Chronic transplantation of olfactory ensheathing cells promotes partial recovery after complete spinal cord transection in the rat

The goal of this study was to ascertain whether olfactory ensheathing cells (OECs) were able to promote axonal regeneration and functional recovery when transplanted 45 days after complete transection of the thoracic spinal cord in adult rats. OECs promoted partial restitution of supraspinal pathways evaluated by motor evoked potentials and modest recovery of hindlimb movements. In addition, OEC grafts reduced lumbar reflex hyperexcitability from the first month after transplantation. Histological results revealed that OECs facilitated corticospinal and raphespinal axons regrowth through the injury site and into the caudal spinal cord segments. Interestingly, raphespinal but not corticospinal fibers regenerated long distances through the gray matter and reached the lower lumbar segments (L5) of the spinal cord. However, delayed OEC grafts failed to reduce posttraumatic astrogliosis. In conclusion, the beneficial effects found in the present study further support the use of OECs for treating chronic spinal cord injuries.     

Motor recovery and anatomical evidence of axonal regrowth in spinal cord-repaired adult rats

Behavioral assessments of hindlimb motor recovery and anatomical assessments of extended axons of long spinal tracts were conducted in adult rats following complete spinal cord transection. Rats were randomly divided into 3 groups: 1) sham control group (laminectomy only; n = 12); 2) transection-only group, spinal cord transection at T8 (n = 20); and 3) experimental treatment group, spinal cord transection at T8, with peripheral nerve grafts (PNG) and application of acidic fibroblast growth factor (aFGF) (n = 14). The locomotor behavior and stepping of all rats were analyzed over a 6-month survival time using the Basso, Beattie, Bresnahan (BBB) open field locomotor test and the contact placing test. Immunohistochemistry for serotonin (5-HT), anterograde tracing with biotinylated dextran amine (BDA), and retrograde tracing with fluoro-gold were used to evaluate the presence of axons below the damage site following treatment. When compared with the transection-only group, the nerve graft with the aFGF group showed 1) significant improvement in hindlimb locomotion and stepping, 2) the presence of 5-HT-labeled axons below the lesion site at lumbar cord level (these were interpreted as regenerated axons from the raphe nuclei), 3) the presence of anterograde BDA labeling of corticospinal tract axons at the graft site and below, and 4) fluoro-gold retrograde labeling of neuron populations in motor cortex and in red nucleus, reticulospinal nuclei, raphe nuclei, and vestibular nuclei. We conclude that peripheral nerve grafts and aFGF treatments facilitate the regrowth of the spinal axons and improve hindlimb function in a T-8 spinal cord-transected rat model.     

Regeneration of corticospinal axons in the rat.

In the rat, a few long descending motor tracts capable of carrying an impulse and causing a propagated impulse in the ipsilateral sciatic nerve will regenerate after complete spinal cord transection. In this experiment such regeneration was found in both treated and control animals. Orthograde axonal transport of tritiated proline injected into the motor cortex labels only the corticospinal tracts in the rat spinal cord. Scintillation counts of measured lengths of spinal cord can be used as a measure of the number of labeled corticospinal axons. Comparison of radioactivity per unit length of measured cord segments taken from above and below the site of a previous spinal cord transection can give a reliable estimate of the number of labeled axons that regenerated and crossed the site of injury. Using this test we have demonstrated that some corticospinal axons had regenerated six months after spinlal cord transection in control animals, animals made tolerant to degenerating spinal cord antigens, and animals treated with cyclophosphamide. A group treated with a single 75 mg per kilogram dose of cyclophosphamide 24 hours after spinal cord transection showed the best evidence of corticospinal tract regeneration.     

The fate of severed corticospinal axons

The potential for regeneration of severed corticospinal axons was examined by labeling these axons with horseradish peroxidase following thoracic spinal cord transections in mice. Shortly after severance, the proximal ends of corticospinal axons formed terminal bulbs that persisted for weeks and were associated with axonal retraction. There were few signs of corticospinal axonal sprouting or elongation. By 2 months after injury, corticospinal axons near the transection site showed an increased number of probable labeled terminals in the adjacent gray matter. These new terminals may contribute to the persistence of many corticospinal axons near the injury site long after a spinal cord transection.     

