Search

We reported a 61-year-old man presented with 10-month progressing left sciatic neuropathy and 10-day right facial neuropathy. Serum amphiphysin-IgG was positive. 18 F-FDG PET/CT of the whole body showed no signs of malignancy. Treatment with plasma exchange and oral prednisone relieved the symptoms. Nine months later, right hemiparesis and seizure of right limbs developed. 18 F-FDG and 18 F-PBR06 (18 kDa translocator protein, TSPO) radioligand PET/MRI of the whole body revealed intense uptake in the intracranial lesions. Intracranial lymphoma was diagnosed by stereotactic needle brain biopsy. Mononeuropathies could be paraneoplastic syndromes. TSPO shows high uptake in intracranial lymphoma on 18 F-PBR06 PET images., Competing Interests: Declaration of Competing Interest All authors claimed that there were no conflicts of interest in this case., (Copyright © 2023. Published by Elsevier B.V.)

Interleukin-4 (IL-4) has garnered interest as a cytokine that mediates regeneration across multiple tissues including peripheral nerve. Within nerve, we previously showed endogenous IL-4 was critical to regeneration across nerve gaps. Here, we determined a generalizable role of IL-4 in nerve injury and regeneration. In wild-type (WT) mice receiving a sciatic nerve crush, IL-4 expressing cells preferentially accumulated within the injured nerve compared to affected sites proximal, such as dorsal root ganglia (DRGs), or distal muscle. Immunohistochemistry and flow cytometry confirmed that eosinophils (CD45+, CD11b+, CD64-, Siglec-F+) were sources of IL-4 expression. Examination of targets for IL-4 within nerve revealed macrophages, as well as subsets of neurons expressed IL-4R, while Schwann cells expressed limited IL-4R. Dorsal root ganglia cultures were exposed to IL-4 and demonstrated an increased proportion of neurons that extended axons compared to cultures without IL-4 (control), as well as longer myelinated axons compared to cultures without IL-4. The role of endogenous IL-4 during nerve injury and regeneration in vivo was assessed following a sciatic nerve crush using IL-4 knockout (KO) mice. Loss of IL-4 affected macrophage accumulation within injured nerve compared to WT mice, as well as shifted macrophage phenotype towards a CD206- phenotype with altered gene expression. Furthermore, this loss of IL-4 delayed initial axon regeneration from the injury crush site and subsequently delayed functional recovery and re-innervation of neuromuscular junctions compared to wild-type mice. Given the role of endogenous IL-4 in nerve regeneration, exogenous IL-4 was administered daily to WT mice following a nerve crush to examine regeneration. Daily IL-4 administration increased early axonal extension and CD206+ macrophage accumulation but did not alter functional recovery compared to untreated mice. Our data demonstrate IL-4 promotes nerve regeneration and recovery after injury., (Copyright © 2021 Elsevier Inc. All rights reserved.)

One of the changes brought about by Wallerian degeneration distal to nerve injury is disintegration of axonal mitochondria and consequent leakage of mitochondrial DNA (mtDNA)-the natural ligand for the toll-like receptor 9 (TLR9). RT-PCR and immunohistochemical or Western blot analyses were used to detect TLR9 mRNA and protein respectively in the lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) ipsilateral and contralateral to a sterile unilateral sciatic nerve compression or transection. The unilateral sciatic nerve lesions led to bilateral increases in levels of both TLR9 mRNA and protein not only in the lumbar but also in the remote cervical DRG compared with naive or sham-operated controls. This upregulation of TLR9 was linked to activation of the Nuclear Factor kappa B (NFκB) and nuclear translocation of the Signal Transducer and Activator of Transcription 3 (STAT3), implying innate neuronal immune reaction and a pro-regenerative state in uninjured primary sensory neurons of the cervical DRG. The relationship of TLR9 to the induction of a pro-regenerative state in the cervical DRG neurons was confirmed by the shorter lengths of regenerated axons distal to ulnar nerve crush following a previous sciatic nerve lesion and intrathecal chloroquine injection compared with control rats. The results suggest that a systemic innate immune reaction not only triggers the regenerative state of axotomized DRG neurons but also induces a pro-regenerative state further along the neural axis after unilateral nerve injury.

Aims: Inflammation plays a key role in peripheral nerve adhesion and often leads to severe pain and nerve dysfunction. Minocycline was reported to have potent anti-inflammatory effects and might be a promising drug to prevent or attenuate peripheral nerve adhesion. The present study aimed to clarify whether minocycline contributes to nerve adhesion protection and its underlying mechanism., Materials and Methods: Rats with sciatic nerve adhesion induced by glutaraldehyde glue (GG) were intraperitoneally injected with minocycline or saline every 12 h for 7 consecutive days. After that, the adhesion score, Ashcroft score, demyelination, macrophage polarization and inflammatory factors in peripheral nerve adhesion tissues or tissues in sham group were determined with histological staining, western blot and real time-PCR. Murine macrophage RAW264.7 cells were stimulated by LPS alone or together with minocycline at different concentrations and time duration to study the mechanism of minocycline in alleviating nerve adhesion., Key Findings: We found that minocycline treatment reduced the adhesion score, Ashcroft score, the growth of scar tissue, demyelination, and macrophage recruitment. Moreover, minocycline significantly and dose-dependently promoted regulatory macrophage polarization but decreased pro-inflammatory macrophage polarization. Furthermore, mechanism studies showed that TAK1 and its downstream pathway p38/JNK/ERK1/2/p65 were inhibited by minocycline, which led to lower IL-1β and TNFα expression, but increased IL-10 expression., Significance: Altogether, these results suggest that minocycline is highly effective against peripheral nerve adhesion through anti-fibrosis, anti-inflammation, and myelination protection, making it a highly promising candidate for treating adhesion-related disorders., (Copyright © 2021. Published by Elsevier Inc.)

