Newts have the remarkable ability to regenerate their spinal cords as adults. Their spinal cords regenerate with the regenerating tail after tail amputation, as well as after a gap-inducing spinal cord injury (SCI), such as a complete transection. While most studies on newt spinal cord regeneration have focused on events occurring after tail amputation, less attention has been given to events occurring after an SCI, a context that is more relevant to human SCI. Our goal was to use modern labeling and imaging techniques to observe axons regenerating across a complete transection injury and determine how cells and the extracellular matrix in the injury site might contribute to the regenerative process.
We identify stages of axon regeneration following a spinal cord transection and find that axon regrowth across the lesion appears to be enabled, in part, because meningeal cells and glia form a permissive environment for axon regeneration. Meningeal and endothelial cells regenerate into the lesion first and are associated with a loose extracellular matrix that allows axon growth cone migration. This matrix, paradoxically, consists of both permissive and inhibitory proteins. Axons grow into the injury site next and are closely associated with meningeal cells and glial processes extending from cell bodies surrounding the central canal. Later, ependymal tubes lined with glia extend into the lesion as well. Finally, the meningeal cells, axons, and glia move as a unit to close the gap in the spinal cord. After crossing the injury site, axons travel through white matter to reach synaptic targets, and though ascending axons regenerate, sensory axons do not appear to be among them. This entire regenerative process occurs even in the presence of an inflammatory response.
These data reveal, in detail, the cellular and extracellular events that occur during newt spinal cord regeneration after a transection injury and uncover an important role for meningeal and glial cells in facilitating axon regeneration. Given that these cell types interact to form inhibitory barriers in mammals, identifying the mechanisms underlying their permissive behaviors in the newt will provide new insights for improving spinal cord regeneration in mammals.
While many studies have focused on modulating the immune response and enhancing axonal regeneration after spinal cord injury (SCI), there is limited work being performed on evaluating the role of glial scar in SCI. We sought to evaluate the effects of glial scar resection in contusion models and dorsal hemisection models of SCI. At one week postinjury, 2mm of glial scar was excised from specimens in one of the two groups from each injury model. Functional outcome was measured weekly using the Basso, Beattie, Bresnahan (BBB) Locomotor Rating Scale along with histologic evaluation of spinal cord tracts to determine axonal regeneration. Within the dorsal hemisection model, there was no significant difference in recovery for animals that underwent glial scar excision versus animals that did not have scar excision (p=0.61). Animals subjected to the contusion model, however, demonstrated lower BBB scores in the glial resection group during the earlier postoperative periods (<4 weeks; p<0.05). Histological analysis revealed no axons within the glial resection contusion model, and moderate axonal growth within the nonresection contusion group and both hemisection groups (p>0.05 for differences among the three groups). While glial scar may serve to stabilize the preserved axonal tracts and thereby permit modest recovery in a contusion model of SCI, it may be of less importance with a dorsal hemisection model. These experiments highlight that basic biologic processes following SCI may vary tremendously based on the injury mechanism and that the role of glial scar in spinal cord regeneration must be elucidated.
axon regeneration; astrocyte; glial scar; spinal cord injury
After spinal cord injury (SCI), the formation of glial scar contributes to the failure of injured adult axons to regenerate past the lesion. Increasing evidence indicates that olfactory ensheathing cells (OECs) implanted into spinal cord are found to migrate into the lesion site and induce axons regeneration beyond glial scar and resumption of functions. However, little is known about the mechanisms of OECs migrating from injection site to glial scar/lesion site.
Methods and Findings
In the present study, we identified a link between OECs migration and reactive astrocytes in glial scar that was mediated by the tumor necrosis factor-α (TNF-α). Initially, the Boyden chamber migration assay showed that both glial scar tissue and reactive astrocyte-conditioned medium promoted OECs migration in vitro. Reactive astrocyte-derived TNF-α and its type 1 receptor TNFR1 expressed on OECs were identified to be responsible for the promoting effect on OECs migration. TNF-α-induced OECs migration was demonstrated depending on activation of the extracellular signal-regulated kinase (ERK) signaling cascades. Furthermore, TNF-α secreted by reactive astrocytes in glial scar was also showed to attract OECs migration in a spinal cord hemisection injury model of rat.
These findings showed that TNF-α was released by reactive astrocytes in glial scar and attracted OECs migration by interacting with TNFR1 expressed on OECs via regulation of ERK signaling. This migration-attracting effect of reactive astrocytes on OECs may suggest a mechanism for guiding OECs migration into glial scar, which is crucial for OECs-mediated axons regrowth beyond the spinal cord lesion site.
