Axon Regeneration In Peripheral Nerves Research Articles

Fibroblast exosomal TFAP2C induced by chitosan oligosaccharides promotes peripheral axon regeneration via the miR-132-5p/CAMKK1 axis.

Chitosan and its degradation product, oligosaccharides, have been shown to facilitate peripheral nerve regeneration. However, the underlying mechanisms are not well understood. In this study, we analyzed the protein expression profiles in sciatic nerves after injury using proteomics. A group of proteins related to exosome packaging and transport is up-regulated by chitosan oligosaccharides (COS), implying that exosomes are involved in COS-induced peripheral nerve regeneration. In fact, exosomes derived from fibroblasts (f-EXOs) treated with COS significantly promoted axon extension and regeneration. Exosomal protein identification and functional studies, revealed that TFAP2C is a key factor in neurite outgrowth induced by COS-f-EXOs. Furthermore, we showed that TFAP2C targets the pri-miRNA-132 gene and represses miR-132-5p expression in dorsal root ganglion neurons. Camkk1 is a downstream substrate of miR-132-5p that positively affects axon extension. In rats, miR-132-5p antagomir stimulates CAMKK1 expression and improves axon regeneration and functional recovery in sciatic nerves after injury. Our data reveal the mechanism for COS in axon regeneration, that is COS induce fibroblasts to produce TFAP2C-enriched EXOs, which are then transferred into axons to promote axon regeneration via miR-132-5p/CAMKK1. Moreover, these results show a new facet of fibroblasts in axon regeneration in peripheral nerves.

Mitochondrial Bioenergetic, Photobiomodulation and Trigeminal Branches Nerve Damage, What's the Connection? A Review.

Background: Injury of the trigeminal nerve in oral and maxillofacial surgery can occur. Schwann cell mitochondria are regulators in the development, maintenance and regeneration of peripheral nerve axons. Evidence shows that after the nerve injury, mitochondrial bioenergetic dysfunction occurs and is associated with pain, neuropathy and nerve regeneration deficit. A challenge for research is to individuate new therapies able to normalise mitochondrial and energetic metabolism to aid nerve recovery after damage. Photobiomodulation therapy can be an interesting candidate, because it is a technique involving cell manipulation through the photonic energy of a non-ionising light source (visible and NIR light), which produces a nonthermal therapeutic effect on the stressed tissue. Methods: The review was based on the following questions: (1) Can photo-biomodulation by red and NIR light affect mitochondrial bioenergetics? (2) Can photobiomodulation support damage to the trigeminal nerve branches? (preclinical and clinical studies), and, if yes, (3) What is the best photobiomodulatory therapy for the recovery of the trigeminal nerve branches? The papers were searched using the PubMed, Scopus and Cochrane databases. This review followed the ARRIVE-2.0, PRISMA and Cochrane RoB-2 guidelines. Results and conclusions: The reliability of photobiomodulatory event strongly bases on biological and physical-chemical evidence. Its principal player is the mitochondrion, whether its cytochromes are directly involved as a photoacceptor or indirectly through a vibrational and energetic variation of bound water: water as the photoacceptor. The 808-nm and 100 J/cm2 (0.07 W; 2.5 W/cm2; pulsed 50 Hz; 27 J per point; 80 s) on rats and 800-nm and 0.2 W/cm2 (0.2 W; 12 J/cm2; 12 J per point; 60 s, CW) on humans resulted as trustworthy therapies, which could be supported by extensive studies.

Functional Polymer‐Based Nerve Guide Conduits to Promote Peripheral Nerve Regeneration

AbstractBridging critical‐sized defects in peripheral nerves to achieve functional recovery is a challenge for orthopedic and hand surgeons. Inadequate regeneration of peripheral nerve axons often results in long‐term partial or total sensory and/or motor impairment. Currently, the best treatment available for long‐gap peripheral nerve regeneration is autologous nerve transplantation, while the successful implementation of this approach requires for secondary surgery and donor nerves. The nerve guide conduit (NGC) serves as an alternative to autograft of nerve, as it connects the proximal and distal ends of nerve defects and provides physical and biochemical guidances for axon regeneration. Functionalized NGCs enhance nerve regeneration by providing neuroprotection, antioxidation, vascular regeneration enhancement, and immune regulatory effects. In this review, the authors summarize the latest advances in functional polymer‐based NGCs for peripheral nerve regeneration and present the perspectives on the development of peripheral NGCs for potential clinical applications.

