Axonal regeneration by chronically injured supraspinal neurons can be enhanced by exposure to insulin-like growth factor, basic fibroblast growth factor or transforming growth factor beta

To test whether known growth factors could promote the regenerative reponse of chronically injured neurons, we exposed the injured adult rat spinal cord to insulin-like growth factor 1 (IGF-1), basic fibroblast growth factor (bFGF) or transforming growth factor beta 1 + 2 (TGFβs) 1 month after creation of a hemisection lesion. At 1 week later an autologous peripheral nerve graft was apposed to the rostral cavity wall and 1 month later Nuclear Yellow (NY) was used to retrogradely label neurons that had grown an axon into the graft. Neurons capable of axonal regeneration after a long term (5 weeks) injury were double labeled with True Blue (TB, provided at the time of hemisection lesion) and NY. Exposure to any of the three growth factors, compared to a PBS-treated control, resulted in a significant increase in the total number of regenerating supraspinal neurons, with the greatest increase after treatment with TGFβs. Treatment with TGFβs or bFGF led to a significant increase in the number of regenerating neurons in 6 out of 7 major regions (excluding the motor cortex) contributing to descending spinal pathways. Treatment with IGF-1 promoted significant regeneration only by reticular formation neurons. These results indicate that exposure to specific growth factors can enhance axonal regeneration by chronically injured neurons, thus overcoming one significant challenge to the repair of long standing structural damage to the spinal cord. ? 1996 Elsevier Science Ireland Ltd. All rights reserved.     

Peripheral nerve grafts after cervical spinal cord injury in adult cats

Peripheral nerve grafts (PNG) into the rat spinal cord support axon regeneration after acute or chronic injury, with synaptic reconnection across the lesion site and some level of behavioral recovery. Here, we grafted a peripheral nerve into the injured spinal cord of cats as a preclinical treatment approach to promote regeneration for eventual translational use. Adult female cats received a partial hemisection lesion at the cervical level (C7) and immediate apposition of an autologous tibial nerve segment to the lesion site. Five weeks later, a dorsal quadrant lesion was performed caudally (T1), the lesion site treated with chondroitinase ABC 2 days later to digest growth inhibiting extracellular matrix molecules, and the distal end of the PNG apposed to the injury site. After 4-20 weeks, the grafts survived in 10/12 animals with several thousand myelinated axons present in each graft. The distal end of 9/10 grafts was well apposed to the spinal cord and numerous axons extended beyond the lesion site. Intraspinal stimulation evoked compound action potentials in the graft with an appropriate latency illustrating normal axonal conduction of the regenerated axons. Although stimulation of the PNG failed to elicit responses in the spinal cord distal to the lesion site, the presence of c-Fos immunoreactive neurons close to the distal apposition site indicates that regenerated axons formed functional synapses with host neurons. This study demonstrates the successful application of a nerve grafting approach to promote regeneration after spinal cord injury in a non-rodent, large animal model.     

Chondroitinase ABC promotes axonal regeneration of Clarke's neurons after spinal cord injury

We examined whether enzymatic digestion of chondroitin sulfate (CS) promoted the axonal regeneration of neurons in Clarke's nucleus (CN) into a peripheral nerve (PN) graft following injury of the spinal cord. After hemisection at T11, a segment of PN graft was implanted at the lesion site. Either vehicle, brain-derived neurotrophic factor (BDNF) or chondroitinase ABC was applied at the implantation site. The postoperative survival period was 4 weeks. Treatment with vehicle or BDNF did not promote the axonal regeneration of CN neurons into the PN graft. Application of 2.5 unit/ml chondroitinase ABC resulted in a significant increase (12.8%) in the number of regenerated CN neurons. Double labeling with Fluoro-Gold and NADPH-diaphorase histochemistry showed that the regenerated CN neurons did not express nitric oxide synthase (NOS). Our results suggest that CS is inhibitory to the regeneration of CN neurons following injury of the spinal cord.     

