Glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor sustain the axonal regeneration of chronically axotomized motoneurons in vivo

In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.     

FK506 increases peripheral nerve regeneration after chronic axotomy but not after chronic schwann cell denervation

Poor functional recovery after peripheral nerve injury is attributable, at least in part, to chronic motoneuron axotomy and chronic Schwann cell (SC) denervation. While FK506 has been shown to accelerate the rate of nerve regeneration following a sciatic nerve crush or immediate nerve repair, for clinical application, it is important to determine whether the drug is effective after chronic nerve injuries. Two models were employed in the same adult rats using cross-sutures: chronic axotomy and chronic denervation of SCs. For chronic axotomy, a chronically (2 months) injured proximal tibial (TIB) was sutured to a freshly cut common peroneal (CP) nerve. For chronic denervation, a chronically (2 months) injured distal CP nerve was sutured to a freshly cut TIB nerve. Rats were given subcutaneous injections of FK506 or saline (5 mg/kg/day) for 3 weeks. In the chronic axotomy model, FK506 doubled the number of regenerated motoneurons identified by retrograde labeling (from 205 to 414 TIB motoneurons) and increased the numbers of myelinated axons (from 57 to 93 per 1000 microm2) and their myelin sheath thicknesses (from 0.42 to 0.78 microm) in the distal nerve stump. In contrast, after chronic denervation, FK506 did not improve the reduced capacity of SCs to support axonal regeneration. Taken together, the results suggest that FK506 acts directly on the neuron (as opposed to the denervated distal nerve stump) to accelerate and promote axonal regeneration of neurons whose regenerative capacity is significantly reduced by chronic axotomy.     

Experimental strategies to promote functional recovery after peripheral nerve injuries

The capacity of Schwann cells (SCs) in the peripheral nervous system to support axonal regeneration, in contrast to the oligodendrocytes in the central nervous system, has led to the misconception that peripheral nerve regeneration always restores function. Here, we consider how prolonged periods of time that injured neurons remain without targets during axonal regeneration (chronic axotomy) and that SCs in the distal nerve stumps remain chronically denervated (chronic denervation) progressively reduce the number of motoneurons that regenerate their axons. We demonstrate the effectiveness of low-dose, brain-derived neurotrophic and glial-derived neurotrophic factors to counteract the effects of chronic axotomy in promoting axonal regeneration. High-dose brain-derived neurotrophic factor (BDNF) on the other hand, acting through the p75 receptor, inhibits axonal regeneration and may be a factor in stopping regenerating axons from forming neuromuscular connections in skeletal muscle. The immunophilin, FK506, is also effective in promoting axonal regeneration after chronic axotomy. Chronic denervation of SCs (>1 month) severely deters axonal regeneration, although the few motor axons that do regenerate to reinnervate muscles become myelinated and form enlarged motor units in the reinnervated muscles. We found that in vitro incubation of chronically denervated SCs with transforming growth factor-beta re-established their growth-supportive phenotype in vivo, consistent with the idea that the interaction between invading macrophages and denervated SCs during Wallerian degeneration is essential to sustain axonal regeneration by promoting the growth-supportive SC phenotype. Finally, we consider the effectiveness of a brief period of 20 Hz electrical stimulation in promoting the regeneration of axons across the surgical gap after nerve repair.     

Effects of short- and long-term Schwann cell denervation on peripheral nerve regeneration, myelination, and size

