Transcriptional genes of lysosome-associated membrane protein 2A in sciatic nerve injuries by bioinformatics.

Recent studies have shown that autophagy is activated in response to nerve damage and occurs simultaneously with the initial stages of Schwann cell-mediated demyelination. Although several studies have reported that macroautophagy is involved in the peripheral nerve, the role of chaperone-mediated autophagy (CMA) has not yet been investigated in peripheral nerve injury. The present study investigates the role of CMA in the sciatic nerve. Using a mouse model of sciatic nerve injury, the authors employed immunofluorescence analysis to observe the expression of LAMP2A, a critical marker for CMA. RNA sequencing was performed to observe the transcriptional profile of Lamp2a in Schwann cells. Bioinformatics analysis was carried out to observe the hub genes associated with Lamp2a . Expression of Lamp2a , a key gene in CMA, increased following sciatic nerve injury, based on an immunofluorescence assay. To identify differentially expressed genes using Lamp2a , RNA sequence analysis was conducted using rat Schwann cells overexpressing Lamp2a . The nine hub genes ( Snrpf, Polr1d, Snip1, Aqr, Polr2h, Ssbp1, Mterf3, Adcy6 , and Sbds ) were identified using the CytoHubba plugin of Cytoscape. Functional analysis revealed that Lamp2a overexpression affected the transcription levels of genes associated with mitotic spindle organization and mRNA splicing via the spliceosome. In addition, Polr1d and Snrpf1 were downregulated throughout postnatal development but elevated following sciatic nerve injury, according to a bioinformatics study. CMA may be an integral pathway in sciatic nerve injury via mRNA splicing.

Genome-wide Analysis of Histone H3 Lysine 27 Trimethylation Profiles in Sciatic Nerve of Chronic Constriction Injury Rats.

The histone H3 lysine 27 trimethylation (H3K27me3) is one of the most important chromatin modifications, which is associated with injury-activated gene expression in Schwann cells (SCs). However, the alteration of genome-wide H3K27me3 enrichments in the development of neuropathic pain is still unknown. Here, we applied the chromatin immunoprecipitation sequencing (ChIP-seq) approach to identify the alteration of differential enrichments of H3K27me3 in chronic constriction injury (CCI) sciatic nerve of rats and potential molecular mechanisms underlying the development of neuropathic pain. Our results indicated that CCI increased the numbers of SCs displaying H3K27 methyltransferase enhancer of zeste homolog 2 (EZH2) and H3K27me3 in the sciatic nerve. ChIP-seq data showed that CCI significantly changed H3K27me3 enrichments on gene promoters in the sciatic nerve. Bioinformatics analyses exhibited that genes gaining H3K27me3 were mostly associated with regulation of cell proliferation, response to stress and oxidation-reduction process. Genes losing this mark were enriched in neuronal generation, and MAPK, cAMP as well as ERBB signaling pathways. Importantly, IL1A, CCL2, NOS2, S100A8, BDNF, GDNF, ERBB3 and C3 were identified as key genes in neuropathic pain. CCI led to significant upregulation of key genes in the sciatic nerve. EZH2 inhibitor reversed CCI-induced increases of H3K27me3 and key gene protein levels, which were accompanied by relieved mechanical allodynia and thermal hyperalgesia in CCI rats. These results indicate that genes with differential enrichments of H3K27me3 in SCs function in various cellular processes and pathways, and many are linked to neuropathic pain after peripheral nerve injury.

Identification of Epigenetic Interactions between MicroRNA-30c-5p and DNA Methyltransferases in Neuropathic Pain.