Neurotrophins BDNF and NT-3 promote axonal re-entry into the distal host spinal cord through Schwann cell-seeded mini-channels

To promote axonal regeneration in the injured adult spinal cord, a two-phase repair strategy was employed to (i) bridge a spinal cord hemilesion cavity with a grafted Schwann cell (SC)-seeded mini-channel, and (ii) promote axonal re-entry into the distal cord by infusing two neurotrophins, BDNF and/or NT-3, directly into the distal cord parenchyma. Here we report that infusion of two neurotrophins, delivered alone or in combination, effectively promotes axonal outgrowth from SC-seeded mini-channels into the distal host spinal cord. When an anterogradely transported marker, PHA-L or BDA, was injected into the spinal cord 3 mm rostral to the graft, a large number of axons was observed to regenerate from the SC graft into the distal cord in neurotrophin-treated groups. A subpopulation of these axons was found to grow up to 6 mm within the distal spinal cord. These axons, which were confined mainly within the grey matter, arborized and formed structures which resemble terminal boutons. In channels containing no SCs, the infusion of neurotrophins did not promote axonal ingrowth from the proximal cord stump. In cases which received SC grafts but no neurotrophin infusion, axonal re-entry into the distal cord was limited. Thus, the present study demonstrates that regenerating axons not only cross a lesion site when a permissive cellular bridge is provided but also penetrate into the distal host spinal cord and elongate for a distance of several cord segments after the infusion of two neurotrophins. The latter event is prerequisite for establishment of appropriate connections between regenerating axons and target neurons and thus, functional recovery.     

Growth associated protein 43 and neurofilament immunolabeling in the transected lumbar spinal cord of lizard indicates limited axonal regeneration

Previous cytological studies on the transected lumbar spinal cord of lizards have shown the presence of differentiating glial cells,few neurons and axons in the bridge region between the proximal and distal stumps of the spinal cord in some cases.A limited number of axons(20-50)can cross the bridge and re-connect the caudal stump of the spinal cord with small neurons located in the rostral stump of the spinal cord.This axonal regeneration appears to be related to the recovery of hind-limb movements after initial paralysis.The present study extends previous studies and shows that after transection of the lumbar spinal cord in lizards,a glial-connective tissue bridge that reconnects the rostral and caudal stumps of the interrupted spinal cord is formed at 11-34 days post-injury.Following an initial paralysis some recovery of hindlimb movements occurs within 1-3 months post-injury.Immunohistochemical and ultrastructural analysis for a growth associated protein 43(GAP-43)of 48-50 k Da shows that sparse GAP-43 positive axons are present in the proximal stump of the spinal cord but their number decreased in the bridge at 11-34 days post-transection.Few immunolabeled axons with a neurofilament protein of 200-220 k Da were seen in the bridge at 11-22 days post-transection but their number increased at 34 days and 3 months post-amputation in lizards that have recovered some hindlimb movements.Numerous neurons in the rostral and caudal stumps of the spinal cord were also labeled for GAP43,a cytoplasmic protein that is trans-located into their axonal growth cones.This indicates that GAP-43 biosynthesis is related to axonal regeneration and sprouting from neurons that were damaged by the transection.Taken together,previous studies that utilized tract-tracing technique to label the present observations confirm that a limited axonal re-connection of the transected spinal cord occurs 1-3 months post-injury in lizards.The few regenerating-sprouting axons within the bridge reconnect the caudal with the rostral stumps of the spinal cord,and likely contribute to activate the neural circuits that sustain the limited but important recovery of hind-limb movements after initial paralysis.The surgical procedures utilized in the study followed the regulations on animal care and experimental procedures under the Italian Guidelines(art.5,DL 116/92).     

