Role of transforming growth factor-βin peripheral nerve regeneration

Injuries caused by trauma and neurodegenerative diseases can damage the peripheral nervous system and cause functional deficits.Unlike in the central nervous system,damaged axons in peripheral nerves can be induced to regenerate in response to intrinsic cues after reprogramming or in a growth-promoting microenvironment created by Schwann cells.However,axon regeneration and repair do not automatically result in the restoration of function,which is the ultimate therapeutic goal but also a major clinical challenge.Transforming growth factor(TGF)is a multifunctional cytokine that regulates various biological processes including tissue repair,embryo development,and cell growth and differentiation.There is accumulating evidence that TGF-βfamily proteins participate in peripheral nerve repair through various factors and signaling pathways by regulating the growth and transformation of Schwann cells;recruiting specific immune cells;controlling the permeability of the blood-nerve barrier,thereby stimulating axon growth;and inhibiting remyelination of regenerated axons.TGF-βhas been applied to the treatment of peripheral nerve injury in animal models.In this context,we review the functions of TGF-βin peripheral nerve regeneration and potential clinical applications.

Chemokine platelet factor 4 accelerates peripheral nerve regeneration by regulating Schwann cell activation and axon elongation

Schwann cells in peripheral nerves react to traumatic nerve injury by attempting to grow and regenerate.Howeve r,it is unclear what factors play a role in this process.In this study,we searched a GEO database and found that expression of platelet factor 4 was markedly up-regulated after sciatic nerve injury.Platelet factor is an important molecule in cell apoptosis,diffe rentiation,survival,and proliferation.Further,polymerase chain reaction and immunohistochemical staining confirmed the change in platelet factor 4 in the sciatic nerve at different time points after injury.Enzyme-linked immunosorbent assay confirmed that platelet factor 4 was secreted by Schwann cells.We also found that silencing platelet factor 4 decreased the proliferation and migration of primary cultured Schwann cells,while exogenously applied platelet factor 4 stimulated Schwann cell prolife ration and migration and neuronal axon growth.Furthermore,knocking out platelet factor 4 inhibited the prolife ration of Schwann cells in injured rat sciatic nerve.These findings suggest that Schwann cell-secreted platelet factor 4 may facilitate peripheral nerve repair and regeneration by regulating Schwann cell activation and axon growth.Thus,platelet factor 4 may be a potential therapeutic target for traumatic peripheral nerve injury.

A functional tacrolimus-releasing nerve wrap for enhancing nerve regeneration following surgical nerve repair

Axonal regeneration following surgical nerve repair is slow and often incomplete,resulting in poor functional recovery which sometimes contributes to lifelong disability.Currently,there are no FDA-approved therapies available to promote nerve regeneration.Tacrolimus accelerates axonal regeneration,but systemic side effects presently outweigh its potential benefits for peripheral nerve surgery.The authors describe herein a biodegradable polyurethane-based drug delivery system for the sustained local release of tacrolimus at the nerve repair site,with suitable properties for scalable production and clinical application,aiming to promote nerve regeneration and functional recovery with minimal systemic drug exposure.Tacrolimus is encapsulated into co-axially electrospun polycarbonate-urethane nanofibers to generate an implantable nerve wrap that releases therapeutic doses of bioactive tacrolimus over 31 days.Size and drug loading are adjustable for applications in small and large caliber nerves,and the wrap degrades within 120 days into biocompatible byproducts.Tacrolimus released from the nerve wrap promotes axon elongation in vitro and accelerates nerve regeneration and functional recovery in preclinical nerve repair models while off-target systemic drug exposure is reduced by 80%compared with systemic delivery.Given its surgical suitability and preclinical efficacy and safety,this system may provide a readily translatable approach to support axonal regeneration and recovery in patients undergoing nerve surgery.