Regenerating descending axons preferentially reroute to the gray matter in the presence of a general macrophage/microglial reaction caudal to a spinal transection in adult zebrafish

We analyzed pathway choices of regenerating, mostly supraspinal, descending axons in the spinal cord of adult zebrafish and the cellular changes in the spinal cord caudal to a lesion site after complete spinal transection. Anterograde tracing (by application of the tracer rostral to the spinal lesion site) showed that significantly more descending axons (74%) regenerated in the spinal gray matter of the caudal spinal cord than would be expected from random growth. Retrograde tracing (by application of the tracer caudal to the spinal lesion site) showed that, rostral to the lesion, most of these axons (80%) extended into the major white matter tracts. Thus, ventral descending tracts often were devoid of labeled axons caudal to a spinal lesion but contained many axons rostral to the lesion in the same animals, indicating a pathway switch of descending axons from the white matter to the gray matter. Ascending axons of spinal neurons were not observed regrowing to the rostral tracer application site; therefore, they most likely did not contribute to the axonal populations analyzed. A macrophage/microglia response within 2 days of spinal cord transection, along with phagocytosis of myelin, was observed caudal to the transection by immunohistochemistry and electron microscopy. Nevertheless, caudal to the lesion, descending tracts in the white matter were filled with myelin debris during the time of axonal regrowth, at least up to 6 weeks postlesion. We suggest that the spontaneous regeneration of axons of supraspinal origin after spinal cord transection in adult zebrafish may be due in part to the axons' ability to negotiate novel pathways in the spinal cord gray matter.     

L1 CAM expression is increased surrounding the lesion site in rats with complete spinal cord transection as neonates

L1 is a cell adhesion molecule associated with axonal outgrowth, fasciculation, and guidance during development and injury. In this study, we examined the long-term effects of spinal cord injury with and without exercise on the re-expression of L1 throughout the rat spinal cord. Spinal cords from control rats were compared to those from rats receiving complete mid-thoracic spinal cord transections at postnatal day 5, daily treadmill step training for up to 8 weeks, or both transection and step training. Three months after spinal cord transection, we observed substantially higher levels of L1 expression by both Western blot analysis and immunocytochemistry in rats with and without step training. Higher expression levels of L1 were seen in the dorsal gray matter and in the dorsal lateral funiculus both above and below the lesion site. In addition, L1 was re-expressed on the descending fibers of the corticospinal tract above the lesion. L1-labeled axons also expressed GAP-43, a protein associated with axon outgrowth and regeneration. Treadmill step training had no effect on L1 expression in either control or transected rats despite the fact that spinal transected rats displayed improved stepping patterns indicative of spinal learning. Thus, spinal cord transection at an early age induced substantial L1 expression on axons near the lesion site, but was not additionally augmented by exercise.     

Sodium channel blockade with phenytoin protects spinal cord axons, enhances axonal conduction, and improves functional motor recovery after contusion SCI

Accumulation of intracellular sodium through voltage-gated sodium channels (VGSCs) is an important event in the cascade leading to anatomic degeneration of spinal cord axons and poor functional outcome following traumatic spinal cord injury (SCI). In this study, we hypothesized that phenytoin, a sodium channel blocker, would result in protection of axons with concomitant improvement of functional recovery after SCI. Adult male Sprague-Dawley rats underwent T9 contusion SCI after being fed normal chow or chow containing phenytoin; serum levels of phenytoin were within therapeutic range at the time of injury. At various timepoints after injury, quantitative assessment of lesion volumes, axonal degeneration, axonal conduction, and functional locomotor recovery were performed. When compared to controls, phenytoin-treated animals demonstrated reductions in the degree of destruction of gray and white matter surrounding the lesion epicenter, sparing of axons within the dorsal corticospinal tract (dCST) and dorsal column (DC) system rostral to the lesion site, and within the dorsolateral funiculus (DLF) caudal to the lesion site, and enhanced axonal conduction across the lesion site. Improved performance in measures of skilled locomotor function was observed in phenytoin-treated animals. Based on these results, we conclude that phenytoin provides neuroprotection and improves functional outcome after experimental SCI, and that it merits further examination as a potential treatment strategy in human SCI.     

