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Introduction: Damage to the corneal nerves can result in discomfort and chronic pain, profoundly impacting the quality of life of patients. Development of novel in vitro method is crucial to better understand corneal nerve regeneration and to find new treatments for the patients. Existing in vitro models often overlook the physiology of primary sensory neurons, for which the soma is separated from the nerve endings. Methods: To overcome this limitation, our novel model combines a compartmentalized microfluidic culture of trigeminal ganglion neurons from adult mice with live-imaging and automated 3D image analysis offering robust way to assess axonal regrowth after axotomy. Results: Physical axotomy performed by a two-second aspiration led to a reproducible 70% axonal loss and altered the phenotype of the neurons, increasing the number of substance P-positive neurons 72h post-axotomy. To validate our new model, we investigated axonal regeneration after exposure to pharmacological compounds. We selected various targets known to enhance or inhibit axonal regrowth and analyzed their basal expression in trigeminal ganglion cells by scRNAseq. NGF/GDNF, insulin, and Dooku-1 (Piezo1 antagonist) enhanced regrowth by 81, 74 and 157%, respectively, while Yoda-1 (Piezo1 agonist) had no effect. Furthermore, SARM1-IN-2 (Sarm1 inhibitor) inhibited axonal regrowth, leading to only 6% regrowth after 72h of exposure (versus 34% regrowth without any compound). Discussion: Combining compartmentalized trigeminal neuronal culture with advanced imaging and analysis allowed a thorough evaluation of the extent of the axotomy and subsequent axonal regrowth. This innovative approach holds great promise for advancing our understanding of corneal nerve injuries and regeneration and ultimately improving the quality of life for patients suffering from sensory abnormalities, and related conditions. [ABSTRACT FROM AUTHOR]

Abstract Background Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. Methods Ccr2 knock-out (KO) and Ccr2 gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. Results Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2 gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2 gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2 gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. Conclusions Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response.

Abstract Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the axon and transported back to the soma. This transport is essential since the final degradation of the vesicular content occurs only close to or in the soma. Here, we established an in vivo live-imaging model in the rat optic nerve using viral vector mediated LC3-labeling and two-photon-microscopy to analyze axonal transport of AVs. Under basal conditions in vivo, 50% of the AVs are moving with a majority of 85% being transported in the retrograde direction. Transport velocity is higher in the retrograde than in the anterograde direction. A crush lesion of the optic nerve results in a rapid breakdown of retrograde axonal transport while the anterograde transport stays intact over several hours. Close to the lesion site, the formation of AVs is upregulated within the first 6 h after crush, but the clearance of AVs and the levels of lysosomal markers in the adjacent axon are reduced. Expression of p150Glued, an adaptor protein of dynein, is significantly reduced after crush lesion. In vitro, fusion and colocalization of the lysosomal marker cathepsin D with AVs are reduced after axotomy. Taken together, we present here the first in vivo analysis of axonal AV transport in the mammalian CNS using live-imaging. We find that axotomy leads to severe defects of retrograde motility and a decreased clearance of AVs via the lysosomal system.

Neurons pose a particular challenge to degradative processes like autophagy due to their long and thin processes. Autophagic vesicles (AVs) are formed at the tip of the axon and transported back to the soma. This transport is essential since the final degradation of the vesicular content occurs only close to or in the soma. Here, we established an in vivo live-imaging model in the rat optic nerve using viral vector mediated LC3-labeling and two-photon-microscopy to analyze axonal transport of AVs. Under basal conditions in vivo, 50% of the AVs are moving with a majority of 85% being transported in the retrograde direction. Transport velocity is higher in the retrograde than in the anterograde direction. A crush lesion of the optic nerve results in a rapid breakdown of retrograde axonal transport while the anterograde transport stays intact over several hours. Close to the lesion site, the formation of AVs is upregulated within the first 6 h after crush, but the clearance of AVs and the levels of lysosomal markers in the adjacent axon are reduced. Expression of p150Glued, an adaptor protein of dynein, is significantly reduced after crush lesion. In vitro, fusion and colocalization of the lysosomal marker cathepsin D with AVs are reduced after axotomy. Taken together, we present here the first in vivo analysis of axonal AV transport in the mammalian CNS using live-imaging. We find that axotomy leads to severe defects of retrograde motility and a decreased clearance of AVs via the lysosomal system. [ABSTRACT FROM AUTHOR]

Background: Since the 1990s, evidence has accumulated that macrophages promote peripheral nerve regeneration and are required for enhancing regeneration in the conditioning lesion (CL) response. After a sciatic nerve injury, macrophages accumulate in the injury site, the nerve distal to that site, and the axotomized dorsal root ganglia (DRGs). In the peripheral nervous system, as in other tissues, the macrophage response is derived from both resident macrophages and recruited monocyte-derived macrophages (MDMs). Unresolved questions are: at which sites do macrophages enhance nerve regeneration, and is a particular population needed. Methods: Ccr2 knock-out (KO) and Ccr2gfp/gfp knock-in/KO mice were used to prevent MDM recruitment. Using these strains in a sciatic CL paradigm, we examined the necessity of MDMs and residents for CL-enhanced regeneration in vivo and characterized injury-induced nerve inflammation. CL paradigm variants, including the addition of pharmacological macrophage depletion methods, tested the role of various macrophage populations in initiating or sustaining the CL response. In vivo regeneration, measured from bilateral proximal test lesions (TLs) after 2 d, and macrophages were quantified by immunofluorescent staining. Results: Peripheral CL-enhanced regeneration was equivalent between crush and transection CLs and was sustained for 28 days in both Ccr2 KO and WT mice despite MDM depletion. Similarly, the central CL response measured in dorsal roots was unchanged in Ccr2 KO mice. Macrophages at both the TL and CL, but not between them, stained for the pro-regenerative marker, arginase 1. TL macrophages were primarily CCR2-dependent MDMs and nearly absent in Ccr2 KO and Ccr2gfp/gfp KO mice. However, there were only slightly fewer Arg1+ macrophages in CCR2 null CLs than controls due to resident macrophage compensation. Zymosan injection into an intact WT sciatic nerve recruited Arg1+ macrophages but did not enhance regeneration. Finally, clodronate injection into Ccr2gfp KO CLs dramatically reduced CL macrophages. Combined with the Ccr2gfp KO background, depleting MDMs and TL macrophages, and a transection CL, physically removing the distal nerve environment, nearly all macrophages in the nerve were removed, yet CL-enhanced regeneration was not impaired. Conclusions: Macrophages in the sciatic nerve are neither necessary nor sufficient to produce a CL response. [ABSTRACT FROM AUTHOR]