A novel flexible nerve guidance conduit promotes nerve regeneration while providing excellent mechanical properties

Autografting is the gold standard for surgical repair of nerve defects>5 mm in length;however,autografting is associated with potential complications at the nerve donor site.As an alternative,nerve guidance conduits may be used.The ideal conduit should be flexible,resistant to kinks and lumen collapse,and provide physical cues to guide nerve regeneration.We designed a novel flexible conduit using electrospinning technology to create fibers on the innermost surface of the nerve guidance conduit and employed melt spinning to align them.Subsequently,we prepared disordered electrospun fibers outside the aligned fibers and helical melt-spun fibers on the outer wall of the electrospun fiber lumen.The presence of aligned fibers on the inner surface can promote the extension of nerve cells along the fibers.The helical melt-spun fibers on the outer surface can enhance resistance to kinking and compression and provide stability.Our novel conduit promoted nerve regeneration and functional recovery in a rat sciatic nerve defect model,suggesting that it has potential for clinical use in human nerve injuries.

FK506 contributes to peripheral nerve regeneration by inhibiting neuroinflammatory responses and promoting neuron survival

FK506(Tacrolimus)is a systemic immunosuppressant approved by the U.S.Food and Drug Administration.FK506 has been shown to promote peripheral nerve regeneration,however,its precise mechanism of action and its pathways remain unclear.In this study,we established a rat model of sciatic nerve injury and found that FK506 improved the morphology of the injured sciatic nerve,increased the numbers of motor and sensory neurons,reduced inflammatory responses,markedly improved the conduction function of the injured nerve,and promoted motor function recovery.These findings suggest that FK506 promotes peripheral nerve structure recovery and functional regeneration by reducing the intensity of inflammation after neuronal injury and increasing the number of surviving neurons.

Runx2 regulates peripheral nerve regeneration to promote Schwann cell migration and re-myelination

Runx2 is a major regulator of osteoblast differentiation and function;however,the role of Runx2 in peripheral nerve repair is unclea r.Here,we analyzed Runx2expression following injury and found that it was specifically up-regulated in Schwann cells.Furthermore,using Schwann cell-specific Runx2 knocko ut mice,we studied peripheral nerve development and regeneration and found that multiple steps in the regeneration process following sciatic nerve injury were Runx2-dependent.Changes observed in Runx2 knoc kout mice include increased prolife ration of Schwann cells,impaired Schwann cell migration and axonal regrowth,reduced re-myelination of axo ns,and a block in macrophage clearance in the late stage of regeneration.Taken together,our findings indicate that Runx2 is a key regulator of Schwann cell plasticity,and therefore peripheral nerve repair.Thus,our study shows that Runx2 plays a major role in Schwann cell migration,re-myelination,and peripheral nerve functional recovery following injury.

Blockade of Rho-associated kinase prevents inhibition of axon regeneration of peripheral nerves induced by anti-ganglioside antibodies

Anti-ganglioside antibodies are associated with delayed/poor clinical recovery in Guillain-Barrèsyndrome,mostly related to halted axon regeneration.Cross-linking of cell surface gangliosides by anti-ganglioside antibodies triggers inhibition of nerve repair in in vitro and in vivo paradigms of axon regeneration.These effects involve the activation of the small GTPase Rho A/ROCK signaling pathways,which negatively modulate growth cone cytoskeleton,similarly to well stablished inhibitors of axon regeneration described so far.The aim of this work was to perform a proof of concept study to demonstrate the effectiveness of Y-27632,a selective pharmacological inhibitor of ROCK,in a mouse model of axon regeneration of peripheral nerves,where the passive immunization with a monoclonal antibody targeting gangliosides GD1a and GT1b was previously reported to exert a potent inhibitory effect on regeneration of both myelinated and unmyelinated fibers.Our results demonstrate a differential sensitivity of myelinated and unmyelinated axons to the pro-regenerative effect of Y-27632.Treatment with a total dosage of 9 mg/kg of Y-27632 resulted in a complete prevention of anti-GD1a/GT1b monoclonal antibody-mediated inhibition of axon regeneration of unmyelinated fibers to skin and the functional recovery of mechanical cutaneous sensitivity.In contrast,the same dose showed toxic effects on the regeneration of myelinated fibers.Interestingly,scale down of the dosage of Y-27632 to 5 mg/kg resulted in a significant although not complete recovery of regenerated myelinated axons exposed to anti-GD1a/GT1b monoclonal antibody in the absence of toxicity in animals exposed to only Y-27632.Overall,these findings confirm the in vivo participation of Rho A/ROCK signaling pathways in the molecular mechanisms associated with the inhibition of axon regeneration induced by anti-GD1a/GT1b monoclonal antibody.Our findings open the possibility of therapeutic pharmacological intervention targeting Rho A/Rock pathway in immune neuropathies associated with the presence of anti-ganglioside antibodies and delayed or incomplete clinical recovery after injury in the peripheral nervous system.

