Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the injured spinal cord of adult rats – possible roles of brain-derived neurotrophic factor, TrkB and p75 neurotrophin receptor

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague–Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.     

Immunological myelin disruption does not alter expression of regeneration-associated genes in intact or axotomized rubrospinal neurons

The inability of axotomized neurons to regenerate within the CNS has been partially attributed to a number of inhibitory factors associated with CNS myelin that are extrinsic to the severed neurons. However, some neurons are capable of limited regeneration after injury and this ability has been shown to correlate with the expression of certain regeneration-associated genes (RAGs) intrinsic to injured neurons. It has therefore been postulated that neutralization of inhibitory factors, as well as the induction of an appropriate neuronal cell body response, would facilitate improved regrowth of injured CNS axons. In previous studies we have shown that immunological removal of myelin from the spinal cord facilitates axonal regeneration by rubrospinal neurons, as indicated by retrograde transport of a fluorescent dye placed distal to the site of injury. Here, we investigated whether the immunological focal removal of spinal cord myelin, following a thoracic spinal cord injury, concomitantly stimulated an increase in the expression of RAGs in rubrospinal neurons. In situ hybridization for Talpha-1 tubulin and GAP-43 at days 7, 14, and 21 revealed no significant increase in gene expression in rubrospinal neurons following immunological demyelination. The ability of various neuronal populations to sprout or slowly regrow without expressing the previously characterized cell body response is reviewed. We conclude that the recently demonstrated regeneration of rubrospinal tract, after immunologically directed spinal cord demyelination, is the result of either axonal sprouting or slow axonal regrowth without the increased expression of RAGs characteristic for fast axon regeneration.     

Rubrospinal neurons fail to respond to brain-derived neurotrophic factor applied to the spinal cord injury site 2 months after cervical axotomy

Numerous experimental therapies to promote axonal regeneration have shown promise in animal models of acute spinal cord injury, but their effectiveness is often found to diminish with a delay in administration. We evaluated whether brain-derived neurotrophic factor (BDNF) application to the spinal cord injury site 2 months after cervical axotomy could promote a regenerative response in chronically axotomized rubrospinal neurons. BDNF was applied to the spinal cord in three different concentrations 2 months after cervical axotomy of the rubrospinal tract. The red nucleus was examined for reversal of neuronal atrophy, GAP43 and Talpha1 tubulin mRNA expression, and trkB receptor immunoreactivity. A peripheral nerve transplant paradigm was used to measure axonal regeneration into peripheral nerve transplants. Rubrospinal axons were anterogradely traced and trkB receptor immunohistochemistry performed on the injured spinal cord. We found that BDNF treatment did not reverse rubrospinal neuronal atrophy, nor promote GAP-43 and Talpha1 tubulin mRNA expression, nor promote axonal regeneration into peripheral nerve transplants. TrkB receptor immunohistochemistry demonstrated immunoreactivity on the neuronal cell bodies, but not on anterogradely labeled rubrospinal axons at the injury site. These findings suggest that the poor response of rubrospinal neurons to BDNF applied to the spinal cord injury site 2 months after cervical axotomy is not related to the dose of BDNF administered, but rather to the loss of trkB receptors on the injured axons over time. Such obstacles to axonal regeneration will be important to identify in the development of therapeutic strategies for chronically injured individuals.     

Microarray analysis suggests the involvement of proteasomes,lysosomes, and matrix metalloproteinases in the response of motor neurons to root avulsion

We used microarray analysis of RNA expression from punch samples from ventral horn of spinal cord to identify alterations in gene expression in motor neurons 3 days after proximal spinal root avulsion, a traumatic injury that results in the death of 80% of the motor neurons. This analysis identified the anticipated increases in expression of genes coding for proteins involved in the apoptosis cascades and abortive cell cycle re-entry, as well as decreases in expression of genes coding for proteins related to neuronal functional activity, including groups of genes related to energy metabolism, transporter proteins, ion channels, and receptors. It was also found that cathepsins, metalloproteinases, and proteasome-related protein products were highly up-regulated in motor neurons following axotomy. Each of these products represent pathways that have been implicated in other models of neuronal damage, but which have not previously been described as a response to axotomy.     