Introduction/Aims: Daily intramuscular injections of fibroblast growth factor 2 (FGF2) but not of brain‐derived neurotrophic factor (BDNF) significantly improve whisking behavior and mono‐innervation of the rat levator labii superioris (LLS) muscle 56 days after buccal nerve transection and suture (buccal–buccal anastomosis, BBA). We explored the dose–response of BDNF, FGF2, and insulin growth factor 2 (IGF2) on the same parameters, asking whether higher doses of BDNF would promote recovery. Methods: After BBA, growth factors were injected (30 μL volume) daily into the LLS muscle over 14, 28, or 56 days. At 56 days, video‐based motion analysis of vibrissal whisking was performed and the extent of mono‐ and poly‐reinnervation of the reinnervated neuromuscular junctions (NMJs) of the muscle determined with immunostaining of the nerve with β‐tubulin and histochemical staining of the endplates with Alexa Fluor 488‐conjugated α‐bungarotoxin. Results: The dose–response curve demonstrated significantly higher whisking amplitudes and corresponding increased mono‐innervation of the NMJ in the reinnervated LLS muscle at concentrations of 20–30 μg/mL BDNF administered daily for 14–28 days after BBA surgery. In contrast, high doses of IGF2 and FGF2, or doses of 20 and 40 μg/mL of BDNF administered for 14–56 days had no effect on either whisking behavior or in reducing poly‐reinnervation of endplates in the muscle. Discussion: These data suggest that the re‐establishment of mono‐innervation of whiskerpad muscles and the improved motor function by injections of BDNF into the paralyzed vibrissal musculature after facial nerve injury have translation potential and promote clinical application. [ABSTRACT FROM AUTHOR]

IntroductionDamage to the corneal nerves can result in discomfort and chronic pain, profoundly impacting the quality of life of patients. Development of novel in vitro method is crucial to better understand corneal nerve regeneration and to find new treatments for the patients. Existing in vitro models often overlook the physiology of primary sensory neurons, for which the soma is separated from the nerve endings.MethodsTo overcome this limitation, our novel model combines a compartmentalized microfluidic culture of trigeminal ganglion neurons from adult mice with live–imaging and automated 3D image analysis offering robust way to assess axonal regrowth after axotomy.ResultsPhysical axotomy performed by a two-second aspiration led to a reproducible 70% axonal loss and altered the phenotype of the neurons, increasing the number of substance P-positive neurons 72 h post-axotomy. To validate our new model, we investigated axonal regeneration after exposure to pharmacological compounds. We selected various targets known to enhance or inhibit axonal regrowth and analyzed their basal expression in trigeminal ganglion cells by scRNAseq. NGF/GDNF, insulin, and Dooku-1 (Piezo1 antagonist) enhanced regrowth by 81, 74 and 157%, respectively, while Yoda-1 (Piezo1 agonist) had no effect. Furthermore, SARM1-IN-2 (Sarm1 inhibitor) inhibited axonal regrowth, leading to only 6% regrowth after 72 h of exposure (versus 34% regrowth without any compound).DiscussionCombining compartmentalized trigeminal neuronal culture with advanced imaging and analysis allowed a thorough evaluation of the extent of the axotomy and subsequent axonal regrowth. This innovative approach holds great promise for advancing our understanding of corneal nerve injuries and regeneration and ultimately improving the quality of life for patients suffering from sensory abnormalities, and related conditions.

Chronic pain occurs at epidemic levels throughout the population. Hypersensitivity to touch, is a cardinal symptom of chronic pain. Despite dedicated research for over a century, quantifying this hypersensitivity has remained impossible at scale. To address these issues, we developed the Chainmail Sensitivity Test (CST). Our results show that control mice spend significantly more time on the chainmail portion of the device than mice subject to neuropathy. Treatment with gabapentin abolishes this difference. CST-derived data correlate well with von Frey measurements and quantify hypersensitivity due to inflammation. Our study demonstrates the potential of the CST as a standardized tool for assessing mechanical hypersensitivity in mice with minimal operator input. [ABSTRACT FROM AUTHOR]

Vascular endothelial growth factor (VEGF) is well known for its angiogenic activity, but recent evidence has revealed a neuroprotective action of this factor on injured or diseased neurons. In the present review, we summarize the most relevant findings that have contributed to establish a link between VEGF deficiency and neuronal degeneration. At issue, 1) mutant mice with reduced levels of VEGF show adult-onset muscle weakness and motoneuron degeneration resembling amyotrophic lateral sclerosis (ALS), 2) administration of VEGF to different animal models of motoneuron degeneration improves motor performance and ameliorates motoneuronal degeneration, and 3) there is an association between low plasmatic levels of VEGF and human ALS. Altogether, the results presented in this review highlight VEGF as an essential motoneuron neurotrophic factor endowed with promising therapeutic potential for the treatment of motoneuron disorders. [ABSTRACT FROM AUTHOR]

Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for biochemical studies, and (iii) the measurement of neurite markers and metabolites after axotomy. LUHMES-based spheroids, plated in culture dishes, extended neurites of several thousand µm length, while all somata remained aggregated. These cultures allowed an easy microscopic observation of live or fixed neurites. Neurite-only cultures (NOC) were produced by cutting out the still-aggregated somata. The potential application of such cultures was exemplified by determinations of their protein and RNA contents. For instance, the mitochondrial TOM20 protein was highly abundant, while nuclear histone H3 was absent. Similarly, mitochondrial-encoded RNAs were found at relatively high levels, while the mRNA for a histone or the neuronal nuclear marker NeuN (RBFOX3) were relatively depleted in NOC. Another potential use of NOC is the study of neurite degeneration. For this purpose, an algorithm to quantify neurite integrity was developed. Using this tool, we found that the addition of nicotinamide drastically reduced neurite degeneration. Also, the chelation of Ca2+ in NOC delayed the degeneration, while inhibitors of calpains had no effect. Thus, NOC proved to be suitable for biochemical analysis and for studying degeneration processes after a defined cut injury. [ABSTRACT FROM AUTHOR]

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.

Neurotrauma is among the main causes of human disability and mortality. However, the mechanisms that mediate the survival and death of cells in the peripheral nervous system are still not fully understood. The transcription factors p53 and E2F1 are the master regulators of basic cellular functions, including DNA repair, cell cycle, metabolism, and apoptosis. Overexpression of p53 and E2F1, shown in a number of experimental models of peripheral nerve injury, suggests an important role of these proteins in the pathogenesis of neurotrauma. This review discusses the epigenetic mechanisms of p53 and E2F1 activation and regulation, which may contribute to the survival or death of neurons and glial cells after traumatic injury. Prospects for further studies of the mechanisms of regulation of the p53 and E2F1 proteins, including those involving histone deacetylases, for the development of neuroprotectors are considered. [ABSTRACT FROM AUTHOR]

