Physiology of Nerve Regeneration: Key Factors Affecting Clinical Outcomes.

Functional recovery after peripheral nerve injuries is disappointing despite surgical advances in nerve repair. This review summarizes the relatively short window of opportunity for successful nerve regeneration due to the decline in the expression of growth-associated genes and in turn, the decline in regenerative capacity of the injured neurons and the support provided by the denervated Schwann cells, and the atrophy of denervated muscles. Brief, low-frequency electrical stimulation and post-injury exercise regimes ameliorate these deficits in animal models and patients, but the misdirection of regenerating nerve fibers compromises functional recovery and remains an important area of future research.

Convergent and divergent transcriptional reprogramming of motor and sensory neurons underlying response to peripheral nerve injury.

INTRODUCTION: Motor neurons differ from sensory neurons in aspects including origins and surrounding environment. Understanding the similarities and differences in molecular response to peripheral nerve injury (PNI) and regeneration between sensory and motor neurons is crucial for developing effective drug targets for CNS regeneration. However, genome-wide comparisons of molecular changes between sensory and motor neurons following PNI remains limited. OBJECTIVES: This study aims to investigate genome-wide convergence and divergence of injury response between sensory and motor neurons to identify novel drug targets for neural repair. METHODS: We analyzed two large-scale RNA-seq datasets of in situ captured sensory neurons (SNs) and motoneurons (MNs) upon PNI, retinal ganglion cells and spinal cord upon CNS injury. Additionally, we integrated these with other related single-cell level datasets. Bootstrap DESeq2 and WGCNA were used to detect and explore co-expression modules of differentially expressed genes (DEGs). RESULTS: We found that SNs and MNs exhibited similar injury states, but with a delayed response in MNs. We identified a conserved regeneration-associated module (cRAM) with 274 shared DEGs. Of which, 47% of DEGs could be changed in injured neurons supported by single-cell resolution datasets. We also identified some less-studied candidates in cRAM, including genes associated with transcription, ubiquitination (Rnf122), and neuron-immune cells cross-talk. Further in vitro experiments confirmed a novel role of Rnf122 in axon growth. Analysis of the top 10% of DEGs with a large divergence suggested that both extrinsic (e.g., immune microenvironment) and intrinsic factors (e.g., development) contributed to expression divergence between SNs and MNs following injury. CONCLUSIONS: This comprehensive analysis revealed convergent and divergent injury response genes in SNs and MNs, providing new insights into transcriptional reprogramming of sensory and motor neurons responding to axonal injury and subsequent regeneration. It also identified some novel regeneration-associated candidates that may facilitate the development of strategies for axon regeneration.

Brief Electrical Stimulation Promotes Recovery after Surgical Repair of Injured Peripheral Nerves.

Injured peripheral nerves regenerate their axons in contrast to those in the central nervous system. Yet, functional recovery after surgical repair is often disappointing. The basis for poor recovery is progressive deterioration with time and distance of the growth capacity of the neurons that lose their contact with targets (chronic axotomy) and the growth support of the chronically denervated Schwann cells (SC) in the distal nerve stumps. Nonetheless, chronically denervated atrophic muscle retains the capacity for reinnervation. Declining electrical activity of motoneurons accompanies the progressive fall in axotomized neuronal and denervated SC expression of regeneration-associated-genes and declining regenerative success. Reduced motoneuronal activity is due to the withdrawal of synaptic contacts from the soma. Exogenous neurotrophic factors that promote nerve regeneration can replace the endogenous factors whose expression declines with time. But the profuse axonal outgrowth they provoke and the difficulties in their delivery hinder their efficacy. Brief (1 h) low-frequency (20 Hz) electrical stimulation (ES) proximal to the injury site promotes the expression of endogenous growth factors and, in turn, dramatically accelerates axon outgrowth and target reinnervation. The latter ES effect has been demonstrated in both rats and humans. A conditioning ES of intact nerve days prior to nerve injury increases axonal outgrowth and regeneration rate. Thereby, this form of ES is amenable for nerve transfer surgeries and end-to-side neurorrhaphies. However, additional surgery for applying the required electrodes may be a hurdle. ES is applicable in all surgeries with excellent outcomes.