Spinal cord injury (SCI) in mammals leads to a robust inflammatory response followed by the formation of a glial and connective tissue scar that comprises a barrier to axonal regeneration. The inbred MRL/MpJ mouse strain exhibits reduced inflammation after peripheral injury and shows true regeneration without tissue scar formation following an ear punch wound. We hypothesized that following SCI, the unique genetic wound healing traits of this strain would result in reduced glial and connective tissue scar formation, increased axonal growth, and improved functional recovery. Adult MRL/MpJ and C57BL/6J mice were subjected to a mid-thoracic spinal contusion and the distribution of axon profiles and selected cellular and extracellular matrix components was compared at 1, 2, 4 and 6 weeks post-injury. Recovery of hind-limb locomotor function was assessed over the same time period. The MRL/MpJ mice exhibited robust axon growth within the lesion, beginning at 4 weeks post-injury. This growth was accompanied by reduced macrophage staining at 1, 2, 4 and 6 weeks post-injury, decreased chondroitin sulfate proteoglycan staining at 1–2 weeks and increased laminin staining throughout the lesion at 2–6 weeks post-injury. Paradoxically, the extent of locomotor recovery was impaired in the MRL/MpJ mice. Close examination of the chronic lesion site revealed evidence of ongoing degeneration both within and surrounding the lesion site. Thus, the regenerative genetic wound healing traits of the MRL/MpJ mice contribute to the evolution of a lesion environment that supports enhanced axon growth after SCI. However, this response occurs at the expense of meaningful functional recovery.
regeneration; degeneration; contusion; inflammation; astrocyte; glial scar
Previous studies have presented evidence which indicates that the regeneration of axons in the spinal cord occurs following spinal cord transection in young rats. However, in a transection-regeneration model, the completeness of the transection is often a matter of dispute. We established a method for shortening the rat spine and spinal cord to provide a spinal cord injury (SCI) model in which there was no doubt about whether the axonal transection was complete. In the future, this model may be applied to the chronic period of complete paralysis following SCI. Adult, female Wistar rats (220–250g) were used in the study. The spinal cord was exposed and a 4-mm-long segment of the spinal cord was removed at Th8. Subsequently, the Th7/8 and Th8/9 discs were cut between the stumps of the spinal cord to remove the Th8 vertebra. The stitches which had been passed through the 7th and 9th ribs bilaterally were tied gradually to bring together the stumps of the spinal cord. Almost all the rats survived until the end of the experiment. Uncoordinated movements of the hind limbs in locomotion were observed at 4 weeks after surgery. However coordinated movements of the hind limbs in locomotion were not observed until the end of the experiment. After 12 weeks, an intracardiac perfusion was performed to remove the thoracic spine and the spinal cord. There were no signs of infection. The bone fusion of the Th7 and Th9 vertebrae was observed to be complete in all specimens and the alignment of the thoracic spine was maintained. The spinal canal was also correctly reconstituted. The stumps of the spinal cord were connected. Light microscopy of the cord showed that scar tissue intervened at the connection site. Cavitation inhibiting the axonal regeneration was also observed. This model was also made on the assumption that glial scar tissue inhibits axonal regeneration in chronic SCI. Axonal regeneration was not observed across the transected spinal cord in this model. Attempts should be made to minimize the damage to the spinal cord and the surgery time for successful axonal regeneration to occur. The model developed in this study may be useful in the study of axonal regeneration in SCI.
spinal cord injury; animal model; rat
The formation of glial scars after spinal cord injury (SCI) is one of the factors inhibiting axonal regeneration. Glial scars are mainly composed of reactive astrocytes overexpressing intermediate filament (IF) proteins such as glial fibrillary acidic protein (GFAP) and vimentin. In the current study, we delivered small interfering RNAs (siRNAs) targeting these IF proteins to SCI model rats using photomechanical waves (PMWs), and examined the restoration of motor function in the rats. PMWs are generated by irradiating a light-absorbing material with 532-nm nanosecond laser pulses from a Q-switched Nd:YAG laser. PMWs can site-selectively increase the permeability of the cell membrane for molecular delivery. Rat spinal cord was injured using a weight-drop device and the siRNA(s) solutions were intrathecally injected into the vicinity of the exposed SCI, to which PMWs were applied. We first confirmed the substantial uptake of fluorescence-labeled siRNA by deep glial cells; then we delivered siRNAs targeting GFAP and vimentin into the lesion. The treatment led to a significant improvement in locomotive function from five days post-injury in rats that underwent PMW-mediated siRNA delivery. This was attributable to the moderate silencing of the IF proteins and the subsequent decrease in the cavity area in the injured spinal tissue.
