demyelinating Autoimmune Diseases, CNS; Disease Progression; Hindlimb; Image Processing, Computer-Assisted; Immunotherapy, Active; Lymphoid Tissue; Mice; Mice, Transgenic; Myelin Basic Protein; Nerve Growth Factors; Polymerase Chain Reaction; Receptors, Antigen, T-Cell; Recovery of Function; Reflex; RNA, Messenger; Spinal Cord; Spinal Cord Injuries

Spinal cord injury and its devastating consequences are the subject of intensive research aimed at reversing or at least minimizing functional loss. Research efforts focus on either attenuating the post-injury spread of damage (secondary degeneration) or inducing some regeneration. In most of these studies, as well as in clinical situations, evaluation of the state of the injured spinal cord poses a serious difficulty. To address this problem, we carried out a diffusion-weighted MRI experiment and developed an objective routine for quantifying anisotropy in injured rat spinal cords. Rats were subjected to a contusive injury of the spinal cord caused by a controlled weight drop. Untreated control rats were compared with rats treated with T cells specific to the central nervous system self-antigen myelin basic protein, a form of therapy recently shown to be neuroprotective. After the rats were killed their excised spinal cords were fixed in formalin and imaged by multislice spin echo MRI, using two orthogonal diffusion gradients. Apparent diffusion coefficient (ADC) values and anisotropy ratio (AI) maps were extracted on a pixel-by-pixel basis. The calculated sum of AI values (SAI) for each slice was defined as a parameter representing the total amount of anisotropy. The mean-AI and SAI values increased gradually with the distance from the site of the lesion. At the site itself, the mean-AI and SAI values were significantly higher in the spinal cords of the treated animals than in the controls (P = 0.047, P = 0.028, respectively). These values were consistent with the score of functional locomotion. The difference was also manifested in the AI maps, which revealed well-organized neural structure in the treated rats but not in the controls. The SAI values, AI histograms, and AI maps proved to be useful parameters for quantifying injury and recovery in an injured spinal cord. These results encourage the development of diffusion anisotropy MRI as a helpful approach for quantifying the extent of secondary degeneration and measuring recovery after spinal cord injury. Magn Reson Med 45:1-9, 2001.

Topics: Animals; Anisotropy; Female; Image Processing, Computer-Assisted; Immunotherapy; Magnetic Resonance Imaging; Myelin Basic Protein; Rats; Rats, Inbred Lew; Recovery of Function; Spinal Cord; Spinal Cord Injuries; T-Lymphocytes

Primary damage caused by injury to the CNS is often followed by delayed degeneration of initially spared neurons. Studies in our laboratory have shown that active or passive immunization with CNS myelin-associated self-antigens can reduce this secondary loss. Here we show, using four experimental paradigms in rodents, that CNS trauma spontaneously evokes a beneficial T cell-dependent immune response, which reduces neuronal loss. (1) Survival of retinal ganglion cells in rats was significantly higher when optic nerve injury was preceded by an unrelated CNS (spinal cord) injury. (2) Locomotor activity of rat hindlimbs (measured in an open field using a locomotor rating scale) after contusive injury of the spinal cord (T8) was significantly better (by three to four score grades) after passive transfer of myelin basic protein (MBP)-activated splenocytes derived from spinally injured rats than in untreated injured control rats or rats similarly treated with splenocytes from naive animals or with splenocytes from spinally injured rats activated ex vivo with ovalbumin or without any ex vivo activation. (3) Neuronal survival after optic nerve injury was 40% lower in adult rats devoid of mature T cells (caused by thymectomy at birth) than in normal rats. (4) Retinal ganglion cell survival after optic nerve injury was higher (119 +/- 3.7%) in transgenic mice overexpressing a T cell receptor (TcR) for MBP and lower (85 +/- 1.3%) in mice overexpressing a T cell receptor for the non-self antigen ovalbumin than in matched wild types. Taken together, the results imply that CNS injury evokes a T cell-dependent neuroprotective response.

