Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord

We have investigated the effects of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) on the intraspinal regeneration of anterogradely labeled axotomized ascending primary sensory fibers in the adult rat. These fibers were allowed to grow across a predegenerated peripheral nerve graft and back into the thoracic spinal cord. In control animals that had been infused with vehicle for two weeks into the dorsal column, 3 mm rostral to the nerve graft, essentially no fibers had extended from the nerve graft back into the spinal cord. The number of sensory fibers in the rostral end of the nerve graft was not significantly different between control and neurotrophin-infused animals. With infusion of NGF, 37+/-2% of the fibers at the rostral end of the graft had grown up to 0.5 mm into the dorsal column white matter, 30+/-2% up to 1 mm, 19+/-3% up to 2 mm and 8+/-2% up to 3 mm, i.e., the infusion site. With infusion of NT-3, sensory fiber outgrowth was similar to that seen with NGF, but with BDNF fewer fibers reached farther distances into the cord. Infusion of a mixture of all three neurotrophins did not increase the number of regenerating sensory fibers above that seen after infusion of the individual neurotrophins. These findings suggest that injured ascending sensory axons are responsive to all three neurotrophins and confirm our previous findings that neurotrophic factors can promote regeneration in the adult central nervous system.     

Functional repair after dorsal root rhizotomy using nerve conduits and neurotrophic molecules

Functional recovery after large excision of dorsal roots is absent because of both the limited regeneration capacity of the transected root, and the inability of regenerating sensory fibers to traverse the dorsal root entry zone. In this study, bioresorbable guidance conduits were used to repair 6-mm dorsal root lesion gaps in rats, while neurotrophin-encoding adenoviruses were used to elicit regeneration into the spinal cord. Polyester conduits with or without microfilament bundles were implanted between the transected ends of lumbar dorsal roots. Four weeks later, adenoviruses encoding NGF or GFP were injected into the spinal cord along the entry zone of the damaged dorsal roots. Eight weeks after injury, nerve regeneration was observed through both types of implants, but those containing microfilaments supported more robust regeneration of calcitonin gene-related peptide (CGRP)-positive nociceptive axons. NGF overexpression induced extensive regeneration of CGRP(+) fibers into the spinal cord from implants showing nerve repair. Animals that received conduits containing microfilaments combined with spinal NGF virus injections showed the greatest recovery in nociceptive function, approaching a normal level by 7-8 weeks. This recovery was reversed by recutting the dorsal root through the centre of the conduit, demonstrating that regeneration through the implant, and not sprouting of intact spinal fibers, restored sensory function. This study demonstrates that a combination of PNS guidance conduits and CNS neurotrophin therapy can promote regeneration and restoration of sensory function after severe dorsal root injury.     

Grafting of choroid plexus ependymal cells promotes the growth of regenerating axons in the dorsal funiculus of rat spinal cord: a preliminary report

Nerve regeneration in the central nervous system has been studied by grafting various tissues and cells. In the present study, we demonstrated that choroid plexus ependymal cells can promote nerve regeneration when grafted into spinal cord lesions. The choroid plexus was excised from the fourth ventricle of adult rats (Wistar), minced into small fragments, and grafted into the dorsal funiculus at the C2 level in adult rat spinal cord from the same strain. Electron microscopy and fluorescence histochemistry showed that ependymal cells of the grafted choroid plexus intimately interacted with growing axons, serving to support the massive growth of regenerating axons. CGRP-positive fibers closely interacted with grafted ependymal cells. HRP injection at the sciatic nerve showed that numerous HRP-labeled regenerating fibers from the fasciculus gracilis extended into the graft 7 days after grafting. This regenerating axons from the fasciculus gracilis was maintained for at least 10 months, with some axons elongating rostrally into the dorsal funiculus. Evoked potentials of long duration were recorded at a level ca. 5 mm rostral to the lesion in the rats 8 to 10 months after grafting. These findings indicate that choroid plexus ependymal cells have the ability to facilitate axonal growth in vivo, suggesting that they may be a promising candidate as graft for the promotion of nerve regeneration in the spinal cord.     

Sensory afferents regenerated into dorsal columns after spinal cord injury remain in a chronic pathophysiological state

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.     