Hypertrophic scarring and poor intrinsic axon growth capacity constitute major obstacles for spinal cord repair. These processes are tightly regulated by microtubule dynamics. We found that moderate microtubule stabilization decreased scar formation after spinal cord injury (SCI) in rodents via various cellular mechanisms, including dampening of TFG-β signalling. It prevented the accumulation of chondroitin sulfate proteoglycans (CSPGs) and rendered the lesion site permissive for axon regeneration of growth competent sensory neurons. Additionally, microtubule stabilization promoted growth of CNS axons of the Raphe-spinal tract and led to functional improvement. Thus, microtubule stabilization reduces fibrotic scarring and enhances the capacity of axons to grow. Manipulation of microtubules may offer the basis for a multi-targeted therapy after SCI.
Traumatic spinal cord injury (SCI) results in the formation of a fibrous scar acting as a growth barrier for regenerating axons at the lesion site. We have previously shown (Klapka et al., 2005) that transient suppression of the inhibitory lesion scar in rat spinal cord leads to long distance axon regeneration, retrograde rescue of axotomized cortical motoneurons, and improvement of locomotor function. Here we applied a systemic approach to investigate for the first time specific and dynamic alterations in the cortical gene expression profile following both thoracic SCI and regeneration-promoting anti-scarring treatment (AST). In order to monitor cortical gene expression we carried out microarray analyses using total RNA isolated from layer V/VI of rat sensorimotor cortex at 1–60 days post-operation (dpo). We demonstrate that cortical neurons respond to injury by massive changes in gene expression, starting as early as 1 dpo. AST, in turn, results in profound modifications of the lesion-induced expression profile. The treatment attenuates SCI-triggered transcriptional changes of genes related to inhibition of axon growth and impairment of cell survival, while upregulating the expression of genes associated with axon outgrowth, cell protection, and neural development. Thus, AST not only modifies the local environment impeding spinal cord regeneration by reduction of fibrous scarring in the injured spinal cord, but, in addition, strikingly changes the intrinsic capacity of cortical pyramidal neurons toward enhanced cell maintenance and axonal regeneration.
anti-scarring treatment; axonal regeneration; axotomy; corticospinal tract; lesion scar; microarray; sensorimotor cortex; spinal cord injury
Traumatic spinal cord injury (SCI) induces tissue damage and results in the formation of a cavity that inhibits axonal regrowth. Filling this cavity with a growth-permissive substrate would likely promote regeneration and repair. Single-walled carbon nanotubes functionalized with polyethylene glycol (SWNT-PEG) have been shown to increase the length of selected neurites in vitro. We hypothesized that administration of SWNT-PEG after experimental SCI will promote regeneration of axons into the lesion cavity and functional recovery of the hindlimbs. To evaluate this hypothesis, complete transection SCI was induced at the T9 vertebral level in adult female rats. One week after transection, the epicenter of the lesion was injected with 25 μL of either vehicle (saline), or 1 μg/mL, 10 μg/mL, or 100 μg/mL of SWNT-PEG. Behavioral analysis was conducted before injury, before treatment, and once every 7 days for 28 days after treatment. At 28 days post-injection the rats were euthanized and spinal cord tissue was extracted. Immunohistochemistry was used to detect the area of the cyst, the extent of the glial scar, and axonal morphology. We found that post-SCI administration of SWNT-PEG decreased lesion volume, increased neurofilament-positive fibers and corticospinal tract fibers in the lesion, and did not increase reactive gliosis. Additionally, post-SCI administration of SWNT-PEG induced a modest improvement in hindlimb locomotor recovery without inducing hyperalgesia. These data suggest that SWNT-PEG may be an effective material to promote axonal repair and regeneration after SCI.
axonal regeneration; gliosis; graft; nanofibers; nanomaterials
Axon regeneration after experimental spinal cord injury (SCI) can be promoted by combinatorial treatments that increase the intrinsic growth capacity of the damaged neurons and reduce environmental factors that inhibit axon growth. A prior peripheral nerve conditioning lesion is a well established means of increasing the intrinsic growth state of sensory neurons whose axons project within the dorsal columns of the spinal cord. Combining such a prior peripheral nerve conditioning lesion with the infusion of antibodies that neutralize the growth-inhibitory effects of the NG2 chondroitin sulfate proteoglycan promotes sensory axon growth through the glial scar and into the white matter of the dorsal columns. The physiological properties of these regenerated axons, particularly in the chronic SCI phase, have not been established. Here we examined the functional status of regenerated sensory afferents in the dorsal columns after SCI. Six months post-injury, we located and electrically mapped functional sensory axons that had regenerated beyond the injury site. The regenerated axons had reduced conduction velocity, decreased frequency-following ability, and increasing latency to repetitive stimuli. Many of the axons that had regenerated into the dorsal columns rostral to the injury site were chronically demyelinated. These results demonstrate that regenerated sensory axons remain in a chronic pathophysiological state and emphasize the need to restore normal conduction properties to regenerated axons after spinal cord injury.