Assisted peripheral nerve recovery by KMUP-1, an activator of large-conductance Ca2+-activated potassium channel, in a rat model of sciatic nerve crush injury

Axonal regeneration in peripheral nerves after injury is a complicated process. Numerous cytokines, growth factors, channels, kinases, and receptors are involved, and matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis subsequent to nerve injury. In this study, the effect of KMUP-1, an activator of large-conductance Ca(2+)-activated potassium channel, on functional recovery, myelinated axon growth, and immunoreactivity of MMP-9 was evaluated in rats subjected to sciatic nerve crush injury. A total of 144 male Sprague-Dawley rats were divided into the following six groups (n = 24/group): group 1, sham-operated; group 2, sciatic nerve injury without treatment; group 3, injured and vehicle-treated; group 4, injured and treated with 1 mM KMUP-1 by topical application; group 5, injured and treated with 10 mM KMUP-1; group 6, injured and treated with 50 mM KMUP-1. Functional recovery was evaluated using walking track analysis at 1, 2, 3, and 4 weeks (n = 6/group at each time point) after injury. In addition, the number of myelinated axons and MMP-9 in the nerve was also examined. Animals subjected to sciatic nerve crush injury had decreased motor function, a reduced number of myelinated axons, and increased MMP-9 in the nerve. Treatment with KMUP-1 concentration-dependently improved functional recovery, increased the number of myelinated axons, and decreased MMP-9. These results suggest that KMUP-1 may be a novel agent for assisting peripheral nerve recovery after injury. The beneficial effect is probably due to known ability of the compound in activating the nitric oxide/cGMP/protein kinase G pathway.

The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves

The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.

Neurotrophin‐4/5 is implicated in the enhancement of axon regeneration produced by treadmill training following peripheral nerve injury

The role of neurotrophin-4/5 (NT-4/5) in the enhancement of axon regeneration in peripheral nerves produced by treadmill training was studied in mice. Common fibular nerves of animals of the H strain of thy-1-YFP mice, in which a subset of axons in peripheral nerves is marked by the presence of yellow fluorescent protein, were cut and surgically repaired using nerve grafts from non-fluorescent mice. Lengths of profiles of fluorescent regenerating axons were measured using optical sections made through whole mounts of harvested nerves. Measurements from mice that had undergone 1 h of daily treadmill training at modest speed (10 m/min) were compared with those of untrained (control) mice. Modest treadmill training resulted in fluorescent axon profiles that were nearly twice as long as controls at 1, 2 and 4 week survival times. Similar enhanced regeneration was found when cut nerves of wild type mice were repaired with grafts from NT-4/5 knockout mice or grafts made acellular by repeated freezing/thawing. No enhancement was produced by treadmill training in NT-4/5 knockout mice, irrespective of the nature of the graft used to repair the cut nerve. Much as had been observed previously for the effects of brief electrical stimulation, the effects of treadmill training on axon regeneration in cut peripheral nerves are independent of changes produced in the distal segment of the cut nerve and depend on the promotion of axon regeneration by changes in NT-4/5 expression by cells in the proximal nerve segment.