Neuropathic pain is a prevalent and severe chronic syndrome, often refractory to treatment, whose development and maintenance may involve epigenetic mechanisms. We previously demonstrated a causal relationship between miR-30c-5p upregulation in nociception-related neural structures and neuropathic pain in rats subjected to sciatic nerve injury. Furthermore, a short course of an miR-30c-5p inhibitor administered into the cisterna magna exerts long-lasting antiallodynic effects via a TGF-ß1-mediated mechanism. Herein, we show that miR-30c-5p inhibition leads to global DNA hyper-methylation of neurons in the lumbar dorsal root ganglia and spinal dorsal horn in rats subjected to sciatic nerve injury. Specifically, the inhibition of miR-30-5p significantly increased the expression of the novo DNA methyltransferases DNMT3a and DNMT3b in those structures. Furthermore, we identified the mechanism and found that miR-30c-5p targets the mRNAs of DNMT3a and DNMT3b. Quantitative methylation analysis revealed that the promoter region of the antiallodynic cytokine TGF-ß1 was hypomethylated in the spinal dorsal horn of nerve-injured rats treated with the miR-30c-5p inhibitor, while the promoter of Nfyc, the host gene of miR-30c-5p, was hypermethylated. These results are consistent with long-term protection against neuropathic pain development after nerve injury. Altogether, our results highlight the key role of miR-30c-5p in the epigenetic mechanisms' underlying neuropathic pain and provide the basis for miR-30c-5p as a therapeutic target.

Transgenic Schwann cells overexpressing POU6F1 promote sciatic nerve regeneration within acellular nerve allografts.

Objective.Acellular nerve allograft (ANA) is an effective surgical approach used to bridge the sciatic nerve gap. The molecular regulators of post-surgical recovery are not well-known. Here, we explored the effect of transgenic Schwann cells (SCs) overexpressing POU domain class 6, transcription factor 1 (POU6F1) on sciatic nerve regeneration within ANAs. We explored the functions of POU6F1 in nerve regeneration by using a cell model of H2O2-induced SCs injury and transplanting SCs overexpressing POU6F1 into ANA to repair sciatic nerve gaps.Approach.Using RNA-seq, Protein-Protein Interaction network analysis, gene ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes pathway analysis, we identified a highly and differentially expressed transcription factor, POU6F1, following ANA treatment of sciatic nerve gap. Expressing a high degree of connectivity, POU6F1 was predicted to play a role in peripheral nervous system myelination.Main results.To test the role of POU6F1 in nerve regeneration after ANA, we infected SCs with adeno-associated virus-POU6F1, demonstrating that POU6F1 overexpression promotes proliferation, anti-apoptosis, and migration of SCsin vitro. We also found that POU6F1 significantly upregulated JNK1/2 and c-Jun phosphorylation and that selective JNK1/2 inhibition attenuated the effects of POU6F1 on proliferation, survival, migration, and JNK1/2 and c-Jun phosphorylation. The direct interaction of POU6F1 and activated JNK1/2 was subsequently confirmed by co-immunoprecipitation. In rat sciatic nerve injury model with a 10 mm gap, we confirmed the pattern of POU6F1 upregulation and co-localization with transplanted SCs. ANAs loaded with POU6F1-overexpressing SCs demonstrated the enhanced survival of transplanted SCs, axonal regeneration, myelination, and functional motor recovery compared to the ANA group loaded by SCs-only in line within vitrofindings.Significance.This study identifies POU6F1 as a novel regulator of post-injury sciatic nerve repair, acting through JNK/c-Jun signaling in SCs to optimize therapeutic outcomes in the ANA surgical approach.

scRNA-seq generates a molecular map of emerging cell subtypes after sciatic nerve injury in rats.

Patients with peripheral nerve injury, viral infection or metabolic disorder often suffer neuropathic pain due to inadequate pharmacological options for relief. Developing novel therapies has been challenged by incomplete mechanistic understanding of the cellular microenvironment in sensory nerve that trigger the emergence and persistence of pain. In this study, we report a high resolution transcriptomics map of the cellular heterogeneity of naïve and injured rat sensory nerve covering more than 110,000 individual cells. Annotation reveals distinguishing molecular features of multiple major cell types totaling 45 different subtypes in naïve nerve and an additional 23 subtypes emerging after injury. Ligand-receptor analysis revealed a myriad of potential targets for pharmacological intervention. This work forms a comprehensive resource and unprecedented window into the cellular milieu underlying neuropathic pain and demonstrates that nerve injury is a dynamic process orchestrated by multiple cell types in both the endoneurial and epineurial nerve compartments.

