Invasion of lesion territory by regenerating fibers after spinal cord injury in adult macaque monkeys.

In adult macaque monkeys subjected to an incomplete spinal cord injury (SCI), corticospinal (CS) fibers are rarely observed to grow in the lesion territory. This situation is little affected by the application of an anti-Nogo-A antibody which otherwise fosters the growth of CS fibers rostrally and caudally to the lesion. However, when using the Sternberger monoclonal-incorporated antibody 32 (SMI-32), a marker detecting a non-phosphorylated neurofilament epitope, numerous SMI-32-positive (+) fibers were observed in the spinal lesion territory of 18 adult macaque monkeys; eight of these animals had received a control antibody infusion intrathecally for 1month after the injury, five animals an anti-Nogo-A antibody, and five animals received an anti-Nogo-A antibody together with brain-derived neurotrophic factor (BDNF). These fibers occupied the whole dorso-ventral axis of the lesion site with a tendency to accumulate on the ventral side, and their trajectories were erratic. Most of these fibers (about 87%) were larger than 1.3μm and densely SMI-32 (+) stained. In the undamaged spinal tissue, motoneurons form the only large population of SMI-32 (+) neurons which are densely stained and have large diameter axons. These data therefore suggest that a sizeable proportion of the fibers seen in the lesion territory originate from motoneurons, although fibers of other origins could also contribute. Neither the presence of the antibody neutralizing Nogo-A alone, nor the presence of the antibody neutralizing Nogo-A combined with BDNF influenced the number or the length of the SMI-32 (+) fibers in the spinal lesion area. In summary, our data show that after a spinal cord lesion in adult monkeys, the lesion site is colonized by fibers, a large portion of which presumably originate from motoneurons.

Schwann cells for spinal cord repair

The complex nature of spinal cord injury appears to demand a multifactorial repair strategy. One of the components that will likely be included is an implant that will fill the area of lost nervous tissue and provide a growth substrate for injured axons. Here we will discuss the role of Schwann cells (SCs) in cell-based, surgical repair strategies of the injured adult spinal cord. We will review key studies that showed that intraspinal SC grafts limit injury-induced tissue loss and promote axonal regeneration and myelination, and that this response can be improved by adding neurotrophic factors or anti-inflammatory agents. These results will be compared with several other approaches to the repair of the spinal cord. A general concern with repair strategies is the limited functional recovery, which is in large part due to the failure of axons to grow across the scar tissue at the distal graft-spinal cord interface. Consequently, new synaptic connections with spinal neurons involved in motor function are not formed. We will highlight repair approaches that did result in growth across the scar and discuss the necessity for more studies involving larger, clinically relevant types of injuries, addressing this specific issue. Finally, this review will reflect on the prospect of SCs for repair strategies in the clinic.

Neurodegeneration and Increased Production of Nitrotyrosine, Nitric Oxide Synthase, IFN-gamma and S100 beta Protein in the Spinal Cord of IL-12p40-Deficient Mice Infected with Trypanosoma cruzi