Poor functional recovery after peripheral nerve injury has been generally attributed to inability of denervated muscles to accept reinnervation and recover from denervation atrophy. However, deterioration of the Schwann cell environment may play a more vital role. This study was undertaken to evaluate the effects of chronic denervation on the capacity of Schwann cells in the distal nerve stump to support axonal regeneration and to remyelinate regenerated axons. We used a delayed cross-suture anastomosis technique in which the common peroneal (CP) nerve in the rat was denervated for 0-24 weeks before cross-suture of the freshly axotomized tibial (TIB) and chronically denervated CP nerve stumps. Motor neurons were backlabeled with either fluoro-ruby or fluorogold 12 months later, to identify and count TIB motor neurons that regenerated axons into chronically denervated CP nerve stumps. Number, size, and myelination of regenerated sensory and motor axons were determined using light and electron microscopy. We found that short-term denervation of < or =4 weeks did not affect axonal regeneration but more prolonged denervation profoundly reduced the numbers of backlabeled motor neurons and axons in the distal nerve stump. Yet, atrophic Schwann cells retained their capacity to remyelinate regenerated axons. In fact, the axons were larger and well myelinated by long-term chronically denervated Schwann cells. These findings demonstrate a progressive inability of chronically denervated Schwann cells to support axonal regeneration and yet a sustained capacity to remyelinate the axons which do regenerate. Thus, axonal interaction can effectively switch the nonmyelinating phenotype of atrophic Schwann cells back into the myelinating phenotype.     

Chronic Schwann cell denervation and the presence of a sensory nerve reduce motor axonal regeneration

Motor axonal regeneration is compromised by chronic distal nerve stump denervation, induced by delayed repair or prolonged regeneration distance, suggesting that the pathway for regeneration is progressively impaired with time and/or distance. In the present experiments, we tested the impacts of (i) chronic distal sensory nerve stump denervation on axonal regeneration and (ii) sensory or motor innervation of a nerve graft on the ability of motoneurons to regenerate their axons from the opposite end of the graft. Using the motor and sensory branches of rat femoral nerve and application of neuroanatomical tracers, we evaluated the numbers of regenerated femoral motoneurons and nerve fibers when motoneurons regenerated (i) into freshly cut and 2-month chronically denervated distal sensory nerve stump, (ii) alone into a 4-cm-long distally ligated sensory autograft (MGL) and, (iii) concurrently as sensory (MGS) or motor (MGM) nerves regenerated into the same autograft from the opposite end. We found that all (315 +/- 24: mean +/- SE) the femoral motoneurons regenerated into a freshly cut distal sensory nerve stump as compared to 254 +/- 20 after 2 months of chronic denervation. Under the MGL condition, 151 +/- 5 motoneurons regenerated, which was not significantly different from the MGM group (134 +/- 13) but was significantly reduced to 99 +/- 2 in the MGS group (P < 0.05). The number of regenerated nerve fibers was 1522 +/- 81 in the MGL group, 888 +/- 18 in the MGM group, and 516 +/- 44 in the MGS group, although the high number of nerve fibers in the MGL group was due partly to the elaboration of multiple sprouts. Nerve fiber number and myelination were reduced in the MGS group and increased in the MGM group. These results demonstrate that both chronic denervation and the presence of sensory nerve axons reduced desired motor axonal regeneration into sensory pathways. A common mechanism may involve reduced responsiveness of sensory Schwann cells within the nerve graft or chronically denervated distal nerve stump to regenerating motor axons. The findings confirm that motor regeneration is optimized by avoiding even short-term denervation. They also imply that repairing pure motor nerves (without their cutaneous sensory components) to distal nerve stumps should be considered clinically when motor recovery is the main desired outcome.     

Influence of the axotomy to cell body distance in rat rubrospinal and spinal motoneurons: differential regulation of GAP-43, tubulins, and neurofilament-M.