Topics: Animals; Autoimmunity; Cell Survival; Cells, Cultured; Disease Models, Animal; Female; Guinea Pigs; Hindlimb; Immunity, Cellular; Immunization, Passive; Interleukin-10; Male; Mice; Mice, Inbred Strains; Mice, Transgenic; Myelin Basic Protein; Nerve Crush; Optic Nerve Injuries; Ovalbumin; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Receptors, Antigen, T-Cell; Retinal Ganglion Cells; Spinal Cord Injuries; Spleen; Thymectomy; Wounds, Nonpenetrating

Spinal cord injury results in a massive loss of neurons, and thus of function. We recently reported that passive transfer of autoimmune T cells directed against myelin-associated antigens provides acutely damaged spinal cords with effective neuroprotection. The therapeutic time window for the passive transfer of T cells was found to be at least 1 week. Here we show that posttraumatic T cell-based active vaccination is also neuroprotective. Immunization with myelin-associated antigens such as myelin basic protein (MBP) significantly promoted recovery after spinal cord contusion injury in the rat model. To reduce the risk of autoimmune disease while retaining the benefit of the immunization, we vaccinated the rats immediately after severe incomplete spinal cord injury with MBP-derived altered peptide ligands. Immunization with these peptides resulted in significant protection from neuronal loss and thus in a reduced extent of paralysis, assessed by an open-field behavioral test. Retrograde labeling of the rubrospinal tracts and magnetic resonance imaging supported the behavioral results. Further optimization of nonpathogenic myelin-derived peptides can be expected to lead the way to the development of an effective therapeutic vaccination protocol as a strategy for the prevention of total paralysis after incomplete spinal cord injury.

Topics: Adjuvants, Immunologic; Amino Acid Substitution; Animals; Autoantigens; Autoimmune Diseases; Contusions; Cordotomy; Exploratory Behavior; Female; Guinea Pigs; Immunotherapy, Active; Locomotion; Magnetic Resonance Imaging; Male; Myelin Basic Protein; Paraplegia; Peptide Fragments; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Safety; Single-Blind Method; Spinal Cord Injuries; Time Factors

The reaction of oligodendrocyte progenitor cells (OPCs) after spinal cord injury (SCI) is poorly understood. In this study, we examined oligodendroglial reactions after contusion SCI in adult rats by immunohistochemistry. OPCs were identified by staining with monoclonal antibodies (mAbs) A2B5 and O4. Each of the A2B5-, O4-positive OPCs and galactocerebroside-positive oligodendrocytes dramatically increased in the lesion of the dorsal posterior funiculus. Bromodeoxyuridine (BrdU) incorporation studies showed that most O4-positive cells in the lesion were labeled with BrdU, suggesting that these OPCs were proliferative. In contrast, the expression of myelin basic protein was decreased in the lesion compared with controls that received laminectomy only. From the injured cord, OPCs were isolated by immunopanning with mAb A2B5. We observed an increased number of OPCs from the injured spinal cords compared with those isolated from controls and unoperated animals. After several days in culture, the OPCs from the lesion expressed galactocerebroside. These results suggest that OPCs are induced and can differentiate following SCI in the adult rat.

Topics: Animals; Antibodies; Antigens, Differentiation; Astrocytes; Bromodeoxyuridine; Cell Division; Cells, Cultured; Disease Models, Animal; Female; Fluorescent Antibody Technique; Glial Fibrillary Acidic Protein; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Oligodendroglia; Rats; Rats, Wistar; Recovery of Function; Spinal Cord; Spinal Cord Injuries; Stem Cells; Wound Healing