Axotomized bulbospinal neurons express c-Jun after cervical spinal cord injury

In several central nervous system neuronal populations, axotomy triggers the upregulation of regeneration-associated genes such as c-Jun, which determines neurons ability to regenerate axon in a growth-permissive environment. We analyzed the expression of c-Jun in rat ventral medullary neurons after cervical hemisection in order to investigate their intrinsic regenerative potential. Maximal expression of c-Jun was observed 7 days after injury mainly in axotomized medullary neurons located in the gigantocellularis nucleus, the raphe nucleus and, although less intensively, in the rostral ventral respiratory group. This suggests that after high cervical injury, a large number of medullary neurons projecting to the spinal cord become competent for axonal regeneration, although this regenerating potential may not be equivalent between the various neuronal populations.     

Epigenetic Regulation of Axon Outgrowth and Regeneration in CNS Injury: The First Steps Forward

Inadequate axonal sprouting and lack of regeneration limit functional recovery following neurologic injury, such as stroke, brain, and traumatic spinal cord injury. Recently, the enhancement of the neuronal regenerative program has led to promising improvements in axonal sprouting and regeneration in animal models of axonal injury. However, precise knowledge of the essential molecular determinants of this regenerative program remains elusive, thus limiting the choice of fully effective therapeutic strategies. Given that molecular regulation of axonal outgrowth and regeneration requires carefully orchestrated waves of gene expression, both temporally and spatially, epigenetic changes may be an ideal regulatory mechanism to address this unique need. While recent evidence suggests that epigenetic modifications could contribute to the regulation of axonal outgrowth and regeneration following axonal injury in models of stroke, and spinal cord and optic nerve injury, a number of unanswered questions remain. Such questions require systematic investigation of the epigenetic landscape between regenerative and non-regenerative conditions for the potential translation of this knowledge into regenerative strategies in human spinal and brain injury, as well as stroke.     

Developmental and regional expression of NADPH-diaphorase/nitric oxide synthase in spinal cord neurons correlates with the emergence of limb motor networks in metamorphosing Xenopus laevis

Metamorphosis in anuran amphibians requires a complete transformation in locomotor strategy from undulatory tadpole swimming to adult quadrupedal propulsion. The underlying reconfiguration of spinal networks may be influenced by various neuromodulators including nitric oxide, which is known to play an important role in CNS development and plasticity in diverse species, including metamorphosis of amphibians. Using NADPH-diaphorase (NADPH-d) staining and neuronal nitric oxide synthase (nNOS) immunofluorescence labelling, the expression and developmental distribution of NOS-containing neurons in the spinal cord and brainstem were analysed in all metamorphic stages of Xenopus laevis. Wholemount preparations of the spinal cord from early stages of metamorphosis (coincident with emergence of the fore- and hindlimb buds) revealed two clusters of NOS-positive neurons interspersed with areas devoid of stained somata. These cells were distributed in three topographic subgroups, the most ventral of which had axonal projections that crossed the ventral commissure. Motoneurons innervating the fore- and hindlimb buds were retrogradely labelled with horseradish peroxidase (HRP) to determine their position in relation to the two NOS-expressing cord regions. Limb motoneurons and NOS-positive cells did not overlap, indicating that during early stages of metamorphosis nitrergic neurons are excluded from regions where spinal limb circuits are forming. As metamorphosis progresses, NOS expression became distributed along the length of the spinal cord together with an increase in the number and intensity of labelled cells and fibers. NOS expression reached a peak as the forelimbs emerge then declined. These findings are consistent with a role for nitric oxide (NO) in the developmental transition from undulatory swimming to quadrupedal locomotion.     

