Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Local delivery of glial cell line-derived neurotrophic factor improves facial nerve regeneration after late repair.

OBJECTIVES/HYPOTHESIS: Facial nerve regeneration is limited in some clinical situations: in long grafts, by aged patients, and when the delay between nerve lesion and repair is prolonged. This deficient regeneration is due to the limited number of regenerating nerve fibers, their immaturity and the unresponsiveness of Schwann cells after a long period of denervation. This study proposes to apply glial cell line-derived neurotrophic factor (GDNF) on facial nerve grafts via nerve guidance channels to improve the regeneration. METHODS: Two situations were evaluated: immediate and delayed grafts (repair 7 months after the lesion). Each group contained three subgroups: a) graft without channel, b) graft with a channel without neurotrophic factor; and c) graft with a GDNF-releasing channel. A functional analysis was performed with clinical observation of facial nerve function, and nerve conduction study at 6 weeks. Histological analysis was performed with the count of number of myelinated fibers within the graft, and distally to the graft. Central evaluation was assessed with Fluoro-Ruby retrograde labeling and Nissl staining. RESULTS: This study showed that GDNF allowed an increase in the number and the maturation of nerve fibers, as well as the number of retrogradely labeled neurons in delayed anastomoses. On the contrary, after immediate repair, the regenerated nerves in the presence of GDNF showed inferior results compared to the other groups. CONCLUSIONS: GDNF is a potent neurotrophic factor to improve facial nerve regeneration in grafts performed several months after the nerve lesion. However, GDNF should not be used for immediate repair, as it possibly inhibits the nerve regeneration.

Critical role for the lactate transporter, monocarboxylate transporter 1 (mct1), in the regeneration of peripheral nerves

Axons, and particularly regenerating axons, have high metabolic needs in order to maintain critical functions such as axon transport and membrane depolarization. Though some of the required energy likely comes form extracellular glucose and ATP generated in the soma, we and others hypothesize that some of the energy may be supplied by lactate. Unlike glucose that requires glycolytic enzymes to produce pyruvate, lactate can be converted directly to pyruvate by lactate dehydrogenase and transported into mitochondria for oxidative metabolism. In order to be transported into or out of cells, lactate requires specific monocarboxylate transporters (MCTs), the most abundant of which is MCT1. If MCT1 and lactate are critical for nerve function and regeneration, we hypothesize that MCT1 heterozygote null mice, which appear phenotypically normal despite having approximately 40% MCT1 as compared to wildtype littermate mice, would have reduced capacity for repair following nerve injury. To investigate this, adult MCT1 heterozygote null mice or wild-type mice underwent unilateral sciatic nerve crush in the proximal thigh. We found that regeneration of the sciatic nerve, as measured by recovery of compound muscle action potentials (CMAP) in the lateral plantar muscles following proximal sciatic nerve stimulation, was delayed from a median of 21 days in wildtype mice to 38.5 days in MCT1 heterozygote mice. In fact, half of the MCT1 heterozygote null mice had no recovery of CMAP by the endpoint of the study at 42 days, while all of the wild-type mice had recovered. In addition, the maximal amplitude of CMAP recovery in MCT1 heterozygote mull mice was reduced from a mean of 3 mV to 0.5 mV. As would be expected, the denervated gastrocnemius muscle of MCT1 heterozygote null mice remained atrophic at 42 days compared to wild-type mice. Our experiments show that lactate supplied through MCT1 is necessary for nerve regeneration. Experiments are underway to determine whether loss of MCT1 prevents nerve regrowth directly due to reduced energy supply to axons or indirectly by dysfunctional Schwann cells normally dependent on lactate supply through MCT1.

Thyroid hormone in biodegradable nerve guides stimulates sciatic nerve regeneration: a potential therapeutic approach for human peripheral nerve injuries.

