Conditioning lesions enhance axonal regeneration of descending brain neurons in spinal-cord-transected larval lamprey

In larval lamprey, with increasing recovery times after a transection of the rostral spinal cord, there is a gradual recovery of locomotor behavior, and descending brain neurons regenerate their axons for progressively greater distances below the transection site. In the present study, spinal cord "conditioning lesions" (i.e., transections) were performed in the spinal cord at 30% body length (BL; normalized distance from the head) or 50% BL. After various "lesion delay times" (D), a more proximal spinal cord "test lesion" (i.e., transection) was performed at 10% BL, and then, after various recovery times (R), horseradish peroxidase was applied to the spinal cord at 20% BL to determine the extent of axonal regeneration of descending brain neurons. Conditioning lesions at 30% BL, lesion delay times of 2 weeks, and recovery times of 4 weeks (D-R = 2-4 group) resulted in a significant enhancement of axonal regeneration for the total numbers of descending brain neurons as well as neurons in certain brain cell groups compared to control animals without conditioning lesions. Experiments with hemiconditioning lesions, which reduce interanimal variability, confirmed that conditioning lesions do significantly enhance axonal regeneration and indicate that axotomy rather than diffusible factors released at the injury site is primarily involved in this enhancement. Results from the present study suggest that conditioning lesions "prime" descending brain neurons via cell body responses and enhance subsequent axonal regeneration, probably by reducing the initial delay and/or increasing the initial rate of axonal outgrowth.     

Cyclic AMP elevates tubulin expression without increasing intrinsic axon growth capacity

Exposing rat dorsal root ganglion (DRG) neurons to dibutyryl cAMP (db-cAMP) enables central branches to regenerate in the spinal cord by nullifying the ability of CNS myelin to inhibit elongation. A conditioning lesion (CL) promotes similar regeneration of central branches in the spinal cord by increasing neuronal cAMP levels. It is a matter of speculation whether any of the other effects of a CL are triggered by elevated cAMP. We found that like a CL, intraganglionic injection of db-cAMP increases the expression of growth-associated tubulin isotypes. However, unlike a CL, db-cAMP does not increase the velocity at which tubulin is delivered to the tips of growing axons by slow component b (SCb). db-cAMP also fails to increase intrinsic axon growth capacity enough to raise the rate of regeneration of peripheral branches in the sciatic nerve or enable central branches to elongate long distances in an environment free of all CNS inhibitors of elongation (i.e., a peripheral nerve graft transplanted into the spinal cord at the site of dorsal column transection). Thus, the increase in cAMP induced by a CL induces some, but not all, of the changes that may be necessary to increase intrinsic axon growth capacity.     

Electrical stimulation of intact peripheral sensory axons in rats promotes outgrowth of their central projections

A lesion of a peripheral nerve before a second injury (conditioning lesion, CL), enhances peripheral and central regeneration of dorsal root ganglion (DRG) axons. This effect is mediated by elevated neuronal cAMP. Here we wanted to investigate whether electrical stimulation (ES) of an intact nerve, which has been shown to accelerate peripheral axon outgrowth, is also effective in promoting axon regeneration of injured DRG axons in vitro and of the central DRG axons in vivo and, whether this effect is mediated by elevation of cAMP. For the in vitro assay, the intact sciatic nerve of adult rats was stimulated at 20 Hz for 1 h, 7 days before harvest and primary culture of DRG neurons on a growth permissive substrate. In the in vivo study, the central axons of the lumbosacral DRGs were cut in the Th8 dorsal column, and the sciatic nerve was either cut or left intact, and subjected to 1 h ES at 20 Hz or 200 Hz. In vitro, ES increased neurite outgrowth 4-fold as compared to non-stimulated DRG neurons. In vivo, ES at 20 Hz significantly increased axon outgrowth into the central lesion site as compared to the Sham control. The 20 Hz ES was as effective as the CL in increasing axon outgrowth into the lesion site but not in promoting axonal elongation even though 20 Hz ES increased intracellular cAMP levels in DRG neurons as effectively as the CL. Thus elevation of cAMP may account for the central axonal outgrowth after ES and a CL.     