Choroid plexus epithelial cells represent a continuation of, and have the same origin as, ventricular ependymal cells, and are regarded as modified ependymal cells. To extend previous studies of the use of choroid plexus ependymal cell (CPEC) grafting for nerve regeneration in the spinal cord, we investigated the capacity of cultured choroid plexus ependymal cells to differentiate into other types of glial cells in the spinal cord tissue. The choroid plexuses were excised from the fourth ventricle of green fluorescent protein (GFP)-transgenic mice and the cells were dissociated and cultured for 4-6 weeks. CPECs were harvested from the monolayer cultures and injected into the pre-lesioned spinal cords of wild-type mice of the same strain using a Hamilton syringe. One week after injection, some GFP-positive transplanted cells became immunohistochemically positive for glial fibrillary acidic protein (GFAP) but negative for neurofilament and myelin basic protein. All the GFAP-positive transplanted cells were negative for vimentin. Two weeks after grafting, immunoelectron microscopy showed that the GFP-positive transplanted cells that had gained GFAP immunoreactivity contained numerous bundles of intermediate filaments, a morphological characteristic similar to that of astrocytes, and were in close contact with adjacent host tissue. These results indicate that, when grafted into the spinal cord, at least some cultured choroid plexus ependymal cells have the capacity to differentiate into astrocytes.

Topics: Animals; Astrocytes; Brain Tissue Transplantation; Cell Differentiation; Cell Lineage; Cells, Cultured; Choroid Plexus; Ependyma; Excitatory Amino Acid Transporter 2; Glial Fibrillary Acidic Protein; Graft Survival; Green Fluorescent Proteins; Immunohistochemistry; Indicators and Reagents; Luminescent Proteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microscopy, Electron; Myelin Basic Protein; Neurofilament Proteins; Spinal Cord; Spinal Cord Injuries; Tubulin; Vimentin

Autoimmune T cells against central nervous system myelin associated peptide reduce the spread of damage and promote recovery in injured rat spinal cord, findings that might lead to neuroprotective cell therapy without risk of autoimmune disease.

Topics: Animals; Autoimmunity; Central Nervous System; Magnetic Resonance Imaging; Myelin Basic Protein; Nerve Degeneration; Rats; Spinal Cord Injuries; T-Lymphocytes

In order to determine if a specific response is induced after spinal cord injury, we performed a kinetic search for IgG antibodies against various spinal cord antigenic preparations in a rat contusion model. Even though spinal cord injured animals showed two reactive bands, these could be originated by the reaction of natural antibodies, since they were also observed before lesion. Thus, these antibodies would not be of relevance in the pathogenic events of spinal cord injury in this rat model. Our findings do not demonstrate the existence of a specific IgG response against spinal cord constituents after injury.

Topics: Animals; Antibody Formation; Antigens; Autoantibodies; Contusions; Immunoglobulin G; Myelin Basic Protein; Rats; Rats, Long-Evans; Spinal Cord; Spinal Cord Injuries

Demyelination contributes to the loss of function consequent to central nervous system (CNS) injury. Enhanced remyelination through transplantation of myelin-producing cells may offer a pragmatic approach to restoring meaningful neurological function. An unlimited source of cells suitable for such transplantation therapy can be derived from embryonic stem (ES) cells, which are both pluripotent and genetically flexible. In this paper we show that oligodendrocyte cultures can be reliably produced from retinoic acid-induced ES cells and that these oligodendrocytes can myelinate axons in vitro. Methods were further developed for generating highly enriched cultures of oligodendrocytes through an additional culturing step, producing an intermediate "oligosphere" stage. To test whether ES cells can survive, migrate, and differentiate into mature myelin-producing cells in areas of demyelination in the adult CNS, ES cells were transplanted into the dorsal columns of adult rat spinal cord 3 days after chemical demyelination. In the demyelination site, large numbers of ES cells survived and differentiated primarily into mature oligodendrocytes that were capable of myelinating axons. Furthermore, when oligosphere cells were transplanted into the spinal cords of myelin-deficient shiverer (shi/shi) mutant mice, the ES cell-derived oligodendrocytes migrated into the host tissue, produced myelin and myelinated host axons. These studies demonstrate the ability of ES cell-derived oligodendrocytes to myelinate axons in culture and to replace lost myelin in the injured adult CNS. Transplantation of ES cells may be a practical approach to treatment of primary and secondary demyelinating diseases in the adult CNS.