Neurodegeneration is a complex progressive pathological process leading to the neuronal death, which is induced by various external and internal factors. Neurodegenerative diseases, injuries of the central and peripheral nervous system, mental disorders, and a number of other pathological conditions, accompanied by functional and structural degradation of neurons and their death, is a serious problem in the global healthcare system, as due to these diseases millions of people around the world become disabled or die every year. The situation is complicated by the lack of selective, clinically effective neuroprotective drugs. It has been shown that nitric oxide (NO) and hydrogen sulfide (H2S) are actively involved in neurodegeneration and cell death of neurons and glia, but their role is not completely clear. This review considers NO- and H2S-dependent signaling mechanisms underlying the pathogenesis of neurodegenerative processes. The prospects for further studies of the role of NO and H2S in the nervous tissue under conditions of pathological conditions associated with neurodegeneration are considered. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is one of the leading causes of disability and death worldwide. It is characterized by various molecular–cellular events, with the main ones being apoptosis and damage to axons. To date, there are no clinically effective neuroprotective drugs. In this study, we examined the role of hydrogen sulfide (H2S) in the localization and expression of the key pro-apoptotic protein p53, as well as cell death in the nervous tissue in TBI and axotomy. We used a fast donor (sodium sulphide, Na2S) H2S and a classic inhibitor (aminooxyacetic acid, AOAA) of cystathionine β-synthase (CBS), which is a key enzyme in H2S synthesis. These studies were carried out on three models of neurotrauma in vertebrates and invertebrates. As a result, it was found that Na2S exhibits a pronounced neuroprotective effect that reduces the number of TUNEL-positive neurons and glial cells in TBI and apoptotic glia in axotomy. This effect could be realized through the Na2S-dependent decrease in the level of p53 in the cells of the nervous tissue of vertebrates and invertebrates, which we observed in our study. We also observed the opposite effect when using AOAA, which indicates the important role of CBS in the regulation of p53 expression and death of neurons and glial cells in TBI and axotomy. [ABSTRACT FROM AUTHOR]

The microtubule-associated protein Tau is highly enriched in axons of brain neurons where it regulates axonal outgrowth, plasticity, and transport. Efficient axonal Tau sorting is critical since somatodendritic Tau missorting is a major hallmark of Alzheimer's disease and other tauopathies. However, the molecular mechanisms of axonal Tau sorting are still not fully understood. In this study, we aimed to unravel to which extent anterograde protein transport contributes to axonal Tau sorting. We developed a laser-based axotomy approach with single-cell resolution and combined it with spinning disk confocal microscopy enabling multi live-cell monitoring. We cultivated human iPSC-derived cortical neurons and mouse primary forebrain neurons in specialized chambers allowing reliable post-fixation identification and Tau analysis. Using this approach, we achieved high post-axotomy survival rates and observed axonal regrowth in a subset of neurons. When we assessed somatic missorting and phosphorylation levels of endogenous human or murine Tau at different time points after axotomy, we surprisingly did not observe somatic Tau accumulation or hyperphosphorylation, regardless of their regrowing activity, consistent for both models. These results indicate that impairment of anterograde transit of Tau protein and acute axonal damage may not play a role for the development of somatic Tau pathology. In sum, we developed a laser-based axotomy model suitable for studying the impact of different Tau sorting mechanisms in a highly controllable and reproducible setting, and we provide evidence that acute axon loss does not induce somatic Tau accumulation and AT8 Tau phosphorylation. UV laser-induced axotomy of human iPSC-derived and mouse primary neurons results in decreased somatic levels of endogenous Tau and AT8 Tau phosphorylation. [ABSTRACT FROM AUTHOR]

Chronic pain occurs at epidemic levels throughout the population. Hypersensitivity to touch, is a cardinal symptom of chronic pain. Despite dedicated research for over a century, quantifying this hypersensitivity has remained impossible at scale. To address these issues, we developed the Chainmail Sensitivity Test (CST). Our results show that control mice spend significantly more time on the chainmail portion of the device than mice subject to neuropathy. Treatment with gabapentin abolishes this difference. CST-derived data correlate well with von Frey measurements and quantify hypersensitivity due to inflammation. Our study demonstrates the potential of the CST as a standardized tool for assessing mechanical hypersensitivity in mice with minimal operator input.

The convergence of transcriptional and epigenetic changes in the peripheral nervous system (PNS) reshapes the spatiotemporal gene expression landscape in response to nerve transection. The control of these molecular programs exhibits sexually dimorphic characteristics that remain not sufficiently characterized. In the present study, we recorded genome-wide and sex-dependent early-phase transcriptional changes in regenerating (proximal) sciatic nerve 24 h after axotomy. Male nerves exhibited more extensive transcriptional changes with male-dominant upregulation of cytoskeletal binding and structural protein genes. Regulation of mRNAs encoding ion and ionotropic neurotransmitter channels displayed prominent sexual dimorphism consistent with sex-specific mRNA axonal transport in an early-phase regenerative response. Protein kinases and axonal transport genes showed sexually dimorphic regulation. Genes encoding components of synaptic vesicles were at high baseline expression in females and showed post-injury induction selectively in males. Predictive bioinformatic analyses established patterns of sexually dimorphic regulation of neurotrophic and immune genes, including activation of glial cell line-derived neurotrophic factor Gfra1 receptor and immune checkpoint cyclin D1 (Ccnd1) potentially linked to X-chromosome encoded tissue inhibitor of matrix metallo proteinases 1 (Timp1). Regulatory networks involving Olig1, Pou3f3/Oct6, Myrf, and Myt1l transcription factors were linked to sex-dependent reprogramming in regenerating nerves. Differential expression patterns of non-coding RNAs motivate a model of sexually dimorphic nerve regenerative responses to injury determined by epigenetic factors. Combined with our findings in the corresponding dorsal root ganglia (DRG), unique early-phase sex-specific molecular triggers could enrich the mechanistic understanding of peripheral neuropathies.

Sexual dimorphism is a powerful yet understudied factor that influences the timing and efficiency of gene regulation in axonal injury and repair processes in the peripheral nervous system. Here, we identified common and distinct biological processes in female and male degenerating (distal) nerve stumps based on a snapshot of transcriptional reprogramming 24 h after axotomy reflecting the onset of early phase Wallerian degeneration (WD). Females exhibited transcriptional downregulation of a larger number of genes than males. RhoGDI, ERBB, and ERK5 signaling pathways increased activity in both sexes. Males upregulated genes and canonical pathways that exhibited robust baseline expression in females in both axotomized and sham nerves, including signaling pathways controlled by neuregulin and nerve growth factors. Cholesterol biosynthesis, reelin signaling, and synaptogenesis signaling pathways were downregulated in females. Signaling by Rho Family GTPases, cAMP-mediated signaling, and sulfated glycosaminoglycan biosynthesis were downregulated in both sexes. Estrogens potentially influenced sex-dependent injury response due to distinct regulation of estrogen receptor expression. A crosstalk of cytokines and growth hormones could promote sexually dimorphic transcriptional responses. We highlighted prospective regulatory activities due to protein phosphorylation, extracellular proteolysis, sex chromosome-specific expression, major urinary proteins (MUPs), and genes involved in thyroid hormone metabolism. Combined with our earlier findings in the corresponding dorsal root ganglia (DRG) and regenerating (proximal) nerve stumps, sex-specific and universal early phase molecular triggers of WD enrich our knowledge of transcriptional regulation in peripheral nerve injury and repair.