Expression of insulin-like growth factors and corresponding binding proteins (IGFBP 1–6) in rat spinal cord and peripheral nerve after axonal injuries

Insulin-like growth factors (IGFs) exert trophic effects on several different cell types in the nervous system, including spinal motoneurons. After peripheral nerve injury, the increased expression of IGFs in the damaged nerve has been suggested to facilitate axonal regeneration. Here we have examined the expression pattern of mRNAs encoding IGF-1 and and -2, IGF binding proteins (IGFBPs) 1–6 in the rat spinal cord and peripheral nerve in three lesion models affecting lumbar motoneurons, i.e., sciatic nerve transection, ventral root avulsion, and a cut lesion in the ventral funiculus of the spinal cord. The expression was also studied in enriched Schwann cell and astrocyte cultures. The injured sciatic nerve expressed IGF-1 and IGF-2 as well as IGFBP-4 and IGFBP-5, whereas central nervous system (CNS) scar tissue expressed IGF-1, IGFBP-2, and IGFBP-5. IGFBP-6 mRNA was strongly upregulated in spinal motoneurons after all three types of lesions. IGFBP-6-like immunoreactivity was present in motoneuron cell bodies, dendrites in the ventral horn, and axons in the sciatic nerve. In line with the in vivo findings, cultured Schwann cells expressed IGF-1, IGF-2, IGFBP-4, and IGFBP-5 mRNAs, whereas cultured astrocytes expressed IGF-1, IGFBP-2, and IGFBP-5 mRNAs. These findings show that IGF-1 is available for lesioned motoneurons both after peripheral and central axonal lesions, whereas there are clear differences in the expression patterns for IGF-2 and some of the binding proteins in CNS and peripheral nervous system (PNS) scar tissue. The robust upregulation of IGFBP-6 mRNA in lesioned motoneurons suggests that this binding protein may be of special relevance for the severed cells. J. Comp. Neurol. 400:57–72, 1998. © 1998 Wiley-Liss, Inc.     

Motor axonal regeneration after partial and complete spinal cord transection

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

Axonal regeneration from injured dorsal roots into the spinal cord of adult rats.

Injury to the central processes of primary sensory neurons produces less profound changes in the expression of growth-related molecules and less vigorous axonal regeneration than does injury to their peripheral processes. The left L4, L5, and L6 dorsal roots of deeply anaesthetized adult Sprague-Dawley rats were severed and reanastomosed, and in some animals, the ipsilateral sciatic nerve was crushed to increase the expression of growth-related molecules. After between 28 days and three months, the sciatic nerve of most animals was injected with transganglionic tracers and the animals were killed 2-3 days later. Other animals were perfused for electron microscopy. Very few regenerating axons entered the spinal cord of the rats without sciatic nerve injuries. Labelled axons, however, were always found in the spinal cord of rats with sciatic nerve injuries. They often entered the cord around blood vessels, ran rostrally within the superficial dorsal horn, and avoided the degenerating white matter. The animals with a conditioning sciatic nerve crush had many more myelinated axons around the dorsal root entry zone (DREZ) and on the surface of the cord. Thus, a conditioning lesion of their peripheral processes increased the ability of the central processes of myelinated A fibres to regenerate, including to sites (such as lamina II) they do not normally occupy. Astrocytes, oligodendrocytes, and meningeal fibroblasts in and around the DREZ may have inhibited regeneration in that region, but growth of the axons into the deep grey matter and degenerated dorsal column was also blocked.     

