Peripheral Nerve Injury Model Research Articles

Porcine decellularized nerve matrix hydrogel attenuates neuroinflammation after peripheral nerve injury by inhibiting the TLR4/MyD88/NF-κB axis

Peripheral nerve injury causes severe neuroinflammation and has become a global medical challenge. Previous research has demonstrated that porcine decellularized nerve matrix hydrogel exhibits excellent biological properties and tissue specificity, highlighting its potential as a biomedical material for the repair of severe peripheral nerve injury; however, its role in modulating neuroinflammation post–peripheral nerve injury remains unknown. Here, we aimed to characterize the anti-inflammatory properties of porcine decellularized nerve matrix hydrogel and their underlying molecular mechanisms. Using peripheral nerve injury model rats treated with porcine decellularized nerve matrix hydrogel, we evaluated structural and functional recovery, macrophage phenotype alteration, specific cytokine expression, and changes in related signaling molecules in vivo. Similar parameters were evaluated in vitro using monocyte/macrophage cell lines stimulated with lipopolysaccharide and cultured on porcine decellularized nerve matrix hydrogel–coated plates in complete medium. These comprehensive analyses revealed that porcine decellularized nerve matrix hydrogel attenuated the activation of excessive inflammation at the early stage of peripheral nerve injury and increased the proportion of the M2 subtype in monocytes/macrophages. Additionally, porcine decellularized nerve matrix hydrogel negatively regulated the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-κB axis both in vivo and in vitro. Our findings suggest that the efficacious anti-inflammatory properties of porcine decellularized nerve matrix hydrogel induce M2 macrophage polarization via suppression of the Toll-like receptor 4/myeloid differentiation factor 88/nuclear factor-?B pathway, providing new insights into the therapeutic mechanism of porcine decellularized nerve matrix hydrogel in peripheral nerve injury.

A microfluidic model of the first sensory synapse for analgesic target discovery.

The synaptic connections between dorsal root ganglia (DRG) and dorsal horn (DH) neurons are a crucial relay point for the transmission of painful stimuli. To delineate how synaptic plasticity may modulate the excitability of DH neurons, we have devised a microfluidic co-culture model that recapitulates the first sensory synapse using postnatal mouse sensory neurons. We show that DRG-DH co-cultures characterize salient features of the in vivo physiology of sensory neurons. Immunocytcochemical experiments of the cultured DH neurons show a co-localization of MAP2 with VGLUT2 and of MAP2 with Synapsin 1, corroborating the glutamatergic identity of the DH neurons and further suggesting the formation of active synapses in this neuronal set. Fluorometric imaging experiments demonstrate the elicitation of calcium responses in DH neurons following the stimulation of DRG cell bodies or axons. Selective NMDA and AMPA receptor blockade appreciably silences DH neuron responses, suggesting that glutamatergic signaling is maintained in vitro. Last, a surrogate model of peripheral nerve injury is introduced in the form of an axotomy, which results in elevated and prolonged calcium responses of DH neurons. Overall, the microfluidic mouse co-cultures provide a method advancement in the study of periphery-to-center pain signaling, where the potential of utilizing the platform for drug target identification is underscored.

In Vitro Models for Peripheral Nerve Regeneration.

Peripheral nerve injury (PNI) resulting in lesions is highly prevalent clinically, but current therapeutic approaches fail to provide satisfactory outcomes in many patients. While peripheral nerves have intrinsic regenerative capacity, the regenerative capabilities of peripheral nerves are often insufficient to restore full functionality. This highlights an unmet need for developing more effective strategies to repair damaged peripheral nerves and improve regenerative success. Consequently, researchers are actively exploring a variety of therapeutic strategies, encompassing the local delivery of trophic factors or bioactive molecules, the design of advanced biomaterials that interact with regenerating axons, and augmentation with nerve guidance conduits or complex prostheses. However, clinical translation of these technologies remains limited, emphasizing the need for continued research on peripheral nerve regeneration modalities that can enhance functional restoration. Experimental models that accurately recapitulate key aspects of peripheral nerve injury and repair biology can accelerate therapeutic development by enabling systematic testing of new techniques. Advancing regenerative therapies for PNI requires bridging the gap between basic science discoveries and clinical application. This review discusses different in vitro models of peripheral nerve injury and repair, including their advantages, limitations, and potential applications.

Establishment of a Magnetically Controlled Scalable Nerve Injury Model.