Guillain-Barré Syndrome (GBS), characterized by peripheral nerve demyelination and axonal damage, is initiated and aggravated through various of immunopathogenesis. Ginsenoside Rd, main active components extracted from ginseng saponins, is known to exhibit immune-regulate functions in many immune-mediated diseases. However, the evidence of preventive effect of Ginsenoside Rd on GBS is lacking. Experimental autoimmune neuritis (EAN) mice, classic model of GBS, were established and treated with GSRd or vehicle. Clinical score and nerve tissue histomorphology were evaluated. Monocytes in peripheral blood and tissue were detected by flow cytometry analysis and immunofluorescence staining. For the in vitro study, GSRd and vehicle were added in the culture medium to assess their regulatory function on monocytes phenotype. In vivo data showed a protective role of GSRd on alleviating symptoms and tissue damage on Day 20 and 25. Administration of GSRd increased non-classical Ly6C lo monocytes in both peripheral blood and injured nerve tissue, and also switched tissue macrophages phenotype into resolution-phase. In vitro study indicated similar role of GSRd on monocytes differentiation status. Transcription factors like Nr4a1 were elevated after GSRd treatment. These findings revealed the protective role of GSRd against EAN, and potential preventive function on GBS patients., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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.

Ablation/segmental loss peripheral nerve injuries (PNIs) exhibit poor functional recovery due to slow and inaccurate outgrowth of regenerating axons. Viable peripheral nerve allografts (PNAs) as growth-guide conduits are immunologically rejected and all anucleated donor/host axonal segments undergo Wallerian degeneration. In contrast, we report that ablation-type sciatic PNIs repaired by neurorrhaphy of viable sciatic PNAs and a polyethylene glycol (PEG)-fusion protocol using PEG immediately restored axonal continuity for many axons, reinnervated/maintained their neuromuscular junctions, and prevented much Wallerian degeneration. PEG-fused PNAs permanently restored many sciatic-mediated behaviors within 2-6 weeks. PEG-fused PNAs were not rejected even though host/donors were neither immunosuppressed nor tissue-matched in outbred female Sprague Dawley rats. Innate and adaptive immune responses to PEG-fused sciatic PNAs were analyzed using electron microscopy, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction for morphological features, T cell and macrophage infiltration, major histocompatibility complex (MHC) expression, apoptosis, expression of cytokines, chemokines, and cytotoxic effectors. PEG-fused PNAs exhibited attenuated innate and adaptive immune responses by 14-21 days postoperatively, as evidenced by (a) many axons and cells remaining viable, (b) significantly reduced infiltration of cytotoxic and total T cells and macrophages, (c) significantly reduced expression of inflammatory cytokines, chemokines, and MHC proteins, (d) consistently low apoptotic response. Morphologically and/or biochemically, PEG-fused sciatic PNAs often resembled sciatic autografts or intact sciatic nerves. In brief, PEG-fused PNAs are an unstudied, perhaps unique, example of immune tolerance of viable allograft tissue in a nonimmune-privileged environment and could greatly improve the clinical outcomes for PNIs relative to current protocols., (© 2020 Wiley Periodicals LLC.)

Introduction: The immuno-microenvironment of injured nerves adversely affects mesenchymal stem cell (MSC) therapy for neurotmesis. Magnetic resonance imaging (MRI) can be used noninvasively to monitor nerve degeneration and regeneration. The aim of this study was to investigate nerve repair after MSC transplantation combined with microenvironment immunomodulation in neurotmesis by using multiparametric MRI., Methods: Rats with sciatic nerve transection and surgical coaptation were treated with MSCs combined with immunomodulation or MSCs alone. Serial multiparametric MRI examinations were performed over an 8-week period after surgery., Results: Nerves treated with MSCs combined with immunomodulation showed better functional recovery, rapid recovery of nerve T2, fractional anisotropy and radial diffusivity values, and more rapid restoration of the fiber tracks than nerves treated with MSCs alone., Discussion: Transplantation of MSCs in combination with immunomodulation can exert a synergistic repair effect on neurotmesis, which can be monitored by multiparametric MRI., (© 2020 Wiley Periodicals, Inc.)

Neuropathic pain is a widespread and debilitating chronic pain and the treatment remains a clinical challenge. Isoorientin (3',4',5,7-tetrahydroxy-6-C-glucopyranosyl flavone) is a natural flavonoid-like compound that exhibits antioxidant and anti-inflammatory activities; however, its effect on neuropathic pain remains unclear. Our study aimed to evaluate the antinociceptive effect of isoorientin in neuropathic pain mouse models induced by chronic constriction injury (CCI). In our study, the mice with CCI were administered with 7.5, 15, and, 30 mg/kg isoorientin for 8 consecutive days. Behavioral parameters were assayed on days 0, 7, 8, 10, 12, and 14 post-CCI surgery. Electrophysiological, histopathological, and biochemical indices were analyzed on day 14. Immunofluorescence was utilized to examine matrix metalloproteinase-9 (MMP-9) and glial cell activation, and proinflammatory cytokine expression levels were detected via Western blot. It is obvious that the treatment of Isoorientin remarkably ameliorated hyperalgesia and allodynia, increased sensory nerve conduction velocities, and restored CCI-induced sciatic nerve damage in mice. Isoorientin treatment significantly increased the total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD) and catalase (CAT) levels, and decreased the malondialdehyde (MDA) concentrations. Isoorientin also suppressed MMP-9 and glial cell activation, and downregulated tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) expression levels. Therefore, this study provided a novel approach for neuropathic pain treatment and new insights into the pharmacological action of isoorientin., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toll-like receptors (TLRs) are extremely significant pattern recognition receptors. When nerve injury occurs, a variety of inflammatory factors are generated, leading to an exceedingly complex micro-environment. TLRs recognize damage-associated molecular patterns. To investigate the correlation between TLR4 and recovery after sciatic nerve injury, the model of sciatic nerve injury was conducted using TLR4-mutated mice (C3H/HeJ) and wild mice (C3H/HeN). Our goal was to identify short-stage and long-stage changes after sciatic nerve injury, mainly by checking the expression changes of inflammation factors in the short-stage and the differences in the recovery of the injured sciatic nerve in the long-stage. The results show that the increase of changes in the HeN group of IL-1β, IL-6, TNF-α and MCP-1 are more obvious than in the HeJ group, with caspase1 expression higher and Nlrp3 expression lower in the former group. Further results reveal intense inflammation occurred in the HeN group showing more neutrophils and macrophages. Nlrp3 and caspase1 showed little difference by Immunohistochemistry, with Nlrp6 expression differing between the HeJ group and the HeN group. The results led us to conclude that better recovery of the injured sciatic nerve occurred in the HeJ group because the expression of GAP-43 and p75NTR was higher and had a better SFI figure. TLR4 mutation can decrease the expression of inflammatory factors and enhance the speed of recovery after sciatic nerve injury. The changes in the expression of Nlrp6, which are related to the TLR4 mutation, may influence recovery of the injured sciatic nerve. Further studies will be conducted to confirm these results., (Copyright © 2017. Published by Elsevier B.V.)