spinal cord injury; demyelination; pathophysiology; NG2; regeneration; myelin; conduction velocity
Bone marrow mesenchymal stem cells (MSCs) are one of the potential tools for treatment of the spinal cord injury; however, the survival and differentiation of MSCs in an injured spinal cord still need to be improved. In the present study, we investigated whether Governor Vessel electro-acupuncture (EA) could efficiently promote bone marrow mesenchymal stem cells (MSCs) survival and differentiation, axonal regeneration and finally, functional recovery in the transected spinal cord.
The spinal cords of adult Sprague-Dawley (SD) rats were completely transected at T10, five experimental groups were performed: 1. sham operated control (Sham-control); 2. operated control (Op-control); 3. electro-acupuncture treatment (EA); 4. MSCs transplantation (MSCs); and 5. MSCs transplantation combined with electro-acupuncture (MSCs+EA). After 2-8 weeks of MSCs transplantation plus EA treatment, we found that the neurotrophin-3 (NT-3), cAMP level, the differentiation of MSCs, the 5-HT positive and CGRP positive nerve fibers in the lesion site and nearby tissue of injured spinal cord were significantly increased in the MSCs+EA group as compared to the group of the MSCs transplantation or the EA treated alone. Furthermore, behavioral test and spinal cord evoked potentials detection demonstrated a significantly functional recovery in the MSCs +EA group.
These results suggest that EA treatment may promote grafted MSCs survival and differentiation; MSCs transplantation combined with EA treatment could promote axonal regeneration and partial locomotor functional recovery in the transected spinal cord in rats and indicate a promising avenue of treatment of spinal cord injury.
Reports based primarily on anatomical evidence suggest that olfactory ensheathing glia (OEG) transplantation promotes axon regeneration across a complete spinal cord transection in adult rats. Based on functional, electrophysiological, and anatomical assessments, we found that OEG promoted axon regeneration across a complete spinal cord transection and that this regeneration altered motor responses over time. At 7 months after transection, 70% of OEG-treated rats showed motor-evoked potentials in hindlimb muscles after transcranial electric stimulation. Furthermore, a complete spinal cord retransection performed 8 months after injury demonstrated that this axon regeneration suppressed locomotor performance and decreased the hypersensitive hindlimb withdrawal response to mechanical stimulation. OEG transplantation alone promoted reorganization of lumbosacral locomotor networks and, when combined with long-term training, enhanced some stepping measures. These novel findings demonstrate that OEG promote regeneration of mature axons across a complete transection and reorganization of spinal circuitry, both of which contribute to sensorimotor function.
Due to the varied and numerous changes in spinal cord tissue following injury, successful treatment for repair may involve strategies combining neuroprotection (pharmacological prevention of some of the damaging intracellular cascades that lead to secondary tissue loss), axonal regeneration promotion (cell transplantation, genetic engineering to increase growth factors, neutralization of inhibitory factors, reduction in scar formation), and rehabilitation. Our goal has been to find effective combination strategies to improve outcome after injury to the adult rat thoracic spinal cord. Combination interventions tested have been implantation of Schwann cells (SCs) plus neuroprotective agents and growth factors administered in various ways, olfactory ensheathing cell (OEC) implantation, chondroitinase addition, or elevation of cyclic AMP. The most efficacious strategy in our hands for the acute complete transection/SC bridge model, including improvement in locomotion [Basso, Beattie, Bresnahan Scale (BBB)], is the combination of SCs, OECs, and chondroitinase administration (BBB 2.1 vs 6.6, 3 times more myelinated axons in the SC bridge, increased serotonergic axons in the bridge and beyond, and significant correlation between the number of bridge myelinated axons and functional improvement). We found the most successful combination strategy for a subacute spinal cord contusion injury (12.5–mm, 10–g weight, MASCIS impactor) to be SCs and elevation of cyclic AMP (BBB 10.4 vs 15, significant increases in white matter sparing, in myelinated axons in the implant, and in responding reticular formation and red and raphe nuclei, and a significant correlation between the number of serotonergic fibers and improvement in locomotion). Thus, in two injury paradigms, these combination strategies as well as others studied in our laboratory have been found to be more effective than SCs alone and suggest ways in which clinical application may be developed.