Intraspinal microinjection of chondroitinase ABC following injury promotes axonal regeneration out of a peripheral nerve graft bridge

Chondroitin sulfate proteoglycans (CSPG) within the glial scar formed after central nervous system (CNS) injury are thought to play a crucial role in regenerative failure. We previously showed that delivery of the CSPG-digesting enzyme chondroitinase ABC (ChABC) via an osmotic minipump allowed axonal regeneration and functional recovery in a peripheral nerve graft (PNG)-bridging model. In this study, we sought to overcome the technical limitations associated with minipumps by microinjecting ChABC directly into the distal lesion site in the PN bridging model. Microinjection of ChABC immediately rostral and caudal to an injury site resulted in extensive CSPG digestion. We also demonstrate that this delivery technique is relatively atraumatic and does not result in a noticeable inflammatory response. Importantly, microinjections of ChABC into the lesion site permitted more regenerating axons to exit a PNG and reenter spinal cord tissue than saline injections. These results are similar to our previous findings when ChABC was delivered via a minipump and suggest that microinjecting ChABC is an effective method of delivering the potentially therapeutic enzyme directly to an injury site.     

Differential effects of neurotrophins on neuronal survival and axonal regeneration after spinal cord injury in adult rats

Spinal cord injury (SCI) induces retrograde cell death in descending pathways, which can be prevented by long-term intrathecal infusion of neurotrophins (Novikova et al. [2000] Eur J Neurosci 12:776-780). The present study investigates whether the same treatment also leads to improved regeneration of the injured tracts. After cervical SCI in adult rats, a peripheral nerve graft was attached to the rostral wall of the lesion cavity. The animals were treated by local application into the cavity of Gelfoam soaked in (1) phosphate buffered saline (untreated controls) or (2) a mixture of the neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) (local treatment), or by intrathecal infusion of BDNF + NT-3 for (3) 2 weeks (short-term treatment) or (4) 5-8 weeks (long-term treatment). Despite a very strong survival effect, long-term treatment failed to stimulate ingrowth of descending tracts into the nerve graft. In comparison with untreated controls, the latter treatment also caused 35% reduction in axonal sprouting of descending pathways rostral to the lesion site and 72% reduction in the number of spinal cord neurons extending axons into the nerve graft. Local and short-term treatments neither prevented retrograde cell death nor enhanced regeneration of descending tracts, but induced robust regeneration of spinal cord neurons into the nerve graft. These results indicate that the signal pathways promoting neuronal survival and axonal regeneration, respectively, in descending tracts after SCI respond differently to neurotrophic stimuli and that efficient rescue of axotomized tract neurons is not a sufficient prerequisite for regeneration.     

Peripheral nerve graft and neurotrophic factors enhance neuronal survival and expression of nitric oxide synthase in Clarke's nucleus after hemisection of the spinal cord in adult rat.

The present study examined the effects of peripheral nerve (PN) graft and neurotrophic factors on the expression of nitric oxide synthase (NOS) and the survival of Clarke's nucleus (CN) neurons at the first lumbar spinal segment (L1) 15 days after hemisection of the spinal cord at T11. Normal intact CN neurons did not express NOS. Forty-one percent of the ipsilateral CN neurons survived after hemisection at T11, and 48% of the surviving neurons expressed NOS. Transplantation of PN graft at the lesion site promoted the survival of CN neurons to 71% and increased the expression of NOS to 70%. Continuous infusion of brain-derived neurotrophic factor, ciliary neurotrophic factor, and neurotrophic-3, but not glial cell-derived neurotrophic factor, at the lesion site enhanced the survival of CN neurons to about 65%. Among the surviving neurons about 70% were NOS-positive. These results indicated that transplantation of autologous PN graft or continuous infusion of neurotrophic factors could enhance the survival of axotomized CN neurons. In addition, the survival-promoting function of the neurotrophic agents was coincided with the upregulation of the expression of NOS. However, whether the upregulation of NOS expression in injured CN neurons is related to the rescue function or is a side effect of the neurotrophic factors is not clear and needed further investigation.     

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.     