Peripheral nerve sheath tumors are benign tumors that have the potential to transform into malignant peripheral nerve sheath tumors (MPNSTs). Interleukin-13 receptor alpha 2 (IL13Rα2) is a cancer associated receptor expressed in glioblastoma and other invasive cancers. We analyzed IL13Rα2 expression in several MPNST cell lines including the STS26T cell line, as well as in several peripheral nerve sheath tumors to utilize the IL13Rα2 receptor as a target for therapy. In our studies, we demonstrated the selective expression of IL13Rα2 in several peripheral nerve sheath tumors by immunohistochemistry (IHC) and immunoblots. We established a sciatic nerve MPNST mouse model in NIH III nude mice using a luciferase transfected STS26T MPNST cell line. Similarly, analysis of the mouse sciatic nerves after tumor induction revealed significant expression of IL13Rα2 by IHC when compared to a normal sciatic nerve. IL13 conjugated liposomal doxorubicin was formulated and shown to bind and internalized in the MPNST cell culture model demonstrating cytotoxic effect. Our subsequent in vivo investigation in the STS26T MPNST sciatic nerve tumor model indicated that IL13 conjugated liposomal doxorubicin (IL13LIPDXR) was more effective in inhibiting tumor progression compared to unconjugated liposomal doxorubicin (LIPDXR). This further supports that IL13 receptor targeted nanoliposomes is a potential approach for treating MPNSTs.

Interleukin-8 (IL-8) is a pro-inflammatory cytokine that has been shown to play a role in inflammatory and autoimmune disorders. The objective of the present study was to assess the levels of IL-8 in rat serum, dorsal root ganglion (DRG) and the sciatic nerve following four different forms of sciatic nerve injury. The models used to induce the injury included partial sciatic ligation (PSL), chronic constriction injury (CCI), perineural inflammation (neuritis) and complete sciatic transection (CST). Mechanical and thermal hyperalgesia were detected by measuring withdrawal responses from a mechanical stimulus and withdrawal latency from thermal stimulation. Enzyme-linked immunosorbent assays (ELISA) was used to assess the IL-8 levels in the affected and contralateral sciatic nerves. Rats exposed to PSL and neuritis developed significant nociceptive response (mechanical and thermal hyperalgesia) in the affected side at three days post-surgery whereas the CCI group at eight days post-surgery. No mechanical or thermal hyperalgesia was observed in rats exposed to CST at either three or eight days postsurgery. Additionally, IL-8 levels were significantly increased in the injured sciatic nerve at 3 and 8days following PSL and neuritis as well as at 8days following CCI when compared to naïve animals. A significant up regulation of IL-8 levels was observed in the ipsilateral DRG at 3 and 8days following CST compared to naïve animals. The serum IL-8 levels remained unchanged in all models of nerve damage. The results of this study suggest that IL-8's role in the neuropathic pain etiology may be specific to nerve injury type., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations., New Methods: We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols., Conclusions: We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The precise mechanisms underlying the efficacy of intravenous immunoglobulin (IVIg) in autoimmune neurological disorders including Guillain-Barré syndrome (GBS) are not known. Anti-ganglioside antibodies have been reported to be pathogenic in some variants of GBS, and we have developed passive transfer animal models to study anti-ganglioside antibody mediated-endoneurial inflammation and associated neuropathological effects and to evaluate the efficacy of new therapeutic approaches. Some studies indicate that IVIg's anti-inflammatory activity resides in a minor sialylated IVIg (sIVIg) fractions and is dependent on an innate Th2 response via binding to a specific ICAM3-grabbing nonintegrin related 1 receptor (SIGN-R1). Therefore the efficacy of IVIg, IVIg fractions with various IgG Fc sialylation status, and the involvement of Th2 pathway were examined in one of our animal model of antibody-mediated inhibition of axonal regeneration. We demonstrate that both IVIg and sIVIg ameliorated anti-glycan antibody mediated-pathological effect, whereas, the unsialylated fractions of IVIg were not beneficial in our model. Tenfold lower doses of sIVIg compared to whole IVIg provided equivalent efficacy in our studies. Moreover, we found that whole IVIg and sIVIg significantly upregulates the gene expression of IL-33, which itself can provide protection from antibody-mediated nerve injury in our model. Our results support that the SIGN-R1-Th2 pathway is involved in the anti-inflammatory effects of IVIg on endoneurium in our model and elements of this pathway including IL-33 can provide novel therapeutics in inflammatory neuropathies., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Prior studies of aging and neuropathic injury have focused on senescent animals compared to young adults, while changes in middle age, particularly in the dorsal root ganglia (DRG), have remained largely unexplored. 14 neuroimmune mRNA markers, previously associated with peripheral nerve injury, were measured in multiplex assays of lumbar spinal cord (LSC), and DRG from young and middle-aged (3, 17 month) naïve rats, or from rats subjected to chronic constriction injury (CCI) of the sciatic nerve (after 7 days), or from aged-matched sham controls. Results showed that CD2, CD3e, CD68, CD45, TNF-α, IL6, CCL2, ATF3 and TGFβ1 mRNA levels were substantially elevated in LSC from naïve middle-aged animals compared to young adults. Similarly, LSC samples from older sham animals showed increased levels of T-cell and microglial/macrophage markers. CCI induced further increases in CCL2, and IL6, and elevated ATF3 mRNA levels in LSC of young and middle-aged adults. Immunofluorescence images of dorsal horn microglia from middle-aged naïve or sham rats were typically hypertrophic with mostly thickened, de-ramified processes, similar to microglia following CCI. Unlike the spinal cord, marker expression profiles in naïve DRG were unchanged across age (except increased ATF3); whereas, levels of GFAP protein, localized to satellite glia, were highly elevated in middle age, but independent of nerve injury. Most neuroimmune markers were elevated in DRG following CCI in young adults, yet middle-aged animals showed little response to injury. No age-related changes in nociception (heat, cold, mechanical) were observed in naïve adults, or at days 3 or 7 post-CCI. The patterns of marker expression and microglial morphologies in healthy middle age are consistent with development of a para-inflammatory state involving microglial activation and T-cell marker elevation in the dorsal horn, and neuronal stress and satellite cell activation in the DRG. These changes, however, did not affect the establishment of neuropathic pain.