Enhanced axonal regeneration and functional recovery of the injured sciatic nerve in a rat model by lithium-loaded electrospun nanofibrous scaffolds

Increasing evidence indicates that engineered nerve grafts have great potential for the regeneration of peripheral nerve injuries(PNIs).While most studies have focused only on the topographical features of the grafts,we have considered both the biophysical and biochemical manipulations in our applied nanoscaffold.To achieve this,we fabricated an electrospun nanofibrous scaffold(ENS)containing polylactide nanofibers loaded with lithium(Li)ions,a Wnt/β-catenin signaling activator.In addition,we seeded human adipose-derived mesenchymal stem cells(hADMSCs)onto this engineered scaffold to examine if their differentiation toward Schwann-like cells was induced.We further examined the efficacy of the scaffolds for nerve regeneration in vivo via grafting in a PNI rat model.Our results showed that Li-loaded ENSs gradually released Li within 11 d,at concentrations ranging from 0.02 to(3.64±0.10)mmol/L,and upregulated the expression of Wnt/β-catenin target genes(cyclinD1 and c-Myc)as well as those of Schwann cell markers(growth-associated protein 43(GAP43),S100 calcium binding protein B(S100B),glial fibrillary acidic protein(GFAP),and SRY-box transcription factor 10(SOX10))in differentiated hADMSCs.In the PNI rat model,implantation of Li-loaded ENSs with/without cells improved behavioral features such as sensory and motor functions as well as the electrophysiological characteristics of the injured nerve.This improved function was further validated by histological analysis of sciatic nerves grafted with Li-loaded ENSs,which showed no fibrous connective tissue but enhanced organized myelinated axons.The potential of Li-loaded ENSs in promoting Schwann cell differentiation of hADMSCs and axonal regeneration of injured sciatic nerves suggests their potential for application in peripheral nerve tissue engineering.

Harnessing endothelial cells and vascularization strategies for nerve regeneration

Peripheral nerves are essential components of the human body’s communication system,transmitting signals between the central nervous system and various body parts.Damage resulting from trauma or disease can result in debilitating sensory and motor deficits.Nerve injuries,particularly those resulting in significant gaps in the nerve tissue,pose a formidable challenge for clinicians and researchers.Despite their limitations,including limited availability and donor site morbidity,nerve autografts remain the clinical gold standard for treating nerve injuries.

Role of pituitary adenylate cyclase-activating polypeptide in peripheral nerve regeneration:a cellular and molecular perspective

Neuro regeneration is a very complex phenomenon characterized by the generation of new neurons and synapses,involving connections between adjacent cells and axonal projections.Neuroregeneration supplies additional longterm resources to replace those altered by the injury and ensure lasting functional recovery.