Syntenin‐a promotes spinal cord regeneration following injury in adult zebrafish

In contrast to mammals, adult zebrafish recover locomotor function after spinal cord injury, in part due to the capacity of the central nervous system to repair severed connections. To identify molecular cues that underlie regeneration, we conducted mRNA expression profiling and found that syntenin‐a expression is upregulated in the adult zebrafish spinal cord caudal to the lesion site after injury. Syntenin is a scaffolding protein involved in mammalian cell adhesion and movement, axonal outgrowth, establishment of cell polarity, and protein trafficking. It could thus be expected to be involved in supporting regeneration in fish. Syntenin‐a mRNA and protein are expressed in neurons, glia and newly generated neural cells, and upregulated caudal to the lesion site on days 6 and 11 following spinal cord injury. Treatment of spinal cord‐injured fish with two different antisense morpholinos to knock down syntenin‐a expression resulted in significant inhibition of locomotor recovery at 5 and 6 weeks after injury, when compared to control morpholino‐treated fish. Knock‐down of syntenin‐a reduced regrowth of descending axons from brainstem neurons into the spinal cord caudal to the lesion site. These observations indicate that syntenin‐a is involved in regeneration after traumatic insult to the central nervous system of adult zebrafish, potentially leading to novel insights into the cellular and molecular mechanisms that require activation in the regeneration‐deficient mammalian central nervous system.     

Effects of repetitive conditioning crush lesions on regeneration of the rat sciatic nerve

The effect of repetitive conditioning lesions was tested on regeneration of the rat sciatic nerve. The nerve was conditioned by crush lesions one, two or three times with an interval of 2 or 4 days between each successive lesion. Axonal elongation was measured 3 days after a final test crush lesion. Two conditioning lesions stimulated axonal elongation more than one, while a third conditioning lesion had no further effect on axonal outgrowth. However, if the number of conditioning lesions were varied within a constant conditioning interval, outgrowth after the test lesion was the same. This suggests that the conditioning interval and not the number of conditioning lesions determined the outgrowth after a test lesion. When the conditioning lesion(s) and the test lesion were made at the same place, outgrowth was longer than if the lesions were spatially separated. Incorporation of [3H]thymidine in the regenerated nerve segment showed that proliferation of non-neuronal cells was initiated by each lesion. By counting the number of cell nuclei this proliferation was shown to correspond to an increase of cells in the regenerating nerve. It is therefore possible that the greater number of non-neuronal cells in the distal nerve segment accounts for the enhanced conditioning lesion effect in nerves where the conditioning and test lesions are made at the same place.     

Nerve growth factor-hypersecreting Schwann cell grafts augment and guide spinal cord axonal growth and remyelinate central nervous system axons in a phenotypically appropriate manner that correlates with expression of L1.

Schwann cells contribute to efficient axonal regeneration after peripheral nerve injury and, when grafted to the central nervous system (CNS), also support a modest degree of central axonal regeneration. This study examined (1) whether Schwann cells grafted to the CNS exhibit normal patterns of differentiation and association with spinal axons and what signals putatively modulate these interactions, and (2) whether Schwann cells overexpressing neurotrophic factors enhance axonal regeneration. Thus, primary Schwann cells were transduced to hypersecrete human nerve growth factor (NGF) and were grafted to spinal cord injury sites in adult rats. Comparisons were made to nontransfected Schwann cells. From 3 days to 6 months later, grafted Schwann cells exhibited a phenotypic and temporal course of differentiation that matched patterns normally observed after peripheral nerve injury. Schwann cells spontaneously aligned into regular spatial arrays within the cord, appropriately remyelinated coerulospinal axons that regenerated into grafts, and appropriately ensheathed but did not myelinate sensory axons extending into grafts. Coordinate expression of the cell adhesion molecule L1 on Schwann cells and axons correlated with establishment of appropriate patterns of axon-Schwann cell ensheathment. Transduction of Schwann cells to overexpress NGF robustly increased axonal growth but did not otherwise alter the nature of interactions with growing axons. These findings suggest that signals expressed on Schwann cells that modulate peripheral axonal regeneration and myelination are also recognized in the CNS and that the modification of Schwann cells to overexpress growth factors significantly augments their capacity to support extensive axonal growth in models of CNS injury.     