Axotomized motoneurons regenerate their axons regardless of whether axotomy occurs proximally or distally from their cell bodies. In contrast, regeneration of rubrospinal axons into peripheral nerve grafts has been detected after cervical but not after thoracic injury of the rubrospinal tract. By using in situ hybridization (ISH) combined with reliable retrograde tracing methods, we compared regeneration-associated gene expression after proximal and distal axotomy in spinal motoneurons versus rubrospinal neurons. Regardless of whether they were axotomized at the iliac crest (proximal) or popliteal fossa (distal), sciatic motoneurons underwent highly pronounced changes in ISH signals for Growth Associated Protein 43 (GAP-43) (10-20x increase) and neurofilament M (60-85% decrease). In contrast, tubulin ISH signals substantially increased only after proximal axotomy (3-5x increase). To compare these changes in gene expression with those of axotomized rubrospinal neurons, the rubrospinal tract was transected at the cervical (proximal) or thoracic (distal) levels of the spinal cord. Cervically axotomized rubrospinal neurons showed three- to fivefold increases in ISH signals for GAP-43 and tubulins (only transient) and a 75% decrease for neurofilament-M. In sharp contrast, thoracic axotomy had only marginal effects. After implantation of peripheral nerve transplants into the spinal cord injury sites, retrograde labeling with the sensitive retrograde tracer Fluoro-Gold identified regenerating rubrospinal neurons only after cervical axotomy. Furthermore, rubrospinal neurons specifically regenerating into the transplants were hypertrophied and expressed high levels of GAP-43 and tubulins. Taken together, these data support the concept that, even if central nervous system (CNS) axons are presented with a permissive/supportive environment, appropriate cell body responses to injury are a prerequisite for CNS axonal regeneration.     

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

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

Regeneration‐associated genes decline in chronically injured rat sciatic motoneurons

Chronic nerve injuries are notorious for their poor regenerative outcomes. Here, we addressed the question of whether the established reduced ability of injured motoneurons to regenerate their axons with time of disconnection with targets (chronic axotomy) is associated with a failure of injured motoneurons to express and sustain their expression of regeneration‐associated genes. Sciatic motoneurons were prevented from regenerating by ligation of the transected nerves (chronic axotomy), and then subjected to a second nerve transection (acute axotomy) to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to delayed nerve repair. The expression of α₁‐tubulin, actin and GAP‐43 mRNA was analysed in axotomized sciatic motoneurons by the use of in situ hybridization followed by autoradiography and silver grain quantification. The expression of these regeneration‐associated genes by naive (acutely) axotomized motoneurons declined exponentially, to reach baseline levels within 6 months. These chronically injured motoneurons responded to a refreshment axotomy by elevating the expression of the genes to the same levels as in acutely (i.e. for the first time) axotomized sciatic motoneurons. However, the expression of these declined more rapidly than after acute axotomy. We conclude that a progressive decline in the expression of the regeneration‐associated genes in chronically axotomized motoneurons and the even more rapid decline in their expression in response to a refreshment axotomy may explain why the regenerative capacity of chronically axotomized neurons declines with time.     

A decline in glial cell-line-derived neurotrophic factor expression is associated with impaired regeneration after long-term Schwann cell denervation.

In the peripheral nervous system, regeneration of motor and sensory axons into chronically denervated distal nerve segments is impaired compared to regeneration into acutely denervated nerves. In order to find possible causes for this phenomenon we examined the changes in the expression pattern of the glial cell-line-derived neurotrophic factor (GDNF) family of growth factors and their receptors in chronically denervated rat sciatic nerves as a function of time with or without regeneration. Among the GDNF family of growth factors, only GDNF mRNA expression was rapidly upregulated in Schwann cells as early as 48 h after denervation. This upregulation peaked at 1 week and then declined to minimal levels by 6 months of denervation. The changes in the protein expression paralleled the changes in the expression of the GDNF mRNA. The mRNAs for receptors GFRalpha-1 and GFRalpha-2 were upregulated only after maximal GDNF upregulation and remained elevated as late as 6 months. There were no significant changes in the expression of GFRalpha-3 or the tyrosine kinase coreceptor, RET. When we examined the expression of GDNF in a delayed regeneration paradigm, there was no upregulation in the distal chronically denervated tibial nerve even when the freshly axotomized peroneal branch of the sciatic nerve was sutured to the distal tibial nerve. This study suggests that one of the reasons for impaired regeneration into chronically denervated peripheral nerves may be the inability of Schwann cells to maintain important trophic support for both motor and sensory neurons.     