Animal models of peripheral nerve injury (PNI) serve as the fundamental basis for the investigations of nerve injury, regeneration, and neuropathic pain. The injury properties of such models, including the intensity and duration, significantly influence the subsequent pathological changes, pain development, and therapeutic efficacy. However, precise control over the intensity and duration of nerve injury remains challenging within existing animal models, thereby impeding accurate and comparative assessments of relevant cases. Here, a new model that provides quantitative and off-body controllable injury properties via a magnetically controlled clamp, is presented. The clamp can be implanted onto the rat sciatic nerve and exert varying degrees of compression under the control of an external magnetic field. It is demonstrated that this model can accurately simulate various degrees of pathology of human patients by adjusting the magnetic control and reveal specific pathological changes resulting from intensity heterogeneity that are challenging to detect previously. The controllability and quantifiability of this model may significantly reduce the uncertainty of central response and inter-experimenter variability, facilitating precise investigations into nerve injury, regeneration, and pain mechanisms.

Induced pluripotent stem cell-derived mesenchymal stem cells enhance acellular nerve allografts to promote peripheral nerve regeneration by facilitating angiogenesis.

Previous research has demonstrated the feasibility of repairing nerve defects through acellular allogeneic nerve grafting with bone marrow mesenchymal stem cells. However, adult tissue-derived mesenchymal stem cells encounter various obstacles, including limited tissue sources, invasive acquisition methods, cellular heterogeneity, purification challenges, cellular senescence, and diminished pluripotency and proliferation over successive passages. In this study, we used induced pluripotent stem cell-derived mesenchymal stem cells, known for their self-renewal capacity, multilineage differentiation potential, and immunomodulatory characteristics. We used induced pluripotent stem cell-derived mesenchymal stem cells in conjunction with acellular nerve allografts to address a 10 mm-long defect in a rat model of sciatic nerve injury. Our findings reveal that induced pluripotent stem cell-derived mesenchymal stem cells exhibit survival for up to 17 days in a rat model of peripheral nerve injury with acellular nerve allograft transplantation. Furthermore, the combination of acellular nerve allograft and induced pluripotent stem cell-derived mesenchymal stem cells significantly accelerates the regeneration of injured axons and improves behavioral function recovery in rats. Additionally, our in vivo and in vitro experiments indicate that induced pluripotent stem cell-derived mesenchymal stem cells play a pivotal role in promoting neovascularization. Collectively, our results suggest the potential of acellular nerve allografts with induced pluripotent stem cell-derived mesenchymal stem cells to augment nerve regeneration in rats, offering promising therapeutic strategies for clinical translation.

Nerve Regeneration Potential of Antioxidant-Modified Black Phosphorus Quantum Dots in Peripheral Nerve Injury.

Peripheral nerve injury is a major societal concern. Black phosphorus (BP) has inherent advantages over cell-based therapies in regenerative medicine. However, controlling spontaneous degradation and size-dependent cytotoxicity remains challenging and poses difficulties for clinical translation. In this study, we constructed zero-dimensional BP quantum dots (QDs) modified with antioxidant β-carotene and comprehensively investigated them in Schwann cells (SCs) to elucidate their potential for peripheral nerve repair. In vitro experiments demonstrated that BPQD@β-carotene has an inappreciable toxicity and good biocompatibility, favoring neural regrowth, angiogenesis, and inflammatory regulation of SCs. Furthermore, the PI3K/Akt and Ras/ERK1/2 signaling pathways were activated in SCs at the genetic, protein, and metabolite levels. The BPQD@β-carotene-embedded GelMA/PEGDA scaffold enhanced functional recovery by promoting axon remyelination and regeneration and facilitating intraneural angiogenesis in peripheral nerve injury models of rats and beagle dogs. These results contribute to advancing knowledge of BP nanomaterials in tissue regeneration and show significant potential for application in translational medicine.