Objective(s): In this study, the impact of thiamine (Thi), N-acetyl cysteine (NAC), and dexamethasone (DEX) were investigated in axotomized rats, as a model for neural injury.Materials and Methods: Sixty-five axotomized rats were divided into two different experimental approaches, the first experiments included five study groups (n=5): intrathecal Thi (Thi.it), intraperitoneal (Thi), NAC, DEX, and control. Cell survival was assessed in L5DRG in the 4th week by histological assessment. In the second study, 40 animals were engaged to assess Bcl-2, Bax, IL-6, and TNF-α expression in L4-L5DRG in the 1st and 2nd weeks after sural nerve axotomy under treatment of these agents (n=10).Results: Ghost cells were observed in morphological assessment of L5DRG sections, and following stereological analysis, the volume and neuronal cell counts significantly were improved in the NAC and Thi.it groups in the 4th week (P

PurposeTo evaluate the integrative potential of neural stem cells (NSCs) with the visual system and characterize effects on the survival and axonal regeneration of axotomized retinal ganglion cells (RGCs).MethodsFor in vitro studies, primary, postnatal rat RGCs were directly cocultured with human NSCs or cultured in NSC-conditioned media before their survival and neurite outgrowth were assessed. For in vivo studies, human NSCs were transplanted into the transected rat optic nerve, and immunohistology of the retina and optic nerve was performed to evaluate RGC survival, RGC axon regeneration, and NSC integration with the injured visual system.ResultsIncreased neurite outgrowth was observed in RGCs directly cocultured with NSCs. NSC-conditioned media demonstrated a dose-dependent effect on RGC survival and neurite outgrowth in culture. NSCs grafted into the lesioned optic nerve modestly improved RGC survival following an optic nerve transection (593 ± 164 RGCs/mm2 vs. 199 ± 58 RGCs/mm2; P < 0.01). Additionally, RGC axonal regeneration following an optic nerve transection was modestly enhanced by NSCs transplanted at the lesion site (61.6 ± 8.5 axons vs. 40.3 ± 9.1 axons, P < 0.05). Transplanted NSCs also differentiated into neurons, received synaptic inputs from regenerating RGC axons, and extended axons along the transected optic nerve to incorporate with the visual system.ConclusionsHuman NSCs promote the modest survival and axonal regeneration of axotomized RGCs that is partially mediated by diffusible NSC-derived factors. Additionally, NSCs integrate with the injured optic nerve and have the potential to form neuronal relays to restore retinofugal connections.

Background: Peripheral nerve injury (PNI) is the most common type of nerve trauma yet, while injured motoneurons exhibit a robust capacity for regeneration, behavioral recovery is protracted and typically poor. Neurotherapeutic approaches to PNI and repair have primarily focused on the enhancement of axonal regeneration, in terms of rate, axonal sprouting, and reconnection connectivity. Both electrical stimulation (ES) and treatment with androgens [e.g., testosterone propionate (TP)] have been demonstrated to enhance axonal sprouting, regeneration rate and functional recovery following PNI. To date, very little work has been done to examine the effects of ES and/or TP on dendritic morphology and organization within the spinal cord after PNI. Objective: The objective of the current study was to examine the impact of treatment with TP and ES, alone or in combination, on the dendritic arbor of spinal motoneurons after target disconnection via sciatic nerve crush injury in the rat. Methods: Rats received a crush injury to the sciatic nerve. Following injury, some animals received either (1) no further treatment beyond implantation with empty Silastic capsules, (2) electrical nerve stimulation immediately after injury, (3) implantation with Silastic capsules filled with TP, or (4) electrical nerve stimulation immediately after injury as well as implantation with TP. All of these groups of axotomized animals also received bi-weekly electromyography (EMG) testing. Additional groups of intact untreated animals as well as a group of injured animals who received no further treatment or EMG testing were also included. Eight weeks after injury, motoneurons innervating the anterior tibialis muscle were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Results: After nerve crush and ES and/or TP treatment, motoneurons innervating the anterior tibialis underwent marked dendritic hypertrophy. Surprisingly, this dendritic hypertrophy occurred in all animals receiving repeated bi-weekly EMG testing, regardless of treatment. When the EMG testing was eliminated, the dendritic arbor extent and distribution after nerve crush in the treated groups did not significantly differ from intact untreated animals. Conclusions: The ability of repeated EMG testing to so dramatically affect central plasticity following a peripheral nerve injury was unexpected. It was also unexpected that gonadal steroid hormones and/or ES, two neurotherapeutic approaches with demonstrated molecular/behavioral changes consistent with peripheral improvements in axonal repair and target reconnection, do not appear to impact central plasticity in a similar manner. The significance of peripheral EMG testing and resulting central plasticity reorganization remains to be determined. [ABSTRACT FROM AUTHOR]

Simple Summary: It has remained obscure whether microglia proliferate in the diseased or injured brain. This study analyzed the mitotic ability of microglia in vivo in a rat facial nerve axotomy model in which the blood cells do not enter the parenchyma. Microglia were found to proliferate in the axotomized facial nucleus at 5–7 days post-insult. They enhanced the levels of macrophage colony-stimulating factor (M-CSF) as a mitotic factor, its receptor cFms, and cell-cycle-related proteins, such as proliferating cell nuclear antigen (PCNA) and cyclins in vivo. In the M-CSF-dependent proliferation system in vitro, c-Jun N-terminal kinase (JNK) and p38 were shown to function in the induction of PCNA/cyclins and cFms in microglia, respectively. These analyses revealed that some specific signaling cascades are linked to the proliferative reaction of microglia. Microglia in the axotomized facial nucleus were considered to exhibit a mitotic ability to rescue injured motoneurons as neurotrophic cells. Although microglia exist as a minor glial cell type in the normal state of the brain, they increase in number in response to various disorders and insults. However, it remains unclear whether microglia proliferate in the affected area, and the mechanism of the proliferation has long attracted the attention of researchers. We analyzed microglial mitosis using a facial nerve transection model in which the blood–brain barrier is left unimpaired when the nerves are axotomized. Our results showed that the levels of macrophage colony-stimulating factor (M-CSF), cFms (the receptor for M-CSF), cyclin A/D, and proliferating cell nuclear antigen (PCNA) were increased in microglia in the axotomized facial nucleus (axotFN). In vitro experiments revealed that M-CSF induced cFms, cyclin A/D, and PCNA in microglia, suggesting that microglia proliferate in response to M-CSF in vivo. In addition, M-CSF caused the activation of c-Jun N-terminal kinase (JNK) and p38, and the specific inhibitors of JNK and p38 arrested the microglial mitosis. JNK and p38 were shown to play roles in the induction of cyclins/PCNA and cFms, respectively. cFms was suggested to be induced through a signaling cascade of p38-mitogen- and stress-activated kinase-1 (MSK1)-cAMP-responsive element binding protein (CREB) and/or p38-activating transcription factor 2 (ATF2). Microglia proliferating in the axotFN are anticipated to serve as neuroprotective cells by supplying neurotrophic factors and/or scavenging excite toxins and reactive oxygen radicals. [ABSTRACT FROM AUTHOR]