Spinal cord injuries; Schwann cells; Olfactory ensheathing cells; Axonal regeneration; Chondroitinase; Cyclic adenosine monophosphate
Following the initial acute stage of spinal cord injury, a cascade of cellular and inflammatory responses will lead to progressive secondary damage of the nerve tissue surrounding the primary injury site. The degeneration is manifested by loss of neurons and glial cells, demyelination and cyst formation. Injury to the mammalian spinal cord results in nearly complete failure of the severed axons to regenerate. We have previously demonstrated that the antioxidants N-acetyl-cysteine (NAC) and acetyl-L-carnitine (ALC) can attenuate retrograde neuronal degeneration after peripheral nerve and ventral root injury. The present study evaluates the effects of NAC and ALC on neuronal survival, axonal sprouting and glial cell reactions after spinal cord injury in adult rats. Tibial motoneurons in the spinal cord were pre-labeled with fluorescent tracer Fast Blue one week before lumbar L5 hemisection. Continuous intrathecal infusion of NAC (2.4 mg/day) or ALC (0.9 mg/day) was initiated immediately after spinal injury using Alzet 2002 osmotic minipumps. Neuroprotective effects of treatment were assessed by counting surviving motoneurons and by using quantitative immunohistochemistry and Western blotting for neuronal and glial cell markers 4 weeks after hemisection. Spinal cord injury induced significant loss of tibial motoneurons in L4–L6 segments. Neuronal degeneration was associated with decreased immunostaining for microtubular-associated protein-2 (MAP2) in dendritic branches, synaptophysin in presynaptic boutons and neurofilaments in nerve fibers. Immunostaining for the astroglial marker GFAP and microglial marker OX42 was increased. Treatment with NAC and ALC rescued approximately half of the motoneurons destined to die. In addition, antioxidants restored MAP2 and synaptophysin immunoreactivity. However, the perineuronal synaptophysin labeling was not recovered. Although both treatments promoted axonal sprouting, there was no effect on reactive astrocytes. In contrast, the microglial reaction was significantly attenuated. The results indicate a therapeutic potential for NAC and ALC in the early treatment of traumatic spinal cord injury.
Trauma to the adult CNS initiates multiple processes including primary and secondary axotomy, inflammation, and glial scar formation that have devastating effects on neuronal regeneration. After spinal cord injury, the infiltration of phagocytic macrophages coincides with long distance axonal retraction from the initial site of injury, a deleterious phenomenon known as axonal dieback. We have previously shown that activated macrophages directly induce long distance retraction of dystrophic axons in an in vitro model of the glial scar. We hypothesized that treatments which are primarily thought to increase neuronal regeneration following spinal cord injury may in fact derive a portion of their beneficial effects from inhibition of macrophage-mediated axonal retraction. We analyzed the effects of protease inhibition, substrate modification, and neuronal preconditioning on macrophage-axon interactions using our established in vitro model. General inhibition of matrix metalloproteinases and specific inhibition of MMP-9 prevented macrophage-induced axonal retraction despite significant physical interactions between the two cell types, whereas inhibition of MMP-2 had no effect. Chondroitinase ABC-mediated digestion of the aggrecan substrate also prevented macrophage-induced axonal retraction in the presence of extensive macrophage-axon interactions. The use of a conditioning lesion to stimulate intrinsic neuronal growth potential in the absence of substrate modification likewise prevented macrophage-induced axonal retraction in vitro and in vivo following spinal cord injury. These data provide valuable insight into the cellular and molecular mechanisms underlying macrophage-mediated axonal retraction and demonstrate modifications that can alleviate the detrimental effects of this unfavorable phenomenon on the post-lesion CNS.
Spinal cord injury; Inflammation; Growth Cone; Proteoglycan; Macrophage; Regeneration
Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia and to limited axonal regeneration after injury. Here we report that transplantation of human dental pulp stem cells into the completely transected adult rat spinal cord resulted in marked recovery of hind limb locomotor functions. Transplantation of human bone marrow stromal cells or skin-derived fibroblasts led to substantially less recovery of locomotor function. The human dental pulp stem cells exhibited three major neuroregenerative activities. First, they inhibited the SCI-induced apoptosis of neurons, astrocytes, and oligodendrocytes, which improved the preservation of neuronal filaments and myelin sheaths. Second, they promoted the regeneration of transected axons by directly inhibiting multiple axon growth inhibitors, including chondroitin sulfate proteoglycan and myelin-associated glycoprotein, via paracrine mechanisms. Last, they replaced lost cells by differentiating into mature oligodendrocytes under the extreme conditions of SCI. Our data demonstrate that tooth-derived stem cells may provide therapeutic benefits for treating SCI through both cell-autonomous and paracrine neuroregenerative activities.