Axonal Regeneration: Underlying Molecular Mechanisms and Potential Therapeutic Targets.

Axons in the peripheral nervous system have the ability to repair themselves after damage, whereas axons in the central nervous system are unable to do so. A common and important characteristic of damage to the spinal cord, brain, and peripheral nerves is the disruption of axonal regrowth. Interestingly, intrinsic growth factors play a significant role in the axonal regeneration of injured nerves. Various factors such as proteomic profile, microtubule stability, ribosomal location, and signalling pathways mark a line between the central and peripheral axons' capacity for self-renewal. Unfortunately, glial scar development, myelin-associated inhibitor molecules, lack of neurotrophic factors, and inflammatory reactions are among the factors that restrict axonal regeneration. Molecular pathways such as cAMP, MAPK, JAK/STAT, ATF3/CREB, BMP/SMAD, AKT/mTORC1/p70S6K, PI3K/AKT, GSK-3ß/CLASP, BDNF/Trk, Ras/ERK, integrin/FAK, RhoA/ROCK/LIMK, and POSTN/integrin are activated after nerve injury and are considered significant players in axonal regeneration. In addition to the aforementioned pathways, growth factors, microRNAs, and astrocytes are also commendable participants in regeneration. In this review, we discuss the detailed mechanism of each pathway along with key players that can be potentially valuable targets to help achieve quick axonal healing. We also identify the prospective targets that could help close knowledge gaps in the molecular pathways underlying regeneration and shed light on the creation of more powerful strategies to encourage axonal regeneration after nervous system injury.

Neurotherapeutic Potential of Cervus elaphus Sibericus on Axon Regeneration and Growth Cone Reformation after H2O2-Induced Injury in Rat Primary Cortical Neurons.

Cervus elaphus sibericus (CES), commonly known as deer antler, has been used as a medicinal herb because of its various pharmacological activities, including its anti-infective, anti-arthritic, anti-allergic, and anti-oxidative properties. However, the precise mechanisms by which CES functions as a potent anti-oxidative agent remain unknown; particularly, the effects of CES on cortical neurons and its neurobiological mechanism have not been examined. We used primary cortical neurons from the embryonic rat cerebral cortex and hydrogen peroxide to induce oxidative stress and damage in neurons. After post-treatment of CES at three concentrations (10, 50, and 200 µg/mL), the influence of CES on the neurobiological mechanism was assessed by immunocytochemistry, flow cytometry, and real-time PCR. CES effectively prevented neuronal death caused by hydrogen peroxide-induced damage by regulating oxidative signaling. In addition, CES significantly induced the expression of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated genes. We also observed newly processing elongated axons after CES treatment under oxidative conditions. In addition, filopodia tips generally do not form a retraction bulb, called swollen endings. Thus, CES shows therapeutic potential for treating neurological diseases by stimulating neuron repair and regeneration.

Apamin Enhances Neurite Outgrowth and Regeneration after Laceration Injury in Cortical Neurons.

Apamin is a minor component of bee venom and is a polypeptide with 18 amino acid residues. Although apamin is considered a neurotoxic compound that blocks the potassium channel, its neuroprotective effects on neurons have been recently reported. However, there is little information about the underlying mechanism and very little is known regarding the toxicological characterization of other compounds in bee venom. Here, cultured mature cortical neurons were treated with bee venom components, including apamin, phospholipase A2, and the main component, melittin. Melittin and phospholipase A2 from bee venom caused a neurotoxic effect in dose-dependent manner, but apamin did not induce neurotoxicity in mature cortical neurons in doses of up to 10 µg/mL. Next, 1 and 10 µg/mL of apamin were applied to cultivate mature cortical neurons. Apamin accelerated neurite outgrowth and axon regeneration after laceration injury. Furthermore, apamin induced the upregulation of brain-derived neurotrophic factor and neurotrophin nerve growth factor, as well as regeneration-associated gene expression in mature cortical neurons. Due to its neurotherapeutic effects, apamin may be a promising candidate for the treatment of a wide range of neurological diseases.