A conditioning lesion of the peripheral axons of dorsal root ganglion cells accelerates regeneration of only their peripheral axons

Axotomy of the peripheral axon of dorsal root ganglion (DRG) cells is known to result in chromatolysis and changes in protein synthesis in DRG cells. We investigated whether a stimulus produced by peripheral branch axotomy would affect the regenerative properties of both the central and peripheral axon of the DRG cell equally. To examine this question, a conditioning crush lesion was made distally on the sciatic nerve 2 weeks prior to a testing lesion of either the dorsal root or peripheral branch axon near the DRG. Fast axonal transport of radioactive proteins was used to assess regeneration of DRG axons. In the adult rat, leading peripheral branch axons normally regenerate at a rate of 4.4 mm/day. If a conditioning lesion of the sciatic nerve is made 2 weeks before the test lesion, the rate of peripheral branch axonal regeneration increases by 25% to 5.5 mm/day. This effect is not limited to the fastest growing axons in the nerve since a population of more slowly growing axons also exhibits accelerated outgrowth in response to a prior peripheral axotomy. In contrast to this, the fastest growing central branch axons of DRG cells, which normally regenerate at a rate of 2.5 mm/day, are not significantly affected by a prior peripheral axotomy. A population of more slowly growing axons in the dorsal root also does not exhibit accelerated outgrowth in response to a peripheral conditioning lesion. The results of these experiments indicate that changes in the DRG neuron's metabolism induced by prior axotomy of its peripheral axon do not affect the regenerative properties of both axons equally. This raises the possibility that accelerated axonal outgrowth in only one axonal branch results from a differentially regulated supply of proteins to the two axons by the DRG cell body.     

Growth factors and combinatorial therapies for CNS regeneration

There has been remarkable progress in the last 20 years in understanding mechanisms that underlie the success of axonal regeneration in the peripheral nervous system, and the failure of axonal regeneration in the central nervous system. Following the identification of these underlying mechanisms, several distinct therapeutic approaches have been tested in in vivo models of spinal cord injury (SCI) to enhance central axonal structural plasticity, including the therapeutic administration of neurotrophic factors. While several tested mechanisms apparently enhance axonal growth, more recent, properly controlled studies indicate that experimental approaches to combine therapies that target distinct neural mechanisms achieve greater axonal growth than therapies applied in isolation. The search for combination therapies that optimize axonal growth after SCI continues.     

Induced expression of polysialic acid in the spinal cord promotes regeneration of sensory axons

After spinal cord injury axonal regeneration is prevented by glial scar formation. In this study we examined whether induced expression of polysialic acid (PSA) in the lesion site would render the glial scar permissive to axonal regeneration after dorsal column transection. PSA was induced by lentiviral vector-mediated expression of polysialyltransferase (LV/PST). PSA expression increased astrocyte infiltration and permitted the penetration of regenerating axons across the caudal border of the lesion and into the lesion cavity. In LV/PST-injected animals with a peripheral nerve-conditioning lesion, 20 times more axons grew into the lesion cavity than those LV/GFP-injected plus conditioning lesion, and some axons grew across the cavity and extended to the rostral cord, while in LV/GFP group most ascending axons terminated at the caudal border of the lesion. Our result suggests that induced expression of PSA can provide a favorable environment for axonal regeneration.     