Autophagy-targeting modulation to promote peripheral nerve regeneration

Nerve regeneration following traumatic peripheral nerve injuries and neuropathies is a complex process modulated by diverse factors and intricate molecular mechanisms.Past studies have focused on factors that stimulate axonal outgrowth and myelin regeneration.However,recent studies have highlighted the pivotal role of autophagy in peripheral nerve regeneration,particularly in the context of traumatic injuries.Consequently,autophagy-targeting modulation has emerged as a promising therapeutic approach to enhancing peripheral nerve regeneration.Our current understanding suggests that activating autophagy facilitates the rapid clearance of damaged axons and myelin sheaths,thereby enhancing neuronal survival and mitigating injury-induced oxidative stress and inflammation.These actions collectively contribute to creating a favorable microenvironment for structural and functional nerve regeneration.A range of autophagyinducing drugs and interventions have demonstrated beneficial effects in alleviating peripheral neuropathy and promoting nerve regeneration in preclinical models of traumatic peripheral nerve injuries.This review delves into the regulation of autophagy in cell types involved in peripheral nerve regeneration,summarizing the potential drugs and interventions that can be harnessed to promote this process.We hope that our review will offer novel insights and perspectives on the exploitation of autophagy pathways in the treatment of peripheral nerve injuries and neuropathies.

miRNA-21-5p is an important contributor to the promotion of injured peripheral nerve regeneration using hypoxia-pretreated bone marrow-derived neural crest cells

Our previous study found that rat bone marrow–derived neural crest cells(acting as Schwann cell progenitors)have the potential to promote long-distance nerve repair.Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication.Nevertheless,the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear.To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves,we collected conditioned culture medium from hypoxia-pretreated neural crest cells,and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation.The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells.We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells.Subsequently,to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons,we used a microfluidic axonal dissociation model of sensory neurons in vitro,and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons,which was greatly dependent on loaded miR-21-5p.Finally,we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb,as well as muscle tissue morphology of the hind limbs,were obviously restored.These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p.miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome.This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves,and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

A hyaluronic acid granular hydrogel nerve guidance conduit promotes regeneration and functional recovery of injured sciatic nerves in rats

A hyaluronic acid granular hydrogel can promote neuronal and astrocyte colony formation and axonal extension in vitro,suggesting that the hydrogel can simulate an extracellular matrix structure to promote neural regeneration.However,in vivo experiments have not been conducted.In this study,we transplanted a hyaluronic acid granular hydrogel nerve guidance conduit to repair a 10-mm long sciatic nerve gap.The Basso,Beattie,and Bresnahan locomotor rating scale,sciatic nerve compound muscle action potential recording,Fluoro-Gold retrograde tracing,growth related protein 43/S100 immunofluorescence staining,transmission electron microscopy,gastrocnemius muscle dry/wet weight ratio,and Masson’s trichrome staining results showed that the nerve guidance conduit exhibited similar regeneration of sciatic nerve axons and myelin sheath,and recovery of the electrophysiological function and motor function as autologous nerve transplantation.The conduit results were superior to those of a bulk hydrogel or silicone tube transplant.These findings suggest that tissue-engineered nerve conduits containing hyaluronic acid granular hydrogels effectively promote the morphological and functional recovery of the injured sciatic nerve.The nerve conduits have the potential as a material for repairing peripheral nerve defects.

Hallmarks of peripheral nerve function in bone regeneration

Skeletal tissue is highly innervated.Although different types of nerves have been recently identified in the bone,the crosstalk between bone and nerves remains unclear.In this review,we outline the role of the peripheral nervous system(PNS)in bone regeneration following injury.We first introduce the conserved role of nerves in tissue regeneration in species ranging from amphibians to mammals.We then present the distribution of the PNS in the skeletal system under physiological conditions,fractures,or regeneration.Furthermore,we summarize the ways in which the PNS communicates with bone-lineage cells,the vasculature,and immune cells in the bone microenvironment.Based on this comprehensive and timely review,we conclude that the PNS regulates bone regeneration through neuropeptides or neurotransmitters and cells in the peripheral nerves.An in-depth understanding of the roles of peripheral nerves in bone regeneration will inform the development of new strategies based on bone-nerve crosstalk in promoting bone repair and regeneration.

Platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury

The effect of platelet-rich plasma on nerve regeneration remains controversial.In this study,we established a rabbit model of sciatic nerve small-gap defects with preserved epineurium and then filled the gaps with platelet-rich plasma.Twenty-eight rabbits were divided into the following groups(7 rabbits/group):model,low-concentrati on PRP(2.5-3.5-fold concentration of whole blood platelets),medium-concentration PRP(4.5-6.5-fold concentration of whole blood platelets),and high-concentration PRP(7.5-8.5-fold concentration of whole blood platelets).Electrophysiological and histomorphometrical assessments and proteomics analysis we re used to evaluate regeneration of the sciatic nerve.Our results showed that platelet-rich plasma containing 4.5-6.5-and 7.5-8.5-fold concentrations of whole blood platelets promoted repair of sciatic nerve injury.Proteomics analysis was performed to investigate the possible mechanism by which platelet-rich plasma promoted nerve regeneration.Proteomics analysis showed that after sciatic nerve injury,platelet-rich plasma increased the expression of integrin subunitβ-8(ITGB8),which participates in angiogenesis,and differentially expressed proteins were mainly enriched in focal adhesion pathways.Additionally,two key proteins,ribosomal protein S27 a(RSP27 a)and ubiquilin 1(UBQLN1),which were selected after protein-protein interaction analysis,are involved in the regulation of ubiquitin levels in vivo.These data suggest that platelet-rich plasma promotes peripheral nerve regeneration after sciatic nerve injury by affecting angiogenesis and intracellular ubiquitin levels.

Translational bioengineering strategies for peripheral nerve regeneration:opportunities,challenges,and novel concepts

Peripheral nerve injuries remain a challenging problem in need of better treatment strategies.Despite best efforts at surgical reconstruction and postoperative rehabilitation,patients are often left with persistent,debilitating motor and sensory deficits.There are currently no therapeutic strategies proven to enhance the regenerative process in humans.A clinical need exists for the development of technologies to promote nerve regeneration and improve functional outcomes.Recent advances in the fields of tissue engineering and nanotechnology have enabled biomaterial scaffolds to modulate the host response to tissue repair through tailored mechanical,chemical,and conductive cues.New bioengineered approaches have enabled targeted,sustained delivery of protein therapeutics with the capacity to unlock the clinical potential of a myriad of neurotrophic growth factors that have demonstrated promise in enhancing regenerative outcomes.As such,further exploration of combinatory strategies leveraging these technological advances may offer a pathway towards clinically translatable solutions to advance the care of patients with peripheral nerve injuries.This review first presents the various emerging bioengineering strategies that can be applied for the management of nerve gap injuries.We cover the rationale and limitations for their use as an alternative to autografts,focusing on the approaches to increase the number of regenerating axons crossing the repair site,and facilitating their growth towards the distal stump.We also discuss the emerging growth factor-based therapeutic strategies designed to improve functional outcomes in a multimodal fashion,by accelerating axonal growth,improving the distal regenerative environment,and preventing end-organs atrophy.

Repair and regeneration of peripheral nerve injuries that ablate branch points

The peripheral nervous system has an extensive branching organization, and peripheral nerve injuries that ablate branch points present a complex challenge for clinical repair. Ablations of linear segments of the PNS have been extensively studied and routinely treated with autografts, acellular nerve allografts, conduits, wraps, and nerve transfers. In contrast, segmental-loss peripheral nerve injuries, in which one or more branch points are ablated so that there are three or more nerve endings, present additional complications that have not been rigorously studied or documented. This review discusses:(1) the branched anatomy of the peripheral nervous system,(2) case reports describing how peripheral nerve injuries with branched ablations have been surgically managed,(3) factors known to influence regeneration through branched nerve structures,(4) techniques and models of branched peripheral nerve injuries in animal models, and(5) conclusions regarding outcome measures and studies needed to improve understanding of regeneration through ablated branched structures of the peripheral nervous system.