Background/Aim: Chagas` disease is caused by Trypanosoma cruzi and occurs in most Latin American countries. The protozoan may colonize the central nervous system (CNS) of immune-compromised human hosts, thus causing neuronal disorders. Systemic control of the intracellular forms of the parasite greatly depends on the establishment of a TH1 response and subsequent nitric oxide (NO) release. At the CNS, it is known that low concentrations of NO promote neuronal survival and growth, while high concentrations exert toxic effects and neuron death. Accounting for NO production by astrocytes is the glia-derived factor S100 beta, which is overproduced in some neurodegenerative diseases. In the current work, we studied the expression of NO, interferon (IFN)-gamma and S100 beta in the spinal cord tissue of IL-12p40KO mice infected with T. cruzi, a model of neurodegenerative process. Methods: IL-12p40KO and wild-type (WT) female mice infected with T. cruzi Sylvio X10/4 (10(5) trypomastigotes, intraperitoneally) were euthanized when IL-12p40KO individuals presented limb paralysis. Spinal cord sections were submitted to immunohistochemical procedures for localization of neurofilament, laminin, nitrotyrosine, NO synthases (NOS), IFN-gamma and S100 beta. The total number of neurons was estimated by stereological analysis and the area and intensity of immunoreactivities were assessed by microdensitometric/morphometric image analysis. Results: No lesion was found in the spinal cord sections of WT mice, while morphological disarrangements, many inflammatory foci, enlarged vessels, amastigote nests and dying neurons were seen at various levels of IL-12p40KO spinal cord. Compared to WT mice, IL-12p40KO mice presented a decrement on total number of neurons (46.4%, p<0.05) and showed increased values of immunoreactive area for nitrotyrosine (239%, p<0.01) and NOS (544%, p<0.001). Moreover, the intensity of nitrotyrosine (16%, p<0.01), NOS (38%, p<0.05) and S100 beta (21%, p<0.001) immunoreactivities were also augmented. No IFN-gamma labeled cells were seen in WT spinal cord tissue, contrary to IL-12p40KO tissue that displayed inflammatory infiltrating cells and also some parenchymal cells positively labeled.Conclusion: We suggest that overproduction of NO may account for neuronal death at the spinal cord of T. cruzi-infected IL-12p40KO mice and that IFN-gamma and S100 beta may contribute to NOS activation in the absence of IL-12. Copyright (C) 2009 S. Karger AG, Basel

Proteome analysis of spinal cord during the clinical course of monophasic experimental autoimmune encephalomyelitis

The induction of autoimmune encephalomyelitis (EAE) in Lewis rats results in a period of exacerbation followed by complete recovery. Therefore, this model is widely used for studying the evolution of multiple sclerosis. In the present investigation, differentially expressed proteins in the spinal cord of Lewis rats during the evolution of EAE were assessed using the combination of 2DE and MALDI-TOF MS. The majority of the differentially expressed proteins were identified during the acute phase of EAE, in relation to naive control animals. On the other hand, recovered rats presented a similar protein expression pattern in comparison with the naive ones. This observation can be explained, at least in part, by the intense catabolism existent in acute phase due to nervous tissue damage. In recovered rats, we have described the upregulation of proteins that are apparently involved in the recovery of damaged tissue, such as light and medium neurofilaments, glial fibrillary acidic protein, tubulins subunits, and quaking protein. These proteins are involved mainly in cell growth, myelination, and remyelination as well as in astrocyte and oligodendrocyte maturation. The present study has demonstrated that the inflammatory response, characterized by an increase of the proliferative response and infiltration of autoreactive T lymphocytes in the central nervous system, occurs simultaneously with neurodegeneration.

Gene expression profiling in anti-CD11d mAb-treated spinal cord-injured rats

Acute administration of a mononclonal antibody (mAb) raised against the CD11d subunit of the leukocyte CD11d/CD18 integrin after spinal cord injury (SCI) in the rat greatly improves neurological outcomes. We have profiled gene expression in anti-CD11d and isotyped-matched control mAb-treated rats after SCI. Microarray analysis demonstrated reduced expression of pro-inflammatory cytokines and increased expression of inflammatory mediators that promote wound healing and the expression of scar proteins predicted to improve nerve growth. These changes in gene expression may reflect changes in the types of macrophages that populate the lesions in anti-CD11d mAb-treated rats.

Brain fiber tract plasticity in experimental spinal cord injury: diffusion tensor imaging.

Diffusion tensor imaging (DTI) and immunohistochemistry were performed in spinal cord injured rats to understand the basis for activation of multiple regions in the brain observed in functional magnetic resonance imaging (fMRI) studies. The measured fractional anisotropy (FA), a scalar measure of diffusion anisotropy, along the region encompassing corticospinal tracts (CST) indicates significant differences between control and injured groups in the 3 to 4 mm area posterior to bregma that correspond to internal capsule and cerebral peduncle. Additionally, DTI-based tractography in injured animals showed increased number of fibers that extend towards the cortex terminating in the regions that were activated in fMRI. Both the internal capsule and cerebral peduncle demonstrated an increase in GFAP-immunoreactivity compared to control animals. GAP-43 expression also indicates plasticity in the internal capsule. These studies suggest that the previously observed multiple regions of activation in spinal cord injury are, at least in part, due to the formation of new fibers.