Neuronal activity regulated pentraxin (Narp) is a secreted protein implicated in regulating synaptic plasticity via its association with the extracellular surface of AMPA receptors. We found robust Narp immunostaining in dorsal root ganglia (DRG) that is largely restricted to small diameter neurons, and in the superficial layers of the dorsal horn of the spinal cord. In double staining studies of DRG, we found that Narp is expressed in both IB4- and CGRP-positive neurons, markers of distinct populations of nociceptive neurons. Although a panel of standard pain behavioral assays were unaffected by Narp deletion, we found that Narp knockout mice displayed an exaggerated microglia/macrophage response in the dorsal horn of the spinal cord to sciatic nerve transection 3days after surgery compared with wild type mice. As other members of the pentraxin family have been implicated in regulating innate immunity, these findings suggest that Narp, and perhaps other neuronal pentraxins, also regulate inflammation in the nervous system., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The widespread plant volatile beta-caryophyllene (BCP) was recently identified as a natural selective agonist of the peripherally expressed cannabinoid receptor 2 (CB₂). It is found in relatively high concentrations in many spices and food plants. A number of studies have shown that CB₂ is critically involved in the modulation of inflammatory and neuropathic pain responses. In this study, we have investigated the analgesic effects of BCP in animal models of inflammatory and neuropathic pain. We demonstrate that orally administered BCP reduced inflammatory (late phase) pain responses in the formalin test in a CB₂ receptor-dependent manner, while it had no effect on acute (early phase) responses. In a neuropathic pain model the chronic oral administration of BCP attenuated thermal hyperalgesia and mechanical allodynia, and reduced spinal neuroinflammation. Importantly, we found no signs of tolerance to the anti-hyperalgesic effects of BCP after prolonged treatment. Oral BCP was more effective than the subcutaneously injected synthetic CB₂ agonist JWH-133. Thus, the natural plant product BCP may be highly effective in the treatment of long lasting, debilitating pain states. Our results have important implications for the role of dietary factors in the development and modulation of chronic pain conditions., (© 2013 Published by Elsevier B.V. and ECNP.)

Subsequent to peripheral nerve compression and irritation, pathophysiological processes take place within nervous and immune systems. Here, we utilized a multimodal approach to comprehend peripheral and central soft tissue changes as well as alterations occurring in systemic analytes following unilateral chronic constriction injury (CCI) of the sciatic nerve in rodents. Using magnetic resonance imaging and [18F]-2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography, we demonstrated robust structural abnormalities and enhanced FDG uptake within the injured nerve and surrounding muscle, respectively. To assess whether central morphological changes were induced by nerve injury, diffusion tenor imaging was performed. A decrease in fractional anisotropy in primary motor cortex contralateral to the injury site was observed. Evaluation of a panel of circulating cytokines, chemokines, and growth factors showed decreased levels of interleukin-1β and Fractalkine in CCI animals. Area under the receiver operating curve (ROC) calculations of analyte levels, imaging, and behavioral end points ranged from 0.786 to 1, where behavioral and peripheral imaging end points (eg, FDG uptake in muscle) were observed to have the highest discriminatory capabilities (maximum area under ROC = 1) between nerve injury and sham conditions. Lastly, performance of correlation analysis involving all analyte, behavioral, and imaging data provided an understanding of the overall association amongst these end points, and importantly, a distinction in correlation patterns was observed between CCI and sham conditions. These findings demonstrate the multidimensional pathophysiology of sciatic nerve injury and how a combined analyte, behavioral, and imaging assessment can be implemented to probe this complexity., (Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: The infusion or decoction of Mirabilis jalapa leaves is used in traditional medicine in Brazil to treat inflammatory and painful diseases. Thus, the present study was designed to investigate whether the leaf ethyl acetate (Eta) fraction from Mirabilis jalapa exhibits antinociceptive effect in clinically relevant pain models in mice. Furthermore, we have investigated the role of cholinergic system in the antinociceptive action produced by Eta in mice., Materials and Methods: The effect of Eta administered orally (10mg/kg, p.o.) in mice was verified on the painful hypersensitivity (mechanical allodynia) in models of chronic inflammation (subcutaneous injection of complete Freund's Adjuvant-CFA in the plantar surface of the right hind paw), postoperative (paw surgical incision) and neuropathic (partial sciatic nerve ligation) pain. In the chronic inflammation model, we further verified the effect of Eta treatment on paw edema and interleukin-1β (IL-1β) levels. We also investigated the role of muscarinic and nicotinic receptors in the antiallodynic action produced by Eta as well as the possible action of Eta on in vitro and ex vivo acetylcholinesterase activity in CFA treated animals. Furthermore, we verified the effect of Eta treatment on the parameters of liver and kidney lesion (level of urea, and activity of aspartate aminotransferase and alanine aminotransferase)., Results: Eta produced marked reduction in the allodynia caused by CFA, surgical incision and partial sciatic nerve ligation. However, Eta did not alter the paw edema or the increase of IL-1β levels produced by CFA. The antinociceptive effect of Eta was reversed by the pre-treatment of animals with the antagonists of muscarinic (atropine, 5mg/kg, s.c) or nicotinic (mecamylamine, 0.001mg/kg, s.c.) receptors. Eta did not alter in vitro acetylcholinesterase activity in blood or spinal cord samples, but it reversed the increase in the acetylcholinesterase activity observed in the spinal cord samples from mice injected with CFA. Moreover, Eta did not alter the indicators of liver or kidney lesion., Conclusions: Based on its use in traditional medicine, the results of the present study confirmed the antinociceptive properties of Eta in clinically relevant pain models. Also its effect on the CFA-induced chronic inflammation seems to be related to acetylcholinesterase inhibition and cholinergic system., (© 2013 Elsevier Ireland Ltd. All rights reserved.)