Inflammation near the nerve cell body enhances axonal regeneration

Although crushed axons in a dorsal spinal root normally regenerate more slowly than peripheral axons, their regeneration can be accelerated by a conditioning lesion to the corresponding peripheral nerve. These and other observations indicate that injury to peripheral sensory axons triggers changes in their nerve cell bodies that contribute to axonal regeneration. To investigate mechanisms of activating nerve cell bodies, an inflammatory reaction was provoked in rat dorsal root ganglia (DRG) through injection of Corynebacterium parvum. This inflammation enhanced regeneration in the associated dorsal root, increasing 4-fold the number of regenerating fibers 17 d after crushing; peripheral nerve regeneration was not accelerated. A milder stimulation of dorsal root regeneration was detected after direct injection of isogenous macrophages into the ganglion. It is concluded that changes favorable to axonal regeneration can be induced by products of inflammatory cells acting in the vicinity of the nerve cell body. Satellite glial cells and other unidentified cells in lumbar DRG were shown by thymidine radioautography to proliferate after sciatic nerve transection or injection of C. parvum into the ganglia. Intrathecal infusion of mitomycin C suppressed axotomy-induced mitosis of satellite glial cells but did not impede axonal regeneration in the dorsal root or the peripheral nerve. Nevertheless, the similarity in reactions of satellite glial cells during 2 processes that activate neurons adds indirect support to the idea that non-neuronal cells in the DRG might influence regenerative responses of primary sensory neurons.     

IL-6 promotes regeneration and functional recovery after cortical spinal tract injury by reactivating intrinsic growth program of neurons and enhancing synapse formation

Most neurons in adult mammalian central nervous system (CNS) fail to regenerate their axons after injury. Peripherally conditioned primary sensory neurons have an increased capacity to regenerate their central processes. Recent studies demonstrate that a conditioning lesion increased intrinsic growth capability is associated with the up-regulation of a group of growth-associated genes, one of the most established is interleukin-6 (IL-6). However, the cellular and molecular mechanisms by which IL-6 exerts its beneficial effect on axonal regeneration and functional recovery remain to be elucidated. The purpose of this study is to further investigate the molecular mechanisms of IL-6 in promoting regeneration and functional recovery after spinal cord injury (SCI). Here, we demonstrate that in vitro administration of IL-6 enhances neurite outgrowth of neurons on an inhibitory substrate myelin proteins, accompanied by increased expression of growth-associated genes GAP-43, SPRR1A and Arginase I. In vivo, intrathecal delivery of IL-6 for 7 days after cortical spinal tract injury induces synaptic rearrangements of sprouting axons and increases the expression of mTOR in neurons surrounding the lesion site, accompanied by improved functional recovery. In conclusion, our results show that IL-6 increases the expression of growth-associated genes and induces the expression of mTOR in lesion adjacent neurons, resulting in reactivating the intrinsic growth program of neurons to promote axonal regrowth and functional recovery after SCI.     

The nerve conditioning lesion: A strategy to enhance nerve regeneration

By altering the intrinsic metabolism of the cell, including the upregulation of regeneration‐associated genes (RAGs) and the production of structural proteins for axonal outgrowth, the conditioning lesion sets up an environment highly conducive to regeneration. In this review, we assess 40 years of research to provide a comprehensive overview of the conditioning lesion literature, directed at (1) discussing the mechanisms of and barriers to nerve regeneration that can be mitigated by the conditioning lesion, (2) describing the cellular and molecular pathways implicated in the conditioning lesion effect, and (3) deliberating on how these insights might be applied clinically. The consequential impact on regeneration is profound, with a conditioned nerve demonstrating longer neurite extensions in vitro, enhanced expression of RAGs within the dorsal root ganglia, early assembly and transportation of cytoskeletal elements, accelerated axonal growth, and improved functional recovery in vivo. Although this promising technique is not yet feasible to be performed in humans, there are potential strategies, such as conditioning electrical stimulation that may be explored to allow nerve conditioning in a clinically safe and well‐tolerated manner. Ann Neurol 2018;83:691–702     

Amino acid incorporation into rat spinal cord and brain after simultaneous and interval sciatic nerve lesions