Spared Nerve Injury Causes Sexually Dimorphic Mechanical Allodynia and Differential Gene Expression in Spinal Cords and Dorsal Root Ganglia in Rats.

Neuropathic pain is more prevalent in women. However, females are under-represented in animal experiments, and the mechanisms of sex differences remain inadequately understood. We used the spared nerve injury (SNI) model in rats to characterize sex differences in pain behaviour, unbiased RNA-Seq and proteomics to study the mechanisms. Male and female rats were subjected to SNI- and sham-surgery. Mechanical and cold allodynia were assessed. Ipsilateral lumbar dorsal root ganglia (DRG) and spinal cord (SC) segments were collected for RNA-seq analysis with DESeq2 on Day 7. Cerebrospinal fluid (CSF) samples for proteomic analysis and DRGs and SCs for analysis of IB-4 and CGRP, and IBA1 and GFAP, respectively, were collected on Day 21. Females developed stronger mechanical allodynia. There were no differences between the sexes in CGRP and IB-4 in the DRG or glial cell markers in the SC. No CSF protein showed change following SNI. DRG and SC showed abundant changes in gene expression. Sexually dimorphic responses were found in genes related to T-cells (cd28, ctla4, cd274, cd4, prf1), other immunological responses (dpp4, c5a, cxcr2 and il1b), neuronal transmission (hrh3, thbs4, chrna4 and pdyn), plasticity (atf3, c1qc and reg3b), and others (bhlhe22, mcpt1l, trpv6). We observed significantly stronger mechanical allodynia in females and numerous sexually dimorphic changes in gene expression following SNI in rats. Several genes have previously been linked to NP, while some are novel. Our results suggest gene targets for further studies in the development of new, possibly sex-specific, therapies for NP.

Delayed manipulation of regeneration within injured peripheral axons.

While several new translational strategies to enhance regrowth of peripheral axons have been identified, combined approaches with different targets are rare. Moreover, few have been studied after a significant delay when growth programs are already well established and regeneration-related protein expression has waned. Here we study two approaches, Rb1 (Retinoblastoma 1) knockdown that targets overall neuron plasticity, and near nerve insulin acting as a growth factor. Both are validated to boost regrowth only at the outset of regeneration. We show that local delivery of Rb1 siRNA alone, with electroporation to an area of prior sciatic nerve injury generated knockdown of Rb1 mRNA in ipsilateral lumbar dorsal root ganglia. While mice treated with Rb1-targeted siRNA, compared with scrambled control siRNA, starting 2 weeks after the onset of regeneration, had only limited behavioural or electrophysiological benefits, they had enhanced reinnervation of epidermal axons. We next confirmed that intrinsic Rb1 knockdown combined with exogenous insulin had dramatic synergistic impacts on the growth patterns of adult sensory neurons studied in vitro, prompting analysis of a combined approach in vivo. Using an identical delayed post-injury protocol, we noted that added insulin not only augmented epidermal reinnervation rendered by Rb1 knockdown alone but also improved indices of mechanical sensation and motor axon recovery. The findings illustrate that peripheral neurons that are well into attempted regrowth retain their responsiveness to both intrinsic and exogenous approaches that improve their recovery. We also identify a novel local approach to manipulate gene expression and outcome in regrowing axons.

Transcriptomic signature of painful human neurofibromatosis type 2 schwannomas.

Schwannomas are benign neoplasms that can cause gain- and loss-of-function neurological phenotypes, including severe, intractable pain. To investigate the molecular mechanisms underlying schwannoma-associated pain we compared the RNA sequencing profile of painful and non-painful schwannomas from NF2 patients. Distinct segregation of painful and non-painful tumors by gene expression patterns was observed. Differential expression analysis showed the upregulation of fibroblast growth factor 7 (FGF7) in painful schwannomas. Behavioral support for this finding was observed using a xenograft human NF2-schwannoma model in nude mice. In this model, over-expression of FGF7 in intra-sciatically implanted NF2 tumor cells generated pain behavior compared with controls.