Objective(s): In this study, the impact of thiamine (Thi), N-acetyl cysteine (NAC), and dexamethasone (DEX) were investigated in axotomized rats, as a model for neural injury. Materials and Methods: Sixty-five axotomized rats were divided into two different experimental approaches, the first experiments included five study groups (n=5): intrathecal Thi (Thi.it), intraperitoneal (Thi), NAC, DEX, and control. Cell survival was assessed in L5DRG in the 4th week by histological assessment. In the second study, 40 animals were engaged to assess Bcl-2, Bax, IL-6, and TNF-α expression in L4-L5DRG in the 1st and 2nd weeks after sural nerve axotomy under treatment of these agents (n=10). Results: Ghost cells were observed in morphological assessment of L5DRG sections, and following stereological analysis, the volume and neuronal cell counts significantly were improved in the NAC and Thi.it groups in the 4th week (P<0.05). Although Bcl-2 expression did not show significant differences, Bax was reduced in the Thi group (P=0.01); and the Bcl-2/Bax ratio increased in the NAC group (1st week, P<0.01). Furthermore, the IL-6 and TNF-α expression decreased in the Thi and NAC groups, on the 1st week of treatment (P≤0.05 and P<0.01). However, in the 2nd week, the IL-6 expression in both Thi and NAC groups (P<0.01), and the TNF-α expression in the DEX group (P=0.05) were significantly decreased. Conclusion: The findings may classify Thi in the category of peripheral neuroprotective agents, in combination with routine medications. Furthermore, it had strong cell survival effects as it could interfere with the destructive effects of TNF-α by increasing Bax. [ABSTRACT FROM AUTHOR]

Regeneration and functional recovery of the damaged nerve are challenging due to the need for effective therapeutic drugs, biomaterials, and approaches. The poor outcome of the treatment of nerve injury stems from the incomplete understanding of axonal biology and interactions between neurons and the surrounding environment, such as glial cells and extracellular matrix. Microfluidic devices, in combination with various injury techniques, have been applied to test biological hypotheses in nerve injury and nerve regeneration. The microfluidic devices provide multiple advantages over the in vitro cell culture on a petri dish and in vivo animal models because a specific part of the neuronal environment can be manipulated using physical and chemical interventions. In addition, single‐cell behavior and interactions between neurons and glial cells can be visualized and quantified on microfluidic platforms. In this article, current in vitro nerve injury models on a chip that mimics in vivo axonal injuries and the regeneration process of axons are summarized. The microfluidic‐based nerve injury models could enhance the understanding of the physiological and pathophysiological mechanisms of nerve tissues and simultaneously serve as powerful drug and biomaterial screening platforms. [ABSTRACT FROM AUTHOR]

Peripheral nerve injury induces genome-wide transcriptional reprogramming of first-order neurons and auxiliary cells of dorsal root ganglia (DRG). Accumulating experimental evidence suggests that onset and mechanistic principles of post-nerve injury processes are sexually dimorphic. We examined largely understudied aspects of early transcriptional events in DRG within 24 h after sciatic nerve axotomy in mice of both sexes. Using high-depth RNA sequencing (>50 million reads/sample) to pinpoint sexually dimorphic changes related to regeneration, immune response, bioenergy, and sensory functions, we identified a higher number of transcriptional changes in male relative to female DRG. In males, the decline in ion channel transcripts was accompanied by the induction of innate immune cascades via TLR, chemokine, and Csf1-receptor axis and robust regenerative programs driven by Sox, Twist1/2, and Pax5/9 transcription factors. Females demonstrated nerve injury-specific transcriptional co-activation of the actinin 2 network. The predicted upstream regulators and interactive networks highlighted the role of novel epigenetic factors and genetic linkage to sex chromosomes as hallmarks of gene regulation post-axotomy. We implicated epigenetic X chromosome inactivation in the regulation of immune response activity uniquely in females. Sexually dimorphic regulation of MMP/ADAMTS metalloproteinases and their intrinsic X-linked regulator Timp1 contributes to extracellular matrix remodeling integrated with pro-regenerative and immune functions. Lexis1 non-coding RNA involved in LXR-mediated lipid metabolism was identified as a novel nerve injury marker. Together, our data identified unique early response triggers of sex-specific peripheral nerve injury regulation to gain mechanistic insights into the origin of female- and male-prevalent sensory neuropathies.

Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for biochemical studies, and (iii) the measurement of neurite markers and metabolites after axotomy. LUHMES-based spheroids, plated in culture dishes, extended neurites of several thousand µm length, while all somata remained aggregated. These cultures allowed an easy microscopic observation of live or fixed neurites. Neurite-only cultures (NOC) were produced by cutting out the still-aggregated somata. The potential application of such cultures was exemplified by determinations of their protein and RNA contents. For instance, the mitochondrial TOM20 protein was highly abundant, while nuclear histone H3 was absent. Similarly, mitochondrial-encoded RNAs were found at relatively high levels, while the mRNA for a histone or the neuronal nuclear marker NeuN (RBFOX3) were relatively depleted in NOC. Another potential use of NOC is the study of neurite degeneration. For this purpose, an algorithm to quantify neurite integrity was developed. Using this tool, we found that the addition of nicotinamide drastically reduced neurite degeneration. Also, the chelation of Ca2+ in NOC delayed the degeneration, while inhibitors of calpains had no effect. Thus, NOC proved to be suitable for biochemical analysis and for studying degeneration processes after a defined cut injury.

Chronic, often intractable, pain is caused by neuropathic conditions such as traumatic peripheral nerve injury (PNI) and spinal cord injury (SCI). These conditions are associated with alterations in gene and protein expression correlated with functional changes in somatosensory neurons having cell bodies in dorsal root ganglia (DRGs). Most studies of DRG transcriptional alterations have utilized PNI models where axotomy-induced changes important for neural regeneration may overshadow changes that drive neuropathic pain. Both PNI and SCI produce DRG neuron hyperexcitability linked to pain, but contusive SCI produces little peripheral axotomy or peripheral nerve inflammation. Thus, comparison of transcriptional signatures of DRGs across PNI and SCI models may highlight pain-associated transcriptional alterations in sensory ganglia that do not depend on peripheral axotomy or associated effects such as peripheral Wallerian degeneration. Data from our rat thoracic SCI experiments were combined with meta-analysis of published whole-DRG RNA-seq datasets from prominent rat PNI models. Striking differences were found between transcriptional responses to PNI and SCI, especially in regeneration-associated genes (RAGs) and long noncoding RNAs (lncRNAs). Many transcriptomic changes after SCI also were found after corresponding sham surgery, indicating they were caused by injury to surrounding tissue, including bone and muscle, rather than to the spinal cord itself. Another unexpected finding was of few transcriptomic similarities between rat neuropathic pain models and the only reported transcriptional analysis of human DRGs linked to neuropathic pain. These findings show that DRGs exhibit complex transcriptional responses to central and peripheral neural injury and associated tissue damage. Although only a few genes in DRG cells exhibited similar changes in expression across all the painful conditions examined here, these genes may represent a core set whose transcription in various DRG cell types is sensitive to significant bodily injury, and which may play a fundamental role in promoting neuropathic pain. [ABSTRACT FROM AUTHOR]