Intraspinal transplantation of hNT neurons in the lesioned adult rat spinal cord

BACKGROUND: The role of neural transplantation as a restorative strategy for spinal cord injury continues to be intensely investigated. Ideally, the tissue source for transplantation must be readily available, free of disease and able to survive and mature following implantation into the adverse environment created by the injury. We have studied the use of a commercially available cell line of cultured human neurons (hNT neurons) as a tissue source for neural transplantation in spinal cord injury. METHODS: Following a left lateral thoracic hemisection, 54 immunosuppressed, female Wistar rats were randomly allocated into different treatment groups; hemisection only or hemisection and hNT cell transplantation (via a bridge, double or triple graft). Grafting occurred three days after spinal cord injury. After thirteen weeks the animals were sacrificed and tissue sections were stained with human neuron specific enolase and human specific neural cell adhesion molecule. RESULTS: Immunohistochemical evidence of graft survival was displayed in 66.7% of the surviving, grafted animals. Fibre outgrowth, greatest in the bridge and triple grafts, was observed in both rostral and caudal directions essentially bridging the lesion. Double grafts were smaller, displaying less fibre outgrowth, which did not cross the lesion. Long fibre outgrowth was evident up to 2 cm from the graft as assessed by tracing and immunohistochemical studies. CONCLUSIONS: Bridge and triple grafts displayed greater growth and enabled the hNT graft to essentially bridge the lesion. This suggests that hNT neurons have the potential to structurally reconnect the proximal and distal spinal cord across the region of injury.     

Collateral branching in axonal projections to spinal cord from paramedian reticular nucleus neurons

Experiments were done in cats to identify neurons in the paramedian reticular nucleus (PRN) sending collateral axons to the region of the intermediolateral nucleus (IML) at different levels of the thoracic cord by using lectin-conjugated horseradish peroxidase (HRP) and double-labeling fluorochrome histochemistry to retrogradely label PRN neurons. Injections of Fast blue (FB) into the spinal cord at the T2 level centered in the region of the IML were coupled with injections of Nuclear yellow (NY) into the ipsilateral cord at either the T4 or T7 levels centered in the region of the IML. Neurons in the PRN retrogradely labeled after diffusion of HRP into the region of the IML at the T2 level were observed throughout the rostrocaudal extent of the ventral PRN. In addition, a few labeled neurons were noted in the ventral portion of the dorsal PRN. About 40% of the neurons in the PRN which were labeled with FB after an injection at the T2 level were also labeled with NY injected into the cord in further caudal segments. These data suggest that the PRN may exert its influence on the cardiovascular system partly through collateral axonal branches to widely separated populations of sympathetic preganglionic neurons in different spinal segmental levels.     

Both regenerating and late-developing pathways contribute to transplant-induced anatomical plasticity after spinal cord lesions at birth.

Fetal spinal cord transplants prevent the retrograde cell death of immature axotomized central nervous system (CNS) neurons and provide a terrain which supports axonal elongation in the injured immature spinal cord. The current experiments were designed to determine whether the axons which grow across the site of the neonatal lesion and transplant are derived from axotomized neurons and are therefore regenerating or whether the axons which grow across the transplant are late-growing axons that have not been axotomized directly. We have used an experimental paradigm of midthoracic spinal cord lesion plus transplant at birth and temporally spaced retrograde tracing with the fluorescent tracers fast blue (FB) and diamidino yellow (DY) to address this issue. Fast blue was placed into the site of a spinal cord hemisection in rat pups less than 48 h old. After 3-6 h to allow uptake and transport of the tracer, the source of fast blue was removed by aspiration and the lesion was enlarged to an "over-hemisection." A transplant of Embryonic Day 14 fetal spinal cord tissue was placed into the lesion site. The animals survived 3-6 weeks prior to the injection of the second tracer (DY) bilaterally into the host spinal cord caudal to the lesion plus transplant. Neurons with late-developing axons would not be exposed to the first dye (FB), but could only be exposed to the second tracer, diamidino yellow. Thus, neurons with a diamidino yellow-labeled nucleus are interpreted as "late-developing" neurons. Neurons axotomized by midthoracic spinal cord lesion at birth could be exposed to the first tracer, fast blue. If after axotomy they regrew caudal to the transplant, they could be labeled by the second tracer as well. We interpret these double-labeled neurons as regenerating neurons. If neurons labeled with fast blue and axotomized by the spinal cord hemisection either failed to regenerate or grew into the transplant but not caudal to it, they would be labeled only by the first dye. We have examined the pattern and distribution of single (FB or DY)- and double (FB + DY)-labeled neurons in the sensorimotor cortex, red nucleus, locus coeruleus, and raphe nuclei. The sensorimotor cortex contains only DY-labeled neurons. The red nucleus contains both FB- and FB + DY-labeled neurons.(ABSTRACT TRUNCATED AT 400 WORDS)     