Role of Myc Proto-Oncogene as a Transcriptional Hub to Regulate the Expression of Regeneration-Associated Genes following Preconditioning Peripheral Nerve Injury.

Preconditioning peripheral nerve injury enhances the intrinsic growth capacity of DRGs sensory axons by inducing transcriptional upregulation of the regeneration-associated genes (RAGs). However, it is still unclear how preconditioning injury leads to the orchestrated induction of many RAGs. The present study identified Myc proto-oncogene as a transcriptional hub gene to regulate the expression of a distinct subset of RAGs in DRGs following the preconditioning injury. We demonstrated that c-MYC bound to the promoters of certain RAGs, such as Jun, Atf3, and Sprr1a, and that Myc upregulation following SNI preceded that of the RAGs bound by c-MYC. Marked DNA methylation of the Myc exon 3 sequences was implicated in the early transcriptional activation and accompanied by open histone marks. Myc deletion led to a decrease in the injury-induced expression of a distinct subset of RAGs, which were highly overlapped with the list of RAGs that were upregulated by Myc overexpression. Following dorsal hemisection spinal cord injury in female rats, Myc overexpression in DRGs significantly prevented the retraction of the sensory axons in a manner dependent on its downstream RAG, June Our results suggest that Myc plays a critical role in axon regeneration via its transcriptional activity to regulate the expression of a spectrum of downstream RAGs and subsequent effector molecules. Identification of more upstream hub transcription factors and the epigenetic mechanisms specific for individual hub transcription factors would advance our understanding of how the preconditioning injury induces orchestrated upregulation of RAGs.

Functional Electrical Stimulation and the Modulation of the Axon Regeneration Program.

Neural injury in mammals often leads to persistent functional deficits as spontaneous repair in the peripheral nervous system (PNS) is often incomplete, while endogenous repair mechanisms in the central nervous system (CNS) are negligible. Peripheral axotomy elicits growth-associated gene programs in sensory and motor neurons that can support reinnervation of peripheral targets given sufficient levels of debris clearance and proximity to nerve targets. In contrast, while damaged CNS circuitry can undergo a limited amount of sprouting and reorganization, this innate plasticity does not re-establish the original connectivity. The utility of novel CNS circuitry will depend on effective connectivity and appropriate training to strengthen these circuits. One method of enhancing novel circuit connectivity is through the use of electrical stimulation, which supports axon growth in both central and peripheral neurons. This review will focus on the effects of CNS and PNS electrical stimulation in activating axon growth-associated gene programs and supporting the recovery of motor and sensory circuits. Electrical stimulation-mediated neuroplasticity represents a therapeutically viable approach to support neural repair and recovery. Development of appropriate clinical strategies employing electrical stimulation will depend upon determining the underlying mechanisms of activity-dependent axon regeneration and the heterogeneity of neuronal subtype responses to stimulation.

Alteration in global DNA methylation status following preconditioning injury influences axon growth competence of the sensory neurons.

Preconditioning peripheral nerve injury primes the sensory neurons in the dorsal root ganglia (DRGs) to acquire axon regeneration competence. Transcription of a large set of regeneration-associated-genes (RAGs) contributes to the enhanced intrinsic axonal regeneration capacity. However, the mechanism underlying the coordinated upregulation of RAGs orchestrated by preconditioning injury is unclear. We sought to determine potential influence of DNA methylation change on transcriptional activation of RAGs in the L4-L6 DRGs following sciatic nerve injury. Genome-wide sequencing revealed that about 20% of the methylated DNA fragments were differentially methylated, and >3000 genes contained differentially methylated regions. Not only demethylation but also increased methylation was observed to a similar extent. The change in the global DNA methylation did not correlate with the gene expression level of most genes, including the well-documented RAGs. However, pharmacological inhibition or activation of DNA methylation markedly attenuated the axon growth capacity of the preconditioned DRG neurons. Pharmacological perturbation of DNA methylation resulted in simultaneous downregulation of many highly overlapping non-transcription factor RAGs, which was accompanied by a concurrent, robust upregulation of SOCS3 and Serpine1. Overexpression of SOCS3 and Serpine1 in the DRG neurons overrode injury-induced axon growth competence, corroborating their roles as the negative regulators of axon regeneration. We conclude that the injury-induced global alteration of DNA methylome strongly influences the axon growth competence in preconditioned DRG neurons. Our results also suggest a possibility that perturbing DNA methylome changes might lead to the upregulation of negative regulator RAGs thereby attenuating axon growth capacity.

