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Reactive oxygen species (ROS) contribute to tissue damage and remodelling mediated by the inflammatory response after injury. Here we show that ROS, which promote axonal dieback and degeneration after injury, are also required for axonal regeneration and functional recovery after spinal injury. We find that ROS production in the injured sciatic nerve and dorsal root ganglia requires CX3CR1-dependent recruitment of inflammatory cells. Next, exosomes containing functional NADPH oxidase 2 complexes are released from macrophages and incorporated into injured axons via endocytosis. Once in axonal endosomes, active NOX2 is retrogradely transported to the cell body through an importin-β1–dynein-dependent mechanism. Endosomal NOX2 oxidizes PTEN, which leads to its inactivation, thus stimulating PI3K–phosporylated (p-)Akt signalling and regenerative outgrowth. Challenging the view that ROS are exclusively involved in nerve degeneration, we propose a previously unrecognized role of ROS in mammalian axonal regeneration through a NOX2–PI3K–p-Akt signalling pathway. Hervera et al. show that extracellular vesicles containing NOX2 complexes are released from macrophages and incorporated into injured axons, leading to axonal regeneration through PI3K–p-Akt signalling.

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C high monocytes infiltrate the nerve first and rapidly give way to Ly6C negative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages ‘eat’ apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient ( Csf2 -/- ) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair.

The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better toolswould greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Central and peripheral nerve injuries can lead to permanent paralysis and organ dysfunction. In recent years, many cell and exosome implantation techniques have been developed in an attempt to restore function after nerve injury with promising but generally unsatisfactory clinical results. Clinical outcome may be enhanced by bio-scaffolds specifically fabricated to provide the appropriate three-dimensional (3D) conduit, growth-permissive substrate, and trophic factor support required for cell survival and regeneration. In rodents, these scaffolds have been shown to promote axonal regrowth and restore limb motor function following experimental spinal cord or sciatic nerve injury. Combining the appropriate cell/exosome and scaffold type may thus achieve tissue repair and regeneration with safety and efficacy sufficient for routine clinical application. In this review, we describe the efficacies of bio-scaffolds composed of various natural polysaccharides (alginate, chitin, chitosan, and hyaluronic acid), protein polymers (gelatin, collagen, silk fibroin, fibrin, and keratin), and self-assembling peptides for repair of nerve injury. In addition, we review the capacities of these constructs for supporting in vitro cell-adhesion, mechano-transduction, proliferation, and differentiation as well as the in vivo properties critical for a successful clinical outcome, including controlled degradation and re-absorption. Finally, we describe recent advances in 3D bio-printing for nerve regeneration.

Paclitaxel (PTX) is an antineoplastic agent commonly used in the treatment of solid tumors and is known to cause dose-limiting peripheral neurotoxicity. This study was performed to evaluate the protective effect of curcumin (CUR) against PTX-induced spinal cord and sciatic nerve injuries in rats. The rats were administered PTX (2 mg/kg, BW) intraperitoneally for the first 5 consecutive days followed by administration of CUR (100 and 200 mg/kg, BW daily in corn oil) orally for 10 days. Our results showed that CUR significantly reduced mRNA expression levels of NF-κB, TNF-α, IL-6, iNOS and GFAP whereas caused an increase in levels of Nrf2, HO-1 and NQO1 in the spinal cord and sciatic nerve of PTX-induced rats. In addition, CUR suppressed the activation of apoptotic and autophagic pathways by increasing Bcl-2 and Bcl-xL, and decreasing p53, caspase-3, Apaf-1, LC3A, LC3B and beclin-1 mRNA expression levels. The results showed that CUR also maintained the spinal cord and sciatic nerve histological architecture and integrity by both LFB staining and H&E staining. Immunohistochemical expressions of 8-OHdG, caspase-3 and LC3B in the PTX-induced spinal cord tissue were decreased after administration of CUR. Taken together, our findings demonstrated that CUR has protective effects on PTX-induced spinal cord and sciatic nerve injuries in rats.