Effects of Upslope Treadmill Exercise On Axon Regeneration in Peripheral Nerves

Treadmill exercise after sciatic nerve injury enhances subsequent axon regeneration. However, this enhancement is weighted more towards motor axons previously innervating ankle extensor rather than flexor muscles. We hypothesized that a more exuberant regeneration of ankle flexor axons, and a more balanced enhancement of axon regeneration could be achieved by manipulating the mechanical demands of the training paradigm. PURPOSE: To compare the effects of level (Lv) and upslope (Us) treadmill exercise, during which there is more intense ankle flexor activation, on enhancing axon regeneration following peripheral nerve injury. METHODS: Different retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial (Tib) and common fibular (CF) nerves, the primary conduits for ankle extensor and ankle flexor motor axons, respectively, 2 weeks after transection and repair of the mouse sciatic nerve. Retrogradely labeled motoneurons (MNs) were studied in untreated (UT) control mice, and two groups of treadmill-trained mice (10 m/minute, 1 hour/day, 5 days/week, 2 weeks), one running at Lv and the other walking Us on a 20 deg incline. Paired and unpaired t-tests were used to compare slopes and treatment groups. RESULTS: More labeled MNs were found in both trained groups, but Us trained mice had 15-fold (p=0.01) and 1.5-fold (p=0.01) increases compared to UT mice and Lv trained mice, respectively. Nearly twice as many MNs could be labeled from the CF nerve in Us trained mice than from Lv trained animals (p<0.05). No significant change was found in the number of MNs labeled from the Tib nerve in Us vs Lv trained mice (p=0.16). In Us trained mice, the proportions of MNs labeled from the CF nerve that were found in spinal cord locations reserved for Tib MNs in intact mice was greater than in UT control mice (p=0.01) and Lv trained mice (p<0.05). CONCLUSION: Us training in the first 2 weeks following peripheral nerve injury produces an increased enhancement axon regeneration of MNs that are more active during Us walking. It may be possible to tailor exercise to maximize its rehabilitative potential after peripheral nerve injury.

Diffusion tensor imaging to assess axonal regeneration in peripheral nerves

Development of outcome measures to assess ongoing nerve regeneration in the living animal that can be translated to human can provide extremely useful tools for monitoring the effects of therapeutic interventions to promote nerve regeneration. Diffusion tensor imaging (DTI), a magnetic resonance based technique, provides image contrast for nerve tracts and can be applied serially on the same subject with potential to monitor nerve fiber content. In this study, we examined the use of ex vivo high-resolution DTI for imaging intact and regenerating peripheral nerves in mice and correlated the MRI findings with electrophysiology and histology. DTI was done on sciatic nerves with crush, without crush, and after complete transection in different mouse strains. DTI measures, including fractional anisotropy (FA), parallel diffusivity, and perpendicular diffusivity were acquired and compared in segments of uninjured and crushed/transected nerves and correlated with morphometry. A comparison of axon regeneration after sciatic nerve crush showed a comparable pattern of regeneration in different mice strains. FA values were significantly lower in completely denervated nerve segments compared to uninjured sciatic nerve and this signal was restored toward normal in regenerating nerve segments (crushed nerves). Histology data indicate that the FA values and the parallel diffusivity showed a positive correlation with the total number of regenerating axons. These studies suggest that DTI is a sensitive measure of axon regeneration in mouse models and provide basis for further development of imaging technology for application to living animals and humans.

Regenerative arrest of inflamed peripheral nerves: role of nitric oxide

Inflammation can both support and hinder regeneration. In this work, we asked whether regeneration of peripheral nerve axons is facilitated or interrupted when it proceeds through a zone of local but nondirected inflammation. Regeneration was examined in new nerve bridges forming through conduits connecting transected rat sciatic nerves. The conduits, infused with lipopolysaccharide to generate a sterile and nondirected inflammatory response, had substantial rises in inducible nitric oxide (iNOS) mRNA synthesis. iNOS was expressed within macrophages just beyond the zone of axon regrowth. Under these conditions, there was complete interruption of regenerative bridge formation in all instances without axon regrowth across the transection. In a separate cohort, infusion of a broad-spectrum NOS inhibitor (Nomega-nitro-L-arginine-methyl ester) into the conduit salvaged bridge formation in a proportion (3/8) of rats. Our findings indicate that local inflammatory conditions inhibit regenerative events and that nitric oxide may contribute to these events.

Mechanisms of neuroprotective effect of estrogens associated with vascular endothelial growth factor expression

The results of axonal regeneration in peripheral nerves and of the modulating effect of estrogens on the posttraumatic plasticity of motor neurons through the classical and alternative genomic mechanisms involving the activation of estrogen receptors ERalpha and ERbeta and increased transcription of specific neuroprotective control genes. New mechanisms of the neuroprotective effect of estrogens related to the cross interaction of estrogens with intracellular signaling cascades of many other biologically active compounds, in particular, vascular endothelial growth factor and endothelin, are proposed.