Uptake of nerve growth factor along peripheral and spinal axons of primary sensory neurons

To investigate the distribution of nerve growth factor (NGF) receptors on peripheral and central axons, [125I]NGF was injected into the sciatic nerve or spinal cord of adult rats. Accumulation of [125I]NGF in lumbar dorsal root ganglia was monitored by gamma emission counting and radioautography. [125I]NGF, injected endoneurially in small quantities, was taken into sensory axons by a saturable process and was transported retrogradely to their cell bodies at a maximal rate of 2.5 to 7.5 mm/hr. Because very little [125I]NGF reached peripheral terminals, the results were interpreted to indicate that receptors for NGF are present on nonterminal segments of sensory axons. The specificity and high affinity of NGF uptake were illustrated by observations that negligible amounts of gamma activity accumulated in lumbar dorsal root ganglia after comparable intraneural injection of [125I] cytochrome C or [125I]oxidized NGF. Similar techniques were used to demonstrate avid internalization and retrograde transport of [125I]NGF by intraspinal axons arising from dorsal root ganglia. Following injection of [125I]NGF into lumbar or cervical regions of the spinal cord, neuronal perikarya were clearly labeled in radioautographs of lumbar dorsal root ganglia. Sites for NGF uptake on primary sensory neurons in the adult rat are not restricted to peripheral axon terminals but are extensively distributed along both peripheral and central axons. Receptors on axons provide a mechanism whereby NGF supplied by glia could influence neuronal maintenance or axonal regeneration.     

Brain-derived neurotrophic factor applied to the motor cortex promotes sprouting of corticospinal fibers but not regeneration into a peripheral nerve transplant

Previous experiments from our laboratory have shown that application of brain-derived neurotrophic factor (BDNF) to the red nucleus or the motor cortex stimulates an increase in the expression of regeneration-associated genes in rubrospinal and corticospinal neurons. Furthermore, we have previously shown that BDNF application stimulates regeneration of rubrospinal axons into a peripheral graft after a thoracic injury. The current study investigates whether application of BDNF to the motor cortex will facilitate regeneration of corticospinal neurons into a peripheral nerve graft placed into the thoracic spinal cord. In adult Sprague Dawley rats, the dorsal columns and the corticospinal tract between T9 and T10 were ablated by suction, and a 5-mm-long segment of predegenerated tibial nerve was autograft implanted into the lesion. With an osmotic pump, BDNF was infused directly into the parenchyma of the motor cortex for 14 days. Growth of the corticospinal tract into the nerve graft was then evaluated by transport of an anterograde tracer. Anterogradely labeled corticospinal fibers were not observed in the peripheral nerve graft in animals treated with saline or BDNF. Serotinergic and noradrenergic fibers, as well as peripheral sensory afferents, were observed to penetrate the graft, indicating the viability of the peripheral nerve graft as a permissive growth substrate for these specific fiber types. Although treatment of the corticospinal fibers with BDNF failed to produce regeneration into the graft, there was a distinct increase in the number of axonal sprouts rostral to the injury site. This indicates that treatment of corticospinal neurons with neurotrophins, e.g., BDNF, can be used to enhance sprouting of corticospinal axons within the spinal cord. Whether such sprouting leads to functional recovery after spinal cord injury is currently under investigation.     

Central projections of primary sensory afferents to the spinal dorsal horn in the long-tailed stingray, Himantura fai

The central projections of primary sensory afferents innervating the caudal region of the pectoral fin of the long-tailed stingray (Himantura fai) were labeled by applying the lipophilic carbocyanine dye DiI to the dorsal roots in fixed tissue. These observations were complemented by examination of hemotoxylin and eosin-stained paraffin sections of the dorsal root entry zone, and transmission electron microscopy of the dorsal horn. Transverse sections of the sensory nerve and dorsal root revealed two distinct myelinated axon sizes in the sensory nerve. Although the thick and thin axons do not appear to group together in the sensory nerves and dorsal root, they segregate into a dorsally directed bundle of thin fibers and a more horizontally directed bundle of thick fibers soon after entering the spinal cord. In DiI-labeled horizontal sections, fibers were observed to enter the spinal cord and diverge into rostrally and caudally directed trajectories. Branching varicose axons could be traced in the dorsal horn gray matter in the segment of entry and about half of the adjacent rostral and caudal segments. In transverse and sagittal sections, DiI-labeled afferents were seen to innervate the superficial and, to a lesser extent, deeper laminae of the dorsal horn, but not the ventral horn. Electron microscopy of unlabeled dorsal horn sections revealed a variety of synaptic morphologies including large presynaptic elements (some containing dense-core vesicles) making synaptic contacts with multiple processes in a glomerular arrangement; in this respect, the synaptic ultrastructure is broadly similar to that seen in the dorsal horn of rodents and other mammals.     