Failure of axon regeneration in the mammalian central nervous system (CNS) is due in part to the presence of various inhibitory molecules, including myelin-associated proteins and proteoglycans enriched in glial scars. Here we evaluate whether axon guidance molecules also regulate regenerative growth after injury in adulthood. Wnts are a large family of axon guidance molecules, which can attract ascending axons and repel descending axons along the length of the developing spinal cord. Their expression (all 19 Wnts) is not detectable in normal adult spinal cord by in situ hybridization. However, three of them are clearly reinduced after spinal cord injury. Wnt1 and Wnt5a, encoding potent repellents of the descending corticospinal tract (CST) axons, were robustly and acutely induced broadly in the spinal cord gray matter following unilateral hemisection. Ryk, the conserved repulsive Wnt receptor, was also induced in the lesion area, and Ryk immunoreactivity was found on the lesioned CST axons. Wnt4, which attracts ascending sensory axons in development, was acutely induced in areas closer to the lesion than Wnt1 and Wnt5a. Injection of function-blocking Ryk antibodies into the dorsal bilateral hemisectioned spinal cord either prevented the retraction of CST axons or promoted their regrowth but clearly enhanced the sprouting of CST collateral branches around and beyond the injury site. Therefore, repulsive Wnt signaling may be a cause of cortical spinal tract axon retraction and inhibits axon sprouting following injury.
Wnt1; Wnt5a; Ryk; Spinal cord injury; Paralysis; Axon retraction; Corticospinal tract; Collateral branches
Following CNS injuries, axon growth inhibitors from the myelin and the scar tissue at the injury site are considered major impediments to axon regeneration. The presence of several classes of inhibitors with multiple members in each class suggests functional redundancy in growth inhibition. To test redundancy within the myelin inhibitory pathway, we analyzed raphespinal serotonergic (5-HT) axon regeneration in mice deficient in two major myelin inhibitors, Nogo and MAG, and their common receptor NgR1 (or NgR). Following a complete transection spinal cord injury, there was no significant enhancement of 5-HT axon regeneration beyond the injury site in either Nogo/MAG/NgR1 triple mutants or NgR1 single mutants. Occasional, genotype-independent traversal of 5-HT axons through GFAP-positive tissue bridges at the injury site implicates GFAP-negative lesion areas as especially inhibitory to 5-HT axons. To assess the contribution of class 3 Semaphorins that are expressed by GFAP-negative meningeal fibroblasts at the injury site, we analyzed mice deficient in PlexinA3 and PlexinA4, two key receptors for class 3 Semaphorins, with or without additional NgR1 deletion. No enhanced regeneration of 5-HT or corticospinal axons was detected in PlexinA3/PlexinA4 double mutants or PlexinA3/PlexinA4/NgR1 triple mutants through a complete transection injury. In contrast with previous reports, these data demonstrate that attenuating myelin or Semaphorin-mediated inhibition of axon growth is insufficient to promote 5-HT axon regeneration and further indicate that even attenuating both classes of inhibitory influences is insufficient to promote regeneration of injured axons through a complete transection spinal cord injury.
Axon regeneration; serotonergic axons; myelin-derived axon growth inhibitors; Semaphorins; spinal cord injury; CNS repair
Spinal cord injury increases inhibitory factors that may restrict neurite outgrowth after trauma. The expression of repulsive molecules in reactive astrocytes and the formation of the glial scar at the injury site produce the non-permissive environment for axonal regeneration. However, the mechanism that triggers this astrogliotic response is unknown. The release of nucleotides has been linked to this hypertrophic state.
Our goal is to investigate the temporal profile of P2Y2 nucleotide receptor after spinal cord injury in adult female Sprague–Dawley rats. Molecular biology, immunofluorescence studies, and Western Blots were used to evaluate the temporal profile (2, 4, 7, 14, and 28 days post-injury) of this receptor in rats injured at the T-10 level using the NYU impactor device.