Investigation of Neuropathology after Nerve Release in Chronic Constriction Injury of Rat Sciatic Nerve.

Peripheral compressive neuropathy causes significant neuropathic pain, muscle weakness and prolong neuroinflammation. Surgical decompression remains the gold standard of treatment but the outcome is suboptimal with a high recurrence rate. From mechanical compression to chemical propagation of the local inflammatory signals, little is known about the distinct neuropathologic patterns and the genetic signatures after nerve decompression. In this study, controllable mechanical constriction forces over rat sciatic nerve induces irreversible sensorimotor dysfunction with sustained local neuroinflammation, even 4 weeks after nerve release. Significant gene upregulations are found in the dorsal root ganglia, regarding inflammatory, proapoptotic and neuropathic pain signals. Genetic profiling of neuroinflammation at the local injured nerve reveals persistent upregulation of multiple genes involving oxysterol metabolism, neuronal apoptosis, and proliferation after nerve release. Further validation of the independent roles of each signal pathway will contribute to molecular therapies for compressive neuropathy in the future.

The evolution of dystonia-like movements in TOR1A rats after transient nerve injury is accompanied by dopaminergic dysregulation and abnormal oscillatory activity of a central motor network.

TOR1A is the most common inherited form of dystonia with still unclear pathophysiology and reduced penetrance of 30-40%. ∆ETorA rats mimic the TOR1A disease by expression of the human TOR1A mutation without presenting a dystonic phenotype. We aimed to induce dystonia-like symptoms in male ∆ETorA rats by peripheral nerve injury and to identify central mechanism of dystonia development. Dystonia-like movements (DLM) were assessed using the tail suspension test and implementing a pipeline of deep learning applications. Neuron numbers of striatal parvalbumin+, nNOS+, calretinin+, ChAT+ interneurons and Nissl+ cells were estimated by unbiased stereology. Striatal dopaminergic metabolism was analyzed via in vivo microdialysis, qPCR and western blot. Local field potentials (LFP) were recorded from the central motor network. Deep brain stimulation (DBS) of the entopeduncular nucleus (EP) was performed. Nerve-injured ∆ETorA rats developed long-lasting DLM over 12 weeks. No changes in striatal structure were observed. Dystonic-like ∆ETorA rats presented a higher striatal dopaminergic turnover and stimulus-induced elevation of dopamine efflux compared to the control groups. Higher LFP theta power in the EP of dystonic-like ∆ETorA compared to wt rats was recorded. Chronic EP-DBS over 3 weeks led to improvement of DLM. Our data emphasizes the role of environmental factors in TOR1A symptomatogenesis. LFP analyses indicate that the pathologically enhanced theta power is a physiomarker of DLM. This TOR1A model replicates key features of the human TOR1A pathology on multiple biological levels and is therefore suited for further analysis of dystonia pathomechanism.

HAGLR aggravates neuropathic pain and promotes inflammatory response and apoptosis of lipopolysaccharide-treated SH-SY5Y cells by sequestering miR-182-5p from ATAT1 and activating NLRP3 inflammasome.