It has been already demonstrated that thyroid hormone (T3) is one of the most important stimulating factors in peripheral nerve regeneration. We have recently shown that local administration of T3 in silicon tubes at the level of the transected rat sciatic nerve enhanced axonal regeneration and improved functional recovery. Silicon, however, cannot be used in humans because it causes a chronic inflammatory reaction. Therefore, in order to provide future clinical applications of thyroid hormone in human peripheral nerve lesions, we carried out comparative studies on the regeneration of transected rat sciatic nerve bridged either by biodegradable P(DLLA-(-CL) or by silicon nerve guides, both guides filled with either T3 or phosphate buffer. Our macroscopic observation revealed that 85% of the biodegradable guides allowed the expected regeneration of the transected sciatic nerve. The morphological, morphometric and electrophysiological analysis showed that T3 in biodegradable guides induces a significant increase in the number of myelinated regenerated axons (6862 +/- 1831 in control vs. 11799 +/- 1163 in T3-treated). Also, T3 skewed the diameter of myelinated axons toward larger values than in controls. Moreover, T3 increases the compound muscle action potential amplitude of the flexor and extensor muscles of the treated rats. This T3 stimulation in biodegradable guides was equally well to that obtained by using silicone guides. In conclusion, the administration of T3 in biodegradable guides significantly improves sciatic nerve regeneration, confirming the feasibility of our technique to provide a serious step towards future clinical application of T3 in human peripheral nerve injuries.

Thyroid hormone enhances transected axonal regeneration and muscle reinnervation following rat sciatic nerve injury.

Improvement of nerve regeneration and functional recovery following nerve injury is a challenging problem in clinical research. We have already shown that following rat sciatic nerve transection, the local administration of triiodothyronine (T3) significantly increased the number and the myelination of regenerated axons. Functional recovery is a sum of the number of regenerated axons and reinnervation of denervated peripheral targets. In the present study, we investigated whether the increased number of regenerated axons by T3-treatment is linked to improved reinnervation of hind limb muscles. After transection of rat sciatic nerves, silicone or biodegradable nerve guides were implanted and filled with either T3 or phosphate buffer solution (PBS). Neuromuscular junctions (NMJs) were analyzed on gastrocnemius and plantar muscle sections stained with rhodamine alpha-bungarotoxin and neurofilament antibody. Four weeks after surgery, most end-plates (EPs) of operated limbs were still denervated and no effect of T3 on muscle reinnervation was detected at this stage of nerve repair. In contrast, after 14 weeks of nerve regeneration, T3 clearly enhanced the reinnervation of gastrocnemius and plantar EPs, demonstrated by significantly higher recovery of size and shape complexity of reinnervated EPs and also by increased acetylcholine receptor (AChRs) density on post synaptic membranes compared to PBS-treated EPs. The stimulating effect of T3 on EP reinnervation is confirmed by a higher index of compound muscle action potentials recorded in gastrocnemius muscles. In conclusion, our results provide for the first time strong evidence that T3 enhances the restoration of NMJ structure and improves synaptic transmission.

Deficiency in monocarboxylate transporter 1 (MCT1) in mice delays regeneration of peripheral nerves following sciatic nerve crush.

Peripheral nerve regeneration following injury occurs spontaneously, but many of the processes require metabolic energy. The mechanism of energy supply to axons has not previously been determined. In the central nervous system, monocarboxylate transporter 1 (MCT1), expressed in oligodendroglia, is critical for supplying lactate or other energy metabolites to axons. In the current study, MCT1 is shown to localize within the peripheral nervous system to perineurial cells, dorsal root ganglion neurons, and Schwann cells by MCT1 immunofluorescence in wild-type mice and tdTomato fluorescence in MCT1 BAC reporter mice. To investigate whether MCT1 is necessary for peripheral nerve regeneration, sciatic nerves of MCT1 heterozygous null mice are crushed and peripheral nerve regeneration was quantified electrophysiologically and anatomically. Compound muscle action potential (CMAP) recovery is delayed from a median of 21days in wild-type mice to greater than 38days in MCT1 heterozygote null mice. In fact, half of the MCT1 heterozygote null mice have no recovery of CMAP at 42days, while all of the wild-type mice recovered. In addition, muscle fibers remain 40% more atrophic and neuromuscular junctions 40% more denervated at 42days post-crush in the MCT1 heterozygote null mice than wild-type mice. The delay in nerve regeneration is not only in motor axons, as the number of regenerated axons in the sural sensory nerve of MCT1 heterozygote null mice at 4weeks and tibial mixed sensory and motor nerve at 3weeks is also significantly reduced compared to wild-type mice. This delay in regeneration may be partly due to failed Schwann cell function, as there is reduced early phagocytosis of myelin debris and remyelination of axon segments. These data for the first time demonstrate that MCT1 is critical for regeneration of both sensory and motor axons in mice following sciatic nerve crush.