Light and electron microscopic localization of B-50 (GAP43) in the rat spinal cord during transganglionic degenerative atrophy and regeneration.

Crush or transection of a peripheral nerve is known to induce transganglionic degenerative atrophy (TDA) in the segmentally related, ipsilateral Rolando substance of the spinal cord. When the lost peripheral connectivity is reestablished, the consecutive regenerative synaptoneogenesis results in restoration of the circuitry in the formerly deteriorated upper dorsal horn. Enhanced expression of the growth-associated protein (GAP43) B-50 occurs during neuronal differentiation, axon outgrowth, and peripheral nerve regeneration. This study documents changes in immunocytochemical distribution of B-50 in the regions of the lumbar spinal cord which are segmentally related to the axotomized sciatic nerve. At the light microscopic level, a weak B-50 immunoreactivity (BIR) is present in the neuropil of the upper dorsal horn of control animals. After unilateral transection and ligation of the sciatic nerve, BIR increased in the ipsilateral upper dorsal horn at 17 days postinjury, but decreased again after 24 days with respect to the contralateral side. Differences between effects of crush and transection were prominent in combined crush-cut experiments as well (i.e., after unilateral crush and contralateral transection and ligation of the sciatic nerve). Electron microscopic studies show that in the uninjured and injured spinal cord, BIR is detected in axons and axon terminals, but not all are stained. After transection of the sciatic nerve, BIR is found in afflicted primary sensory axon terminals, including those contacting substantia gelatinosa neurons and in axon terminals undergoing glial phagocytosis. The localization of BIR seen after crushing the sciatic nerve is similar. However, at 24 days after crush, BIR is detected also in axonal growth cones. In the ventral horn of control animals, synaptic boutons impinging upon motor neurons exhibited weak BIR. At 17 days after unilateral transection of the sciatic nerve, the pericellular BIR surrounding motor neurons is decreased at the ipsilateral with respect to the contralateral side, whereas 24 days after crush injury it increased considerably. Our results show that peripheral nerve injury inducing TDA also affects BIR distribution in the spinal gray matter. Successful regeneration of the peripheral nerve after crush lesion is associated with enhanced expression of B-50 in growth cones of sprouting central axons. The neuroplastic response of B-50 is in line with a function of B-50 in axonal sprouting and reactive synaptogenesis.     

Distribution of paired immunoglobulin-like receptor B in the nervous system related to regeneration difficulties after unilateral lumbar spinal cord injury

Paired immunoglobulin-like receptor B(Pir B) is a functional receptor of myelin-associated inhibitors for axonal regeneration and synaptic plasticity in the central nervous system, and thus suppresses nerve regeneration. The regulatory effect of Pir B on injured nerves has received a lot of attention. To better understand nerve regeneration inability after spinal cord injury, this study aimed to investigate the distribution of Pir B(via immunofluorescence) in the central nervous system and peripheral nervous system 10 days after injury. Immunoreactivity for Pir B increased in the dorsal root ganglia, sciatic nerves, and spinal cord segments. In the dorsal root ganglia and sciatic nerves, Pir B was mainly distributed along neuronal and axonal membranes. Pir B was found to exhibit a diffuse, intricate distribution in the dorsal and ventral regions. Immunoreactivity for Pir B was enhanced in some cortical neurons located in the bilateral precentral gyri. Overall, the findings suggest a pattern of Pir B immunoreactivity in the nervous system after unilateral spinal transection injury, and also indicate that Pir B may suppress repair after injury.     

Induction of type IV collagen and other basement-membrane-associated proteins after spinal cord injury of the adult rat may participate in formation of the glial scar.