BACKGROUND: Chronic neuropathic pain is characterized by neuroinflammation. Previously, long noncoding RNA (lncRNA) HAGLR was reported to regulate the inflammatory response of SH-SY5Y cells. However, neither the specific function nor the potential mechanism of HAGLR in neuropathic pain has been explored. AIM OF THE STUDY: Our study is aimed to figure out the role of HAGLR in neuropathic pain. METHODS: SH-SY5Y cells were treated with lipopolysaccharide (LPS) to mimic neuron injury in vitro. The chronic constriction injury (CCI) rat models were established by ligation of sciatic nerve to mimic neuropathic pain in vivo. Behavioral assessment assays were performed to determine the effects of HAGLR on hypersensitivity in neuropathic pain. Enzyme-linked immunosorbent assay kits were used for detection of inflammatory cytokines. Flow cytometry analysis and Western blot were applied to detect apoptosis. RESULTS: HAGLR displayed high levels in spinal cords of CCI rats and in LPS treated SH-SY5Y cells. Knockdown of HAGLR inhibited inflammation and neuron apoptosis of LPS treated SH-SY5Y cells. Mechanistically, HAGLR bound with miR-182-5p in SH-SY5Y cells. ATAT1 served as a target of miR-182-5p. HAGLR activated the NLRP3 inflammasome by ATAT1. Rescue assays demonstrated that overexpression of ATAT1 or NLRP3 reversed the suppressive effects of HAGLR silencing on apoptosis and inflammatory response in SH-SY5Y cells and in spinal cords of CCI rats. The inhibitory effects of silenced HAGLR on hypersensitivity in neuropathic pain were also rescued by ATAT1 or NLRP3. CONCLUSIONS: HAGLR aggravates neuropathic pain by sequestering miR-182-5p from ATAT1 and activating NLRP3 inflammasome, which may provide a potential therapeutic target for neuropathic pain treatment.

Arginase-1 deficiency in neural cells does not contribute to neurodevelopment or functional outcomes after sciatic nerve injury.

Arginase-1 (Arg1) is an enzyme controlling the final step of the urea cycle, with highest expression in the liver and lower expression in the lungs, pancreas, kidney, and some blood cells. Arg1 deficiency is an inherited urea cycle disorder presenting with neurological dysfunction including spastic diplegia, intellectual and growth retardation, and encephalopathy. The contribution of Arg1 expression in the central and peripheral nervous system to the development of neurological phenotypes remains largely unknown. Previous studies have shown prominent arginase-1 expression in the nervous system and post-peripheral nerve injury in mice, but very low levels in the naïve state. To investigate neurobiological roles of Arg1, we created a conditional neural (n)Arg1 knockout (KO) mouse strain, with expression eliminated in neuronal and glial precursors, and compared them to littermate controls. Long-term analysis did not reveal any major differences in blood amino acid levels, body weight, or stride gait cycle from 8 to 26-weeks of age. Brain structure measured by magnetic resonance imaging at 16-weeks of age observed only a significant decrease in the volume of the mammillary bodies. We also assessed whether nArg1, which is expressed by sensory neurons after injury, may play a role in regeneration following sciatic nerve crush. Only subtle differences were observed in locomotor and sensory recovery between nArg1 KO and control mice. These results suggest that arginase-1 expression in central and peripheral neural cells does not contribute substantially to the phenotypes of this urea cycle disorder, nor is it likely crucial for post-injury regeneration in this mouse model.

Vitamin C regulates Schwann cell myelination by promoting DNA demethylation of pro-myelinating genes.

Ascorbic acid (vitamin C) is critical for Schwann cells to myelinate peripheral nerve axons during development and remyelination after injury. However, its exact mechanism remains elusive. Vitamin C is a dietary nutrient that was recently discovered to promote active DNA demethylation. Schwann cell myelination is characterized by global DNA demethylation in vivo and may therefore be regulated by vitamin C. We found that vitamin C induces a massive transcriptomic shift (n = 3,848 genes) in primary cultured Schwann cells while simultaneously producing a global increase in genomic 5-hydroxymethylcytosine (5hmC), a DNA demethylation intermediate which regulates transcription. Vitamin C up-regulates 10 pro-myelinating genes which exhibit elevated 5hmC content in both the promoter and gene body regions of these loci following treatment. Using a mouse model of human vitamin C metabolism, we found that maternal dietary vitamin C deficiency causes peripheral nerve hypomyelination throughout early development in resulting offspring. Additionally, dietary vitamin C intake regulates the expression of myelin-related proteins such as periaxin (PRX) and myelin basic protein (MBP) during development and remyelination after injury in mice. Taken together, these results suggest that vitamin C cooperatively promotes myelination through 1) increased DNA demethylation and transcription of pro-myelinating genes, and 2) its known role in stabilizing collagen helices to form the basal lamina that is necessary for myelination.