Hybrid construction of tissue-engineered nerve graft using skin derived precursors induced neurons and Schwann cells to enhance peripheral neuroregeneration

Peripheral nerve injury is a major challenge in clinical treatment due to the limited intrinsic capacity for nerve regeneration. Tissue engineering approaches offer promising solutions by providing biomimetic scaffolds and cell sources to promote nerve regeneration. In the present work, we investigated the potential role of skin-derived progenitors (SKPs), which are induced into neurons and Schwann cells (SCs), and their extracellular matrix in tissue-engineered nerve grafts (TENGs) to enhance peripheral neuroregeneration. SKPs were induced to differentiate into neurons and SCs in vitro and incorporated into nerve grafts composed of a biocompatible scaffold including chitosan neural conduit and silk fibroin filaments. In vivo experiments using a rat model of peripheral nerve injury showed that TENGs significantly enhanced nerve regeneration compared to the scaffold control group, catching up with the autograft group. Histological analysis showed improved axonal regrowth, myelination and functional recovery in animals treated with these TENGs. In addition, immunohistochemical staining confirmed the presence of induced neurons and SCs within the regenerated nerve tissue. Our results suggest that SKP-induced neurons and SCs in tissue-engineered nerve grafts have great potential for promoting peripheral nerve regeneration and represent a promising approach for clinical translation in the treatment of peripheral nerve injury. Further optimization and characterization of these engineered constructs is warranted to improve their clinical applicability and efficacy.

Shell-core structured nanofibers mediate staged anti-inflammatory and pro-neurogenic activities to repair peripheral nerve

The inflammatory reaction significantly impedes the neurogenic process during the restoration of peripheral nerve injury (PNI). Therefore, establishing a non-inflammatory environment is crucial for effective nerve regeneration. This study proposes the use of shell-core structured nanofibers with sequential anti-inflammatory and pro-neurogenic activities to repair PNI. Icariin (ICA), known for its anti-inflammatory effects, was blended with poly(lactic-co-glycolic acid) (PLGA) to form the shell layer’s spinning solution. Concurrently, glial cell-derived neurotrophic factor (GDNF) was combined with graphene oxide (GO) to create the core layer’s spinning solution. These solutions were then subjected to co-axial electrospinning, resulting in shell-core structured GDNF@GO-ICA@PLGA nanofibers. Additionally, a control group of unordered GDNF/GO/ICA/PLGA nanofibers was prepared using conventional electrospinning. The resulting GDNF@GO-ICA@PLGA nanofibers exhibited distinct fibrous structures with a clear shell-core architecture and demonstrated mechanical properties similar to the control group. Notably, the shell-core structured GDNF@GO-ICA@PLGA nanofibers displayed unique staged release kinetics: over 90% ICA was released priorly within the first 0 to 13 days, followed by GDNF release from days 9 to 31. Furthermore, the GDNF@GO-ICA@PLGA nanofibers showed excellent biocompatibility with Schwann cells. In vitro results highlighted the potent anti-inflammatory capabilities of ICA released from the shell layer, while GDNF released from the core layer effectively induced neurogenic differentiation of Schwann cells. The GDNF@GO-ICA@PLGA nanofibers were then processed into a nerve conduit and applied to a 10 mm rat sciatic PNI model. The staged release of ICA and GDNF facilitated by the GDNF@GO-ICA@PLGA nanofibers created a non-inflammatory environment before initiating nerve regeneration, leading to improved PNI restoration. This study underscores the importance of shell-core structured nanofibers in sequentially mediating anti-inflammation and neurogenesis, offering a novel approach for addressing PNI.

Diversity of microglial transcriptional responses during opioid exposure and neuropathic pain.

Microglia take on an altered morphology during chronic opioid treatment. This morphological change is broadly used to identify the activated microglial state associated with opioid side effects, including tolerance and opioid-induced hyperalgesia (OIH). Microglia display similar morphological responses in the spinal cord after peripheral nerve injury (PNI). Consistent with this observation, functional studies have suggested that microglia activated by opioids or PNI engage common molecular mechanisms to induce hypersensitivity. In this article, we conducted deep RNA sequencing (RNA-seq) and morphological analysis of spinal cord microglia in male mice to comprehensively interrogate transcriptional states and mechanistic commonality between multiple models of OIH and PNI. After PNI, we identify an early proliferative transcriptional event across models that precedes the upregulation of histological markers of microglial activation. However, we found no proliferative transcriptional response associated with opioid-induced microglial activation, consistent with histological data, indicating that the number of microglia remains stable during morphine treatment, whereas their morphological response differs from PNI models. Collectively, these results establish the diversity of pain-associated microglial transcriptomic responses and point towards the targeting of distinct insult-specific microglial responses to treat OIH, PNI, or other central nervous system pathologies.

Dexborneol Amplifies Pregabalin's Analgesic Effect in Mouse Models of Peripheral Nerve Injury and Incisional Pain.