The difficulties of injured and degenerated neurons to regenerate neurites and regain functions are more significant than in other body tissues, making neurodegenerative and related diseases hard to cure. Uncovering the secrets of neural regeneration and how this process may be inhibited after injury will provide insights into novel management and potential treatments for these diseases. Caenorhabditis elegans and Drosophila melanogaster are two of the most widely used and well-established model organisms endowed with advantages in genetic manipulation and live imaging to explore this fundamental question about neural regeneration. Here, we review the classical models and techniques, and the involvement and cooperation of subcellular structures during neurite regeneration using these two organisms. Finally, we list several important open questions that we look forward to inspiring future research. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). Methods: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3–4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9–35 ms) were computed for two 7-hexagon groupings—the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. Results: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9–35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. Conclusions: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells. [ABSTRACT FROM AUTHOR]

Upon receiving injury signals, neurons can activate various pathways to reduce harm, initiate neuroprotection, and repair damaged neurite without cell death. Here, we review recent progresses in the study of neurite repair focusing on neuronal cell-autonomous mechanisms, including new findings on ion channels that serve as key regulators to initiate neurite repair and intrinsic signaling pathways and transcriptional and post-transcriptional factors that facilitate neurite repair. We also touch upon reports on how dendrites may be affected upon axotomy and how the regeneration potential in injured neurites might be maximized.

Abstract Background Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods Ccl2 fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2 gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2 gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2 gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

We demonstrated previously that the hypothalamic supraoptic nucleus (SON) undergoes an axonal sprouting response following a unilateral lesion of the hypothalamo-neurohypophysial tract in a 35-day-old rat to repopulate the partially denervated neural lobe (NL). However, no sprouting occurs following the same injury in a 125-day-old rat. We previously reported a significant increase in Thy-1 protein in the SON of a 125-day-old rat compared to a 35-day-old rat in the absence of injury. Thy-1 is a cell surface glycoprotein shown to inhibit axonal outgrowth following injury; however, we did not look at axotomy's effect on Thy-1 in the SON. Therefore, we sought to determine the integrin ligands that bind Thy-1 in the SON and how axotomy impacts Thy-1. Like what others have shown, the co-immunoprecipitation analysis demonstrated that Thy-1 interacts with αvß3 and αvß5 integrin dimers in the SON. We used western blot analysis to examine protein levels of Thy-1 and integrin subunits following injury in the 35- and 125-day-old rat SON and NL. Our results demonstrated that Thy-1 protein levels increase in the lesion SON in a 35-day-old rat. The quantitative dual-fluorescent analysis showed that the increase in Thy-1 in the lesion SON occurred in astrocytes. There was no change in Thy-1 or integrin protein levels following injury in the 125-day-old following injury. Furthermore, the axotomy significantly decreased Thy-1 protein levels in the NL of both 35- and 125-day-old rats. These results provide evidence that Thy-1 protein levels are injury dependent in the magnocellular neurosecretory system. [ABSTRACT FROM AUTHOR]

A spinal cord injury (SCI) damages the axonal projections of neurons residing in the neocortex. This axotomy changes cortical excitability and results in dysfunctional activity and output of infragranular cortical layers. Thus, addressing cortical pathophysiology after SCI will be instrumental in promoting recovery. However, the cellular and molecular mechanisms of cortical dysfunction after SCI are poorly resolved. In this study, we determined that the principal neurons of the primary motor cortex layer V (M1LV), those suffering from axotomy upon SCI, become hyperexcitable following injury. Therefore, we questioned the role of hyperpolarization cyclic nucleotide gated channels (HCN channels) in this context. Patch clamp experiments on axotomized M1LV neurons and acute pharmacological manipulation of HCN channels allowed us to resolve a dysfunctional mechanism controlling intrinsic neuronal excitability one week after SCI. Some axotomized M1LV neurons became excessively depolarized. In those cells, the HCN channels were less active and less relevant to control neuronal excitability because the membrane potential exceeded the window of HCN channel activation. Care should be taken when manipulating HCN channels pharmacologically after SCI. Even though the dysfunction of HCN channels partakes in the pathophysiology of axotomized M1LV neurons, their dysfunctional contribution varies remarkably between neurons and combines with other pathophysiological mechanisms. [ABSTRACT FROM AUTHOR]

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury. [ABSTRACT FROM AUTHOR]

Erinacines derived from Hericium erinaceus have been shown to possess various health benefits including neuroprotective effect against neurodegenerative diseases, yet the underlying mechanism remains unknown. Here we found that erinacine S enhances neurite outgrowth in a cell autonomous fashion. It promotes post-injury axon regeneration of PNS neurons and enhances regeneration on inhibitory substrates of CNS neurons. Using RNA-seq and bioinformatic analyses, erinacine S was found to cause the accumulation of neurosteroids in neurons. ELISA and neurosteroidogenesis inhibitor assays were performed to validate this effect. This research uncovers a previously unknown effect of erinacine S on raising the level of neurosteroids. [ABSTRACT FROM AUTHOR]

Traumatic brain injury (TBI) is one of the leading causes of disability and death worldwide. It is characterized by various molecular–cellular events, with the main ones being apoptosis and damage to axons. To date, there are no clinically effective neuroprotective drugs. In this study, we examined the role of hydrogen sulfide (H2S) in the localization and expression of the key pro-apoptotic protein p53, as well as cell death in the nervous tissue in TBI and axotomy. We used a fast donor (sodium sulphide, Na2S) H2S and a classic inhibitor (aminooxyacetic acid, AOAA) of cystathionine β-synthase (CBS), which is a key enzyme in H2S synthesis. These studies were carried out on three models of neurotrauma in vertebrates and invertebrates. As a result, it was found that Na2S exhibits a pronounced neuroprotective effect that reduces the number of TUNEL-positive neurons and glial cells in TBI and apoptotic glia in axotomy. This effect could be realized through the Na2S-dependent decrease in the level of p53 in the cells of the nervous tissue of vertebrates and invertebrates, which we observed in our study. We also observed the opposite effect when using AOAA, which indicates the important role of CBS in the regulation of p53 expression and death of neurons and glial cells in TBI and axotomy.