Spinal cord regeneration in rats made immunologically unresponsive to CNS antigens.

Seventy days after complete spinal cord transection, both treated and untreated rats showed evidence that some corticospinal axons had regenerated. Rats made immunologically unresponsive to CNS tissue showed no increase in corticospinal regeneration as measured by orthograde axoplasmic flow of tritated proline or retrograde axoplasmic labelling with horeseradish peroxidase. However, treated rats did demonstrate electrophysiological evidence of regeneration of long ascending sensory pathways. Tolerant animals additionally treated with cyclophosphamide showed corticospinal axonal regeneration by tritated proline transport and electrophysiological techniques and also showed electrophysiological evidence of ascending sensory tract regeneration.     

Harnessing neural activity to promote repair of the damaged corticospinal system after spinal cord injury

As most spinal cord injuries(SCIs) are incomplete,an important target for promoting neural repair and recovery of lost motor function is to promote the connections of spared descending spinal pathways with spinal motor circuits.Among the pathways,the corticospinal tract(CST) is most associated with skilled voluntary functions in humans and many animals.CST loss,whether at its origin in the motor cortex or in the white matter tracts subcortically and in the spinal cord,leads to movement impairments and paralysis.To restore motor function after injury will require repair of the damaged CST.In this review,I discuss how knowledge of activity-dependent development of the CST—which establishes connectional specificity through axon pruning,axon outgrowth,and synaptic competition among CST terminals—informed a novel activity-based therapy for promoting sprouting of spared CST axons after injur in mature animals.This therapy,which comprises motor cortex electrical stimulation with and without concurrent trans-spinal direct current stimulation,leads to an increase in the gray matter axon length of spared CST axons in the rat spinal cord and,after a pyramidal tract lesion,restoration of skilled locomotor movements.I discuss how this approach is now being applied to a C4 contusion rat model.     

Contrasting neuropathology and functional recovery after spinal cord injury in developing and adult rats

Conflicting findings exist regarding the link between functional recovery and the regrowth of spinal tracts across the lesion leading to the restoration of functional contacts. In the present study, we investigated whether functional locomotor recovery was attributable to anatomical regeneration at postnatal day 1 (PN1), PN7, PN14 and in adult rats two months after transection injury at the tenth thoracic segment of the spinal cord. The Basso, Beattie, and Bresnahan scores showed that transection led to a failure of hindlimb locomotor function in PN14 and adult rats. However, PN1 and PN7 rats showed a significant level of stepping function after complete spinal cord transection. Unexpectedly, unlike the transected PN14 and adult rats in which the spinal cord underwent limited secondary degeneration and showed a scar at the lesion site, the rats transected at PN1 and PN7 showed massive secondary degeneration both anterograde and retrograde, leaving a >5-mm gap between the two stumps. Furthermore, retrograde tracing with fluorogold (FG) also showed that FG did not cross the transection site in PN1 and PN7 rats as in PN14 and adult rats, and re-transection of the cord caused no apparent loss in locomotor performance in the rats transected at PN1. Thus, these three lines of evidence strongly indicated that the functional recovery after transection in neonatal rats is independent of regrowth of spinal tracts across the lesion site. Our results support the notion that the recovery of locomotor function in developing rats may be due to intrinsic adaptations in the spinal circuitry below the lesion that control hindlimb locomotor activity rather than the regrowth of spinal tracts across the lesion. The difference in secondary degeneration between neonatal and adult rats remains to be explored.     

Acute and delayed transplantation of olfactory ensheathing cells promote partial recovery after complete transection of the spinal cord

The present study was undertaken to determine whether olfactory ensheathing cells (OECs) from the olfactory bulb were capable to promote axonal regeneration and functional recovery when transplanted either acutely or 1 week delayed into the T8 transected rat spinal cord. OEC transplants increased recovery of functional outcomes, as shown electrophysiologically by return of motor evoked potentials and by reduction of hindlimb hyperreflexia, and behaviorally by recovery of movements of hindlimb joints. Axonal regeneration was proven histologically by demonstrating long axonal outgrowth of raphespinal, coerulospinal, and corticospinal tracts within the caudal cord stump. Expression of GFAP and NG2 was down-regulated in perilesional cord segments in transplanted animals, indicating a more suitable environment for axonal regeneration. Overall, earlier recovery and better functional and histological results were observed in rats receiving acute than delayed OEC transplants. The beneficial effects obtained with transplantation after transection are encouraging for the application of OECs in the human injured spinal cord.     