Schwann cells regulate sensory neuron gene expression before and after peripheral nerve injury.

Sensory neurons in the PNS demonstrate substantial capacity for regeneration following injury. Recent studies have identified changes in the transcriptome of sensory neurons, which are instrumental for axon regeneration. The role of Schwann cells (SCs) in mediating these changes remains undefined. We tested the hypothesis that SCs regulate expression of genes in sensory neurons before and after PNS injury by comparing mice in which LDL Receptor-related Protein-1 (LRP1) is deleted in SCs (scLRP1-/- mice) with wild-type (scLRP1+/+ ) littermates. LRP1 is an endocytic and cell-signaling receptor that is necessary for normal SC function and the SC response to nerve injury. scLRP1-/- mice represent a characterized model in which the SC response to nerve injury is abnormal. Adult DRG neurons, isolated from scLRP1-/- mice, with or without a conditioning nerve lesion, demonstrated increased neurite outgrowth when cultured ex vivo, compared with neurons from wild-type mice. Following sciatic nerve crush injury, nerve regeneration was accelerated in vivo in scLRP1-/- mice. These results were explained by transcriptional activation of RAGs in DRG neurons in scLRP1-/- mice prior to nerve injury. Although the presence of abnormal SCs in scLRP1-/- mice primed DRG neurons for repair, nerve regeneration in scLRP1-/- mice resulted in abnormalities in ultrastructure, principally in Remak bundles, and with the onset of neuropathic pain. These results demonstrate the importance of SCs in controlling RAG expression by neurons and the potential for this process to cause chronic pain when abnormal. The SC may represent an important target for preventing pain following PNS injury.

Electrical stimulation as a conditioning strategy for promoting and accelerating peripheral nerve regeneration.

The delivery of a nerve insult (a "conditioning lesion") prior to a subsequent test lesion increases the number of regenerating axons and accelerates the speed of regeneration from the test site. A major barrier to clinical translation is the lack of an ethically acceptable and clinically feasible method of conditioning that does not further damage the nerve. Conditioning electrical stimulation (CES), a non-injurious intervention, has previously been shown to improve neurite outgrowth in vitro. In this study, we examined whether CES upregulates regeneration-associated gene (RAG) expression and promotes nerve regeneration in vivo, similar to a traditional nerve crush conditioning lesion (CCL). Adult rats were divided into four cohorts based on conditioning treatment to the common peroneal (fibular) nerve: i) CES (1h, 20Hz); ii) CCL (10s crush); iii) sham CES (1h, 0Hz); or iv) naïve (unconditioned). Immunofluorescence and qRT-PCR revealed significant RAG upregulation in the dorsal root ganglia of both CES and CCL animals, evident at 3-14days post-conditioning. To mimic a clinical microsurgical nerve repair, all cohorts underwent a common peroneal nerve cut and coaptation one week following conditioning. Both CES and CCL animals increased the length of nerve regeneration (3.8-fold) as well as the total number of regenerating axons (2.2-fold), compared to the sham and naïve-conditioned animals (p<0.001). These data support CES as a non-injurious conditioning paradigm that is comparable to a traditional CCL and is therefore a novel means to potentially enhance peripheral nerve repair in the clinical setting.

Comprehensive mapping of 5-hydroxymethylcytosine epigenetic dynamics in axon regeneration.