Background Chronic pain is often accompanied by short-term memory deficit and depression. Currently, it is believed that short-term memory deficit and depression are consequences of chronic pain. Here, we test the hypothesis that the symptoms might be caused by overproduction of interleukin-1beta (IL-1β) in the injured nerve independent of neuropathic pain following spared nerve injury in rats and mice. Results Mechanical allodynia, a behavioral sign of neuropathic pain, was not correlated with short-term memory deficit and depressive behavior in spared nerve injury rats. Spared nerve injury upregulated IL-1β in the injured sciatic nerve, plasma, and the regions in central nervous system closely associated with pain, memory and emotion, including spinal dorsal horn, hippocampus, prefrontal cortex, nucleus accumbens, and amygdala. Importantly, the spared nerve injury-induced memory deficits, depressive, and pain behaviors were substantially prevented by peri-sciatic administration of IL-1β neutralizing antibody in rats or deletion of IL-1 receptor type 1 in mice. Furthermore, the behavioral abnormalities induced by spared nerve injury were mimicked in naïve rats by repetitive intravenous injection of re combinant rat IL-1β (rrIL-1β) at a pathological concentration as determined from spared nerve injury rats. In addition, microglia were activated by both spared nerve injury and intravenous injection of rrIL-1β and the effect of spared nerve injury was substantially reversed by peri-sciatic administration of anti-IL-1β. Conclusions Neuropathic pain was not necessary for the development of cognitive and emotional disorders, while the overproduction of IL-1β in the injured sciatic nerve following peripheral nerve injury may be a common mechanism underlying the generation of neuropathic pain, memory deficit, and depression.

The regenerative capacity of the peripheral nervous system is closely related to the role that Schwann cells (SCs) play in construction of the basement membrane containing multiple extracellular matrix proteins and secretion of neurotrophic factors, including laminin (LN) and brain-derived neurotrophic factor (BDNF). Here, we developed a self-assembling peptide (SAP) nanofiber hydrogel based on self-assembling backbone Ac-(RADA) -NH (RAD) dual-functionalized with laminin-derived motif IKVAV (IKV) and a BDNF-mimetic peptide epitope RGIDKRHWNSQ (RGI) for peripheral nerve regeneration, with the hydrogel providing a three-dimensional (3D) microenvironment for SCs and neurites. Circular dichroism (CD), atomic force microscopy (AFM), and scanning electron microscopy (SEM) were used to characterize the secondary structures, microscopic structures, and morphologies of self-assembling nanofiber hydrogels. Then the SC adhesion, myelination and neurotrophin secretion were evaluated on the hydrogels. Finally, the SAP hydrogels were injected into hollow chitosan tubes to bridge a 10-mm-long sciatic nerve defect in rats, and gene expression at 1 week, axonal regeneration, target muscular re-innervation, and functional recovery at 12 weeks were assessed. The bioactive peptide motifs were covalently linked to the C-terminal of the self-assembling peptide and the functionalized peptides could form well-defined nanofibrous hydrogels capable of providing a 3D microenvironment similar to native extracellular matrix. SCs displayed improved cell adhesion on hydrogels with both IKV and RGI, accompanied by increased cell spreading and elongation relative to other groups. RSCs cultured on hydrogels with IKV and RGI showed enhanced gene expression of NGF, BDNF, CNTF, PMP22 and NRP2, and decreased gene expression of NCAM compared with those cultured on other three groups after a 7-day incubation. Additionally, the secretion of NGF, BDNF, and CNTF of RSCs was significantly improved on dual-functionalized peptide hydrogels after 3 days. At 1 week after implantation, the expressions of neurotrophin and myelin-related genes in the nerve grafts in SAP and Autograft groups were higher than that in Hollow group, and the expression of S100 in groups containing both IKV and RGI was significantly higher than that in groups containing either IKV or RGI hydrogels, suggesting enhanced SC proliferation. The morphometric parameters of the regenerated nerves, their electrophysiological performance, the innervated muscle weight and remodeling of muscle fibers, and motor function showed that RAD/IKV/RGI and RAD/IKV-GG-RGI hydrogels could markedly improve axonal regeneration with enhanced re-myelination and motor functional recovery through the synergetic effect of IKV and RGI functional motifs. We found that the dual-functionalized SAP hydrogels promoted RSC adhesion, myelination, and neurotrophin secretion and successfully bridged a 10-mm gap representing a sciatic nerve defect in rats . The results demonstrated the synergistic effect of IKVAV and RGI on axonal regrowth and function recovery after peripheral nerve injury.