Long noncoding RNA H19 regulates degeneration and regeneration of injured peripheral nerves

Our previous studies have shown that long noncoding RNA(lncRNA)H19 is upregulated in injured rat sciatic nerve during the process of Wallerian degeneration,and that it promotes the migration of Schwann cells and slows down the growth of dorsal root ganglion axons.However,the mechanism by which lncRNA H19 regulates neural repair and regeneration after peripheral nerve injury remains unclear.In this study,we established a Sprague-Dawley rat model of sciatic nerve transection injury.We performed in situ hybridization and found that at 4–7 days after sciatic nerve injury,lncRNA H19 was highly expressed.At 14 days before injury,adeno-associated virus was intrathecally injected into the L4–L5 foramina to disrupt or overexpress lncRNA H19.After overexpression of lncRNA H19,the growth of newly formed axons from the sciatic nerve was inhibited,whereas myelination was enhanced.Then,we performed gait analysis and thermal pain analysis to evaluate rat behavior.We found that lncRNA H19 overexpression delayed the recovery of rat behavior function,whereas interfering with lncRNA H19 expression improved functional recovery.Finally,we examined the expression of lncRNA H19 downstream target SEMA6D,and found that after lncRNA H19 overexpression,the SEMA6D protein level was increased.These findings suggest that lncRNA H19 regulates peripheral nerve degeneration and regeneration through activating SEMA6D in injured nerves.This provides a new clue to understand the role of lncRNA H19 in peripheral nerve degeneration and regeneration.

Potential application of let-7a antagomir in injured peripheral nerve regeneration

Neurotrophic factors,particularly nerve growth factor,enhance neuronal regeneration.However,the in vivo applications of nerve growth factor are largely limited by its intrinsic disadvantages,such as its short biological half-life,its contribution to pain response,and its inability to cross the blood-brain barrier.Considering that let-7(human miRNA)targets and regulates nerve growth factor,and that let-7 is a core regulator in peripheral nerve regeneration,we evaluated the possibilities of let-7 application in nerve repair.In this study,anti-let-7a was identified as the most suitable let-7 family molecule by analyses of endogenous expression and regulatory relationship,and functional screening.Let-7a antagomir demonstrated biosafety based on the results of in vivo safety assessments and it entered into the main cell types of the sciatic nerve,including Schwann cells,fibroblasts and macrophages.Use of hydrogel effectively achieved controlled,localized,and sustained delivery of let-7a antagomir.Finally,let-7a antagomir was integrated into chitosan conduit to construct a chitosan-hydrogel scaffold tissue-engineered nerve graft,which promoted nerve regeneration and functional recovery in a rat model of sciatic nerve transection.Our study provides an experimental basis for potential in vivo application of let-7a.

Peripheral nerve regeneration through nerve conduits evokes differential expression of growth-associated protein-43 in the spinal cord

Growth-associated protein 43 plays a key role in neurite outgrowth through cytoskeleton remodeling.We have previously demonstrated that structural damage of peripheral nerves induces growth-associated protein 43 upregulation to promote growth cone formation.Conversely,the limited regenerative capacity of the central nervous system due to an inhibitory environment prevents major changes in neurite outgrowth and should be presumably associated with low levels of growth-associated protein 43 expression.However,central alterations due to peripheral nerve damage have never been assessed using the growthassociated protein 43 marker.In this study,we used the tubulization technique to repair 1 cm-long nerve gaps in the rat nerve injury/repair model and detected growth-associated protein 43 expression in the peripheral and central nervous systems.First,histological analysis of the regeneration process confirmed an active regeneration process of the nerve gaps through the conduit from 10 days onwards.The growth-associated protein 43 expression profile varied across regions and follow-up times,from a localized expression to an abundant and consistent expression throughout the regeneration tissue,confirming the presence of an active nerve regeneration process.Second,spinal cord changes were also histologically assessed,and no apparent changes in the structural and cellular organization were observed using routine staining methods.Surprisingly,remarkable differences and local changes appeared in growth-associated protein 43 expression at the spinal cord level,in particular at 20 days post-repair and beyond.Growth-associated protein 43 protein was first localized in the gracile fasciculus and was homogeneously distributed in the left posterior cord.These findings differed from the growth-associated protein 43 pattern observed in the healthy control,which did not express growth-associated protein 43 at these levels.Our results revealed a differential expression in growth-associated protein 43 protein not only in the regenerating nerve tissue but also in the spinal cord after peripheral nerve transection.These findings open the possibility of using this marker to monitor changes in the central nervous system after peripheral nerve injury.