Peripheral neuropathy and insensate limbs and digits cause significant morbidity in diabetic individuals. Previous studies showed that deletion of the receptor for advanced end-glycation products (RAGE) in mice was protective in long-term diabetic neuropathy. Here, we tested the hypothesis that RAGE suppresses effective axonal regeneration in superimposed acute peripheral nerve injury attributable to tissue-damaging inflammatory responses. We report that deletion of RAGE, particularly in diabetic mice, resulted in significantly higher myelinated fiber densities and conduction velocities consequent to acute sciatic nerve crush compared with wild-type control animals. Consistent with key roles for RAGE-dependent inflammation, reconstitution of diabetic wild-type mice with RAGE-null versus wild-type bone marrow resulted in significantly improved axonal regeneration and restoration of function. Diabetic RAGE-null mice displayed higher numbers of invading macrophages in the nerve segments postcrush compared with wild-type animals, and these macrophages in diabetic RAGE-null mice displayed greater M2 polarization. In vitro, treatment of wild-type bone marrow-derived macrophages with advanced glycation end products (AGEs), which accumulate in diabetic nerve tissue, increased M1 and decreased M2 gene expression in a RAGE-dependent manner. Blockade of RAGE may be beneficial in the acute complications of diabetic neuropathy, at least in part, via upregulation of regeneration signals.

Neuropathic pain is a debilitating condition of the somatosensory system caused by pathology of the nervous system. Current drugs treat symptoms but largely fail to target the underlying mechanisms responsible for the pathological changes seen in the central or peripheral nervous system. We investigated the therapeutic effects of PDA-001, a culture expanded placenta-derived adherent cell, in the rat neuritis model. Pain is induced in the model by applying carrageenan to the sciatic nerve trunk, causing perineural inflammation of the sciatic nerve. PDA-001, at doses ranging from 0.4×10(6) to 4×10(6) cells/animal, or vehicle control was intravenously administrated to assess the biological activity of the cells. A dose-dependent effect of PDA-001 on pain relief was demonstrated. PDA-001 at doses of 1×10(6) and 4×10(6), but not 0.4×10(6), reduced mechanical hyperalgesia within 24h following treatment and through day 8 after induction of neuritis. The mechanism underlying PDA-001-mediated reduction of neuroinflammatory pain was also explored. Ex vivo tissue analyses demonstrated that PDA-001 suppressed homing, maturation and differentiation of dendritic cells, thus inhibiting T-cell priming and activation in draining lymph nodes. PDA-001 also reduced interferon gamma and IL-17 in draining lymph nodes and in the ispilateral sciatic nerve, and increased the levels of IL-10 in draining lymph nodes and plasma, pointing to T-cell modulation as a possible mechanism mediating the observed anti-hyperalgesic effects. Furthermore, in the ipsilateral sciatic nerve, significantly less leukocyte infiltration was observed in PDA-001-treated animals. The results suggest that PDA-001may provide a novel therapeutic approach in the management of inflammatory neuropathic pain and similar conditions., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neuroimmune crosstalk in neuropathic pain is a key contributor to pain hypersensitivity following nervous system injury. CD4+CD25+Foxp3+ regulatory T cells (Tregs) are endogenous immune suppressors, reducing T-cell proliferation and proinflammatory cytokine production. Currently, the role of Tregs in neuropathic pain is unknown. In this study, we tested the effects of expanding Tregs on pain hypersensitivity and neuroinflammation in 2 models of neuropathy; sciatic nerve chronic constriction injury and experimental autoimmune neuritis in rats. Following chronic constriction injury, treatment with CD28 superagonist (CD28SupA), a Treg population expander, significantly increased Tregs in the lymphoid tissues, injured sciatic nerve, and lumbar spinal cord of rats. CD28SupA treatment led to a significant reduction in mechanical pain hypersensitivity, alongside a decrease in the numbers of infiltrating T cells, macrophages, and antigen-presenting cells in the sciatic nerve and dorsal root ganglia. In experimental autoimmune neuritis-affected rats, CD28SupA treatment resulted in a significant improvement in disease severity and in mechanical pain hypersensitivity. This was associated with a reduction in the numbers of T cells, macrophages, and antigen-presenting cells in the sciatic nerve and dorsal root ganglia, and reduced activation of microglia and infiltration of T cells in the spinal cord. Furthermore, depletion of Tregs by a CD25 antibody in mice with a partial sciatic nerve ligation resulted in prolonged mechanical pain hypersensitivity. These findings suggest that Tregs play a role in endogenous recovery from neuropathy-induced pain. Thus, this T-cell subset may be specifically targeted to alleviate chronic neuropathic pain., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Serotonin is critically involved in neuropathic pain. However, its role is far from being understood owing to the number of cellular targets and receptor subtypes involved. In a rat model of neuropathic pain evoked by chronic constriction injury (CCI) of the sciatic nerve, we studied the role of 5-HT(2B) receptor in dorsal root ganglia (DRG) and the sciatic nerve. We showed that 5-HT(2B) receptor activation both prevents and reduces CCI-induced allodynia. Intrathecal administration of 5-HT(2B) receptor agonist BW723C86 significantly attenuated established mechanical and cold allodynia; this effect was prevented by co-injection of RS127445, a selective 5-HT(2B) receptor antagonist. A single application of BW723C86 on the sciatic nerve concomitantly to CCI dose-dependently prevented mechanical allodynia and significantly reduced cold allodynia 17 days after CCI. This behavioral effect was accompanied with a marked decrease in macrophage infiltration into the sciatic nerve and, in the DRG, with an attenuated abnormal expression of several markers associated with local neuroinflammation and neuropathic pain. CCI resulted in a marked upregulation of 5-HT(2B) receptor expression in sciatic nerve and DRG. In the latter structure, it was biphasic, consisting of a transient early increase (23-fold), 2 days after the surgery and before the neuropathic pain emergence, followed by a steady (5-fold) increase, that remained constant until pain disappeared. In DRG and sciatic nerve, 5-HT(2B) receptors were immunolocalized on sensory neurons and infiltrating macrophages. Our data reveal a relationship between serotonin, immunocytes, and neuropathic pain development, and demonstrate a critical role of 5-HT(2B) receptors in blood-derived macrophages., (Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Background: The underlying mechanism of exercise on neuropathic pain is not well understood. We investigated whether physical exercise regulates the functional recovery and heat shock protein 72 (Hsp72), tumor necrosis factor-α (TNF-α), and interlukin-1β (IL-1β) expression after chronic constriction injury (CCI) of the sciatic nerve., Methods: Male Sprague-Dawley rats were divided into 7 groups: control, sham operated (SO), SO with swimming or treadmill exercise (SOSE or SOTE), CCI, CCI with swimming or treadmill exercise (CCISE or CCITE). We recorded body weight, thermal withdrawal latency, and mechanical withdrawal threshold as well as Hsp72, TNF-α, and IL-1β expression in sciatic nerve., Results: The body weights in the control and SO groups were heavier than those in the SOSE, SOTE, CCI, CCISE, and CCITE groups. CCI rats with swimming or treadmill exercise showed significant increase in thermal withdrawal latency and mechanical withdrawal threshold when compared with CCI rats without exercise on day 21 after CCI. Both CCISE and CCITE groups demonstrated greater Hsp72 expression and lower TNF-α or IL-1β level than did the CCI group in sciatic nerve on day 21 after CCI., Conclusions: These results suggest that progressive exercise training decreases peripheral neuropathic pain as well as TNF-α and IL-1β overproduction and increases HSP72 expression after CCI of the sciatic nerve.