Grafts of BDNF-producing fibroblasts rescue axotomized rubrospinal neurons and prevent their atrophy

We have reported that intraspinal transplants of fibroblasts genetically modified to express brain-derived neurotrophic factor (BDNF) promote rubrospinal axon regeneration and functional recovery following subtotal cervical hemisection that completely ablated the rubrospinal tract. In the present study we examined whether these transplants could prevent cell loss and/or atrophy of axotomized Red nucleus neurons. Adult rats received a subtotal spinal cord cervical hemisection followed by a graft of unmodified fibroblasts or fibroblasts producing BDNF into the lesion cavity. One or 2 months later, fluorogold was injected several segments caudal to the lesion-transplant site to retrogradely label those Red nucleus neurons whose axons have regenerated. Unmodified fibroblasts failed to protect against either cell loss or atrophy. Neuron counts and soma-size measurements in Nissl-stained preparations showed a 45% loss of recognizable neurons and 40% atrophy of the surviving neurons in the injured Red nucleus. Grafts of BDNF-producing fibroblasts reduced neuron loss to less than 15% and surviving neurons showed only a 20% decrease in mean soma size. Soma size analysis of fluorogold-labeled Red nucleus neurons indicated that the Red nucleus neurons whose axons regenerated caudal to the graft did not atrophy. We conclude that fibroblasts engineered ex vivo to secrete BDNF and grafted into a partial cervical hemisection promote axon regeneration while reducing cell loss and atrophy of neurons in the Red nucleus. These results suggest that transplants of genetically engineered cells could be an important tool for delivery of therapeutic factors that contribute to the repair of spinal cord injury.     

NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord

The regeneration capacity of spinal cord axons is severely limited. Recently, much attention has focused on promoting regeneration of descending spinal cord pathways, but little is known about the regenerative capacity of ascending axons. Here we have assessed the ability of neurotrophic factors to promote regeneration of sensory neurons whose central axons ascend in the dorsal columns. The dorsal columns of adult rats were crushed and either brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin-3 (NT-3) or a vehicle solution was delivered continuously to the lesion site for 4 weeks. Transganglionic labelling with cholera toxin beta subunit (CTB) was used to selectively label large myelinated Abeta fibres. In lesioned rats treated with vehicle, CTB-labelled fibres were observed ascending in the gracile fasciculus, but these stopped abruptly at the lesion site, with no evidence of sprouting or growth into lesioned tissue. No CTB-labelled terminals were observed in the gracile nucleus, indicating that the lesion successfully severed all ascending dorsal column axons. Treatment with BDNF did not promote axonal regeneration. In GDNF-treated rats fibres grew around cavities in caudal degenerated tissue but did not approach the lesion epicentre. NT-3, in contrast, had a striking effect on promoting growth of lesioned dorsal column axons with an abundance of fibre sprouting apparent at the lesion site, and many fibres extending into and beyond the lesion epicentre. Quantification of fibre growth confirmed that only in NT-3-treated rats did fibres grow into the crush site and beyond. No evidence of terminal staining in the gracile nucleus was apparent following any treatment. Thus, although NT-3 promotes extensive growth of lesioned axons, other factors may be required for complete regeneration of these long ascending projections back to the dorsal column nuclei. The intrathecal delivery of NT-3 or other neurotrophic molecules has obvious advantages in clinical applications, as we show for the first time that dorsal column axonal regeneration can be achieved without the use of graft implantation or nerve lesions.     