Noradrenergic innervation of the rat spinal cord caudal to a complete spinal cord transection: Effects of olfactory ensheathing glia

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.     

Prelabeled red nucleus and sensorimotor cortex neurons of the rat survive 10 and 20 weeks after spinal cord transection

To demonstrate definitively the fate of the somata of rubrospinal and corticospinal neurons axotomized by a complete spinal cord transection at T-9, in young adult rats we prelabeled the neurons by injection into the lumbar enlargement of a retrogradely transported fluorescent dye, Fluoro-Gold, and four days later transected the cord. We found no loss in cell number ten or 20 weeks after axotomy. The average size of the neurons in each case is slightly but significantly reduced. These findings unequivocally demonstrate that the somata of long tract neurons of the rubrospinal and corticospinal systems persist in an atrophic and presumably inactive state for at least 20 weeks, and raise the possibility that treatment of spinal cord injury may normalize cell activity and allow long tract regeneration.     

Corticospinal axons persist in cervical and high thoracic regions 10 weeks after a T-9 spinal cord transection

Ten weeks after complete spinal cord transection at T-9, there was a decrease in the volume of the rat corticospinal tract but no loss in the number of axons contained in the cervical (C-2) or high thoracic (T-1) corticospinal tract. The mean area of the myelinated axon profile decreased in spinal cord-transected rats, with fewer axons found in the largest size groups and more in the smaller size groups. The survival of corticospinal axons in the cervical and thoracic cord 10 weeks after cord transection at T-9 indicates that the corticospinal neurons survive at least 10 weeks after cord transection. The fate of axotomized neurons after longer survival times remains to be determined.     

From cortex to cord: motor circuit plasticity after spinal cord injury

Spinal cord injury is associated with chronic sensorimotor deficits due to the interruption of ascending and descending tracts between the brain and spinal cord. Functional recovery after anatomically complete spinal cord injury is limited due to the lack of long-distance axonal regeneration of severed fibers in the adult central nervous system. Most spinal cord injuries in humans, however, are anatomically incomplete.Although restorative treatment options for spinal cord injury remain currently limited, research from experimental models of spinal cord injury have revealed a tremendous capability for both spontaneous and treatment-induced plasticity of the corticospinal system that supports functional recovery. We review recent advances in the understanding of corticospinal circuit plasticity after spinal cord injury and concentrate mainly on the hindlimb motor cortex, its corticospinal projections, and the role of spinal mechanisms that support locomotor recovery. First, we discuss plasticity that occurs at the level of motor cortex and the reorganization of cortical movement representations. Next, we explore downstream plasticity in corticospinal projections. We then review the role of spinal mechanisms in locomotor recovery. We conclude with a perspective on harnessing neuroplasticity with therapeutic interventions to promote functional recovery.     

Further evidence that sparing of function after spinal cord transection in the neonatal rat is not due to axonal generation or regeneration

To test the possibility that a small number of axons may have crossed through the site of injury after midthoracic spinal cord transection in the newborn rat, corticospinal tract axons and ascending spinal axons were traced using autoradiography in chronic neonatal-operated rats. No evidence was found that axons of the labeled nerve tracts passed through or around the site of transection.     

Intercostal nerve implants transduced with an adenoviral vector encoding neurotrophin-3 promote regrowth of injured rat corticospinal tract fibers and improve hindlimb function