Real time RT-PCR showed a significant increase of P2Y2 mRNA after 2 days post-injury that continues throughout 28 days post-injury. Double labeling studies localized P2Y2 immunoreactivity in neuronal cell bodies, axons, macrophages, oligodendrocytes and reactive astrocytes. Immunofluorescence studies also demonstrated a low level of P2Y2 receptor in sham samples, which increased after injury in glial fibrillary acidic protein positive cells. Western Blot performed with contused spinal cord protein samples revealed an upregulation in the P2Y2 42 kDa protein band expression after 4 days post-injury that continues until 28 days post-injury. However, a downregulation of the 62 kDa receptor protein band after 2 days post-injury that continues up to 28 days post-injury was observed.
Therefore, the spatio-temporal pattern of P2Y2 gene expression after spinal cord injury suggests a role in the pathophysiology response generated after trauma.
Astrogliosis; Nucleotide receptor; Regeneration; Glial scar; Trauma; Purinergic
This study was designed to assess a new composite implant to induce regeneration of injured spinal cord in paraplegic rats following complete cord transection. Neuronal xenogeneic cells from biopsies of adult nasal olfactory mucosa (NOM) of human origin, or spinal cords of human embryos, were cultured in two consecutive stages: stationary cultures in a viscous semi-solid gel (NVR-N-Gel) and in suspension on positively charged microcarriers (MCs). A tissue-engineered tubular scaffold, containing bundles of parallel nanofibers, was developed. Both the tube and the nanofibers were made of a biodegradable dextran sulphate–gelatin co-precipitate. The suturable scaffold anchored the implant at the site of injury and provided guidance for the regenerating axons. Implants of adult human NOM cells were implanted into eight rats, from which a 4 mm segment of the spinal cord had been completely removed. Another four rats whose spinal cords had also been transected were implanted with a composite implant of cultured human embryonic spinal cord cells. Eight other cord-transected rats served as a control group. Physiological and behavioral analysis, performed 3 months after implantation, revealed partial recovery of function in one or two limbs in three out of eight animals of the NOM implanted group and in all the four rats that were implanted with cultured human embryonic spinal cord cells. Animals of the control group remained completely paralyzed and did not show transmission of stimuli to the brain. The utilization of an innovative composite implant to bridge a gap resulting from the transection and removal of a 4 mm spinal cord segment shows promise, suggesting the feasibility of this approach for partial reconstruction of spinal cord lesions. Such an implant may serve as a vital bridging station in acute and chronic cases of paraplegia.
Olfactory mucosa; Spinal cord; Transection; Transplantation
In the injured spinal cord, a glial scar forms and becomes a major obstacle to axonal regeneration. Formation of the glial scar involves migration of astrocytes toward the lesion. Matrix metalloproteinases (MMPs), including MMP-9 and MMP-2, govern cell migration through their ability to degrade constituents of the extracellular matrix. Although MMP-9 is expressed in reactive astrocytes, its involvement in astrocyte migration and formation of a glial scar is unknown. Here we found that spinal cord injured, wild-type mice expressing MMPs developed a more severe glial scar and enhanced expression of chondroitin sulfate proteoglycans, indicative of a more inhibitory environment for axonal regeneration/plasticity, than MMP-9 null mice. To determine if MMP-9 mediates astrocyte migration, we conducted a scratch wound assay using astrocytes cultured from MMP-9 null, MMP-2 null, and wild-type mice. Gelatin zymography confirmed the expression of MMP-9 and MMP-2 in wild-type cultures. MMP-9 null astrocytes and wild-type astrocytes, treated with an MMP-9 inhibitor, exhibited impaired migration relative to untreated wild-type controls. MMP-9 null astrocytes showed abnormalities in the actin cytoskeletal organization and function but no detectable untoward effects on proliferation, cellular viability, or adhesion. Interestingly, MMP-2 null astrocytes showed increased migration, which could be attenuated in the presence of an MMP-9 inhibitor. Collectively, our studies provide explicit evidence that MMP-9 is integral to the formation of an inhibitory glial scar and cytoskeleton-mediated astrocyte migration. MMP-9 may thus be a promising therapeutic target to reduce glial scarring during wound healing after spinal cord injury.