Anatomical and physiological assessments of peripheral nerve grafts in the dorsal columns of the spinal cord

Portions of 1 cm length of the sensory radial nerve from the cat forelimb were used to replace an excised portion of the dorsal columns in the upper lumbar spinal cord. Observations were made on the clinical recovery of the animals, and cine recordings were made of their ability to traverse a horizontal ladder 5 months after the grafting procedure. Evoked sensory potential studies performed 6 months after grafting showed that an impulse arising from a stimulus applied to the sciatic nerve could be recorded in the spinal cord caudal to the graft, in the graft and in the spinal cord rostral to the graft in 5 out of 8 animals. Tracing of nerve connections with injection of horseradish peroxidase into the grafts resulted in labelling of nerve cell bodies in dorsal root ganglia and the grey matter of the lumbar spinal cord up to a distance of 10 mm away from the graft. These results confirm that peripheral nerve grafts can provide a satisfactory environment for the regrowth of ascending fibres in the dorsal columns of the spinal cord. However, there is as yet no evidence that the regenerated fibres succeed in forming useful synaptic connections with other nerve cell bodies.     

Reformation of specific synaptic connections by regenerating sensory axons in the spinal cord of the bullfrog

The regrowth of sensory axons into the spinal cord of juvenile bullfrogs was studied after disruption of these fibers in the dorsal root. Within 9 d after the root had been frozen, regenerating sensory axons had reached the spinal cord, as revealed by labeling with horseradish peroxidase. Growth into the spinal cord, however, was much slower. Even several months after denervation, very few fibers had reestablished any of their normal longitudinal projections within the dorsal funiculus. Eventually, however, sensory axons grew across the region and into the dorsal horn. Intracellular recordings from motoneurons revealed that these axons made functional reconnections with spinal neurons. Muscle sensory axons established direct, monosynaptic inputs to motoneurons, whereas cutaneous fibers innervated these neurons polysynaptically. Moreover, sensory afferents from a particular muscle distinguished among different classes of motoneurons, just as in normal frogs. Thus, specific synaptic pathways can be reestablished by regenerating sensory axons if they can reach their appropriate target region within the spinal cord.     

Dorsal column sensory axons lack TrkC and are not rescued by local neurotrophin-3 infusions following spinal cord contusion in adult rats

By reducing the progressive degeneration and disconnection of axons following spinal cord injury the functional outcome should improve. After direct transection of dorsal column sensory axons, neurotrophin-3 (NT-3) treatment can reduce degeneration and promote regeneration of the proximal stumps. Here, we tested in adult rats whether NT-3 infusion at the site of a moderate T9 spinal cord contusion would rescue sensory connections to the gracile nucleus in the medulla. Sensory projections were anterogradely traced bilaterally with injections of cholera toxin B (CTB) into the sciatic nerve 3 days before analysis. Seven days after the contusion plus intrathecal (subarachnoid) vehicle infusion as a control, the CTB-positive innervation of the gracile nucleus was reduced to ∼ 25% of sham-operated rats. Intrathecal infusion of 10 μg/day of NT-3 did not affect this reduced innervation. To ensure good tissue penetration and high concentrations of NT-3 early after the injury, other rats received intraparenchymal infusions of vehicle or NT-3 near the injury site starting 2 days before until 7 days after the injury. This NT-3 treatment also did not affect the reduced innervation. This suggests that local NT-3 treatments cannot protect sensory axons from secondary degeneration after a contusive spinal cord injury. These results are likely because TrkC is not present in axons of the dorsal columns or gracile nucleus, or in other dorsal column cell types, even after the contusion. Together with published results, our data suggest that NT-3 is a peripherally - but not centrally - derived neurotrophic factor for sensory neurons.     