Effect of VEGF treatment on the blood-spinal cord barrier permeability in experimental spinal cord injury: dynamic contrast-enhanced magnetic resonance imaging.

Compromised blood-spinal cord barrier (BSCB) is a factor in the outcome following traumatic spinal cord injury (SCI). Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and vascular permeability. The role of VEGF in SCI is controversial. Relatively little is known about the spatial and temporal changes in the BSCB permeability following administration of VEGF in experimental SCI. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) studies were performed to noninvasively follow spatial and temporal changes in the BSCB permeability following acute administration of VEGF in experimental SCI over a post-injury period of 56 days. The DCE-MRI data was analyzed using a two-compartment pharmacokinetic model. Animals were assessed for open field locomotion using the Basso-Beattie-Bresnahan score. These studies demonstrate that the BSCB permeability was greater at all time points in the VEGF-treated animals compared to saline controls, most significantly in the epicenter region of injury. Although a significant temporal reduction in the BSCB permeability was observed in the VEGF-treated animals, BSCB permeability remained elevated even during the chronic phase. VEGF treatment resulted in earlier improvement in locomotor ability during the chronic phase of SCI. This study suggests a beneficial role of acutely administered VEGF in hastening neurobehavioral recovery after SCI.

Brain-derived neurotrophic factor is an endogenous modulator of nociceptive responses in the spinal cord

The primary sensory neurons that respond to noxious stimulation and project to the spinal cord are known to fall into two distinct groups: one sensitive to nerve growth factor and the other sensitive to glial cell-line-derived neurotrophic factor. There is currently considerable interest in the ways in which these factors may regulate nociceptor properties. Recently, however, it has emerged that another trophic factor—brain-derived neurotrophic factor (BDNF)—may play an important neuromodulatory role in the dorsal horn of the spinal cord. BDNF meets many of the criteria necessary to establish it as a neurotransmitter/neuromodulator in small-diameter nociceptive neurons. It is synthesized by these neurons and packaged in dense core vesicles in nociceptor terminals in the superficial dorsal horn. It is markedly up-regulated in inflammatory conditions in a nerve growth factor-dependent fashion. Postsynaptic cells in this region express receptors for BDNF. Spinal neurons show increased excitability to nociceptive inputs after treatment with exogenous BDNF. There are both electrophysiological and behavioral data showing that antagonism of BDNF at least partially prevents some aspects of central sensitization. Together, these findings suggest that BDNF may be released from primary sensory nociceptors with activity, particularly in some persistent pain states, and may then increase the excitability of rostrally projecting second-order systems. BDNF released from nociceptive terminals may thus contribute to the sensory abnormalities associated with some pathophysiological states, notably inflammatory conditions.

Cryosections of pre-irradiated adult rat spinal cord tissue support axonal regeneration in vitro

Neonatal X-irradiation of central nervous system (CNS) tissue markedly reduces the glial population in the irradiated area. Previous in vivo studies have demonstrated regenerative success of adult dorsal root ganglion (DRG) neurons into the neonatally-irradiated spinal cord. The present study was undertaken to determine whether these results could be replicated in an in vitro environment. The lumbosacral spinal cord of anaesthetised Wistar rat pups, aged between 1 and 5 days, was subjected to a single dose (40 Gray) of X-irradiation. A sham-irradiated group acted as controls. Rats were allowed to reach adulthood before being killed. Their lumbosacral spinal cords were dissected out and processed for sectioning in a cryostat. Cryosections (10 mum-thick) of the spinal cord tissue were picked up on sterile glass coverslips and used as substrates for culturing dissociated adult DRG neurons. After an appropriate incubation period, cultures were fixed in 2% paraformaldehyde and immunolabelled to visualise both the spinal cord substrate using anti-glial fibrillary acidic protein (GFAP) and the growing DRG neurons using anti-growth associated protein (GAP-43). Successful growth of DRG neurites was observed on irradiated, but not on non-irradiated, sections of spinal cord. Thus, neonatal X-irradiation of spinal cord tissue appears to alter its environment such that it can later support, rather than inhibit, axonal regeneration. It is suggested that this alteration may be due, at least in part, to depletion in the number of and/or a change in the characteristics of the glial cells. (C) 2000 ISDN. Published by Elsevier Science Ltd. All rights reserved.