Topics: Animals; Antigens, Differentiation; Biomarkers; Cell Differentiation; Cell Lineage; Cells, Cultured; Demyelinating Diseases; Female; Fetal Tissue Transplantation; Mice; Mice, Neurologic Mutants; Myelin Basic Protein; Myelin Sheath; Nerve Tissue Proteins; Oligodendroglia; Rats; Spinal Cord; Spinal Cord Injuries; Stem Cell Transplantation; Stem Cells

Partial injury to the spinal cord can propagate itself, sometimes leading to paralysis attributable to degeneration of initially undamaged neurons. We demonstrated recently that autoimmune T cells directed against the CNS antigen myelin basic protein (MBP) reduce degeneration after optic nerve crush injury in rats. Here we show that not only transfer of T cells but also active immunization with MBP promotes recovery from spinal cord injury. Anesthetized adult Lewis rats subjected to spinal cord contusion at T7 or T9, using the New York University impactor, were injected systemically with anti-MBP T cells at the time of contusion or 1 week later. Another group of rats was immunized, 1 week before contusion, with MBP emulsified in incomplete Freund's adjuvant (IFA). Functional recovery was assessed in a randomized, double-blinded manner, using the open-field behavioral test of Basso, Beattie, and Bresnahan. The functional outcome of contusion at T7 differed from that at T9 (2.9+/-0.4, n = 25, compared with 8.3+/-0.4, n = 12; p<0.003). In both cases, a single T cell treatment resulted in significantly better recovery than that observed in control rats treated with T cells directed against the nonself antigen ovalbumin. Delayed treatment with T cells (1 week after contusion) resulted in significantly better recovery (7.0+/-1; n = 6) than that observed in control rats treated with PBS (2.0+/-0.8; n = 6; p<0.01; nonparametric ANOVA). Rats immunized with MBP obtained a recovery score of 6.1+/-0.8 (n = 6) compared with a score of 3.0+/-0.8 (n = 5; p<0.05) in control rats injected with PBS in IFA. Morphometric analysis, immunohistochemical staining, and diffusion anisotropy magnetic resonance imaging showed that the behavioral outcome was correlated with tissue preservation. The results suggest that T cell-mediated immune activity, achieved by either adoptive transfer or active immunization, enhances recovery from spinal cord injury by conferring effective neuroprotection. The autoimmune T cells, once reactivated at the lesion site through recognition of their specific antigen, are a potential source of various protective factors whose production is locally regulated.

Topics: Animals; Female; Guinea Pigs; Immunization, Passive; Lymphocyte Transfusion; Myelin Basic Protein; Ovalbumin; Rats; Rats, Inbred Lew; Red Nucleus; Spinal Cord Injuries; T-Lymphocytes; Time Factors

Topics: Animals; Central Nervous System; Humans; Immunotherapy, Active; Lymphocyte Activation; Myelin Basic Protein; Nerve Regeneration; Rats; Spinal Cord Injuries; T-Lymphocytes

Autoimmune inflammation secondary to myelin destruction may play an inhibitory role in restoration of nerve functions in spinal cord injury (SCI). In this study, we demonstrated that T cells recognizing myelin basic protein (MBP) occurred at a high precursor frequency in patients with SCI, which was compatible to that in patients with multiple sclerosis (MS), a disease of presumed autoimmune pathology. The findings suggest of hyperactivity of MBP-reactive T cells in patients with SCI. MBP-reactive T cell lines derived from patients with SCI exhibited a preferential recognition pattern toward the 81-99 and the 151-169 regions of MBP. There were functional differences in the epitope recognition and cytokine profile between two panels of MBP-reactive T cell lines derived from patients with SCI and patients with MS. The study provides new evidence important for further investigation of the role of the inflammatory component in SCI.