The mechanisms underlying axon regeneration in mature neurons are relevant to the understanding of normal nervous system maintenance and for developing therapeutic strategies for injury. Here, we report novel pathways in axon regeneration, identified by extending our previous function-based screen using the C. elegans mechanosensory neuron axotomy model. We identify an unexpected role of the nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme, NMAT-2/NMNAT, in axon regeneration. NMAT-2 inhibits axon regrowth via cell-autonomous and non-autonomous mechanisms. NMAT-2 enzymatic activity is required to repress regrowth. Further, we find differential requirements for proteins in membrane contact site, components and regulators of the extracellular matrix, membrane trafficking, microtubule and actin cytoskeleton, the conserved Kelch-domain protein IVNS-1, and the orphan transporter MFSD-6 in axon regrowth. Identification of these new pathways expands our understanding of the molecular basis of axonal injury response and regeneration.

Recovery of mimic function after facial nerve transection is poor. The successful regrowth of regenerating motor nerve fibers to reinnervate their targets is compromised by (i) poor axonal navigation and excessive collateral branching, (ii) abnormal exchange of nerve impulses between adjacent regrowing axons, namely axonal crosstalk, and (iii) insufficient synaptic input to the axotomized facial motoneurons. As a result, axotomized motoneurons become hyperexcitable but unable to discharge. We review our findings, which have addressed the poor return of mimic function after facial nerve injuries, by testing the hypothesized detrimental component, and we propose that intensifying the trigeminal sensory input to axotomized and electrophysiologically silent facial motoneurons improves the specificity of the reinnervation of appropriate targets. We compared behavioral, functional, and morphological parameters after single reconstructive surgery of the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery, but combined with direct or indirect stimulation of the ipsilateral infraorbital nerve. We found that both methods of trigeminal sensory stimulation, i.e., stimulation of the vibrissal hairs and manual stimulation of the whisker pad, were beneficial for the outcome through improvement of the quality of target reinnervation and recovery of vibrissal motor performance. [ABSTRACT FROM AUTHOR]

The pleiotropic role of the major histocompatibility complex class I (MHC-I) reflects the close association between the nervous and immune systems. In turn, MHC-I upregulation postinjury is associated with a better regenerative outcome in isogenic mice following peripheral nerve damage. In the present work, we compared the time course of neuronal, glial, and sensorimotor recovery (1, 3, 5, 7, and 28 days after lesion—dal) following unilateral sciatic nerve crush in A/J and C57BL/6J mice. The A/J strain showed higher expression of MHC-I (7 dal, ** p < 0.01), Iba-1 (microglial reaction, 7 dal, *** p < 0.001), and GFAP (astrogliosis, 5 dal, * p < 0.05) than the C57BL/6J counterpart. Synaptic coverage (synaptophysin) was equivalent in both strains over time. In addition, mRNA expression of microdissected spinal motoneurons revealed an increase in cytoskeleton-associated molecules (cofilin, shp2, and crmp2, * p < 0.05), but not trkB, in C57BL/6J mice. Gait recovery, studied by the sciatic functional index, was faster in the A/J strain, despite the equivalent results of C57BL/6J at 28 days after injury. A similar recovery was also seen for the nociceptive threshold (von Frey test). Interestingly, when evaluating proprioceptive recovery, C57BL/6J animals showed an enlarged base of support, indicating abnormal ambulation postinjury. Overall, the present results reinforce the role of MHC-I expression in the plasticity of the nervous system following axotomy, which in turn correlates with the variable recovery capacity among strains of mice. [ABSTRACT FROM AUTHOR]

Due to their biomimetic properties, electrospun nanofibers have been widely used in neurobiology studies. However, mechanistic understanding of cell‐nanofiber interactions is challenging based on the current in vitro culture systems due to the lack of control of spatiotemporal patterning of cells and difficulty in monitoring single cell behavior. To overcome these issues, we apply microfluidic technology in combination with electrospun nanofibers for in vitro studies of interactions between neurons and nanofiber materials. We demonstrate a unique nanofiber embedded microfluidic device which contains patterned aligned or random electrospun nanofibers as a new culture system. With this device, we test how different topographies affect axonal growth. Also, we conduct laser based axotomy on neurons cultured on our device to investigate axonal regeneration. The proposed device could be a useful tool for investigating nerve injury mechanisms and high‐throughput screening of biomaterials or drugs for nerve repair. The knowledge obtained using this device can be applicable to design medical devices such as nerve conduits for effective nerve regeneration. [ABSTRACT FROM AUTHOR]

Sexual dimorphism is a powerful yet understudied factor that influences the timing and efficiency of gene regulation in axonal injury and repair processes in the peripheral nervous system. Here, we identified common and distinct biological processes in female and male degenerating (distal) nerve stumps based on a snapshot of transcriptional reprogramming 24 h after axotomy reflecting the onset of early phase Wallerian degeneration (WD). Females exhibited transcriptional downregulation of a larger number of genes than males. RhoGDI, ERBB, and ERK5 signaling pathways increased activity in both sexes. Males upregulated genes and canonical pathways that exhibited robust baseline expression in females in both axotomized and sham nerves, including signaling pathways controlled by neuregulin and nerve growth factors. Cholesterol biosynthesis, reelin signaling, and synaptogenesis signaling pathways were downregulated in females. Signaling by Rho Family GTPases, cAMP-mediated signaling, and sulfated glycosaminoglycan biosynthesis were downregulated in both sexes. Estrogens potentially influenced sex-dependent injury response due to distinct regulation of estrogen receptor expression. A crosstalk of cytokines and growth hormones could promote sexually dimorphic transcriptional responses. We highlighted prospective regulatory activities due to protein phosphorylation, extracellular proteolysis, sex chromosome-specific expression, major urinary proteins (MUPs), and genes involved in thyroid hormone metabolism. Combined with our earlier findings in the corresponding dorsal root ganglia (DRG) and regenerating (proximal) nerve stumps, sex-specific and universal early phase molecular triggers of WD enrich our knowledge of transcriptional regulation in peripheral nerve injury and repair. [ABSTRACT FROM AUTHOR]

Facial motoneuron (FMN) survival is mediated by CD4+ T cells in an interleukin-10 (IL-10)-dependent manner after facial nerve axotomy (FNA), but CD4+ T cells themselves are not the source of this neuroprotective IL-10. The aims of this study were to (1) identify the temporal and cell-specific induction of IL-10 expression in the facial motor nucleus and (2) elucidate the neuroprotective capacity of this expression after axotomy. Immunohistochemistry revealed that FMN constitutively produced IL-10, whereas astrocytes were induced to make IL-10 after FNA. Il10 mRNA co-localized with microglia before and after axotomy, but microglial production of IL-10 protein was not detected. To determine whether any single source of IL-10 was critical for FMN survival, Cre/Lox mouse strains were utilized to selectively knock out IL-10 in neurons, astrocytes, and microglia. In agreement with the localization data reflecting concerted IL-10 production by multiple cell types, no single cellular source of IL-10 alone could provide neuroprotection after FNA. These findings suggest that coordinated neuronal and astrocytic IL-10 production is necessary for FMN survival and has roles in neuronal homeostasis, as well as neuroprotective trophism after axotomy. [ABSTRACT FROM AUTHOR]