Demonstration of the potential for chronically injured neurons to regenerate axons into intraspinal peripheral nerve grafts

Experiments were carried out to determine if neurons damaged by injury to the spinal cord retain the ability to regenerate their axonal process for a prolonged period of time after the initial response to injury and if peripheral nerve (PN) grafts could support the regrowth of these processes. True blue (TB) was injected into one side of the adult rat lumbar spinal cord to label neurons with axons coursing through this region. Seven days later spinal cord tissue surrounding the injection sites was removed by aspiration to create a hemisection cavity 3-4 mm in length. Four weeks later scar tissue lining the lesion cavity was removed prior to grafting 1 cm segments of autologous tibial nerve to the rostral and the caudal surfaces of the cavity wall. The distal end of each graft was ligated and left unapposed to spinal cord tissue. Four weeks later the distal end of each PN graft was exposed to nuclear yellow (NY) to retrogradely label neurons that had grown an axon into the graft. Neurons containing both TB and NY were deemed capable of axonal regeneration while in a chronically injured state. Double-labeled (TB/NY) neurons were found in the ipsilateral spinal cord in laminae IV through X, excluding IX, and in Laminae VI and VII contralateral to the lesion. Most neurons were located within 10 mm of the lesion, with the majority caudal to the lesion. Nearly 50% (range 24-74%) of lumbar dorsal root ganglion neurons containing TB also were labeled with NY.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of adult dorsal root axons into transplants of embryonic spinal cord

Transplants of the embryonic rat spinal cord survive and differentiate in the spinal cords of adult and newborn host rats. Very little is known about the extent to which these homotopic transplants can provide an environment for regeneration of adult host axons that normally terminate in the spinal cord. We have used horseradish peroxidase injury filling and transganglionic transport methods to determine whether transected dorsal roots regenerate into fetal spinal cord tissue grafted into the spinal cords of adult rats. Additional transplants were examined for the presence of calcitonin gene-related peptide-like immunoreactivity, which in the normal dorsal horn is derived exclusively from primary afferent axons. Host animals had one side of the L4-5 spinal cord resected and replaced by a transplant of E14 or E15 spinal cord. Adjacent dorsal roots were sectioned and juxtaposed to the graft. The dorsal roots and their projections into the transplants were then labeled 2-9 months later. The tracing methods that used transport or diffusion of horseradish peroxidase demonstrated that severed host dorsal root axons had regenerated and grown into the transplants. In addition, some donor and host neurons had extended their axons into the periphery to at least the midthigh level as indicated by retrograde labeling following application of tracer to the sciatic nerve. Primary afferent axons immunoreactive for calcitonin gene-related peptide were among those that regenerated into transplants, and the projections shown by this immunocytochemical method exceeded those demonstrated by the horseradish peroxidase tracing techniques. Growth of the host dorsal roots into transplants indicates that fetal spinal cord tissue permits regeneration of adult axotomized neurons that would otherwise be aborted at the dorsal root/spinal cord junction. This transplantation model should therefore prove useful in studying the enhancement and specificity of the regrowth of axons that normally terminate in the spinal cord.     

Central distribution of cervical primary afferents in the rat, with emphasis on proprioceptive projections to vestibular, perihypoglossal, and upper thoracic spinal nuclei

The projections of primary afferents from rostral cervical segments to the brainstem and the spinal cord of the rat were investigated by using anterograde and transganglionic transport techniques. Projections from whole spinal ganglia were compared with those from single nerves carrying only exteroceptive or proprioceptive fibers. Injections of horseradish peroxidase (HRP) or wheat germ agglutinin-horseradish peroxidase conjugate (WGA-HRP) were performed into dorsal root ganglia C2, C3, and C4. Free HRP was applied to the cut dorsal rami C2 and C3, greater occipital nerve, sternomastoid nerve, and to the C1/2 anastomosis, which contains afferents from suboccipital muscles and the atlanto-occipital joint. WGA-HRP injections into ganglia C7 and L5 were performed for comparative purposes. Injections of WGA-HRP or free HRP into rostral cervical dorsal root ganglia and HRP application to C2 and C3 dorsal rami produced labeling in dorsal and ventral horns at the level of entrance, the central cervical nucleus, and in external and main cuneate nuclei. From axons ascending to pontine and descending to upper thoracic spinal levels, medial collaterals were distributed to medial and descending vestibular, perihypoglossal and solitary nuclei, and the intermediate zone and Clarke's nucleus dorsalis in the spinal cord. Lateral collaterals projected mainly to the trigeminal subnucleus interpolaris and to lateral spinal laminae IV and V. Results from HRP application to single peripheral nerves indicated that medial collaterals were almost exclusively proprioceptive, whereas lateral collaterals were largely exteroceptive with a contribution from suboccipital proprioceptive fibers. WGA-HRP injections into dorsal root ganglia C7 and L5 failed to produce significant labeling within vestibular and periphypoglossal nuclei, although they demonstrated classical projection sites within the brainstem and spinal cord. The consistent collateralisation pattern of rostral cervical afferents along their whole rostrocaudal course enables them to contact a variety of precerebellar, vestibulospinal, and preoculomotor neurons. These connections reflect the well-known significance of proprioceptive neck afferents for the control of posture, head position, and eye movements.     