Topics: Adult; Enzyme-Linked Immunosorbent Assay; Epitopes; Humans; Interferon-gamma; Interleukin-10; Interleukin-4; Male; Middle Aged; Multiple Sclerosis; Myelin Basic Protein; Peptide Fragments; Spinal Cord Injuries; T-Lymphocytes; Tumor Necrosis Factor-alpha

We have investigated the temporal and spatial profiles of apoptotic cells in an experimental transection spinal cord injury by the terminal deoxynucleotidyl transferase-mediated biotin-16-2'-deoxyuridine-5'-triphosphate nick-end labeling (TUNEL) method. Twenty-four hours postinjury, a numerous TUNEL-positive cells appeared both rostrally and caudally to the transection site. Those positive cells, however, gradually diminished in number by several days postinjury. In contrast, other TUNEL-positive cells were found scattered within the white matter remote from the lesion by the third day postinjury. These cells were typically embedded in or among vacuolated fibers, where they were identified in close proximity to the vacuolated space enclosed by myelin basic protein (MBP)-positive structures confirmed by TUNEL-MBP double staining. Because of their linear arrangement, these TUNEL-positive cells were considered interfascicular oligodendrocytes, a fact that was confirmed by the finding that some TUNEL-positive cells were also stained with CCI, a cell marker for oligodendrocyte. Electron microscopic studies revealed that the cells expressing apoptotic morphology were invariably encased in a space formed by myelin splitting. Although the biological significance of apoptotic interfascicular oligodendrocytes in the process of wallerian degeneration is yet to be determined, the finding of such profiles localized within degenerating myelin structures suggests that; oligodendrocytes may be "trapped" within rapidly swollen and disintegrating myelin lamellae, which isolates and perhaps predisposes them to death.

Topics: Animals; Apoptosis; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Situ Nick-End Labeling; Male; Myelin Basic Protein; Nerve Fibers, Myelinated; Neurofilament Proteins; Oligodendroglia; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries; Time Factors; Wallerian Degeneration

Regeneration after an injury in the Central Nervous System is dependent on intrinsic and extrinsic factors. Among the latter are the reactions of glial cells. Using the model of total section of adult rat spinal cord, we have studied the spatial and temporal responses of astrocytes and oligodendrocytes to the lesion of spinal cord axons. We studied at molecular and cellular levels the specific markers GFAP (glial fibrillary acidic protein) for the astrocytes, CNP (2'-3' cyclic 3' nucleotide phosphodiesterase) which is principally expressed by immature oligodendrocytes, and MBP (myelin basic protein) implicated later in the myelin compaction, and which is more specific of mature oligodendrocytes. After injury, all astrocytes, but more markedly those of the grey matter, reacted by an increase of GFAP messenger and protein. This increase was very rapid for messenger, and peaked at 3 days. This increase was more protracted for the protein and persisted after 3 weeks. Messenger increase is more marked and more protracted below than above the lesion. Oligodendrocytes also reacted quickly by an increase of CNP and MBP messengers. For CNP, both messenger and protein increased rapidly and returned to control level after 1 week. MBP showed the same time course of changes, with lower and slower decrease above the lesion. Counts of oligodendrocytes showed that the percentage of the less mature form (light oligodendrocytes) increased dramatically above and below the lesion. After 1 week, above the lesion, this percentage was well below that of the control, whereas below the lesion, it reverted to control value. These results indicate that, following a lesion, astrocytes react quickly and intensely, but more so below the lesion; oligodendrocytes resume a sequence of maturation which is eventually completed above the lesion where remyelinisation can occur and which is prematurely interrupted below the lesion. However, intact oligodendrocytes persist below the lesion, where they constitute a potential for remyelinisation of regenerated and/or transplanted axons.