Abstract Background Peripheral nerve injuries are accompanied by inflammatory reactions, over-activation of which may hinder recovery. Among pro-inflammatory pathways, inflammasomes are one of the most potent, leading to release of active IL-1β. Our aim was to understand how inflammasomes participate in central inflammatory reactions accompanying peripheral nerve injury. Methods After axotomy of the sciatic nerve, priming and activation of the NLRP3 inflammasome was examined in cells of the spinal cord. Regeneration of the nerve was evaluated after coaptation using sciatic functional index measurements and retrograde tracing. Results In the first 3 days after the injury, elements of the NLRP3 inflammasome were markedly upregulated in the L4–L5 segments of the spinal cord, followed by assembly of the inflammasome and secretion of active IL-1β. Although glial cells are traditionally viewed as initiators of neuroinflammation, in this acute phase of inflammation, inflammasome activation was found exclusively in affected motoneurons of the ventral horn in our model. This process was significantly inhibited by 5-BDBD, a P2X4 receptor inhibitor and MCC950, a potent NLRP3 inhibitor. Although at later time points the NLRP3 protein was upregulated in microglia too, no signs of inflammasome activation were detected in these cells. Inhibition of inflammasome activation in motoneurons in the first days after nerve injury hindered development of microgliosis in the spinal cord. Moreover, P2X4 or inflammasome inhibition in the acute phase significantly enhanced nerve regeneration on both the morphological and the functional levels. Conclusions Our results indicate that the central reaction initiated by sciatic nerve injury starts with inflammasome activation in motoneurons of the ventral horn, which triggers a complex inflammatory reaction and activation of microglia. Inhibition of neuronal inflammasome activation not only leads to a significant reduction of microgliosis, but has a beneficial effect on the recovery as well.

Although microglia exist as a minor glial cell type in the normal state of the brain, they increase in number in response to various disorders and insults. However, it remains unclear whether microglia proliferate in the affected area, and the mechanism of the proliferation has long attracted the attention of researchers. We analyzed microglial mitosis using a facial nerve transection model in which the blood–brain barrier is left unimpaired when the nerves are axotomized. Our results showed that the levels of macrophage colony-stimulating factor (M-CSF), cFms (the receptor for M-CSF), cyclin A/D, and proliferating cell nuclear antigen (PCNA) were increased in microglia in the axotomized facial nucleus (axotFN). In vitro experiments revealed that M-CSF induced cFms, cyclin A/D, and PCNA in microglia, suggesting that microglia proliferate in response to M-CSF in vivo. In addition, M-CSF caused the activation of c-Jun N-terminal kinase (JNK) and p38, and the specific inhibitors of JNK and p38 arrested the microglial mitosis. JNK and p38 were shown to play roles in the induction of cyclins/PCNA and cFms, respectively. cFms was suggested to be induced through a signaling cascade of p38-mitogen- and stress-activated kinase-1 (MSK1)-cAMP-responsive element binding protein (CREB) and/or p38-activating transcription factor 2 (ATF2). Microglia proliferating in the axotFN are anticipated to serve as neuroprotective cells by supplying neurotrophic factors and/or scavenging excite toxins and reactive oxygen radicals.

Sexual dimorphism is a powerful yet understudied factor that influences the timing and efficiency of gene regulation in axonal injury and repair processes in the peripheral nervous system. Here, we identified common and distinct biological processes in female and male degenerating (distal) nerve stumps based on a snapshot of transcriptional reprogramming 24 h after axotomy reflecting the onset of early phase Wallerian degeneration (WD). Females exhibited transcriptional downregulation of a larger number of genes than males. RhoGDI, ERBB, and ERK5 signaling pathways increased activity in both sexes. Males upregulated genes and canonical pathways that exhibited robust baseline expression in females in both axotomized and sham nerves, including signaling pathways controlled by neuregulin and nerve growth factors. Cholesterol biosynthesis, reelin signaling, and synaptogenesis signaling pathways were downregulated in females. Signaling by Rho Family GTPases, cAMP-mediated signaling, and sulfated glycosaminoglycan biosynthesis were downregulated in both sexes. Estrogens potentially influenced sex-dependent injury response due to distinct regulation of estrogen receptor expression. A crosstalk of cytokines and growth hormones could promote sexually dimorphic transcriptional responses. We highlighted prospective regulatory activities due to protein phosphorylation, extracellular proteolysis, sex chromosome-specific expression, major urinary proteins (MUPs), and genes involved in thyroid hormone metabolism. Combined with our earlier findings in the corresponding dorsal root ganglia (DRG) and regenerating (proximal) nerve stumps, sex-specific and universal early phase molecular triggers of WD enrich our knowledge of transcriptional regulation in peripheral nerve injury and repair.

The convergence of transcriptional and epigenetic changes in the peripheral nervous system (PNS) reshapes the spatiotemporal gene expression landscape in response to nerve transection. The control of these molecular programs exhibits sexually dimorphic characteristics that remain not sufficiently characterized. In the present study, we recorded genome-wide and sex-dependent early-phase transcriptional changes in regenerating (proximal) sciatic nerve 24 h after axotomy. Male nerves exhibited more extensive transcriptional changes with male-dominant upregulation of cytoskeletal binding and structural protein genes. Regulation of mRNAs encoding ion and ionotropic neurotransmitter channels displayed prominent sexual dimorphism consistent with sex-specific mRNA axonal transport in an early-phase regenerative response. Protein kinases and axonal transport genes showed sexually dimorphic regulation. Genes encoding components of synaptic vesicles were at high baseline expression in females and showed post-injury induction selectively in males. Predictive bioinformatic analyses established patterns of sexually dimorphic regulation of neurotrophic and immune genes, including activation of glial cell line-derived neurotrophic factor Gfra1 receptor and immune checkpoint cyclin D1 (Ccnd1) potentially linked to X-chromosome encoded tissue inhibitor of matrix metallo proteinases 1 (Timp1). Regulatory networks involving Olig1, Pou3f3/Oct6, Myrf, and Myt1l transcription factors were linked to sex-dependent reprogramming in regenerating nerves. Differential expression patterns of non-coding RNAs motivate a model of sexually dimorphic nerve regenerative responses to injury determined by epigenetic factors. Combined with our findings in the corresponding dorsal root ganglia (DRG), unique early-phase sex-specific molecular triggers could enrich the mechanistic understanding of peripheral neuropathies. [ABSTRACT FROM AUTHOR]

Background: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. Methods: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2–RFPfl/fl mice. CCL2–CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. Results: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2–RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. Conclusions: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation. [ABSTRACT FROM AUTHOR]