In contrast to central nervous system neurons, dorsal root ganglia (DRG) neurons can switch to a regenerative state after peripheral axotomy. In a screen for chromatin regulators of the regenerative responses in this conditioning lesion paradigm, we identified Tet methylcytosine dioxygenase 3 (Tet3) as upregulated in DRG neurons, along with increased 5-hydroxymethylcytosine (5hmC). We generated genome-wide 5hmC maps in adult DRG, which revealed that peripheral and central axotomy (leading to no regenerative effect) triggered differential 5hmC changes that are associated with distinct signaling pathways. 5hmC was altered in a large set of regeneration-associated genes (RAGs), including well-known RAGs, such as Atf3, Bdnf, and Smad1, that regulate axon growth potential of DRG neurons, thus supporting its role for RAG regulation. Our analyses also predicted HIF-1, STAT, and IRF as potential transcription factors that may collaborate with Tet3 for 5hmC modifications. Intriguingly, central axotomy resulted in widespread 5hmC modifications that had little overlap with those of peripheral axotomy, thus potentially constituting a roadblock for regeneration. Our study revealed 5hmC dynamics as a previously unrecognized epigenetic mechanism underlying the divergent responses after axonal injury.

What makes a RAG regeneration associated?

Regenerative failure remains a significant barrier for functional recovery after central nervous system (CNS) injury. As such, understanding the physiological processes that regulate axon regeneration is a central focus of regenerative medicine. Studying the gene transcription responses to axon injury of regeneration competent neurons, such as those of the peripheral nervous system (PNS), has provided insight into the genes associated with regeneration. Though several individual "regeneration-associated genes" (RAGs) have been identified from these studies, the response to injury likely regulates the expression of functionally coordinated and complementary gene groups. For instance, successful regeneration would require the induction of genes that drive the intrinsic growth capacity of neurons, while simultaneously downregulating the genes that convey environmental inhibitory cues. Thus, this view emphasizes the transcriptional regulation of gene "programs" that contribute to the overall goal of axonal regeneration. Here, we review the known RAGs, focusing on how their transcriptional regulation can reveal the underlying gene programs that drive a regenerative phenotype. Finally, we will discuss paradigms under which we can determine whether these genes are injury-associated, or indeed necessary for regeneration.

Reduced expression of regeneration associated genes in chronically axotomized facial motoneurons.

Chronically axotomized motoneurons progressively fail to regenerate their axons. Since axonal regeneration is associated with the increased expression of tubulin, actin and GAP-43, we examined whether the regenerative failure is due to failure of chronically axotomized motoneurons to express and sustain the expression of these regeneration associated genes (RAGs). Chronically axotomized facial motoneurons were subjected to a second axotomy to mimic the clinical surgical procedure of refreshing the proximal nerve stump prior to nerve repair. Expression of α1-tubulin, actin and GAP-43 was analyzed in axotomized motoneurons using in situ hybridization followed by autoradiography and silver grain quantification. The expression of these RAGs by acutely axotomized motoneurons declined over several months. The chronically injured motoneurons responded to a refreshment axotomy with a re-increase in RAG expression. However, this response to a refreshment axotomy of chronically injured facial motoneurons was less than that seen in acutely axotomized facial motoneurons. These data demonstrate that the neuronal RAG expression can be induced by injury-related signals and does not require acute deprivation of target derived factors. The transient expression is consistent with a transient inflammatory response to the injury. We conclude that transient RAG expression in chronically axotomized motoneurons and the weak response of the chronically axotomized motoneurons to a refreshment axotomy provides a plausible explanation for the progressive decline in regenerative capacity of chronically axotomized motoneurons.

Effect of local application of transforming growth factor-ß at the nerve repair site following chronic axotomy and denervation on the expression of regeneration-associated genes. Laboratory investigation.