Transganglionic regulation of central terminals of dorsal root ganglion cells by nerve growth factor (NGF)

Blockade of axonal transport or transection of the rat sciatic nerve results in transganglionic degenerative atrophy (TDA) of nerve terminals containing fluoride-resistant acid phosphatase (FRAP) in the Rolando substance of the spinal cord. Application of vinblastine (9 micrograms) in a cuff around the sciatic nerve of adult rats blocked the retrograde transport of [125I]NGF in sensory fibers; this amount of vinblastine is identical to the threshold amount that induces TDA. Conversely, application of NGF to the proximal stump of the transected sciatic nerve prevented or delayed the occurrence of TDA as reflected by the maintenance of FRAP in the upper dorsal horn, that otherwise would inevitably disappear following the peripheral nerve lesion. These results suggest that endogenous NGF transported retrogradely in peripheral sensory fibers of the adult rat under normal conditions may be responsible for the regulation of the structural and functional integrity of the central terminals of these FRAP-containing primary sensory neurons and that TDA may be the consequence of the failure of NGF to reach the perikarya of these neurons.     

Axonal regeneration in dorsal spinal roots is accelerated by peripheral axonal transection

Regeneration of crushed axons in rat dorsal spinal roots was measured to investigate the transganglionic influence of an additional peripheral axonal injury. The right sciatic nerve was cut at the hip and the left sciatic nerve was left intact. One week later, both fifth lumbar dorsal roots were crushed and subsequently, regeneration in the two roots was assessed with one of two anatomical techniques. By anterograde tracing with horseradish peroxidase, the maximal rate of axonal regrowth towards the spinal cord was estimated to be 1.0 mm/day on the left and 3.1 mm/day on the right. Eighteen days after crush injury, new, thinly myelinated fibers in the root between crush site and spinal cord were 5-10 times more abundant ipsilateral to the sciatic nerve transection. The central axons of primary sensory neurons regenerate more quickly if the corresponding peripheral axons are also injured.     

Contribution of degeneration of motor and sensory fibers to pain behavior and the changes in neurotrophic factors in rat dorsal root ganglion

To elucidate the role of the degeneration of motor and sensory fibers in neuropathic pain, we examined the pain-related behaviors and the changes of brain-derived neurotrophic factor (BDNF) in the L4/5 dorsal root ganglion (DRG) and the spinal cord after L5 ventral rhizotomy. L5 ventral rhizotomy, producing a selective lesion of motor fibers, produced thermal hyperalgesia and increased BDNF expression in tyrosine kinase A-containing small- and medium-sized neurons in the L5 DRG and their central terminations within the spinal cord, but not in the L4 DRG. Furthermore, L5 ventral rhizotomy up-regulated nerve growth factor (NGF) protein in small to medium diameter neurons in the L5 DRG and also in ED-1-positive cells in the L5 spinal nerve, suggesting that NGF synthesized in the degenerative fibers is transported to the L5 DRG and increases BDNF synthesis. On the other hand, L5 ganglionectomy, producing a selective lesion of sensory fibers, produced heat hypersensitivity and an increase in BDNF and NGF in the L4 DRG. These data indicate that degeneration of L5 sensory fibers distal to the DRG, but not motor fibers, might influence the neighboring L4 nerve fibers and induce neurotrophin changes in the L4 DRG. We suggest that these changes of neurotrophins in the intact primary afferents of neighboring nerves may be one of many complex mechanisms, which can explain the abnormal pain behaviors after nerve injury. The ventral rhizotomy and ganglionectomy models may be useful to investigate the pathophysiological mechanisms of neuropathic pain after Wallerian degeneration in motor or sensory or mixed nerve.     