gelatinase; glial fibrillary acidic protein; chondroitin sulfate proteoglycan; extracellular matrix; actin; Rac1
One important aspect of recovery and repair after spinal cord injury (SCI) lies in the complex cellular interactions at the injury site that leads to the formation of a lesion scar. EphA4, a promiscuous member of the EphA family of repulsive axon guidance receptors, is expressed by multiple cell types in the injured spinal cord, including astrocytes and neurons. We hypothesized that EphA4 contributes to aspects of cell-cell interactions at the injury site after SCI, thus modulating the formation of the astroglial-fibrotic scar. To test this hypothesis, we studied tissue responses to a thoracic dorsal hemisection SCI in an EphA4 mutant mouse line. We found that EphA4 expression, as assessed by β-galactosidase reporter gene activity, is associated primarily with astrocytes in the spinal cord, neurons in the cerebral cortex and, to a lesser extent, spinal neurons, before and after SCI. However, we did not observe any overt reduction of glial fibrillary acidic protein (GFAP) expression in the injured area of EphA4 mutants in comparison with controls following SCI. Furthermore, there was no evident disruption of the fibrotic scar, and the boundary between reactive astrocytes and meningeal fibroblasts appeared unaltered in the mutants, as were lesion size, neuronal survival and inflammation marker expression. Thus, genetic deletion of EphA4 does not significantly alter the astroglial response or the formation of the astroglial-fibrotic scar following a dorsal hemisection SCI in mice. In contrast to what has been proposed, these data do not support a major role for EphA4 in reactive astrogliosis following SCI.
Spinal cord injury; Eph receptor; glial scar; fibrotic scar; astroglial response; meningeal cells; CNS repair
Transplantation of olfactory bulb-derived olfactory ensheathing glia (OEG) combined with step training improves hindlimb locomotion in adult rats with a complete spinal cord transection. Spinal cord injury studies use the presence of noradrenergic (NA) axons caudal to the injury site as evidence of axonal regeneration and we previously found more NA axons just caudal to the transection in OEG- than media-injected spinal rats. We therefore hypothesized that OEG transplantation promotes descending coeruleospinal regeneration that contributes to the recovery of hindlimb locomotion. Now we report that NA axons are present throughout the caudal stump of both media- and OEG-injected spinal rats and they enter the spinal cord from the periphery via dorsal and ventral roots and along large penetrating blood vessels. These results indicate that the presence of NA fibers in the caudal spinal cord is not a reliable indicator of coeruleospinal regeneration. We then asked if NA axons appose cholinergic neurons associated with motor functions, i.e., central canal cluster and partition cells (active during fictive locomotion) and somatic motor neurons (SMNs). We found more NA varicosities adjacent to central canal cluster cells, partition cells, and SMNs in the lumbar enlargement of OEG- than media-injected rats. As non-synaptic release of NA is common in the spinal cord, more associations between NA varicosities and motor-associated cholinergic neurons in the lumbar spinal cord may contribute to the improved treadmill stepping observed in OEG-injected spinal rats. This effect could be mediated through direct association with SMNs and/or indirectly via cholinergic interneurons.
OEC; spinal cord injury; norepinephrine; ChAT; cholinergic neurons; locomotion
Five patients with chronic arachnoiditis and syringomyelia were studied. Three patients had early life meningitis and developed symptoms of syringomyelia eight, 21, and 23 years after the acute infection. One patient had a spinal dural thoracic AVM and developed a thoracic syrinx 11 years after spinal subarachnoid haemorrhage and five years after surgery on the AVM. A fifth patient had tuberculous meningitis with transient spinal cord dysfunction followed by development of a lumbar syrinx seven years later. Arachnoiditis can cause syrinx formation by obliterating the spinal vasculature causing ischaemia. Small cystic regions of myelomalacia coalesce to form cavities. In other patients, central cord ischaemia mimics syringomyelia but no cavitation is present. Scar formation with spinal block leads to altered dynamics of cerebrospinal fluid (CSF) flow and contributes to the formation of spinal cord cystic cavities.
Spinal cord injury (SCI) triggers a sequel of events commonly associated with cell death and dysfunction of glias and neurons surrounding the lesion. Although astrogliosis and glial scar formation have been involved in both damage and repair processes after SCI, their role remains controversial. Our goal was to investigate the effects of the P2 receptors antagonists, PPADS and suramin, in the establishment of the reactive gliosis and the formation of the glial scar. Molecular biology, immunohistochemistry, spared tissue, and locomotor behavioral studies were used to evaluate astrogliosis, in adult female Sprague-Dawley rats treated with P2 antagonists after moderate injury with the NYU impactor device. Semi-quantitative RT-PCR confirmed the presence of P2Y1, P2Y2, P2Y4, P2Y6, P2Y12 and P2X2 receptors in the adult spinal cord. Immunohistochemistry studies confirmed a significant decrease in GFAP labeled cells at the injury epicenter as well as a decrease in spared tissue after treatment with the antagonists. Functional open field testing revealed no significant locomotor score differences between treated and control animals. Our work is consistent with studies suggesting that astrogliosis is an important event after SCI that limits tissue damage and lesion spreading.
Glial Scar; PPADS; Suramin; Spare tissue; Trauma; Purinergic