The incorporation of [³H]lysine into the trichloracetic acid (TCA) precipitable protein and soluble fractions of brain and spinal cord of rats subjected to immediate transection distal and crush proximal or transection distal, two week interval, and crush proximal of the left sciatic nerve has been studied at 5, 11, and 18 days postoperative. In addition, the regeneration of the nerve fibers was measured. A greater rate of (P < 0.05) nerve regeneration was noted 11 and 18 days after interval sciatic nerve lesion compared to the simultaneous lesion. In the amino acid uptake studies, the overall trend of [³H]lysine into protein in brain and spinal cord showed a peak uptake occurring at 11 days for both simultaneous (S) and interval sciatic lesioned animals, although the effect was most pronounced after interval sciatic lesion. The 11-day interval lesioned group showed the highest uptake of amino acid precursor into protein of all groups averaged over all samples. After interval sciatic lesions, the uptake of amino acid into protein showed the relationship, 11 day group > 18 day group > 5 day group. However, no significant differences in uptake into protein in spinal cord and brain were seen between the simultaneous lesion sciatic animals over postoperative days.In brain, differences between the [³H]lysine incorporation into protein of the left (L) and right (R) cortical hemispheres was noted. The incorporation into somatomotor cortex showed a L > R difference which was most pronounced after simultaneous sciatic nerve lesion. Interestingly, the largest R > L difference was in occipital cortex after interval lesions of sciatic nerve.In spinal cord, the protein radioactivity of the L5–6 and S1–4 segments which include most of the affected neurons was greater on the right (unlesioned) side than on the left (sciatic crush) side over all lesioned animals. This effect was most pronounced after interval sciatic lesion. The alteration of central nervous system incorporation of lysine after peripheral nerve (sciatic) lesion and regeneration are discussed.     

A comparative study of glial and non‐neural cell properties for transplant‐mediated repair of the injured spinal cord

Cell transplantation is one strategy for encouraging regeneration after spinal cord injury and a range of cell types have been investigated for their repair potential. However, variations in study design complicate determination of which cells are most effective. In this study we have carried out a direct comparison of the regenerative and integrative properties of several cell preparations following transplantation into the lesioned rat spinal cord. Transplants included: (i) purified olfactory ensheathing cells (OECs) and (ii) fibroblast‐like cells, from olfactory bulb (OBFB‐L), (iii) a 50:50 mixture of (i) and (ii) (OEC/OBFB‐L), (iv) dissociated nasal mucosa (OM), (v) purified peripheral nerve Schwann cells (SCs), (vi) peripheral nerve fibroblasts, and (vii) skin fibroblasts (SF). All transplants supported axonal regeneration: OECs and SCs promoted the greatest regeneration while OBFB‐like cells were least efficient and mixed cell populations were less effective than purified populations. Tract‐tracing experiments demonstrated that none of the cell types promoted regeneration beyond the lesion. Although all cell types prevented cavity formation, the extent of astrocytic hypertrophy [GFAP immunoreactivity (IR) at the transplant/lesion site] differed markedly. OECs and SCs were associated with the least GFAP‐IR, fibroblasts and fibroblast‐like cells resulted in greater GFAP‐IR while hypertrophy surrounding transplants of OM was most extensive. These differences in host‐transplant reactivity were confirmed by transplanting cells into normal spinal cord where the cellular interaction is not complicated by injury. Thus, purified glial cells have advantages for transplant‐mediated repair, combining maximal support for axonal regeneration with a minimal astrocytic reaction around the transplant site. © 2013 Wiley Periodicals, Inc.     

Effects of Neurotransplants and BDNF on the Survival and Regeneration of Injured Adult Spinal Motoneurons

We compared the effects of peripheral nerve grafts, embryonic spinal cord transplants and brain-derived neurotrophic factor (BDNF) on the survival and axon regeneration of adult rat spinal motor neurons undergoing retrograde degeneration after ventral root avulsion. Following implantation into the dorsolateral funiculus of the injured spinal cord segment, neither a peripheral nerve graft nor a combination of peripheral nerve graft with embryonic spinal cord transplant could prevent the retrograde motor neuron degeneration induced by ventral root avulsion. However, intrathecal infusion of BDNF promoted long-term survival of the lesioned motor neurons and induced abundant motor axon regeneration from the avulsion zone along the spinal cord surface towards the BDNF source. A combination of ventral root reconstitution and BDNF treatment might therefore be a promising means for the support of both motor neuron survival and guided motor axon regeneration after ventral root lesions.