Background Chronic neuropathic pain is a frequent sequel to peripheral nerve injury and maladaptive nervous system function. Divanillyl sulfone (DS), a novel structural derivative of 4,4′-dihydroxydibenzyl sulfoxide from a traditional Chinese medicine Gastrodia elata with anti-nociceptive effects, significantly alleviated neuropathic pain following intrathecal injection. Here, we aimed to investigate the underlying mechanisms of DS against neuropathic pain. Methods A chronic constrictive injury (CCI) mouse model of neuropathic pain induced by sciatic nerve ligation was performed to evaluate the effect of DS by measuring the limb withdrawal using Von Frey filament test. Immunofluorescence staining was used to assess the cell localizations and expressions of Iba-1, ASC, NLRP3, and ROS, the formation of autolysosome. The levels of NLRP3-related proteins (caspase-1, NLRP3, and IL-1β), mitophagy-related proteins (LC3, Beclin-1, and p62), and apoptosis-related proteins (Bcl-XL and Bax) were detected by Western blotting. The apoptosis of BV-2 cell and caspase activity were evaluated by flow cytometry. Results DS significantly alleviated the neuropathic pain by increasing the mechanical withdrawal threshold and inhibiting the activation of NLRP3 in CCI-induced model mice. Our findings indicated that DS promoted the mitophagy by increasing the LC3II and Beclin 1 and decreasing the levels of p62 protein in BV-2 cell. This is accompanied by the inhibition of NLRP3 activation, which was shown as inhibited the expression of NLRP3 in lysates as well as the secretion of mature caspase-1 p10 and IL-1β p17 in supernatants in cultured BV-2 microglia. In addition, DS could promote mitophagy-induced improvement of dysfunctional mitochondria by clearing intracellular ROS and restoring mitochondrial membrane potential. Conclusion Together, our findings demonstrated that DS ameliorate chronic neuropathic pain in mice by suppressing NLRP3 inflammasome activation induced by mitophagy in microglia. DS may be a promising therapeutic agent for chronic neuropathic pain.

Autologous nerve transplantation, which is the gold standard for clinical treatment of peripheral nerve injury, still has many limitations. In this study, aligned chitosan fiber hydrogel (ACG) grafted with a bioactive peptide mixture consisting of RGI (Ac-RGIDKRHWNSQGG) and KLT (Ac-KLTWQELYQLKYKGIGG), designated as ACG-RGI/KLT, was used as nerve conduit filler to repair sciatic nerve defects in rats. : Chitosan nanofiber hydrogel was prepared by a combination of electrospinning and mechanical stretching methods, and was then grafted with RGI and KLT, which are peptides mimicking brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF), respectively. The physicochemical properties of ACG-RGI/KLT were fully characterized. , the distribution, proliferation, and secretory activity of Schwann cells were analyzed. Next, the repair potential for 15-mm rat sciatic nerve defects was examined. The recovery of regenerated nerve, muscle, and motor function was evaluated by neuromuscular histology, electrophysiology, and catwalk gait analysis. : We first constructed directionally aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide mixture (ACG-RGI/KLT). ACG-RGI/KLT oriented the Schwann cells, and promoted the proliferation and secretion of neurotrophic factors by Schwann cells. At an early injury stage, ACG-RGI/KLT not only enhanced nerve regeneration, but also promoted vascular penetration. At 12 weeks, ACG-RGI/KLT facilitated nerve regeneration and functional recovery in rats. : Aligned chitosan nanofiber hydrogel grafted with RGI/KLT peptide provides an effective means of repairing sciatic nerve defects and shows great potential for clinical application.

The prognosis of peripheral nerve injury (PNI) is usually poor, and currently, there is no effective treatment for PNI. Studies have shown that exosomes derived from mesenchymal stem cells could promote nerve regeneration by optimizing the function of endogenous Schwann cells (SCs), while the mechanism is unclear. Autophagy, a highly conserved intracellular catabolic process responsible for maintaining cellular homeostasis, has been proved to be involved in the regulation of nerve repair after injury. We explored the effect of exosomes derived from dental pulp stem cells (DPSC-Exos) on the regeneration of myelin sheath in rats with sciatic nerve injury (SNI). In vitro and in vivo experiments were performed to clarify whether the effect of DPSC-Exos is associated with autophagy of SCs and to reveal the mechanism at the molecular level. Our results showed that the SCs of SNI rats exhibited the obvious autophagic characteristics, and the increase of P53 expression was an internal factor of autophagy. Our mechanism research indicated that DPSC-Exos could deliver miR-122-5p from DPSCs into SCs and suppressed the rapamycin (RAPA)-induced autophagy in SCs by inhibiting P53 expression. Rescue experiments showed that both the use of GW4869 and overexpression of exogenous P53 in SCs could reverse the inhibitory effect of DPSCs on the autophagy in SCs from co-culture system. In short, our study indicated that DPSC-Exos could promote the regeneration of the myelin sheath through suppressing the autophagy in SCs caused by PNI via miR-122-5p /P53 pathway; this provides researchers with another option for precise repair of PNI.