Following injury to central nervous tissues, damaged neurons are unable to regenerate their axons spontaneously. Implantation of peripheral nerves into the CNS, however, does result in axonal regeneration into these transplants and is one of the most powerful strategies to promote CNS regeneration. In the present study implantation of peripheral nerve bridges following dorsal hemisection is combined with ex vivo gene transfer with adenoviral vectors encoding neurotrophin-3 (Ad-NT-3) to examine whether this would stimulate regeneration of one of the long descending tracts of the spinal cord, the corticospinal tract (CST), into and beyond the peripheral nerve implant. We chose to use an adenoviral vector encoding NT-3 because CST axons are sensitive to this neurotrophin and Schwann cells in peripheral nerve implants do not express this neurotrophin. At 16 weeks postimplantation of Ad-NT-3-transduced intercostal nerves, approximately three- to fourfold more of the anterogradely traced corticospinal tract fibers had regrown their axons through gray matter below the lesion site when compared to control animals. Regrowth of CST fibers occurred over more than 8 mm distal to the lesion site. No regenerating CST fibers were, however, observed into the transduced peripheral implant. Animals with a peripheral nerve transduced with Ad-NT-3 also exhibited improved function of the hindlimbs when compared to control animals treated with an adenoviral vector encoding LacZ. Thus, transient overexpression of NT-3 in peripheral nerve tissue bridges is apparently sufficient to stimulate regrowth of CST fibers and to promote recovery of hindlimb function, but does not result in regeneration of CST fibers into such transplants. Taken together, combining an established neurotransplantation approach with viral vector-gene transfer promotes the regrowth of injured CST fibers through gray matter and improves the recovery of hindlimb function.     

Motor axonal regeneration after partial and complete spinal cord transection

We subjected rats to either partial midcervical or complete upper thoracic spinal cord transections and examined whether combinatorial treatments support motor axonal regeneration into and beyond the lesion. Subjects received cAMP injections into brainstem reticular motor neurons to stimulate their endogenous growth state, bone marrow stromal cell grafts in lesion sites to provide permissive matrices for axonal growth, and brain-derived neurotrophic factor gradients beyond the lesion to stimulate distal growth of motor axons. Findings were compared with several control groups. Combinatorial treatment generated motor axon regeneration beyond both C5 hemisection and T3 complete transection sites. Yet despite formation of synapses with neurons below the lesion, motor outcomes worsened after partial cervical lesions and spasticity worsened after complete transection. These findings highlight the complexity of spinal cord repair and the need for additional control and shaping of axonal regeneration.     

Recovery from spinal cord injury: a new transection model in the C57Bl/6 mouse

Spinal cord transections in mammalian animal models lead to loss of motor function. In this study, we show that functional recovery from complete transection of the adult mouse spinal cord can in fact occur without any intervention if dural injury along with displacement of the ends of the cut cord and fibroblastic infiltration is minimized. Underlying this function is the expression of GAP-43 in axonal growth cones, axonal extension and bridging of the injury site indicated by biocytin retrograde tracing and neuronal remodeling of both the white matter and the gray matter. Such studies suggest a new murine model for the study of spinal cord regeneration.     

Extensive spinal decussation and bilateral termination of cervical corticospinal projections in rhesus monkeys

To examine neuroanatomical mechanisms underlying fine motor control of the primate hand, adult rhesus monkeys underwent injections of biotinylated dextran amine (BDA) into the right motor cortex. Spinal axonal anatomy was examined using detailed serial‐section reconstruction and modified stereological quantification. Eighty‐seven percent of corticospinal tract (CST) axons decussated in the medullary pyramids and descended through the contralateral dorsolateral tract of the spinal cord. Eleven percent of CST axons projected through the dorsolateral CST ipsilateral to the hemisphere of origin, and 2% of axons projected through the ipsilateral ventromedial CST. Notably, corticospinal axons decussated extensively across the spinal cord midline. Remarkably, nearly 2‐fold more CST axons decussated across the cervical spinal cord midline (≈12,000 axons) than were labeled in all descending components of the CST (≈6,700 axons). These findings suggest that CST axons extend multiple segmental collaterals. Furthermore, serial‐section reconstructions revealed that individual axons descending in either the ipsilateral or contralateral dorsolateral CST can: 1) terminate in the gray matter ipsilateral to the hemisphere of origin; 2) terminate in the gray matter contralateral to the hemisphere of origin; or 3) branch in the spinal cord and terminate on both sides of the spinal cord. These results reveal a previously unappreciated degree of bilaterality and complexity of corticospinal projections in the primate spinal cord. This bilaterality is more extensive than that of the rat CST, and may resemble human CST organization. Thus, augmentation of sprouting of these extensive bilateral CST projections may provide a novel target for enhancing recovery after spinal cord injury. J. Comp. Neurol. 513:151–163, 2009. © 2009 Wiley‐Liss, Inc.