We investigated the spatial and temporal expression of basement-membrane-forming and neurite-outgrowth-supporting matrix proteins after a unilateral dorsal root injury combined with a collagen I/laminin-1 graft and a stab wound lesion to the dorsal horn of the adult rat spinal cord. Ten days after injury, the gamma1 laminin was induced in the reactive glia. At this early stage, the glial cells failed to express type IV collagen and the alpha1 laminin. One month after injury, reactive astrocytes in the dorsal horn of the lesioned side expressed gamma1 laminin, type IV collagen, and the alpha1 laminin whereas astrocytes of the normal spinal cord or the uninjured contralateral dorsal horn were negative. Both astrocytes and neurons of the ipsilateral ventral horn were induced to express laminin-1 and gamma1 laminin. Astrocytes of the ipsilateral ventral horn also expressed type IV collagen. Simultaneously with the changes in expression of the extracellular matrix proteins, the expression pattern of basic fibroblast growth factor (FGF-2) was markedly altered after spinal cord injury. In normal and contralateral spinal cord, FGF-2 was expressed in nerve fibers, but its expression changed from neuronal into glial in the ipsilateral spinal cord within 1 month after injury. Four months after injury, expression of both type IV collagen and the alpha1 laminin had declined, but the astrocytes at the injury site continued expressing the gamma1 laminin. Cultured astrocytes were negative for type IV collagen, but several cytokines, including IL-1beta and TGFbeta1, induced expression of type IV collagen in the astrocytes. These factors also increased deposition of type IV collagen matrix in the glial cultures. These results indicate that type IV collagen and the alpha1 laminin are induced in reactive astrocytes after spinal cord injury in vivo. Induction of type IV collagen in astrocytes in vitro by cytokines indicates that blood-borne or local factors at the injury site may induce the spinal cord glial expression of type IV collagen in vivo. Simultaneous expression of laminin-1 and alpha1 laminin with type IV collagen is known to lead to production of basement membranes. This may hamper the neurite-outgrowth-promoting potential of the gamma1 laminin by initiating formation of the glial scar.     

Gene expression changes after focal stroke, traumatic brain and spinal cord injuries

PURPOSE OF REVIEW: Large-scale gene expression profiling has recently been performed on stroke and spinal cord injuries. These studies provide insights into coordinated patterns of gene expression within the injury and the interrelationships of neurodegenerative and neural repair processes after injury. RECENT FINDINGS: The molecular signals for post-stroke angiogenesis begin within hours of initial cerebral ischemia, with sequential increases in message for initially destabilizing combinations of vascular growth factors and growth factor receptors, followed by growth factor combinations that promote endothelial cell division and stabilization. The overlap in molecular signaling between post-stroke angiogenesis, neurogenesis and axonal sprouting suggests a continuum of vascular and neural reorganization in the tissue adjacent to stroke. Inflammation after injury extends through early and late changes in the cytokine message. SOCS-3, a negative regulator of cytokine signaling, is increased after injury and may be neuroprotective. Components of an adult neuronal growth program have been identified in the peripheral nervous system during axonal regeneration, with overlap to axonal sprouting after stroke. The gene expression profile of the aged brain suggests an altered central nervous system environment that may exacerbate initial injury and impair neural reorganization after stroke and spinal cord injury. SUMMARY: When rigorously tested and independently validated, data from large-scale gene expression analyses provide new insights into the aggregate genetic control of stroke and spinal cord injury, and the interrelationship of important cellular events within the damaged region. These data also highlight novel genes in these processes, and suggest new directions in the investigation of tissue reorganization and repair after central nervous system injury.     

Potential roles of Alzheimer precursor protein A4 and beta-amyloid in survival and function of aged spinal motor neurons after axonal injury

To study the potential role of Alzheimer precursor protein A4 (APP) and beta-amyloid (A/beta) on aging motor neuron survival, expression of APP, A/beta, and choline acetyltransferase (ChaT) were investigated in aged rats after either distal axotomy or root avulsion injury. Approximately 45% in number of total aged spinal motor neuron were normally APP-positive. A/beta-positive neurites were observed normally in the spinal ventral horn of aged rats. After distal axotomy, without apparent neurodegeneration such as cell loss and decreased ChaT-immunoreactivity, increased levels of APP expression were observed in the spinal cords of aged rats post-injury. In contrast, after avulsion, expression of APP and A/beta were downregulated in the spinal ventral horn of aged rats, and marked loss of spinal motor neurons and downregulated ChaT expression were observed. Our data indicate that APP and A/beta might play beneficial roles in neuronal survival of aged spinal motor neurons after axonal injury.     