Misdirection of regenerating motor axons after nerve injury and repair in the rat sciatic nerve model

Misdirection of regenerating axons is one of the factors that can explain the poor results often found after nerve injury and repair. In this study, we quantified the degree of misdirection and the effect on recovery of function after different types of nerve injury and repair in the rat sciatic nerve model; crush injury, direct coaptation, and autograft repair. Sequential tracing with retrograde labeling of the peroneal nerve before and 8 weeks after nerve injury and repair was performed to quantify the accuracy of motor axon regeneration. Digital video analysis of ankle motion was used to investigate the recovery of function. In addition, serial compound action potential recordings and nerve and muscle morphometry were performed. In our study, accuracy of motor axon regeneration was found to be limited; only 71% (± 4.9%) of the peroneal motoneurons were correctly directed 2 months after sciatic crush injury, 42% (± 4.2%) after direct coaptation, and 25% (± 6.6%) after autograft repair. Recovery of ankle motion was incomplete after all types of nerve injury and repair and demonstrated a disturbed balance of ankle plantar and dorsiflexion. The number of motoneurons from which axons had regenerated was not significantly different from normal. The number of myelinated axons was significantly increased distal to the site of injury. Misdirection of regenerating motor axons is a major factor in the poor recovery of nerves that innervate different muscles. The results of this study can be used as basis for developing new nerve repair techniques that may improve the accuracy of regeneration.     

Transforming growth factor-beta and forskolin attenuate the adverse effects of long-term Schwann cell denervation on peripheral nerve regeneration in vivo

Transforming growth factor-beta (TGF-beta) plays a central role in the regulation of Schwann cell (SC) proliferation and differentiation and is essential for the neurotrophic effects of several neurotrophic factors (reviewed by Unsicker and Krieglstein, 2000; Unsicker and Strelau, 2000). However, its role in peripheral nerve regeneration in vivo is not yet understood. Our studies were carried out to characterize (1) the effects of duration of regeneration, and chronic SC denervation on the number of tibial (TIB) motor neurons that regenerated axons over a fixed distance (25 mm into distal common peroneal [CP] nerve stumps), and (2) the effect of in vitro incubation of 6-month chronically denervated sciatic nerve explants with TGF-beta and forskolin on their capacity to support axonal regeneration in vivo. TIB--CP cross-suture in Silastic tubing was used, and regeneration into 0-24-week chronically denervated CP stumps was allowed for either 1.5 or 3 months. Chronically denervated rat sciatic nerve explants (3 x 3 mm(2)) were incubated in vitro with either DMEM and 15% fetal calf serum (D-15) plus TGF-beta/forskolin or D-15 alone for 48 h and placed into a 10-mm Silastic tube that bridged the proximal and distal nerve stumps of a freshly cut TIB nerve. The number of tibial motor neurons that regenerated axons through the explants and 25 mm into the distal nerve stump after 6 months, and TIB regeneration into the CP nerve stumps, were assessed using retrograde tracers, fluorogold, or fluororuby. We found that all tibial motor neurons regenerate their axons 25 mm into 0-4-week denervated CP nerve stumps after a regeneration period of 3 months. Reducing regeneration time to 1.5 months and chronic denervation, reduced the number of motor neurons that regenerated axons over 25 mm. Exposure of 6-month denervated nerve explants to TGF-beta/forskolin increased the number of motor neurons that regenerated through them from 258 +/-13; mean +/- SE to 442 +/- 22. Hence, acute treatment of atrophic SC with TGF-beta can reactivate the growth-permissive SC phenotype to support axonal regeneration.     

A spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression after lumbar ventral root avulsion and implantation

Reimplantation of avulsed rat lumbar spinal ventral roots results in poor recovery of function of the denervated hind limb muscles. In contrast, reimplantation of cervical or sacral ventral roots is a successful repair strategy that results in a significant degree of regeneration. A possible explanation for this difference could be that following lumbar root avulsion, axons have to travel longer distances towards their target muscles, resulting in prolonged denervation of the distal nerve and a diminished capacity to support regeneration. Here we present a detailed spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression following unilateral avulsion and implantation of lumbar ventral roots L3, L4, and L5. Reimplantation prolongs the survival of motoneurons up to one month post-lesion. The first regenerating motor axons entered the reimplanted ventral roots during the first week and large numbers of fibers gradually enter the lumbar plexus between 2 and 4 weeks, indicating that axons enter the reimplanted roots and plexus over an extended period of time. However, motor axon counts show that relatively few axons reach the distal sciatic nerve in the 16 week post-lesion period. The observed initial increase and subsequent decline in expression of glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor correlate with the apparent spatio-temporal decline in the regenerative capacity of motor axons, indicating that the distal nerve is losing its capacity to support regenerating motor axons following prolonged denervation. These findings have important implications for future strategies to promote long-distance regeneration through distal, chronically denervated peripheral nerves.     

Changes in mRNA for choline transporter-like protein following facial nerve transection

When the axon of a motoneuron is transected, axonal regrowth occurs to reconnect it to the correct target. During the regeneration period, a large amount of new membrane synthesis is required for the axons to extend. Choline is an important metabolite in all cells because of the major contribution of phosphatidylcholine and sphingomyelin to the production of membranes. Therefore, choline uptake is necessary for axonal elongation. We cloned rat choline transporter-like protein 1 (rCTL1) as an upregulated gene in the axotomized facial motor nucleus by differential display polymerase chain reaction using adult rat facial nerve axotomy model. rCTL1 belongs to the choline transporter-like protein family, which takes up choline. We investigated the changes in rCTL1 mRNA levels in the facial motor nucleus of adult rats following axotomy by in situ hybridization. In the facial motoneurons signals of rCTL1 mRNA were rarely expressed, were transiently increased following axotomy and gradually returned to the control level. These results suggest that rCTL1 is involved in activated choline uptake for membrane synthesis in motoneurons following nerve transection.     

Canadian Association of Neuroscience review: axonal regeneration in the peripheral and central nervous systems--current issues and advances

Injured nerves regenerate their axons in the peripheral (PNS) but not the central nervous system (CNS). The contrasting capacities have been attributed to the growth permissive Schwann cells in the PNS and the growth inhibitory environment of the oligodendrocytes in the CNS. In the current review, we first contrast the robust regenerative response of injured PNS neurons with the weak response of the CNS neurons, and the capacity of Schwann cells and not the oligodendrocytes to support axonal regeneration. We then consider the factors that limit axonal regeneration in both the PNS and CNS. Limiting factors in the PNS include slow regeneration of axons across the injury site, progressive decline in the regenerative capacity of axotomized neurons (chronic axotomy) and progressive failure of denervated Schwann cells to support axonal regeneration (chronic denervation). In the CNS on the other hand, it is the poor regenerative response of neurons, the inhibitory proteins that are expressed by oligodendrocytes and act via a common receptor on CNS neurons, and the formation of the glial scar that prevent axonal regeneration in the CNS. Strategies to overcome these limitations in the PNS are considered in detail and contrasted with strategies in the CNS.     