Methylprednisolone Administration Improves Axonal Regeneration into Schwann Cell Grafts in Transected Adult Rat Thoracic Spinal Cord

Schwann cell (SC) grafts support the regeneration of axons of numerous spinal cord neurons when placed into transected adult rat midthoracic spinal cord. Clinically, methylprednisolone (MP) has been shown to be neuroprotective if administered within 8 h after spinal cord injury. We investigated whether axonal regrowth into SC grafts is enhanced when MP is administered at the time of spinal cord transection and SC implantation. SCs from adult rat sciatic nerves were purified in culture, suspended in Matrigel, and drawn into semipermeable polymeric channels. MP (30 mg/kg) or vehicle (control) was administered intravenously at 5 min, 2 h, and 4 h to adult Fischer rats after transection at T8 and removal of the next three caudal segments. The rostral cord stump was inserted 1 mm into the channel; the distal end of the channel was capped. Thirty to forty-five days later, the SC/MP group showed large tissue cables in the channels and host cord tissue retained in the rostral end of the channels. Significantly more myelinated axons (1159 ± 308) were present at the 5-mm level in SC/MP grafts (n = 6) than in SC/vehicle cables (355 ± 108,n = 5). More unmyelinated than myelinated axons (approximately 4:1,n = 3) were resolved in the cables by electron microscopy. In the SC/MP group, unlike the SC/vehicle group, serotonergic and noradrenergic fibers were detected immunocytochemically 2.5 and 2.0 mm, respectively, into the graft; astrocytes were also identified at similar distances from the interface. Fast Blue retrograde tracing (SC/MP,n = 4; SC/vehicle,n = 3) showed that more spinal cord neurons (1116 ± 113 vs 284 ± 88, respectively) and spinal cord neurons more distant from the graft (C8 vs C5) responded by extending axons into the graft in the presence of MP. Also, very significantly, supraspinal brain stem neurons extended axons into the graft only when MP was administered (mean 46 vs 0,n = 3). These results indicate that MP improves axonal regeneration from both spinal cord and brain stem neurons into thoracic SC grafts, possibly by reducing secondary host tissue loss adjacent to the graft.     

Peripheral nerve autografts to the rat spinal cord: Studies with axonal tracing methods

In young adult female rats, autologous sciatic nerve segments were transplanted to the thoracic region of the spinal cord. The grafts became well innervated but led to no obvious functional improvement. The origin and termination of axons in the grafts was studied by retrograde neuronal labeling with horseradish peroxidase (HRP) and radioautographic axonal tracing. Studies with HRP indicated that some axons in the grafts originated from intrinsic CNS neurons with their cell bodies in nearby segments of the spinal cord and that others arose from dorsal root ganglia at the level of the grafts and at least 7 segments distal to them. After tritiated amino acids were injected into lumbar dorsal root ganglia, labeled axons could be followed into the grafts but not into the rostral spinal cord stumps. Together with other experimental observations, these results demonstrate a correlation between success or failure of elongation of dorsal root fibers and peripheral or central ensheathment at the axonal tip. The corticospinal tract was studied both with radioautography and retrograde axonal transport of HRP but no extension of its axons into peripheral nerve grafts was detected under these experimental conditions. The findings implicate both neuroglial and axonal factors in the feeble regenerative response usually seen after injury to the spinal cord.     

An in vivo model to quantify motor and sensory peripheral nerve regeneration using bioresorbable nerve guide tubes

An in vivo preparation is presented to study the rate and time course of motor and sensory axonal regeneration. The cut ends of a transected sciatic nerve were inserted into each end of a 5-6 mm non-toxic and bioresorbable nerve guide tube to create a 4 mm nerve gap in adult mice. Subsequently, cell bodies in the ventral spinal cord and L3-L5 dorsal root ganglia that had regenerated axons across the gap were retrogradely labeled with horseradish peroxidase (HRP). The HRP was applied 3 mm distal to the nerve guide and was accessible only to axons that had regenerated through the nerve guide. Labeled cells were counted in 40 micron serial sections at 2, 4 and 6 weeks after initial nerve transection. The results indicate a significant increase in the number of labeled motor and sensory cell bodies over time. By 6 weeks after transection, approximately two thirds as many ventral horn motor cells and one third as many dorsal root ganglion sensory cells were labeled as in control non-transected animals. These data serve as a baseline to compare differential effects of additives to the nerve guide lumen in terms of sensory and motor neuron response.     

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.