Transplantation of bone marrow mesenchymal stem cells reduces lesion volume and induces axonal regrowth of injured spinal cord

It has been demonstrated that transplantation of bone marrow mesenchymal stem cells (BMSCs) improves recovery of injured spinal cord in animal models. However, the mechanism of how BMSCs promote repair of injured spinal cord remains under investigation. The present study investigated the neural differentiation of BMSCs, the lesion volume and axonal regrowth of injured spinal cord after transplantation. Seven days after spinal cord injury, 3 × 10⁵ BMSCs or PBS (control) was delivered into the injury epicenter of the spinal cord. At 8 weeks after spinal cord injury, transplantation of BMSCs reduced the volume of cavity and increased spared white matter as compared to the control. BMSCs did not express the cell marker of neurons, astrocytes and oligodendrocytes in injured spinal cord. Transmission electron microscopic examination displayed an increase in the number of axons in BMSC rats. The effect of BMSCs on growth of neuronal process was further investigated by using a coculture system. The length and the number of neurites from spinal neurons significantly increased when they cocultured with BMSCs. PCR and immunochemical analysis showed that BMSCs expressed brain‐derived neurotrophic factor (BDNF) and glia cell line‐derived neurotrophic factor (GDNF). These findings demonstrate that transplantation of BMSCs reduces lesion volume and promotes axonal regrowth of injured spinal cord.     

Delayed treatment with Rho-kinase inhibitor does not enhance axonal regeneration or functional recovery after spinal cord injury in rats

Axonal regeneration in the central nervous system is blocked by many different growth inhibitory factors. Some of these inhibitors act on neurons by activating RhoA and Rho-kinase, an effector of RhoA. Several studies have shown that Rho-kinase inhibition immediately after spinal cord injury enhances axonal sprouting and functional recovery. In this study, we ask whether delayed treatment with Rho-kinase inhibitor is effective in promoting regeneration and functional recovery. We administered Fasudil, a Rho-kinase inhibitor, locally to the injury site 4 weeks or immediately after contusion of the thoracic spinal cord in rats. Although the immediate treatment significantly stimulated axonal sprouting and recovery of hindlimb function, treatment started 4 weeks after surgery had no effect on fiber sprouting or locomotor recovery. Our findings suggest that RhoA/Rho-kinase alone may not account for the irreversible arrest of axon outgrowth in the chronic stage of injury in the central nervous system.     

Regeneration after spinal nerve root injury

Spinal nerve root avulsion has been considered as a central nervous type of injury and therefore not repaired surgically in man. The possibility for axonal regeneration after root avulsion or root lesion has been investigated in laboratory animals by means of up to date neurophysiological, morphological and tracing techniques. It is shown that, after ventral root avulsion and implantation into the spinal cord, alpha and probably also gamma motoneurons are able to regenerate within the spinal cord for a considerable distance before entering the implanted root and reinnervate previously denervated skeletal muscles. The regenerated neurons were found to respond to afferent activity with excitatory or inhibitory responses, and the regenerated axons could conduct action potentials that elicited muscle twitch responses. After dorsal root injury in the adult animal, regeneration into the spinal cord does not occur. However, regeneration of primary sensory neurons into appropriate locations of the spinal cord can be demonstrated in immature animals.     