Potential Roles of Gene Expression Change in Adult Rat Spinal Motoneurons Following Axonal Injury: A Comparison among c-jun, Low-Affinity Nerve Growth Factor Receptor (LNGFR), and Nitric Oxide Synthase (NOS)

The present study investigates expression of nitric oxide synthase (NOS), immediate early genes (IEGs, c-jun, and c-fos), and low-affinity nerve growth factor receptor (LNGFR) in adult rat spinal motoneurons in response to three conditions of axonal injury: distal axotomy, root avulsion, and root avulsion followed by a peripheral nerve (PN) graft implantation. Expression of c-jun and LNGFR were predominately observed in motoneurons of the distal axotomized segment where most motoneurons survived. In contrast, expression of NOS was exclusively found in motoneurons of the root avulsed segment where most motoneurons died. c-fos was not expressed in motoneurons following either distal axotomy or root avulsion. In animals with PN graft implantation, a double fluorescent labeling technique was used to evaluate motoneuron regeneration. Expression of NOS was completely inhibited in all motoneurons that regenerated into the PN graft, but was not inhibited in those that did not regenerate. Moreover, regenerated motoneurons expressed LNGFR and c-jun while the nonregenerated motoneurons expressed NOS. Results of the present study have shown that motoneurons undergo changes in expression of cellular molecules in response to the axonal injury. The expression of c-jun and LNGFR may be related to the regenerative process while expression of NOS is more likely involved in the degenerative process. The results also show that PN graft implantation can alter the expression of cellular molecules and reduce motoneuron death due to root avulsion. The survival-promoting effects of PN graft implantation (presumably the effects of neurotrophic factors) may be achieved by modifying certain cellular molecules such as NOS.     

Transplantation of embryonic spinal cord neurons to the injured distal nerve promotes axonal regeneration after delayed nerve repair

Peripheral nerve injury (PNI) usually results in poor functional recovery. Nerve repair is the common clinical treatment for PNI but is always obstructed by the chronic degeneration of the distal stump and muscle. Cell transplantation can alleviate the muscle atrophy after PNI, but the subsequent recovery of the locomotive function is seldom described. In this study, we combined cell transplantation and nerve repair to investigate whether the transplantation of embryonic spinal cord cells could benefit the delayed nerve repair. The experiment consisted of 3 stages: transection of the tibial nerve to induce ‘pre‐degeneration’, a second surgery performed 2 weeks later for transplantation of E14 embryonic spinal cord cells or vehicle (culture medium) at the distal end of the injured nerve, and, 3 months later, the removal of the grafted cells and the cross‐suturing of the residual distal end to the proximal end of a freshly cut ipsilateral common peroneal (CP) nerve. Cell survival and fate after the transplantation were investigated, and the functional recovery after the cross‐suturing was compared between the groups. The grafted cells could survive and generate motor neurons, extending axons that were subsequently myelinated and forming synapses with the muscle. After the cross‐suturing, the axonal regeneration from the proximal stump of the injured CP nerve and the functional recovery of the denervated gastrocnemius muscle were significantly promoted in the group receiving the cells. Our study presents a new perspective indicating that the transplantation of embryonic spinal cord neurons may be a valuable therapeutic strategy for PNI.     

Rolipram-induced elevation of cAMP or chondroitinase ABC breakdown of inhibitory proteoglycans in the extracellular matrix promotes peripheral nerve regeneration

The inhibitory growth environment of myelin and extracellular matrix proteoglycans in the central nervous system may be overcome by elevating neuronal cAMP or degrading inhibitory proteoglycans with chondroitinase ABC (ChABC). In this study, we asked whether similar mechanisms operate in peripheral nerve regeneration where effective Wallerian degeneration removes myelin and extracellular proteoglycans slowly. We repaired transected common peroneal (CP) nerve in rats and either elevated cAMP in the axotomized neurons by subcutaneous rolipram, a specific inhibitor of phosphodiesterase IV, and/or promoted degradation of proteoglycans in the distal nerve stump by local ChABC administration. Rolipram treatment significantly increased the number of motoneurons that regenerated axons across the repair site at 1 and 2 weeks, and increased the number of sensory neurons that regenerated axons across the repair site at 2 weeks. Local application of ChABC had a similar effect to rolipram treatment in promoting motor axon regeneration, the effect being no greater when rolipram and ChABC were administered simultaneously. We conclude that blocking inhibitors of axon regeneration by elevating cAMP or degrading proteoglycans in the distal nerve stump promotes peripheral axon regeneration after surgical repair of a transected nerve. It is likely that elevated cAMP is sufficient to encourage axon outgrowth despite the inhibitory growth environment such that simultaneous enzymatic proteoglycan degradation does not promote more axon regeneration than either elevated cAMP or proteoglycan degradation alone.     