SNHG16 aggravates chronic constriction injury-induced neuropathic pain in rats via binding with miR-124-3p and miR-141-3p to upregulate JAG1.

Neuropathic pain is caused by damage to the nervous system. Increasing studies have confirmed that jagged 1 (JAG1) plays a significant role in nervous system diseases. However, the regulatory mechanisms of JAG1 in neuropathic pain remain vague. In this study, a chronic constriction injury (CCI) rat model was performed. JAG1 was found to be upregulated in CCI rats. The recombinant lentiviruses containing sh-JAG1 were injected to the CCI rats for knockdown of JAG1 in rats. JAG1 knockdown improved the mechanical allodynia and thermal hyperalgesia in CCI rats, and decreased the concentrations and mRNA expression of inflammatory cytokines (IL-6, TNF-α and IL-1ß) in spinal cord dorsal horn of CCI rats, suggesting that JAG1 knockdown attenuated neuropathic pain. In addition, we explored for the upstream mechanism of JAG1. Through RNA pull down assay and luciferase reporter assay, we confirmed that miR-124-3p and miR-141-3p bound with JAG1. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG16) was verified to be the upstream molecule of miR-124-3p and miR-141-3p to negatively regulate miR-124-3p and miR-141-3p. SNHG16 positively regulated JAG1 expression through competitively binding with miR-124-3p and miR-141-3p. Moreover, SNHG16 was found to be upregulated in CCI rats. SNHG16 knockdown improved the mechanical allodynia and thermal hyperalgesia as well as reduced the concentrations and mRNA expression of inflammatory cytokines in CCI rats. Finally, SNHG16 was confirmed to aggravate neuropathic pain in CCI rats via upregulating JAG1. In conclusion, this study verified that SNHG16 aggravated neuropathic pain in CCI rats via binding with miR-124-3p and miR-141-3p to upregulate JAG1, which may provide new insights into the development of gene therapy for neuropathic pain.

Silencing Celsr2 inhibits the proliferation and migration of Schwann cells through suppressing the Wnt/ß-catenin signaling pathway.

After a peripheral nerve injury, the remaining Schwann cells undergo proliferation and adopt a migratory phenotype to prepare for the regeneration of nerves. Celsr2 has been reported to play an important role in the development and maintenance of the function of the nervous system. However, the role and mechanism of Celsr2 during peripheral nerve regeneration remain unknown. Here, we showed that after sciatic nerve injury, Celsr2 mRNA and protein were significantly increased in nerve tissues. In addition, silencing Celsr2 decreased the ki67-positve portion and the migration distance of Schwann cells in vivo. In vitro, the results of MTT and EdU staining, transwell and wound healing assays indicated that Celsr2 siRNA-transfected primary Schwann cells showed significant decrease in proliferation and migration compared to that seen in negative control (NC)-transfected cells. Furthermore, we found that Wnt/ß-catenin luciferase activity was reduced, as were the expression of ß-catenin in the nucleus and the mRNA levels of its downstream genes Cyclin D1 and MMP-7 in Celsr2 siRNA-transfected primary Schwann cells. Further investigations showed that silencing Celsr2 inhibited the phosphorylation of GSK3ß. Moreover, specific activators of the Wnt/ß-catenin pathway, LiCl or mutant ß-catenin (S33Y), partially reversed the inhibitory effect of Celsr2 siRNA. Taken together, our data indicated that silencing Celsr2 inhibited Schwann cells migration and proliferation through the suppressing Wnt/ß-catenin pathway, providing a potential target for peripheral nerve regeneration.

microRNA-9 and -29a regulate the progression of diabetic peripheral neuropathy via ISL1-mediated sonic hedgehog signaling pathway.