Pregabalin is a medication primarily used in the treatment of neuropathic pain and anxiety disorders, owing to its gabapentinoid properties. Pregabalin monotherapy faces limitations due to its variable efficacy and dose-dependent adverse reactions. In this study, we conducted a comprehensive investigation into the potentiation of pregabalin's analgesic effects by dexborneol, a neuroprotective bicyclic monoterpenoid compound. We performed animal experiments where pain models were induced using two methods: peripheral nerve injury, involving axotomy and ligation of the tibial and common peroneal nerves, and incisional pain through a longitudinal incision in the hind paw, while employing a multifaceted methodology that integrates behavioral pharmacology, molecular biology, neuromorphology, and lipidomics to delve into the mechanisms behind this potentiation. Dexborneol was found to enhance pregabalin's efficacy by promoting its transportation to the central nervous system, disrupting self-amplifying vicious cycles via the reduction of HMGB1 and ATP release, and exerting significant anti-oxidative effects through modulation of central lipid metabolism. This combination therapy not only boosted pregabalin's analgesic property but also notably decreased its side effects. Moreover, this therapeutic cocktail exceeded basic pain relief, effectively reducing neuroinflammation and glial cell activation-key factors contributing to persistent and chronic pain. This study paves the way for more tolerable and effective analgesic options, highlighting the potential of dexborneol as an adjuvant to pregabalin therapy.

The mutual effect of progesterone and vitamin D in an animal model of peripheral nerve injury.

Experimental and clinical studies have shown the potential role of progesterone in relieving neural injury. In addition, emerging data on vitamin D, a steroid hormone, have shown its neuroprotective properties. This study was designed to evaluate the mutual effect of vitamin D and progesterone on neuropathic pain (NP) in male rats. Chronic constriction injury (CCI) was induced by inserting four ligatures around the sciatic nerve. Hyperalgesia and allodynia (cold and mechanical) were considered positive behavioral scores of NP. After surgery, Sprague Dawley male rats (weighing 200-250 g) were assigned into 7 groups. Vitamin D (250 and 500 units/kg/day, i.p.) and progesterone (4 and 6 mg/kg/day, i.p.) were injected from the 1st day after CCI which continued for 21 days. Moreover, one group received the co-administration of vitamin D (500 units/kg/day, i.p.) and progesterone (6 mg/kg/day, i.p.) from the 1st day until the 21st post-CCI day. Behavioral tests were performed on the 7th, 14th, and 21st days. Daily supplementation with vitamin D (250 and 500 units/kg) did not alter nociception. Progesterone (4 and 6 mg/kg/day) was ineffective on thermal hyperalgesia. In the allodynia test, progesterone significantly decreased pain-related behaviors. The co-administration of vitamin D (500 units/kg/day) with progesterone (6 mg/kg/day) significantly relieved thermal hyperalgesia. Finally, the combination significantly decreased cold and mechanical allodynia. This study showed the mutual effect of progesterone and vitamin D on NP for the first time. Hyperalgesia and allodynia were significantly relieved following co-administration of vitamin D and progesterone.

Rejuvenating fecal microbiota transplant enhances peripheral nerve repair in aged mice by modulating endoneurial inflammation

Peripheral nerve injury (PNI) resulting from trauma or neuropathies can cause significant disability, and its prognosis deteriorates with age. Emerging evidence suggests that gut dysbiosis and reduced fecal short-chain fatty acids (SCFAs) contribute to an age-related systemic hyperinflammation (inflammaging), which hinders nerve recovery after injury. This study thus aimed to evaluate the pro-regenerative effects of a rejuvenating fecal microbiota transplant (FMT) in a preclinical PNI model using aged mice. Aged C57BL/6 mice underwent bilateral crush injuries to their sciatic nerves. Subsequently, they either received FMT from young donors at three and four days after the injury or retained their aged gut microbiota. We analyzed gut microbiome composition and SCFA concentrations in fecal samples. The integrity of the ileac mucosal barrier was assessed by immunofluorescence staining of Claudin-1. Flow cytometry was utilized to examine immune cells and cytokine production in the ileum, spleen, and sciatic nerve. Various assessments, including behavioural tests, electrophysiological studies, and morphometrical analyses, were conducted to evaluate peripheral nerve function and repair following injury. Rejuvenating FMT reversed age-related gut dysbiosis by increasing Actinobacteria, especially Bifidobacteriales genera. This intervention also led to an elevation of gut SCFA levels and mitigated age-related ileac mucosal leakiness in aged recipients. Additionally, it augmented the number of T-helper 2 (Th2) and regulatory T (Treg) cells in the ileum and spleen, with the majority being positive for anti-inflammatory interleukin-10 (IL-10). In sciatic nerves, rejuvenating FMT resulted in increased M2 macrophage counts and a higher IL-10 production by IL-10+TNF-α− M2 macrophage subsets. Ultimately, restoring a youthful gut microbiome in aged mice led to improved nerve repair and enhanced functional recovery after PNI. Considering that FMT is already a clinically available technique, exploring novel translational strategies targeting the gut microbiome to enhance nerve repair in the elderly seems promising and warrants further evaluation.