A light and electron microscopic study of intracellularly HRP-labeled lumbar motoneurons after intramedullary axotomy in the adult cat.

In contrast to many other neurons in the central nervous system, spinal motoneurons in adult cats have been shown to regenerate their axons after an axotomy accomplished within the CNS compartment. This regenerative capacity may be the result of extrinsic influences, or intrinsic properties of the motoneurons themselves, or interactions between extrinsic and intrinsic factors. As part of the effort to establish circumstances of importance for this central regeneration, a detailed analysis of the morphology of lumbar motoneurons was performed 3-11 weeks following a ventral funiculus axotomy. Fourteen large neurons considered to be intramedullarly axotomized alpha motoneurons were labeled intracellularly with horseradish peroxidase. Twelve out of the fourteen analyzed neurons had an axonlike regenerating process. These twelve neurons could, in turn, be separated into two groups, based on the proximity of the axonal lesion and the proximal morphology of the regenerating process. Thus, after a comparatively proximal axotomy, new axons were produced, originating either from the cell soma or from a distal dendritic branch. After a more distal axotomy, but still intramedullarly, it seemed as if the proximal part of the original axon always persisted and subsequently regenerated. Analysis of the relation between the cell soma diameter and the diameter and number of its stem dendrites revealed that dendrites become thinner and also decrease in number after an intramedullary axotomy. In this way, it may be calculated that the total dendritic surface area of lesioned motoneurons will decrease by approximately half. In four neurons, most dendrites had an abnormal appearance in the light microscope with increasing diameter of distal branches. Ultrastructural analysis revealed that such dendrites were surrounded by myelin sheaths. Small filopodia in close relation to axon terminals were found to emerge from the cell membrane of the lesioned motoneurons. Their function may be to establish contact with presynaptic elements and then retract them to the cell membrane. We interpret the morphological changes of the motoneurons as signs of a large capacity for axonal regeneration, even after axotomy in the central nervous system.     

Basic Behavior of Migratory Schwann Cells in Peripheral Nerve Regeneration

In axonal regeneration after a peripheral nerve injury, Schwann cells migrate from the two nerve ends and at last form a continuous tissue cable across the gap which guides the axons toward the bands of Büngner. However, the behavior of migratory Schwann cells and their possible role are obscure. Using a film model in which the proximal stump of a transected nerve in mice was sandwiched between two thin plastic films, we analyzed neural regeneration in the early phase up to the 6th day after axotomy. Regenerating neurites emerged from the nodes of Ranvier adjacent to the axotomized nerve stump within 3 h after axotomy and extended along the parent nerve onto the film. All of the regenerating neurites on the surface of the film consisted of naked axons for at least 2 days after axotomy. Thereafter, Schwann cells from the proximal nerve migrated along a network of the regenerating axons and then closely attached to the axons, ensheathing them. Some of the Schwann cells advanced ahead of the axonal growth cones and were distributed over regions in which axonal extension was not yet present. As calculated from the time course of regenerating neurites, the velocity of axonal regeneration showed two phases: an initial slow phase (77 μm/day) up to the 2nd post-operative day followed by a faster phase (283 μm/day). The first observation of Schwann cells coincided with the onset of the second phase. In addition, the length of regenerating axons on the surface of the film containing many Schwann cells was significantly greater than that on the surface where Schwann cells were not yet present. It meant that migratory Schwann cells stimulated axons to elongate for a longer distance. Furthermore, Schwann cells from a distal stump showed a stronger ability to accelerate the axonal outgrowth than these from a proximal stump.