Role of tumor necrosis factor-alpha in neuronal and glial apoptosis after spinal cord injury

We investigated the role of tumor necrosis factor (TNF)-alpha in the onset of neuronal and glial apoptosis after traumatic spinal cord crush injury in rats. A few TUNEL-positive cells were first observed within and surrounding the lesion area 4 h after injury, with the largest number observed 24-48 h after injury. Double-labeling of cells using cell type-specific markers revealed that TUNEL-positive cells were either neurons or oligodendrocytes. One hour after injury, an intense immunoreactivity to TNF-alpha was observed in neurons and glial cells in the lesion area, but also seen in cells several mm from the lesion site rostrally and caudally. The level of nitric oxide (NO) also significantly increased in the spinal cord 4 h after injury. The injection of a neutralizing antibody against TNF-alpha into the lesion site several min after injury significantly reduced both the level of NO observed 4 h thereafter as well as the number of apoptotic cells observed 24 h after spinal cord trauma. An inhibitor of nitric oxide synthase (NOS), N(G)-monomethyl-l-arginine acetate (l-NMMA), also reduced the number of apoptotic cells. This reduction of apoptotic cells was associated with a decrease in DNA laddering on agarose gel electrophoresis. These results suggest that: (i) TNF-alpha may function as an external signal initiating apoptosis in neurons and oligodendrocytes after spinal cord injury; and (ii) TNF-alpha-initiated apoptosis may be mediated in part by NO as produced by a NOS expressed in response to TNF-alpha.     

Neuronal nitric oxide synthase (nNOS) mRNA is down-regulated, and constitutive NOS enzymatic activity decreased, in thoracic dorsal root ganglia and spinal cord of the rat by a substance P N-terminal metabolite

Nitric oxide (NO) in the spinal cord plays a role in sensory and autonomic activity. Pain induced by acetic acid in the abdominal stretch (writhing) assay and hyperalgesia associated with chronic pain are highly sensitive to NO synthase (NOS) inhibitors. Because substance P (SP) is released and up-regulated in some models of chronic pain, we hypothesized that an accumulation of SP metabolites may influence NOS expression and activity. To test this hypothesis, we examined the effect of intrathecally (i.t.) injected substance P (1-7) [SP(1-7)], the major metabolite of SP in the rat, on neuronal NOS (nNOS) mRNA in the thoracic and lumbar spinal cord, dorsal root ganglia (DRG) and on the corresponding constitutive NOS (cNOS) enzyme activity. Detected using quantitative RT-PCR, nNOS mRNA content in the thoracic spinal cord was decreased 6 h after injection of 5 micromol of SP(1-7) and returned to control 2 days later. In thoracic DRG, nNOS mRNA was reduced 48 h after SP(1-7). The cNOS enzymatic activity in thoracic spinal tissue was gradually decreased to a minimum at 72 h. Down-regulation of NOS by SP(1-7) in the thoracic area appears to be highly associated with capsaicin-sensitive primary afferent neurons. No similar changes in either parameter were measured in the lumbar area after SP(1-7). These data suggest that N-terminal SP fragments, which are known to cause long-term antinociception in the writhing assay, may do so by their ability to down-regulate NO synthesis along nociceptive pathways.     

Effects of Neurotransplants and BDNF on the Survival and Regeneration of Injured Adult Spinal Motoneurons

We compared the effects of peripheral nerve grafts, embryonic spinal cord transplants and brain-derived neurotrophic factor (BDNF) on the survival and axon regeneration of adult rat spinal motor neurons undergoing retrograde degeneration after ventral root avulsion. Following implantation into the dorsolateral funiculus of the injured spinal cord segment, neither a peripheral nerve graft nor a combination of peripheral nerve graft with embryonic spinal cord transplant could prevent the retrograde motor neuron degeneration induced by ventral root avulsion. However, intrathecal infusion of BDNF promoted long-term survival of the lesioned motor neurons and induced abundant motor axon regeneration from the avulsion zone along the spinal cord surface towards the BDNF source. A combination of ventral root reconstitution and BDNF treatment might therefore be a promising means for the support of both motor neuron survival and guided motor axon regeneration after ventral root lesions.