In this study, we tested the hypothesis that overexpression of miR-9 and miR-29a may contribute to DPN development and progression. We performed a meta-analysis of miR expression profile studies in human diabetes mellitus (DM) and the data suggested that miR-9 and miR-29a were highly expressed in patients with DM, which was further verified in serum samples collected from 30 patients diagnosed as DM. Besides, ISL1 was confirmed to be a target gene of miR-9 and miR-29a. Lentivirus-mediated forced expression of insulin gene enhancer binding protein-1 (ISL1) activated the sonic hedgehog (SHH) signaling pathway, increased motor nerve conduction velocity and threshold of nociception, and modulated expression of neurotrophic factors in sciatic nerves in rats with DM developed by intraperitoneal injection of 0.45% streptozotocin, suggesting that ISL1 could delay DM progression and promote neural regeneration and repair after sciatic nerve damage. However, lentivirus-mediated forced expression of miR-9 or miR-29a exacerbated DM and antagonized the beneficial effect of ISL1 on DPN. Collectively, this study revealed potential roles of miR-9 and miR-29a as contributors to DPN development through the SHH signaling pathway by binding to ISL1. Additionally, the results provided an experimental basis for the targeted intervention treatment of miR-9 and miR-29a.

Comparison of the beneficial effects of RS504393, maraviroc and cenicriviroc on neuropathic pain-related symptoms in rodents: behavioral and biochemical analyses.

The latest research highlights the role of chemokine signaling pathways in the development of nerve injury-induced pain. Recent studies have provided evidence for the involvement of CCR2 and CCR5 in the pathomechanism underlying neuropathy. Thus, the aim of our study was to compare the effects of a selective CCR2 antagonist (RS504393), selective CCR5 antagonist (maraviroc) and dual CCR2/CCR5 antagonist (cenicriviroc) and determine whether the simultaneous blockade of both receptors is better than blocking only one of them selectively. All experiments were performed using Wistar rats/Swiss albino mice subjected to chronic constriction injury (CCI) of the sciatic nerve. To assess pain-related reactions, the von Frey and cold plate tests were used. The mRNA analysis was performed using RT-qPCR. We demonstrated that repeated intrathecal administration of the examined antagonists attenuated neuropathic pain in rats 7 days post-CCI. mRNA analysis showed that RS504393 did not modulate the spinal expression of the examined chemokines, whereas maraviroc reduced the CCI-induced elevation of CCL4 level. Cenicriviroc significantly lowered the spinal levels of CCL2-4 and CCL7. At the dorsal root ganglia, strong impacts of RS504393 and cenicriviroc on chemokine expression were observed; both reduced the CCI-induced elevation of CCL2-5 and CCL7 levels, whereas maraviroc decreased only the CCL5 level. Importantly, we demonstrated that a single intrathecal/intraperitoneal injection of cenicriviroc had greater analgesic properties than RS504393 or maraviroc in neuropathic mice. Additionally, we demonstrated that cenicriviroc enhanced opioid-induced analgesia. Based on our results, we suggest that targeting CCR2 and CCR5 simultaneously, is an interesting alternative for neuropathic pain pharmacotherapy.

Prostaglandin D2 synthase modulates macrophage activity and accumulation in injured peripheral nerves.

We have previously reported that prostaglandin D2 Synthase (L-PGDS) participates in peripheral nervous system (PNS) myelination during development. We now describe the role of L-PGDS in the resolution of PNS injury, similarly to other members of the prostaglandin synthase family, which are important for Wallerian degeneration (WD) and axonal regeneration. Our analyses show that L-PGDS expression is modulated after injury in both sciatic nerves and dorsal root ganglia neurons, indicating that it might play a role in the WD process. Accordingly, our data reveals that L-PGDS regulates macrophages phagocytic activity through a non-cell autonomous mechanism, allowing myelin debris clearance and favoring axonal regeneration and remyelination. In addition, L-PGDS also appear to control macrophages accumulation in injured nerves, possibly by regulating the blood-nerve barrier permeability and SOX2 expression levels in Schwann cells. Collectively, our results suggest that L-PGDS has multiple functions during nerve regeneration and remyelination. Based on the results of this study, we posit that L-PGDS acts as an anti-inflammatory agent in the late phases of WD, and cooperates in the resolution of the inflammatory response. Thus, pharmacological activation of the L-PGDS pathway might prove beneficial in resolving peripheral nerve injury.