Nerve allografts provide a temporary scaffold for host nerve regeneration. The need for systemic immunosuppression limits clinical application. Characterization of the immunological mechanisms that induce immune hyporesponsiveness may provide a basis for optimizing immunomodulating regimens. We utilized wild-type and MHC class II-deficient mice, as both recipients and donors. Host treatment consisted of triple costimulatory blockade. Quantitative assessment was made at 3 weeks using nerve histomorphometry, and muscle testing was performed on a subset of animals at 7 weeks. Nerve allograft rejection occurred as long as either the direct or indirect pathways were functional. Indirect antigen presentation appeared to be more important. Nerve allograft rejection occurs in the absence of a normal direct or indirect immune response but may be more dependent on indirect allorecognition. The indirect pathway is required to induce costimulatory blockade immune hyporesponsiveness., (Copyright © 2010 Wiley Periodicals, Inc.)

Unlabelled: Injured optic nerves in the matured central nervous system (CNS), alike injured neurons in other CNS regions, fail to regenerate. Interestingly, activation of inflammatory cells (macrophages) following optic lens injury or implantation of peripheral nerve fragments into the vitreous body, have been previously reported to stimulate retinal ganglion cells (RGCs) to regenerate axons across the injury site and into the distal optic nerve. In addition, the beneficial role of macrophage cells has also been demonstrated in the regeneration of lesioned spinal neurons following sciatic nerve injury. However, it is not known whether these locally activated macrophage cells also contribute to the regeneration of remotely injured neurons within the CNS. Adult Sprague Dawley rats received a conditioning sciatic nerve injury followed by an optic nerve crush (ONC). Retrograde and anterograde tracing results revealed that injured optic axons did not regenerate after peripheral dorsal root ganglion (DRG) lesion, as the beneficial effects of this injury extended only locally. However, a greater inflammatory infiltration/activation was found in injury-combined animals compared to controls, although this was not sufficient to trigger a systemic regenerative response. Proximity of cell body response to injury, accompanied by a timely macrophage activation are critical factors for regeneration of injured CNS neurons to occur. Immune cell surveillance into the CNS compartment was enhanced following peripheral nerve injury., Scope: nervous system development, regeneration and aging., (Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.)

Aims: To determine if specific pro-inflammatory chemokine ligand/receptor pairs expressed in the peripheral nerves of Guillain-Barré syndrome patients are expressed in a severe murine experimental autoimmune neuritis (sm-EAN) model and to determine their cellular localization as a prerequisite to designing potentially therapeutic interventions in vivo., Methods: Sm-EAN was induced in 8-12-week-old female SJL/J mice using bovine peripheral nerve myelin emulsified in complete Freund adjuvant with pertussis toxin and recombinant mouse interleukin-12 acting as co-adjuvants, with appropriate controls. Mice were evaluated for neuromuscular weakness and weighed daily. Dorsal caudal tail and sciatic nerve motor electrophysiological studies were performed at expected maximal severity. Sciatic nerves were harvested and specific chemokine ligand/receptor expression was determined using real-time quantitative reverse transcriptase polymerase chain reaction and indirect fluorescent immunohistochemistry., Results: CCL2/CCR2, CXCL10/CXCR3 and CCL5/CCR1, CCR5 expression was significantly increased in the sciatic nerves of sm-EAN mice compared with controls. CCL2 was expressed on Schwann cells with CCR2 expressed on F4/80+ macrophages and CD3+ T cells. CXCL10 was expressed on endoneurial endothelial cells and within the endoneurial interstitium, with CXCR3 expressed on CD3+ T-lymphocytes. CCL5 co-localized to axons, with CCR1 and CCR5 expression on F4/80+ macrophages and rare CD3+ T cells., Conclusions: This study suggests that CCL2 expressed by Schwann cells and CXCL10 expressed by endoneurial endothelial cells may induce F4/80+ macrophage and CD3+ T cell-mediated inflammation and demyelination in sm-EAN. CCL2-CCR2 and CXCL10-CXCR3 signalling pathways are potential targets for therapeutic intervention in peripheral nerve inflammation.