Molecular control of cell type diversity in the developing spinal cord

1, During embryonic development, a diverse array of neurons and glia are generated at specific positions along the dorsoventral and rostro-caudal axes of the spinal cord from a common pool of precursor cells. 2. This cell type diversity can be distinguished by the spatially and temporally coordinated expression of several transcription factors that are also linked to cell type specification at a very early stage of spinal cord development. 3, Recent studies have started to uncover that the generation of cell type diversity in the developing spinal cord. Moreover, distinct cell types in the spinal cord appear to be determined by the spatially and temporally coordinated expression of transcription factors. 4. The expression of these factors also appears to be controlled by gradients of factors expressed by ventral and dorsal midline cells, namely Sonic hedgehog and members of the transforming growth factor-beta family. 5, Changes in the competence of precursor cells and local cell interactions may also play important roles in cell type specification within the developing spinal cord.

Blockade of interleukin-6 signaling inhibits the classic pathway and promotes an alternative pathway of macrophage activation after spinal cord injury in mice

Background Recent in vivo and in vitro studies in non-neuronal and neuronal tissues have shown that different pathways of macrophage activation result in cells with different properties. Interleukin (IL)-6 triggers the classically activated inflammatory macrophages (M1 phenotype), whereas the alternatively activated macrophages (M2 phenotype) are anti-inflammatory. The objective of this study was to clarify the effects of a temporal blockade of IL-6/IL-6 receptor (IL-6R) engagement, using an anti-mouse IL-6R monoclonal antibody (MR16-1), on macrophage activation and the inflammatory response in the acute phase after spinal cord injury (SCI) in mice. Methods MR16-1 antibodies versus isotype control antibodies or saline alone were administered immediately after thoracic SCI in mice. SC tissue repair was compared between the two groups by Luxol fast blue (LFB) staining for myelination and immunoreactivity for the neuronal markers growth-associated protein (GAP)-43 and neurofilament heavy 200 kDa (NF-H) and for locomotor function. The expression of T helper (Th)1 cytokines (interferon (IFN)-? and tumor necrosis factor-a) and Th2 cytokines (IL-4, IL-13) was determined by immunoblot analysis. The presence of M1 (inducible nitric oxide synthase (iNOS)-positive, CD16/32-positive) and M2 (arginase 1-positive, CD206-positive) macrophages was determined by immunohistology. Using flow cytometry, we also quantified IFN-? and IL-4 levels in neutrophils, microglia, and macrophages, and Mac-2 (macrophage antigen-2) and Mac-3 in M2 macrophages and microglia. Results LFB-positive spared myelin was increased in the MR16-1-treated group compared with the controls, and this increase correlated with enhanced positivity for GAP-43 or NF-H, and improved locomotor Basso Mouse Scale scores. Immunoblot analysis of the MR16-1-treated samples identified downregulation of Th1 and upregulation of Th2 cytokines. Whereas iNOS-positive, CD16/32-positive M1 macrophages were the predominant phenotype in the injured SC of non-treated control mice, MR16-1 treatment promoted arginase 1-positive, CD206-positive M2 macrophages, with preferential localization of these cells at the injury site. MR16-1 treatment suppressed the number of IFN-?-positive neutrophils, and increased the number of microglia present and their positivity for IL-4. Among the arginase 1-positive M2 macrophages, MR16-1 treatment increased positivity for Mac-2 and Mac-3, suggestive of increased phagocytic behavior. Conclusion The results suggest that temporal blockade of IL-6 signaling after SCI abrogates damaging inflammatory activity and promotes functional recovery by promoting the formation of alternatively activated M2 macrophages.