OBJECTIVES: Although peripheral nerves can regenerate after traumatic injury, functional recovery is often suboptimal, especially after injuries to large nerve trunks such as the sciatic nerve or brachial plexus. Current research with animal models suggests that the lack of functional recovery resides in the lack of sufficient mature axons reaching their targets due to the loss of neurotrophic support by Schwann cells in the distal stump of injured nerves. This study was designed to investigate the effect of one-time application of transforming growth factor-ß (TGF-ß) at the repair site of chronically injured nerve. METHODS: The authors used the rat tibial nerve injury and repair model to investigate the effects of application of physiological concentrations of TGF-ß plus forskolin or forskolin alone in vivo at the repair site on gene and protein expression and axon regeneration at 6 weeks after nerve repair. They used gene expression profiling and immunohistochemical analysis of indicative activated proteins in Schwann cells to evaluate the effects of treatments on the delayed repair. They also quantified the regenerated axons distal to the repair site by microscopy of paraffin sections. RESULTS: Both treatment with forskolin only and treatment with TGF-ß plus forskolin resulted in increased numbers of axons regenerated compared with saline-only control. There was robust activation and proliferation of both Schwann cells and macrophages reminiscent of the processes during Wallerian degeneration. The treatment also induced upregulation of genes implicated in cellular activation and growth as detected by gene array. CONCLUSIONS: Addition of TGF-ß plus forskolin to the repair after chronic nerve injury improved axonal regeneration, probably via upregulation of required genes, expression of growth-associated protein, and reactivation of Schwann cells and macrophages. Further studies are required to better understand the mechanism of the positive effect of TGF-ß treatment on old nerve injuries.

A molecular mechanism of optic nerve regeneration in fish: the retinoid signaling pathway.

The fish optic nerve regeneration process takes more than 100 days after axotomy and comprises four stages: neurite sprouting (1-4 days), axonal elongation (5-30 days), synaptic refinement (35-80 days) and functional recovery (100-120 days). We screened genes specifically upregulated in each stage from axotomized fish retina. The mRNAs for heat shock protein 70 and insulin-like growth factor-1 rapidly increased in the retinal ganglion cells soon after axotomy and function as cell-survival factors. Purpurin mRNA rapidly and transiently increased in the photoreceptors and purpurin protein diffusely increased in all nuclear layers at 1-4 days after injury. The purpurin gene has an active retinol-binding site and a signal peptide. Purpurin with retinol functions as a sprouting factor for thin neurites. This neurite-sprouting effect was closely mimicked by retinoic acid and blocked by its inhibitor. We propose that purpurin works as a retinol transporter to supply retinoic acid to damaged RGCs which in turn activates target genes. We also searched for genes involved in the second stage of regeneration. The mRNA of retinoid-signaling molecules increased in retinal ganglion cells at 7-14 days after injury and tissue transglutaminase and neuronal nitric oxide synthase mRNAs, RA-target genes, increased in retinal ganglion cells at 10-30 days after injury. They function as factors for the outgrowth of thick, long neurites. Here we present a retinoid-signaling hypothesis to explain molecular events during the early stages of optic nerve regeneration in fish.

gp130 cytokines are positive signals triggering changes in gene expression and axon outgrowth in peripheral neurons following injury.

Adult peripheral neurons, in contrast to adult central neurons, are capable of regeneration after axonal damage. Much attention has focused on the changes that accompany this regeneration in two places, the distal nerve segment (where phagocytosis of axonal debris, changes in the surface properties of Schwann cells, and induction of growth factors and cytokines occur) and the neuronal cell body (where dramatic changes in cell morphology and gene expression occur). The changes in the axotomized cell body are often referred to as the "cell body response." The focus of the current review is a family of cytokines, the glycoprotein 130 (gp130) cytokines, which produce their actions through a common gp130 signaling receptor and which function as injury signals for axotomized peripheral neurons, triggering changes in gene expression and in neurite outgrowth. These cytokines play important roles in the responses of sympathetic, sensory, and motor neurons to injury. The best studied of these cytokines in this context are leukemia inhibitory factor (LIF) and interleukin (IL)-6, but experiments with conditional gp130 knockout animals suggest that other members of this family, not yet determined, are also involved. The primary gp130 signaling pathway shown to be involved is the activation of Janus kinase (JAK) and the transcription factors Signal Transducers and Activators of Transcription (STAT), though other downstream pathways such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) may also play a role. gp130 signaling may involve paracrine, retrograde, and autocrine actions of these cytokines. Recent studies suggest that manipulation of this cytokine system can also stimulate regeneration by injured central neurons.