Delayed priming promotes CNS regeneration post-rhizotomy in Neurocan and Brevican-deficient mice.

A wealth of literature has provided evidence that reactive tissue at the site of CNS injury is rich in chondroitin sulfate proteoglycans which may contribute to the non-permissive nature of the CNS. We have recently demonstrated using a murine model of human brachial plexus injury that the chondroitin sulfate proteoglycans Neurocan and Brevican are differentially expressed by two subsets of astrocytes in the spinal cord dorsal root entry zone (DREZ) following dorsal root lesion (Beggah et al., Neuroscience 133: 749-762, 2005). However, direct evidence for a growth-inhibitory role of these proteoglycans in vivo is still lacking. We therefore performed dorsal root lesion (rhizotomy) in mice deficient in both Neurocan and Brevican. Rhizotomy in these animals resulted in no significant increase in the number of sensory fibres regenerating through the DREZ compared to genetically matched controls. Likewise, a conditioning peripheral nerve lesion prior to rhizotomy, which increases the intrinsic growth capacity of sensory neurons, enhanced growth to the same extent in transgenic and control mice, indicating that absence of these proteoglycans alone is not sufficient to further promote entry into the spinal cord. In contrast, when priming of the median nerve was performed at a clinically relevant time, i.e. 7 weeks post-rhizotomy, the growth of a subpopulation of sensory axons across the DREZ was facilitated in Neurocan/Brevican-deficient, but not in control animals. This demonstrates for the first time that (i) Neurocan and/or Brevican contribute to the non-permissive environment of the DREZ several weeks after lesion and that (ii) delayed stimulation of the growth program of sensory neurons can facilitate regeneration across the DREZ provided its growth-inhibitory properties are attenuated. Post-injury enhancement of the intrinsic growth capacity of sensory neurons combined with removal of inhibitory chondroitin sulfate proteoglycans may therefore help to restore sensory function and thus attenuate the chronic pain resulting from human brachial plexus injury.

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration.

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.

Changes in nuclear 3,5,3'-triiodothyronine receptor expression in rat dorsal root ganglia and sciatic nerve during development: comparison with regeneration.

The action of the thyroid hormones on responsive cells in the peripheral nervous system requires the presence of nuclear triiodothyronine receptors (NT3R). These nuclear receptors, including both the alpha and beta subtypes of NT3R, were visualized by immunocytochemistry with the specific 2B3 monoclonal antibody. In the dorsal root ganglia (DRG) of rat embryos, NT3R immunoreactivity was first discretely revealed in a few neurons at embryonic day 14 (E14), then strongly expressed by all neurons at E17 and during the first postnatal week; all DRG neurons continued to possess clear NT3R immunostaining, which faded slightly with age. The peripheral glial cells in the DRG displayed a short-lived NT3R immunoreaction, starting at E17 and disappearing from the satellite and Schwann cells by postnatal days 3 and 7 respectively. In the developing sciatic nerve, Schwann cells also exhibited transient NT3R immunoreactivity restricted to a short period ranging from E17 to postnatal day 10; the NT3R immunostaining of the Schwann cells vanished proximodistally along the sciatic nerve, so that the Schwann cells rapidly became free of detectable NT3R immunostaining. However, after the transection or crushing of an adult sciatic nerve, the NT3R immunoreactivity reappeared in the Schwann cells adjacent to the lesion by 2 days, then along the distal segment in which the axons were degenerating, and finally disappeared by 45 days, when the regenerating axons were allowed to re-occupy the distal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathfinding of corticothalamic axons relies on a rendezvous with thalamic projections.