The prevalence and phenotype of activated microglia/macrophages within the spinal cord of the hyperostotic mouse (twy/twy) changes in response to chronic progressive spinalcord compression:implications for human cervical compressive myelopathy

Background:Cervical compressive myelopathy, e.g. due to spondylosis or ossification of the posterior longitudinal ligament is a common cause of spinal cord dysfunction. Although human pathological studies have reported neuronal loss and demyelination in the chronically compressed spinal cord, little is known about the mechanisms involved. In particular, the neuroinflammatory processes that are thought to underlie the condition are poorly understood. The present study assessed the localized prevalence of activated M1 and M2 microglia/macrophages in twy/twy mice that develop spontaneous cervical spinal cord compression, as a model of human disease.Methods:Inflammatory cells and cytokines were assessed in compressed lesions of the spinal cords in 12-, 18- and 24-weeks old twy/twy mice by immunohistochemical, immunoblot and flow cytometric analysis. Computed tomography and standard histology confirmed a progressive spinal cord compression through the spontaneously development of an impinging calcified mass.Results:The prevalence of CD11b-positive cells, in the compressed spinal cord increased over time with a concurrent decrease in neurons. The CD11b-positive cell population was initially formed of arginase-1- and CD206-positive M2 microglia/macrophages, which later shifted towards iNOS- and CD16/32-positive M1 microglia/macrophages. There was a transient increase in levels of T helper 2 (Th2) cytokines at 18 weeks, whereas levels of Th1 cytokines as well as brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and macrophage antigen (Mac) -2 progressively increased.Conclusions:Spinal cord compression was associated with a temporal M2 microglia/macrophage response, which may act as a possible repair or neuroprotective mechanism. However, the persistence of the neural insult also associated with persistent expression of Th1 cytokines and increased prevalence of activated M1 microglia/macrophages, which may lead to neuronal loss and demyelination despite the presence of neurotrophic factors. This understanding of the aetiopathology of chronic spinal cord compression is of importance in the development of new treatment targets in human disease. © 2013 Hirai et al.

Gene therapy strategies for the treatment of spinal cord injury

Spinal cord injury is a complex pathology often resulting in functional impairment and paralysis. Gene therapy has emerged as a possible solution to the problems of limited neural tissue regeneration through the administration of factors promoting axonal growth, while also offering long-term local delivery of therapeutic molecules at the injury site. Of note, gene therapy is our response to the requirements of neural and glial cells following spinal cord injury, providing, in a time-dependent manner, growth substances for axonal regeneration and eliminating axonal growth inhibitors. Herein, we explore different gene therapy strategies, including targeting gene expression to modulate the presence of neurotrophic growth or survival factors and increase neural tissue plasticity. Special attention is given to describing advances in viral and non-viral gene delivery systems, as well as the available routes of gene delivery. Finally, we discuss the future of combinatorial gene therapies and give consideration to the implementation of gene therapy in humans. © 2014 Future Science Ltd.

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Spinal Cord Atrophy Correlates With Disease Duration And Severity In Amyotrophic Lateral Sclerosis.

Our objective was to investigate spinal cord (SC) atrophy in amyotrophic lateral sclerosis (ALS) patients, and to determine whether it correlates with clinical parameters. Forty-three patients with ALS (25 males) and 43 age- and gender-matched healthy controls underwent MRI on a 3T scanner. We used T1-weighted 3D images covering the whole brain and the cervical SC to estimate cervical SC area and eccentricity at C2/C3 level using validated software (SpineSeg). Disease severity was quantified with the ALSFRS-R and ALS Severity scores. SC areas of patients and controls were compared with a Mann-Whitney test. We used linear regression to investigate association between SC area and clinical parameters. Results showed that mean age of patients and disease duration were 53.1 ± 12.2 years and 34.0 ± 29.8 months, respectively. The two groups were significantly different regarding SC areas (67.8 ± 6.8 mm² vs. 59.5 ± 8.4 mm², p < 0.001). Eccentricity values were similar in both groups (p = 0.394). SC areas correlated with disease duration (r = - 0.585, p < 0.001), ALSFRS-R score (r = 0.309, p = 0.044) and ALS Severity scale (r = 0.347, p = 0.022). In conclusion, patients with ALS have SC atrophy, but no flattening. In addition, SC areas correlated with disease duration and functional status. These data suggest that quantitative MRI of the SC may be a useful biomarker in the disease.