Background: Anastomosis of the nerve especially at narrow surgical field and presence of surgical tension is not easily accessible. DuraSeal demonstrates strong adhesive power without producing neurotoxicity. Herein, we evaluate the possibility of DuraSeal as a substitute in the repair of sciatic nerve gap injury., Materials and Methods: The nerve gap model was constructed by excising the sciatic nerve (5mm in length) in Sprague Dawley rats leaving a 5mm nerve defect between nerve stumps. Animals were categorized into four groups: Group I: no treatment; Group II: 4 stitches suture; Group III: nerve approximation fixed by tissue glue; Group IV: nerve approximation fixed by DuraSeal. The motor function assessment included the CatWalk and SFI as well as electrophysiological studies. Nerve continuity and regeneration was examined at 1 and 8 wk after injury. The inflammatory cells, Schwann cell apoptosis, and Schwann cell proliferation were also investigated 1 wk after injury., Results: The achievement of nerve continuity and myelination by DuraSeal approached that of suture demonstrated by crystal violet and Luxol Fast Blue staining at 1 and 8 wk, respectively. Motor function and electrophysiological parameters were restored in DuraSeal and suture group. Early expression of neurofilament and bromodeoxyuridine (BrdU) was also observed in these two groups. There was no statistically significant difference in deposits of macrophages and neutrophil cells or cell apoptosis among these four groups., Conclusions: DuraSeal achieved the same nerve regeneration compared with that of suture and produced better regeneration than that of the tissue glue or without any treatment. The accomplishment of nerve regeneration and continuity without causing neurotoxicity justifies using DuraSeal as a ligature in the anastomosis of nerve gap injury., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Diabetic neuropathy is one of the most frequent complications in diabetes but there are no treatments beyond glucose control, due in part to the lack of an appropriate animal model to assess an effective therapy. This study was undertaken to characterize the degenerative and regenerative responses of peripheral nerves after induced sciatic nerve damage in transgenic rat insulin I promoter / human interferon beta (RIP/IFNbeta) mice made diabetic with a low dose of streptozotocin (STZ) as an animal model of diabetic complications. In vivo, histological and immunohistological studies of cutaneous and sciatic nerves were performed after left sciatic crush. Functional tests, cutaneous innervation, and sciatic nerve evaluation showed pronounced neurological reduction in all groups 2 weeks after crush. All animals showed a gradual recovery but this was markedly slower in diabetic animals in comparison with normoglycemic animals. The delay in regeneration in diabetic RIP/IFNbeta mice resulted in an increase in active Schwann cells and regenerating neurites 8 weeks after surgery. These findings indicate that diabetic-RIP/IFNbeta animals mimic human diabetic neuropathy. Moreover, when these animals are submitted to nerve crush they have substantial deficits in nerve regrowth, similar to that observed in diabetic patients. When wildtype animals were treated with the same dose of STZ, no differences were observed with respect to nontreated animals, indicating that low doses of STZ and the transgene are not implicated in development of the degenerative and regenerative events observed in our study. All these findings indicate that RIP/IFNbeta transgenic mice are a good model for diabetic neuropathy.

In the present study, we investigated the role of the macrophage inflammatory protein-1alpha (MIP-1alpha) in the pathogenesis of neuropathic pain following partial sciatic nerve ligation (PSL) in mice. MIP-1alpha mRNA and its protein were dramatically up-regulated after PSL, and MIP-1alpha was localized on macrophages and Schwann cells in the injured sciatic nerve (SCN). PSL-induced long-lasting tactile allodynia and thermal hyperalgesia were prevented by the perineural injection of anti-MIP-1alpha (2ng). Intraneural (20ng) and perineural (100ng) injection of recombinant MIP-1alpha elicited tactile allodynia and thermal hyperalgesia in sham-operated limb. MIP-1alpha receptors (CCR1 and CCR5) mRNA and their proteins were also up-regulated in the SCN after PSL, and were localized on macrophages and Schwann cells. PSL-induced tactile allodynia was attenuated by perineural injection (0.2nmol) of siRNA against CCR1 and CCR5. On the other hand, PSL-induced thermal hyperalgesia was prevented by siRNA against CCR5, but not CCR1. Interleukin-1beta (IL-1beta) mRNA and its precursor protein in macrophages and Schwann cells were also up-regulated in the SCN after PSL, and PSL-induced neuropathic pain was prevented by the perineural injection of anti-IL-1beta (2ng). PSL-induced IL-1beta up-regulation was suppressed by anti-MIP-1alpha and siRNA against CCR1 and CCR5. Perineural injection of nicotine (20nmol), a macrophage suppressor, prevented PSL-induced neuropathic pain and suppressed MIP-1alpha and IL-1beta expressions. In conclusion, we propose a novel critical molecule MIP-1alpha derived from macrophages and Schwann cells that appears to play a crucial role in the development of neuropathic pain induced by PSL., (Copyright 2010 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

The calpain inhibitor MDL-28710 blocks the early local pro-inflammatory cytokine gene expression in mice after chronic constriction nerve injury (CCI). One-hundred-thirteen wild type mice of C57Bl/6J background received CCI of the right sciatic nerve. Mechanical paw withdrawal thresholds and thermal withdrawal latencies were investigated at baseline and at 1, 3, and 7 days after CCI. Three application regimens were used for MDL-28170: a) single injection 40 min before CCI; b) serial injections of MDL-28170 40 min before and up to day three after CCI; c) sustained application via intraperitoneal osmotic pumps. The control animals received the vehicle DMSO/PEG 400. The tolerable dose of MDL-28170 for mice was 30 mg/kg body weight, higher doses were lethal within the first hours after application. Mechanical withdrawal thresholds and thermal withdrawal latencies were reduced after CCI and did not normalize after single or serial injections, nor with application of MDL-28170 via osmotic pumps. Although the calpain inhibitor MDL-28170 inhibits the early local cytokine upregulation in the sciatic nerve after CCI, pain behavior is not altered. This finding implies that local cytokine upregulation after nerve injury alone is only one factor in the induction and maintenance of neuropathic pain.