Effects of brazilein on PSD-95 protein expression and neurological recovery in mice after sciatic nerve injury.

PURPOSE: To evaluate the local nerve myelin recovery and the expression of PSD-95 protein and mRNA in the L4-L6 segment of the spinal cord after applying Brazilein to sciatic nerve injury BALB/c mice model and investigate the regulatory effects of Brazilein on myelin recovery after peripheral nerve injury. METHODS: A total of 160 BALB/c mice were selected to establish the unilateral sciatic nerve injury model and randomly divided into four groups: saline blank control, Brazilein high-dose, medium-dose, and low-dose. Mice were assessed at different time points (1 w, 2 w, 4 w, 8 w) after sciatic nerve injury for the sciatic functional index (SFI) and sciatic nerve function recovery of the injured side by myelin Luxol Fast Blue (LFB) staining of the sciatic nerve. In addition, immunohistochemistry, real time-PCR, and Western blot were used to detect the PSD-95 expression in the spinal cord L4-L6 segments of the injured sciatic nerve at each time point. RESULTS: The results of SFI and sciatic nerve function recovery, as well as, myelin LFB staining of the injured side indicated that all indexes of the Brazilein middle- and high-dose groups were significantly better than the low-dose and blank control groups at each time point. The PSD-95 expression in the L4-L6 segment of the spinal cord was statistically lower in the high- and medium-dose groups than in the low-dose and blank control groups at 1 w, 2 w, and 4 w, while the differences between the groups were not significant at 8 w. CONCLUSION: Brazilein inhibits PSD-95 activation in the corresponding segment of sciatic nerve spinal cord in BALB/c mice after sciatic nerve injury, thereby inhibiting the excessive expression of free radicals and promoting myelin regeneration.

Sphingosine-1-phosphate receptor 2 modulates pain sensitivity by suppressing the ROS-RUNX3 pathway in a rat model of neuropathy.

Neuropathic pain correlates with a lesion or other dysfunction in the nervous system. Sphingosine-1-phosphate receptor 2 (S1P2) is expressed in the central nervous system and modulates synaptic plasticity. The present study aimed to investigate the role of S1P2 in neuropathic pain caused by chronic constriction injury (CCI). Sprague-Dawley rats were allocated into eight groups (n = 15 for each group): sham, CCI, CCI + green fluorescent protein, CCI + S1P2, CCI + Ctrl-short hairpin RNA (shRNA), CCI + S1P2 shRNA, CCI + S1P2 + CYM-5442, and CCI + S1P2 shRNA + CYM-5442. The CCI model was established via sciatic nerve ligation. S1P2 was overexpressed or knocked down by intrathecal injection of adeno-associated virus-S1P2 (AAV-S1P2) or AAV-S1P2 shRNA. The S1P1 agonist, CYM-5442 (1 mg/kg), was injected intraperitoneally after surgery. S1P2 expression, pain thresholds, apoptosis signaling, inflammation, and oxidative stress in rats were then examined. We found that sciatic nerve injury downregulated S1P2 expression in the spinal cords of rats. S1P2 overexpression enhanced pain thresholds. In contrast, S1P2 knockdown decreased pain thresholds in rats exposed to CCI. CCI and S1P2 silencing increased secretion of interleukin-1ß (IL-1ß), IL-6, and CCL2, whereas S1P2 overexpression decreased. S1P2 impeded CCI-induced reactive oxygen species (ROS) production and runt-related transcription factors 3 (RUNX3) downregulation, and S1P2 knockdown had the opposite effect. S1P2 overexpression suppressed Bax and active caspase 3 expression and promoted Bcl-2 expression, whereas loss of S1P2 reversed their expression. Additionally, S1P1 activation counteracted the effect of S1P2 on pain sensitivity. In conclusion, S1P2 is downregulated in CCI rats and may help modulate neuropathic pain via the ROS/RUNX3 pathway.