Reinnervation of the biceps brachii muscle following cotransplantation of fetal spinal cord and autologous peripheral nerve into the injured cervical spinal cord of the adult rat

In order to compensate the loss of motoneurons resulting from severe spinal cord injury and to reestablish peripheral motor connectivity, solid pieces of fetal spinal cord, taken from embryonic day 14 rat embryos, were transplanted into unilateral aspiration lesions of the cervical spinal cord of adult rats. Concomitantly, one end of a 3.5-cm autologous peripheral nerve graft was put in close contact with the embryonic graft; the other end was sutured to the distal stump of the musculocutaneous nerve which innervate the biceps brachii muscle. The animals were examined 3 and 6 months after surgery. Following intramuscular injection of horseradish peroxidase, retrograde axonal labeling studies indicated that both transplanted and host spinal neurons were able to extend axons all the way through the peripheral nerve graft and nerve stump, up to the reconnected muscles. The labeled cells in the transplant were generally observed close to the intraspinal tip of the peripheral nerve graft. Retrograde axonal tracing, as well as electrophysiological and histological data, demonstrated the sensory and motor reinnervation of the reconnected muscles. This muscular reinnervation was able to reverse the atrophic changes observed in the denervated muscle. In control experiments, the extraspinal end of the peripheral nerve graft was ligatured in order to compare the differentiation of the transplanted neurons and the survival of their growing axons with or without their muscular targets. Six months after both types of surgery, large-size grafted neurons, identified as motoneurons by immunocytochemistry for peripherine and calcitonin gene-related peptide, were only observed in fetal spinal cord transplants which were connected to denervated muscles, thus demonstrating the trophic influence of the muscle target on the survival and differentiation of the transplanted neurons and on the maintenance of the axons they had grown into the peripheral nerve graft.     

NT-3 gene delivery elicits growth of chronically injured corticospinal axons and modestly improves functional deficits after chronic scar resection

Nervous system growth factors promote axonal growth following acute spinal cord injury. In the present experiment, we examined whether delivery of neurotrophic factors after chronic spinal cord injury would also promote axonal growth and influence functional outcomes. Adult Fischer 344 rats underwent mid-thoracic spinal cord dorsal hemisection lesions. Three months later, primary fibroblasts genetically modified to express human neurotrophin-3 (NT-3) were placed in, and distal to, the lesion cavity. Upon sacrifice 3 months later (6 months following the initial lesion), NT-3-grafted animals exhibited significant growth of corticospinal axons up to 15 mm distal to the lesion site and showed a modest but significant 1.5-point improvement in locomotor scores (P < 0.05) on the BBB scale, compared to control-grafted animals. Thus, growth factor gene delivery can elicit growth of corticospinal axons in chronic stages of injury and improves functional outcomes compared to non-growth-factor-treated animals.     

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.     

Histopathological reactions and axonal regeneration in the transected spinal cord of hibernating squirrels