Major outputs of the neocortex are conveyed by corticothalamic axons (CTAs), which form reciprocal connections with thalamocortical axons, and corticosubcerebral axons (CSAs) headed to more caudal parts of the nervous system. Previous findings establish that transcriptional programs define cortical neuron identity and suggest that CTAs and thalamic axons may guide each other, but the mechanisms governing CTA versus CSA pathfinding remain elusive. Here, we show that thalamocortical axons are required to guide pioneer CTAs away from a default CSA-like trajectory. This process relies on a hold in the progression of cortical axons, or waiting period, during which thalamic projections navigate toward cortical axons. At the molecular level, Sema3E/PlexinD1 signaling in pioneer cortical neurons mediates a "waiting signal" required to orchestrate the mandatory meeting with reciprocal thalamic axons. Our study reveals that temporal control of axonal progression contributes to spatial pathfinding of cortical projections and opens perspectives on brain wiring.

Extracellular matrix components in peripheral nerve repair: how to affect neural cellular response and nerve regeneration?

Peripheral nerve injury is a serious problem affecting significantly patients' life. Autografts are the "gold standard" used to repair the injury gap, however, only 50% of patients fully recover from the trauma. Artificial conduits are a valid alternative to repairing peripheral nerve. They aim at confining the nerve environment throughout the regeneration process, and providing guidance to axon outgrowth. Biocompatible materials have been carefully designed to reduce inflammation and scar tissue formation, but modifications of the inner lumen are still required in order to optimise the scaffolds. Biomicking the native neural tissue with extracellular matrix fillers or coatings showed great promises in repairing longer gaps and extending cell survival. In addition, extracellular matrix molecules provide a platform to further bind growth factors that can be released in the system over time. Alternatively, conduit fillers can be used for cell transplantation at the injury site, reducing the lag time required for endogenous Schwann cells to proliferate and take part in the regeneration process. This review provides an overview on the importance of extracellular matrix molecules in peripheral nerve repair.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Role of thyroid hormones and their receptors in peripheral nerve regeneration.

After peripheral nerve injury in adult mammals, reestablishment of functional connections depends on several parameters including neurotrophic factors, the extracellular matrix, and hormones. However, little is known about the contribution of hormones to peripheral nerve regeneration. Thyroid hormones, which are required for the development and maturation of the central nervous system, are also important for the development of peripheral nerves. The action of triiodothyronine (T3) on responsive cells is mediated through nuclear thyroid hormone receptors (TRs) which modulate the expression of specific genes in target cells. Thus, to study the effect of T3, it is first necessary to know whether the target tissues possess TRs. The fact that sciatic nerve cells possess functional TRs suggests that these cells can respond to T3 and, as a consequence, that thyroid hormone may be involved in peripheral nerve regeneration. The silicone nerve guide model provides an excellent system to study the action of local administration of T3. Evidence from such studies demonstrate that animals treated locally with T3 at the level of transection have more complete regeneration of sciatic nerve and better functional recovery. Among the possible regulatory mechanisms by which T3 enhances peripheral nerve regeneration is rapid action on both axotomized neurons and Schwann cells which, in turn, produce a lasting and stimulatory effect on peripheral nerve regeneration. It is probable that T3 up- or down-regulates gene expression of one or more growth factors, extracellular matrix, or cell adhesion molecules, all of which stimulate peripheral nerve regeneration. This could explain the greater effect of T3 on nerve regeneration compared with the effect of any one growth factor or adhesion molecule.