Topics: 2',3'-Cyclic-Nucleotide Phosphodiesterases; Animals; Astrocytes; Blotting, Northern; Female; Glial Fibrillary Acidic Protein; In Situ Hybridization; Myelin Basic Protein; Nerve Regeneration; Oligodendroglia; Rats; Rats, Sprague-Dawley; RNA Probes; RNA, Antisense; RNA, Messenger; Spinal Cord Injuries

The reaction of oligodendrocytes in response to traumatic injury of the CNS are poorly understood. In the present report we studied changes in the expression of a major constituent of CNS myelin, myelin basic protein (MBP), by immunohistochemistry and in situ hybridization from 6 h up to 2 weeks following partial transection of the spinal cord in adult rats. MBP immunohistochemistry showed degeneration of myelin at the lesion center and signs of myelin breakdown in necrotic foci in the dorsal and ventral funiculi proximal and distal to the lesion. In situ hybridization revealed that mRNA for MBP was downregulated at the local lesion site within the first day following injury, probably reflecting oligodendrocytes to undergo cell death. From 2 days on, however, MBP mRNA was conspicuously upregulated at the border of the lesion area. This "reactive" response of surviving oligodendrocytes, as indicated by increased levels of MBP mRNA, peaked around 8 days. At this time, oligodendrocytes displaying strong MBP in situ signal formed stripe-like structures which were oriented radially toward the lesion center and arranged in parallel to neurofilament-positive axons. At around 2 weeks post-injury, MBP mRNA at the border of the lesion area was again downregulated to levels comparable to uninjured controls. These results show that traumatic injury of the spinal cord induces a "reactive" response of surviving oligodendrocytes adjacent to lesion sites. This response might represent an important component of local repair mechanisms.

Topics: Animals; Cordotomy; Gene Expression Regulation; Immunoenzyme Techniques; In Situ Hybridization; Myelin Basic Protein; Nerve Tissue Proteins; Oligodendroglia; Rats; Rats, Inbred Lew; RNA, Messenger; Spinal Cord Injuries; Time Factors

After incomplete traumatic spinal cord injury (SCI), the spared tissue exhibits abnormal myelination that is associated with reduced or blocked axonal conductance. To examine the molecular basis of the abnormal myelination, we used a standardized rat model of incomplete SCI and compared normal uninjured tissue with that after contusion injury. We evaluated expression of mRNA for myelin proteins using in situ hybridization with oligonucleotide probes to proteolipid protein (PLP), the major protein in central myelin; myelin basic protein (MBP), a major component of central myelin and a minor component of peripheral myelin; and protein zero (P0), the major structural protein of peripheral myelin, as well as myelin transcription factor 1 (MYT1). We found reduced expression of PLP and MBP chronically after SCI in the dorsal, lateral, and ventral white matter both rostral and caudal to the injury epicenter. Detailed studies of PLP at 2 months after injury indicated that the density of expressing cells was normal but mRNA per cell was reduced. In addition, P0, normally restricted to the peripheral nervous system, was expressed both at the epicenter and in lesioned areas at least 4 mm rostral and caudal to it. Thus, after SCI, abnormal myelination of residual axons may be caused, at least in part, by changes in the transcriptional regulation of genes for myelin proteins and by altered distribution of myelin-producing cells. In addition, the expression of MYT1 mRNA and protein seemed to be upregulated after SCI in a pattern suggesting the presence of undifferentiated progenitor cells in the chronically injured cord.

Topics: Animals; Blotting, Western; DNA-Binding Proteins; Gene Expression; Immunohistochemistry; In Situ Hybridization; Microscopy, Electron; Myelin Basic Protein; Myelin P0 Protein; Myelin Proteins; Myelin Proteolipid Protein; Oligonucleotide Probes; Rats; Rats, Sprague-Dawley; RNA, Messenger; Schwann Cells; Spinal Cord; Spinal Cord Injuries; Transcription Factors

In order to observe the role of genetically modified Schwann cell (SC) with pSVP0Mcat in the regeneration of injured spinal cord, the cells were implanted into the spinal cord. Ninety SD rats were used to establish a model of hemi-transection of spinal cord at the level of T8, and were divided into three groups, randomly, that is, pSVP0Mcat modified SC implantation (Group A), SC implantation (Group B) and without cell implantation as control (Group C). After three months the presence of axonal regeneration of the injured spinal cord was examined by means of horseradish peroxidase (HRP) retrograde labelling technique and stereography. The results indicated that HRP labelled cells in Group A and B could be found in the superior region of injured spinal cord and the brain stem such as the red nuclei and oculomotor nuclei. The density of ventral hom neurons of the spinal cord and the number of myelinated axons in 100 microns of the white matter was A > B > C group. In brief, the pSVP0Mcat modified SC intraspinal implantation could promote regeneration of the injured spinal cord.