Rab32 facilitates Schwann cell pyroptosis in rats following peripheral nerve injury by elevating ROS levels.

Peripheral nerve injury (PNI) is commonly observed in clinical practice, yet the underlying mechanisms remain unclear. This study investigated the correlation between the expression of a Ras-related protein Rab32 and pyroptosis in rats following PNI, and potential mechanisms have been explored by which Rab32 may influence Schwann cells pyroptosis and ultimately peripheral nerve regeneration (PNR) through the regulation of Reactive oxygen species (ROS) levels. The authors investigated the induction of Schwann cell pyroptosis and the elevated expression of Rab32 in a rat model of PNI. In vitro experiments revealed an upregulation of Rab32 during Schwann cell pyroptosis. Furthermore,the effect of Rab32 on the level of ROS in mitochondria in pyroptosis model has also been studied. Finally, the effects of knocking down the Rab32 gene on PNR were assessed, morphology, sensory and motor functions of sciatic nerves, electrophysiology and immunohistochemical analysis were conducted to assess the therapeutic efficacy. Silencing Rab32 attenuated PNI-induced Schwann cell pyroptosis and promoted peripheral nerve regeneration. Furthermore, our findings demonstrated that Rab32 induces significant oxidative stress by damaging the mitochondria of Schwann cells in the pyroptosis model in vitro. Rab32 exacerbated Schwann cell pyroptosis in PNI model, leading to delayed peripheral nerve regeneration. Rab32 can be a potential target for future therapeutic strategy in the treatment of peripheral nerve injuries.

Platelet-rich plasma-derived exosomes boost mesenchymal stem cells to promote peripheral nerve regeneration

Peripheral nerve injury (PNI) remains a severe clinical problem with debilitating consequences. Mesenchymal stem cell (MSC)-based therapy is promising, but the problems of poor engraftment and insufficient neurotrophic effects need to be overcome. Herein, we isolated platelet-rich plasma-derived exosomes (PRP-Exos), which contain abundant bioactive molecules, and investigated their potential to increase the regenerative capacity of MSCs. We observed that PRP-Exos significantly increased MSC proliferation, viability, and mobility, decreased MSC apoptosis under stress, maintained MSC stemness, and attenuated MSC senescence. In vivo, PRP-Exo-treated MSCs (pExo-MSCs) exhibited an increased retention rate and heightened therapeutic efficacy, as indicated by increased axonal regeneration, remyelination, and recovery of neurological function in a PNI model. In vitro, pExo-MSCs coculture promoted Schwann cell proliferation and dorsal root ganglion axon growth. Moreover, the increased neurotrophic behaviour of pExo-MSCs was mediated by trophic factors, particularly glia-derived neurotrophic factor (GDNF), and PRP-Exos activated the PI3K/Akt signalling pathway in MSCs, leading to the observed phenotypes. These findings demonstrate that PRP-Exos may be novel agents for increasing the ability of MSCs to promote neural repair and regeneration in patients with PNI.

Effectiveness of Low-Level Laser Therapy and Low Intensity Pulsed Ultrasound in Sensory Recovery in Experimentally Induced Peripheral Nerve Injury Rat Model

Background: Peripheral nerve injuries are known to cause significant functional impairment and diminishedsensory recovery, necessitating the exploration of effective therapeutic interventions.Purpose: The purpose of this research is to find the effectiveness of low-level laser therapy (LLLT) and low intensitypulsed ultrasound (LIPUS) sensory recovery in an experimentally induced peripheral nerve injury rat model.Materials and Methods: In this study, 18 adult male wistar rats which are divided into LLLT (n = 6), LIPUS (n = 6),and control (n = 6) groups. All rats underwent a standardized procedure to induce peripheral nerve injury, whilethe control group received sham procedures. Hot-Plate test and Cold-Plate Tests were conducted for pre- andpost-operative evaluation of sensory recovery at POD 7, 14, 21 days.Results: The study’s findings revealed that LLLT exhibited significantly improved sensory recovery compared toLIPUS and control groups on POD 14 and 21, indicating its potential as a promising non-invasive intervention formanaging peripheral nerve injuries (P <0.001).Conclusion: The study recommends that LLLT is more effective when compared with LIPUS in promoting sensoryrecovery and enhancing in a rat model of peripheral nerve injury. Positive outcomes indicate LLLT’s potential asa promising intervention for managing peripheral nerve injuries.