The failure of axonal regeneration in the transected spinal cord of mammals has been attributed to many factors, including an intrinsic lack of regenerative capacity of mature CNS neurons, mechanical obstruction of axonal elongation by glial-connective tissue scars, necrosis of spinal tissue resulting in cavitation, lack of trophic influences sufficient to sustain outgrowth, and contact inhibition resulting from the formation of aberrant synapses. Assessment of the relative importance of each of these factors requires animal models in which one or more of these pathological processes can be eliminated. We therefore examined the effects of spinal transection in the hibernating animal because, during hibernation, collagen formation is depressed while nerve regeneration and slow axonal transport are maintained. Midthoracic spinal transections were performed in hibernating ground squirrels and the spinal cords were examined histologically 1–6 months later. The lesion site was composed primarily of a loose accumulation of macrophages and showed minimal glial and collagenous scarring, or cavitation. There was extensive regeneration of intrinsic spinal cord and dorsal root fibers. These axons grew to the margin of the lesion where they turned abruptly and continued growing along the interface between the lesion and the spinal cord. We conclude (1) that mammalian spinal-cord neurons have considerable regenerative potential; (2) that such mechanical impediments as collagenous and glial scarring, cyst formation, and cavitation cannot provide the sole explanation of why regeneration in the mammalian CNS is abortive; and (3) that specific physical and chemical properties of the cells in the environment of the growth cone regulate the extent and orientation of regenerative axonal outgrowth.     

Peripheral nerve grafting in the spinal cord: a histological and electrophysiological study

Excision of the dorsal columns was used to create a lesion in the spinal cord. The defect was bridged using a peripheral nerve graft. Silver staining showed the grafts being invaded by axons. Neuronal tracing by injecting horseradish peroxidase into the graft showed the origin of these fibres to be in dorsal root ganglia and in the grey matter of the lumbar cord. All of the animals studied showed horseradish peroxidase staining of neurones in the grey matter of the lumbar cord caudal to the graft. Five of the animals had staining of the dorsal root ganglion cells. Electro-physiological assessment was performed 5 to 6 months following the grafting procedure. Evoked potentials were recorded from the graft in six animals and from the dorsal columns above the graft in six animals.     

Attempts to facilitate dorsal column axonal regeneration in a neonatal spinal environment

The response to injury of ascending collaterals of dorsal root axons within the dorsal column (DC) was studied after neonatal spinal overhemisection (OH) made at different levels of the spinal cord. The transganglionic tracer, cholera toxin conjugated to horseradish peroxidase, and the anterograde tracer, biotinylated dextran amine, were used to label dorsal root ganglion cells with peripheral axons contributing to the sciatic nerve. There was no indication of a regenerative attempt by DC axons at acute survival times (3 days and later) after cervical injury, replicating previous work done at chronic survival periods (Lahr and Stelzner [1990] J. Comp. Neurol. 293:377–398). There was also no evidence of DC regeneration after lumbar OH injury even though immunohistochemical studies using the oligodendrocyte markers Rip and myelin basic protein showed few oligodendrocytes in the gracile fasciculus at lumbar levels at birth. Therefore, the lack of myelin in the dorsal funiculus at lumbar levels does not enhance the growth of neonatally axotomized DC axons. In addition, DC axons did not regenerate when presented with fetal spinal tissue implanted into thoracic OH lesions, even though positive control experiments showed that segmental dorsal root axons containing calcition gene-related peptide and corticospinal axons grew into these implants, replicating previous work of others. When a thoracic OH lesion, with or without a fetal spinal implant, was combined with sciatic nerve injury to attempt to stimulate an intracellular regenerative response of DRG neurons, again, no evidence of DC axonal regeneration was detected. Quantitative studies of the L4 and L5 dorsal root ganglia (DRG) showed that OH injury did not result in DRG neuronal loss. However, sciatic nerve injury did result in significant post-axotomy retrograde cell loss of DRG neurons, even in groups receiving thoracic embryonic spinal implants, and is one explanation for the minimal effect of sciatic nerve injury on DC regeneration. Although fetal tissue did not appear to rescue a significant number of DRG neurons, the quantitative analysis showed an enlargement of the largest class of DRG neuron, the class that contributes to the DC projection, in all groups receiving fetal tissue implants. This apparent trophic effect did not affect DC regeneration or neuronal survival after peripheral axotomy. Further studies are needed to determine why DC axons do not regenerate in a neonatal spinal environment or within fetal tissue implants, especially because previous work by others in both the developing and adult spinal cord shows that dorsal root axons will grow within the same type of fetal spinal implant. © 1996 Wiley-Liss, Inc.