Topics: Animals; Female; Gene Transfer Techniques; Genetic Therapy; Male; Myelin Basic Protein; Nerve Regeneration; Random Allocation; Rats; Rats, Sprague-Dawley; Schwann Cells; Spinal Cord; Spinal Cord Injuries

To study injury-induced astrocytic responses associated with regrowth of axons and regeneration of myelin, the method of Collins and colleagues was used to make focal cryogenic lesions in spinal cords of adult rats (Collins et al.: J Neuropathol Exp Neurol 45: 742-757, 1986). The duration of cryogenic injury (CI), the size of the cryode, and its temperature were chosen to destroy all myelin sheaths and axons without producing cavities or hemorrhages. Messenger RNA and peptide distributions of insulin-like growth factor I (IGF-I), IGF-I receptor (IGFR-I), IGF binding protein 2 (IGFBP-2), glial fibrillary acidic protein (GFAP), and myelin basic protein (MBP) were studied 3-56 days after CI by in situ hybridization and immunocytochemistry. At 3 days, vimentin-positive, GFAP-negative astrocyte-like cells in the lesion expressed IGF-I mRNA and peptide and 7 days after CI, both were expressed by typical GFAP-positive, hypertrophic astrocytes, many of which also were vimentin-positive. Levels of IGF-I, IGFBP-2, and GFAP mRNA and peptide were higher in lesion astrocytes after 14 days. They attained maximum levels at 21-28 days before declining to near control levels at 56 days. Decreasing relative levels of oligodendroglial MBP mRNA were found in and around lesions 7-14 days after CI; subsequently, rising levels accompanied remyelination. At 28 and 56 days after CI, some transferrin-positive, oligodendroglia-like cells also were immunostained by anti-IGFR-I. Our findings suggest that early astrocytic production of IGF-I and IGFBP-2 may be involved in the myelin regeneration which occurs in this model of spinal cord injury.

Topics: Animals; Astrocytes; Autoradiography; Axons; Base Sequence; Carrier Proteins; Female; Freezing; Gene Expression; Glial Fibrillary Acidic Protein; Immunohistochemistry; In Situ Hybridization; Insulin-Like Growth Factor Binding Protein 5; Insulin-Like Growth Factor I; Microscopy, Electron; Molecular Sequence Data; Myelin Basic Protein; Myelin Sheath; Nerve Regeneration; Rats; Rats, Sprague-Dawley; RNA, Messenger; Somatomedins; Spinal Cord Injuries

In an embryonic chicken, transection of the thoracic spinal cord prior to embryonic day (E) 13 (of the 21-day developmental period) results in complete neuroanatomical repair and functional locomotor recovery. Conversely, repair rapidly diminishes following a transection on E13-E14 and is nonexistent after an E15 transection. The myelination of fiber tracts within the spinal cord also begins on E13, coincident with the transition from permissive to restrictive repair periods. The onset of myelination can be delayed (dysmyelination) until later in development by the direct injection into the thoracic cord on E9-E12 of a monoclonal antibody to galactocerebroside, plus homologous complement. In such a dysmyelinated embryo, a subsequent transection of the thoracic cord as late as E15 resulted in complete neuroanatomical repair and functional recovery (i.e., extended the permissive period for repair).