Alleviation of neuropathic pain with neuropeptide Y requires spinal Npy1r interneurons that coexpress Grp.

Neuropeptide Y targets the Y1 receptor (Y1) in the spinal dorsal horn (DH) to produce endogenous and exogenous analgesia. DH interneurons that express Y1 (Y1-INs; encoded by Npy1r) are necessary and sufficient for neuropathic hypersensitivity after peripheral nerve injury. However, as Y1-INs are heterogenous in composition in terms of morphology, neurophysiological characteristics, and gene expression, we hypothesized that a more precisely defined subpopulation mediates neuropathic hypersensitivity. Using fluorescence in situ hybridization, we found that Y1-INs segregate into 3 largely nonoverlapping subpopulations defined by the coexpression of Npy1r with gastrin-releasing peptide (Grp/Npy1r), neuropeptide FF (Npff/Npy1r), and cholecystokinin (Cck/Npy1r) in the superficial DH of mice, nonhuman primates, and humans. Next, we analyzed the functional significance of Grp/Npy1r, Npff/Npy1r, and Cck/Npy1r INs to neuropathic pain using a mouse model of peripheral nerve injury. We found that chemogenetic inhibition of Npff/Npy1r-INs did not change the behavioral signs of neuropathic pain. Further, inhibition of Y1-INs with an intrathecal Y1 agonist, [Leu31, Pro34]-NPY, reduced neuropathic hypersensitivity in mice with conditional deletion of Npy1r from CCK-INs and NPFF-INs but not from GRP-INs. We conclude that Grp/Npy1r-INs are conserved in higher order mammalian species and represent a promising and precise pharmacotherapeutic target for the treatment of neuropathic pain.

Quantitative evaluation of rat sciatic nerve degeneration using high-frequency ultrasound

In this study, we evaluated the utility of using high-frequency ultrasound to non-invasively track the degenerative process in a rat model of peripheral nerve injury. Primary analyses explored spatial and temporal changes in quantitative backscatter coefficient (BSC) spectrum-based outcomes and B-mode textural outcomes, using gray level co-occurrence matrices (GLCMs), during the progressive transition from acute to chronic injury. As secondary analyses, correlations among GLCM and BSC spectrum-based parameters were evaluated, and immunohistochemistry were used to suggest a structural basis for ultrasound outcomes. Both mean BSC spectrum-based and mean GLCM-based measures exhibited significant spatial differences across presurgical and 1-month/2-month time points, distal stumps enclosed proximity to the injury site being particularly affected. The two sets of parameters sensitively detected peripheral nerve degeneration at 1-month and 2-month post-injury, with area under the receiver operating charactersitic curve > 0.8 for most parameters. The results also indicated that the many BSC spectrum-based and GLCM-based parameters significantly correlate with each other, and suggested a common structural basis for a diverse set of quantitative ultrasound parameters. The findings of this study suggest that BSC spectrum-based and GLCM-based analysis are promising non-invasive techniques for diagnosing peripheral nerve degeneration.

METTL3-mediated maturation of miR-192-5p targets ATG7 to prevent Schwann cell autophagy in peripheral nerve injury.

The inhibition of miR-192-5p can promote nerve repair in rats with peripheral nerve injury (PNI) but the precise mechanisms underlying this effect remain unclear. Schwann cell (SC) autophagy mediated by autophagy-related gene (ATG) proteins has a key role in PNI but it is uncertain whether miR-192-5p affects the involvement of SC autophagy in PNI. In this study, we investigated the impact of methyltransferase-like protein 3 (METTL3)/miR-192-5p/ATG7 on SC autophagy in a rat PNI model and in an SC oxygen and glucose deprivation model. The results revealed that METTL3 stimulated miR-192-5p maturation via m6A methylation to depress ATG7 and SC autophagy and aggravate PNI. These findings provide a new target and potential basis for the treatment of patients with PNI.