Enhancing axon regeneration in peripheral nerves also increases functionally inappropriate reinnervation of targets

The specificity of reinnervation of peripheral targets by regenerating motor axons was studied in mice by using retrograde fluorescent tracers applied to the cut ends of the tibial and common fibular nerves after transection and surgical repair of the sciatic nerve. When the nerve ends were aligned and secured with fibrin glue, more motoneurons labeled after application of tracer to the common fibular nerve were found in regions of the spinal cord that normally contain only tibial motoneurons. The magnitude of such inappropriate reinnervation did not change at different times after repair. Intentional misalignment of the cut nerve stumps at the time of repair resulted in more extensive inappropriate reinnervation of the different peripheral targets. If the proximal stump of the cut nerve was electrically stimulated at the time of repair, if the distal stump was treated with chondroitinase ABC, or if both protocols were applied, the number of motoneurons labeled was increased. This increase was accompanied by more extensive reinnervation of inappropriate targets than found after untreated nerve repair. Although alterations in the caudorostral distributions of labeled motoneurons observed were not as great as observed after purposeful misalignment of the cut nerve ends, the topographic relationship between the spinal locations of motoneuron somata and the peripheral targets of their axons is disrupted. Enhancement of motor axon regeneration by induction of growth-promoting signaling pathways, reduction in growth inhibition in the environment of regenerating axons, or both, is accompanied by an increase in the amount of functionally inappropriate reinnervation of peripheral targets.

Axon regeneration in peripheral nerves is enhanced by proteoglycan degradation

Regeneration of axons in the peripheral nervous system is enhanced by the removal of glycosaminoglycan side chains (GAGs) of chondroitin sulfate proteoglycans. However, some axons regenerate poorly despite such treatment, suggesting the existence of additional inhibitors. We compared the effects of enzymatic removal of GAGs from chondroitin sulfate proteoglycans versus two other proteoglycan species, heparan sulfate and keratan sulfate proteoglycans, on the regeneration of peripheral axons. Common fibular (CF) nerves of thy-1-YFP-H mice were cut and repaired using short segments of CF nerves harvested from wild-type littermates and pre-treated with a GAG-degrading enzyme for 1 h prior to nerve repair. Axonal regeneration was assayed by measuring the lengths of profiles of YFP + axons in optical sections of the grafted nerves 1 week later. Except for grafts treated with keratanase, more and longer axon profiles were encountered in enzyme-treated grafts than in control grafts. Heparinase III treatments induced the greatest number of axons to enter into the graft. The proportions of axon profiles longer than 1000 μm were greater in grafts treated with chondroitinase ABC or heparinase I, but not with either keratanase or heparinase III. More regenerative sprouts were observed after treatment with heparinase I than any other enzymes. Treatment with a mixture of all four enzymes resulted in an enhancement of axon regeneration which was greater than that observed after treatment with any of the enzymes individually. The effects of chondroitinase ABC and heparinase III were correlated with specific GAG degradation. We believe that enzymatic removal of GAGs is especially effective in promoting the ability of regenerating axons to select their pathway in the distal stump (or nerve graft) and, in the case of chondroitinase ABC or heparinase I, it may also promote growth within that pathway.

Oxidized galectin-1 stimulates macrophages to promote axonal regeneration in peripheral nerves after axotomy.

Various neurotrophic factors that promote axonal regeneration have been investigated in vivo, but the signals that prompt neurons to send out processes in peripheral nerves after axotomy are not well understood. Previously, we have shown oxidized galectin-1 (GAL-1/Ox) promotes initial axonal growth after axotomy in peripheral nerves. However, the mechanism by which GAL-1/Ox promotes axonal regeneration remains unclear and is the subject of the present study. To identify possible target cells of GAL-1/Ox, a fluorescently labeled recombinant human GAL-1/Ox (rhGAL-1/Ox) was incubated with DRG neurons, Schwann cells, and intraperitoneal macrophages from adult rats. Only the cell surfaces of intraperitoneal macrophages bound the rhGAL-1/Ox, suggesting that these cells possess a receptor for GAL-1/Ox. Experiments examining tyrosine phosphorylation revealed that rhGAL-1/Ox stimulated changes in signal transduction pathways in these macrophages. These changes caused macrophages to secrete an axonal growth-promoting factor. This was demonstrated when conditioned media of macrophages stimulated with rhGAL-1/Ox in 48 hr culture strongly enhanced axonal regeneration from transected-nerve sites of DRG explants. Furthermore, activated macrophage-conditioned media also improved Schwann cell migration from the transected-nerve sites. From these results, we propose that axonal regeneration occurs in axotomized peripheral nerves as a result of cytosolic reduced galectin-1 being released from Schwann cells and injured axons, which then becomes oxidized in the extracellular space. Oxidized galectin-1 then stimulates macrophages to secrete a factor that promotes axonal growth and Schwann cell migration, thus enhancing peripheral nerve regeneration.

Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts.

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitally at 2 mm (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

30 mm regeneration of rat sciatic nerve along collagen filaments

This paper describes 30 mm regeneration of peripheral nerve axons along collagen filaments; 31-mm-long collagen filaments or collagen tube were grafted to bridge a 30-mm defect of rat sciatic nerve. The mean number and the diameter of regenerated myelinated axons were 330±227 and 2.7±0.9 μm at the distal end of the collagen-filaments 12 weeks postoperatively; while at the distal end of the tube no axon was found.

Collagen filaments as a scaffold for nerve regeneration

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 μm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 μm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments. © 2001 John Wiley & Sons, Inc. J Biomed Mater Res 56: 400–405, 2001

Collagen filaments as a scaffold for nerve regeneration.

This article describes repair of peripheral nerve defect using collagen filaments instead of tubes. Many tube-shaped nerve guides induce regeneration of severed peripheral nerve axons within a limited distance. Substantial regeneration of nerve axons has not been reported without a tubular conduit. Here we show the regeneration of peripheral nerve axons along filaments of collagen without a tube. Cables of collagen filaments were grafted to repair 20-mm defects of rat sciatic nerves. Nerve autografts and collagen tubes were grafted as controls. The mean number and the mean fiber diameter of regenerated myelinated axons were approximately 4800 and 3.3 microm in the distal end of the nerve autograft at 8 weeks postoperatively while in the distal end of the collagen-filaments nerve guide, they were approximately 5500 and 2.3 microm. Collagen tubes failed to bridge the nerve defect. Histologic studies suggest that nerve axons regenerated substantially along the collagen filaments.

Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties

Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties

Oxidized galectin-1 promotes axonal regeneration in peripheral nerves but does not possess lectin properties.

Galectin-1 has recently been identified as a factor that regulates initial axonal growth in peripheral nerves after axotomy. Although galectin-1 is a well-known beta-galactoside-binding lectin, its potential to promote axonal regeneration as a lectin has not been reported. It is essential that the process of initial repair in peripheral nerves after axotomy is well clarified. We therefore undertook to investigate the relation between the structure and axonal regeneration-promoting activity of galectin-1. Recombinant human galectin-1 secreted into the culture supernatant of transfected COS1 cells (rhGAL-1/COS1) was purified under nonreducing conditions and subjected to structural analysis. Mass spectrometric analysis of peptide fragments from rhGAL-1/COS1 revealed that the secreted protein exists as an oxidized form containing three intramolecular disulfide bonds (Cys2-Cys130, Cys16-Cys88 and Cys42-Cys60). Recombinant human galectin-1 (rhGAL-1) and a galectin-1 mutant in which all six cysteine residues were replaced by serine (CSGAL-1) were expressed in and purified from Escherichia coli for further analysis; the purified rhGAL-1 was subjected to oxidation, which induced the same pattern of disulfide linkages as that observed in rhGAL-1/COS1. Oxidized rhGAL-1 enhanced axonal regeneration from the transected nerve sites of adult rat dorsal root ganglion explants with associated nerve stumps (5.0-5000 pg. mL-1), but it lacked lectin activity. In contrast, CSGAL-1 induced hemagglutination of rabbit erythrocytes but lacked axonal regeneration-promoting activity. These results indicate that galectin-1 promotes axonal regeneration only in the oxidized form containing three intramolecular disulfide bonds, not in the reduced form which exhibits lectin activity.

Oxidized galectin-1 regulates macrophages to promote axonal regeneration in peripheral nerves

Oxidized galectin-1 regulates macrophages to promote axonal regeneration in peripheral nerves