Topics: Age Factors; Animals; Chick Embryo; Electromyography; Fluorescent Antibody Technique; Myelin Basic Protein; Nerve Fibers, Myelinated; Nerve Regeneration; Oligodendroglia; Spinal Cord; Spinal Cord Injuries

Trauma to the spinal cord induces a series of electrophysiological, immunological and biochemical events, but it is still unclear how such reactions are initiated and maintained. Most likely release of neurochemicals, breakdown of microvascular permeability and the formation of vasogenic edema play important roles in the pathophysiology of spinal cord trauma. In an animal model we have focused the attention to the possible involvement of endogenous serotonin, prostaglandins and opioid peptides in the formation of edema and associated disturbances of vascular permeability. The trauma was produced in anesthetized rats by making a focal lesion in the right dorsal horn at the T10-11 segments. This injury resulted in a profound increase in the microvascular permeability to 131I-sodium and an elevation of water content in the rostral T9 and caudal T12 segments as measured 5 h after the onset of the injury. Light microscopy of the perifocal changes in the T9-T12 segments using Nissl stain and immunohistochemistry to glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) showed profound cellular changes which were most severe in the ipsilateral ventral horn. Many nerve cell bodies were shrunken and the tissue had a spongy edematous appearance. There was a marked increase of GFAP immunoreactivity as well as a significant diminution of MBP staining. Pre-treatment with p-chlorophenylalanine (p-CPA, an endogenous serotonin depletor and synthesis inhibitor) or indomethacin (an endogenous prostaglandin synthesis inhibitor) or naloxone (an opioid receptor antagonist) significantly reduced the permeability changes and the edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Blood Pressure; Body Water; Brain Edema; Capillary Permeability; Carbon Dioxide; Fenclonine; Glial Fibrillary Acidic Protein; Indomethacin; Male; Myelin Basic Protein; Naloxone; Oxygen; Partial Pressure; Rats; Rats, Wistar; Spinal Cord; Spinal Cord Injuries

Experimental spinal cord injury was produced in rats by dropping a 10 g weight from 30 cm upon dura-invested exposed spinal cord. Proteolytic activities at neutral (pH 7.6) and acid (pH 5.5 and 3.6) pH were determined in whole homogenate and the cytosolic fraction of the lesion (lumbar) and cervical control segments. The enzyme activity was monitored by SDS-PAGE analysis of the extent of substrate myelin basic protein (MBP) degradation. Activities (neutral and cathepsin B-like) in the sham-operated spinal cord were lower than those of cervical autologous control at 24 h after injury. The increase in neutral proteinase activity was progressive and greater in the lesion than the autologous control. A 61.5% +/- 3.5 loss of MBP was observed at 2 h following injury and increased at 24 h (78.2% +/- 3.4). The loss of MBP coincided with the appearance of several low molecular weight peptides. The cathepsin B-like and cathepsin D activities were also increased in the lesion but to a lesser extent than the neutral proteinase. The neutral proteinase and cathepsin B-like activity were inhibited by leupeptin and not by pepstatin while the converse obtained for cathepsin D activity. The release of neutral proteolytic activity which is nonlysosomal in origin suggests a novel hypothesis for the mechanism of traumatic axon-myelin injury.

Topics: Animals; Catalysis; Cathepsin B; Cathepsin D; Cathepsins; Endopeptidases; Hydrogen-Ion Concentration; In Vitro Techniques; Myelin Basic Protein; Rats; Spinal Cord; Spinal Cord Injuries

Systemic humoral factors have been studied in traumatic, chronic and acute spinal cord injured patients. Antibodies specific to nervous system autoantigens were detected in a majority of the sera obtained from these patients, at different periods after injury. Limited in vitro sprouting of dorsal root ganglia in chicken embryos was observed in the presence of serum from these patients. The possible association between growth inhibiting factors and the presence of antibodies against nervous tissue autoantigens is discussed.

Topics: Adolescent; Adult; Animals; Antibodies; Autoantigens; Chick Embryo; Female; Ganglia, Spinal; Gangliosidoses; Humans; Male; Middle Aged; Myelin Basic Protein; Spinal Cord Injuries

Topics: Animals; Cell Migration Inhibition; Guinea Pigs; Hypersensitivity, Delayed; Lymphocyte Activation; Macrophages; Myelin Basic Protein; Nerve Tissue Proteins; S100 Proteins; Skin Tests; Spinal Cord Injuries