M2 macrophage-derived cathepsin S promotes peripheral nerve regeneration via fibroblast–Schwann cell-signaling relay

BackgroundAlthough peripheral nerves have an intrinsic self-repair capacity following damage, functional recovery is limited in patients. It is a well-established fact that macrophages accumulate at the site of injury. Numerous studies indicate that the phenotypic shift from M1 macrophage to M2 macrophage plays a crucial role in the process of axon regeneration. This polarity change is observed exclusively in peripheral macrophages but not in microglia and CNS macrophages. However, the molecular basis of axonal regeneration by M2 macrophage is not yet fully understood. Herein, we aimed to identify the M2 macrophage-derived axon regeneration factor.MethodsWe established a peripheral nerve injury model by transection of the inferior alveolar nerve (IANX) in Sprague–Dawley rats. Transcriptome analysis was performed on the injured nerve. Recovery from sensory deficits in the mandibular region and histological reconnection of IAN after IANX were assessed in rats with macrophage depletion by clodronate. We investigated the effects of adoptive transfer of M2 macrophages or M2-derived cathepsin S (CTSS) on the sensory deficit. CTSS initiating signaling was explored by western blot analysis in IANX rats and immunohistochemistry in co-culture of primary fibroblasts and Schwann cells (SCs).ResultsTranscriptome analysis revealed that CTSS, a macrophage-selective lysosomal protease, was upregulated in the IAN after its injury. Spontaneous but partial recovery from a sensory deficit in the mandibular region after IANX was abrogated by macrophage ablation at the injured site. In addition, a robust induction of c-Jun, a marker of the repair-supportive phenotype of SCs, after IANX was abolished by macrophage ablation. As in transcriptome analysis, CTSS was upregulated at the injured IAN than in the intact IAN. Endogenous recovery from hypoesthesia was facilitated by supplementation of CTSS but delayed by pharmacological inhibition or genetic silencing of CTSS at the injured site. Adoptive transfer of M2-polarized macrophages at this site facilitated sensory recovery dependent on CTSS in macrophages. Post-IANX, CTSS caused the cleavage of Ephrin-B2 in fibroblasts, which, in turn, bound EphB2 in SCs. CTSS-induced Ephrin-B2 cleavage was also observed in human sensory nerves. Inhibition of CTSS-induced Ephrin-B2 signaling suppressed c-Jun induction in SCs and sensory recovery.ConclusionsThese results suggest that M2 macrophage-derived CTSS contributes to axon regeneration by activating SCs via Ephrin-B2 shedding from fibroblasts.

Polyethylene Glycol Fusion Restores Axonal Continuity and Improves Return of Function in a Rat Median Nerve Denervation Model.

Polyethylene glycol (PEG) can fuse severed closely apposed axolemmas and restore axonal continuity. The authors evaluated the effects of PEG fusion on functional recovery in a rodent forelimb model of peripheral nerve injury. The median nerves of male Lewis rats ( n = 5 per group) were transected and repaired with standard suture repair (SR), SR with PEG (PEG), or SR with PEG and 1% methylene blue (PEG+MB); a sham surgery group was also included. Proximal stimulation produced compound nerve and muscle action potentials recorded distally. The contralateral limb of each animal acted as an internal control for grip strength measurements. Compound nerve and muscle action potentials immediately returned in all PEG and PEG+MB animals, but not in SR animals. The PEG and PEG+MB groups demonstrated earlier return of function by postoperative day (POD) 7 (62.6 ± 7.3% and 50.9 ± 6.7% of contralateral limb grip strength, respectively) compared with the SR group, in which minimal return of function was not measurable until POD 21. At POD 98, the PEG group grip strength recovered to 77.2 ± 2.8% and the PEG+MB grip strength recovered to 79.9 ± 4.4%, compared with 34.9 ± 1.8% recovery in the SR group ( P < 0.05). The PEG and PEG+MB groups reached 50% of the sham group grip strength on POD 3.8 and POD 6.3, respectively, whereas the SR group did not reach 50% grip strength recovery of the sham group throughout the study period. PEG fusion plus neurorrhaphy with or without MB reestablished axonal continuity, shortened recovery time, and augmented functional recovery compared with suture neurorrhaphy alone. PEG fusion with neurorrhaphy may bypass Wallerian degeneration, leading to augmented and shortened functional recovery.