Peripheral nerve injury induces a profound local inflammatory response that involves T cells and macrophages and augments the generation of neuropathic pain. The mechanisms underlying immune cell activation or inhibition in the peripheral nervous system, however, are unknown. The co-inhibitory molecule B7-H1 (PD-L1, CD274) attenuates immune cell proliferation and cytokine production and protects from inflammation-induced tissue damage. We analyzed the temporal gene expression profile of B7-H1 and different cytokines after chronic constriction injury (CCI) of the sciatic nerve, a lesion paradigm inducing neuropathic pain, by quantitative real-time polymerase chain reaction and immunohistochemistry in B7-H1(-/-) mice and wild-type (WT) controls. B7-H1 mRNA was markedly induced in WT nerves after CCI, and macrophages could be identified as major B7-H1 source. The proinflammatory mediators tumor necrosis factor alpha (TNFalpha) and monocyte chemoattractant protein-1 (MCP-1) displayed a strong, but transient expression in degenerating nerves on day 1 after CCI in WT mice, while a biphasic expression peak on day 1 and day 28 was found in B7-H1(-/-) mice. Overall, TNFalpha and MCP-1 levels in B7-H1-deficient nerves dramatically exceeded those in WT controls. In contrast, induction of the anti-inflammatory cytokine interleukin(IL)-10 was restricted to WT nerves. The observation that B7-H1 deficiency enhances inflammation upon CCI was further corroborated by immunohistochemistry showing increased numbers of T cells and macrophages in injured nerves from B7-H1(-/-) mice. Interestingly, mechanical hyperalgesia was more pronounced in the absence of B7-H1. Our study identifies B7-H1 as an important suppressor of the inflammatory response and neuropathic pain occurring after peripheral nerve injury., (Copyright 2009 Elsevier Inc. All rights reserved.)

Experimental autoimmune gray matter disease (EAGMD) is a model of both upper and lower motor neuron degeneration. EAGMD and amyotrophic lateral sclerosis (ALS) possess similar clinical and pathological features. The aim of this study was to find evidence of upper and lower neuronal damage in the EAGMD guinea pigs. The main ultrastructural alterations included abnormal mitochondria and disorganization of neurofilaments in the myelinated nerve fibers of the spinal cord. Swollen mitochondria and dilated endoplasmic reticulum were found in pyramidal cells of the motor cortex. The myelinated fibers in the cerebral peduncle showed atrophied axons and swollen mitochondria. Some motoneurons showed apoptosis-like signs. Pathological changes in the sciatic nerve manifest wallerian-like degeneration. Using immunofluorescence double labeling and confocal laser microscopy, IgG was colocalized with activated microglia in the ventral horn of the spinal cord. We also examined possible evidences of oxidative stress in the EAGMD guinea pig model and the role of p38 mitogen-activated protein kinase (p38MAPK) pathway in motor neuron degeneration. Our findings suggest that nitric oxide and peroxynitrite-mediated oxidative damage may play important roles in the pathogenesis of the neuronal degeneration in the spinal cord. Inflammatory cytokines such as TNF-alpha and IL-1 play important roles in the formation and acceleration of the spinal cord damage. The activation of p38MAPK signal pathway was involved in the development of the motor neuron degeneration of the spinal cord.

Immune-mediated nerve inflammation is involved in many painful states in humans, and causes axonal and behavioral changes in rats. While models of nerve inflammation have been characterized using electrophysiological and behavioral methods, the presence of immune cells has not been fully assessed. We inflamed rat sciatic nerves using complete Freund's adjuvant and quantified the presence of ED-1 macrophages and TCR-alphabeta T-cells for up to 12 weeks. We report that these immune cells are prominent extraneurally up to 12 weeks following the induction of inflammation. This observation does not easily correlate with inflammation-induced axonal mechanical sensitivity, which peaks within 1 week and is resolved after 8 weeks.

Inflammatory response occurs, in general, at the peripheral site of injury or irritation and in the corresponding spinal dorsal horn segments. In this study, we show transient increases in the protein concentrations of interleukins 1beta, 6, 8 and NGF in lumbar DRGs within hours after sciatic mononeuropathy in the rat. Comparable increases, with variation in temporal patterns, were observed after skin inflammation induced by intraplantar injection of endotoxin or complete Freund's adjuvant. The non-matching temporal profiles of cytokines and NGF increases and those of behavioral hypersensitivity, particularly peak values, suggest that the roles of these proteins are not necessarily pro-nociceptive in the DRGs at the chronic phase of neuropathy.

While neuroimmune interactions are increasingly recognized as important in nociceptive processing, the nature and functional significance of these interactions is not well defined. There are multiple reports that the activation of spinal microglia is a critical event in the generation of neuropathic pain behaviors but the mediators of this activation remain disputed. Here we show that the chemokine CCL2, produced by both damaged and undamaged primary sensory neurons in neuropathic pain states in rats, is released in an activity dependent manner from the central terminals of these fibres. We also demonstrate that intraspinal CCL2 in naïve rats leads to activation of spinal microglia and neuropathic pain-like behavior. An essential role for spinal CCL2 is demonstrated by the inhibition of neuropathic pain behavior and microglial activation by a specific neutralising antibody to CCL2 administered intrathecally. Thus, the neuronal expression of CCL2 provides a mechanism for immune activation, which in turn regulates the sensitivity of pain signaling systems in neuropathic pain states.

Neuropathic pain is a clinical manifestation of nerve injury difficult to treat even with potent analgesic compounds. Here, we used different lines of genetically modified mice to clarify the role played by CB(2) cannabinoid receptors in the regulation of the central immune responses leading to the development of neuropathic pain. CB(2) knock-out mice and wild-type littermates were exposed to sciatic nerve injury, and both genotypes developed a similar hyperalgesia and allodynia in the ipsilateral paw. Most strikingly, knock-outs also developed a contralateral mirror image pain, associated with an enhanced microglial and astrocytic expression in the contralateral spinal horn. In agreement, hyperalgesia, allodynia, and microglial and astrocytic activation induced by sciatic nerve injury were attenuated in transgenic mice overexpressing CB(2) receptors. These results demonstrate the crucial role of CB(2) cannabinoid receptor in modulating glial activation in response to nerve injury. The enhanced manifestations of neuropathic pain were replicated in irradiated wild-type mice reconstituted with bone marrow cells from CB(2) knock-outs, thus demonstrating the implication of the CB(2) receptor expressed in hematopoietic cells in the development of neuropathic pain at the spinal cord.

The role of CD11b+ myeloid cells in axonal regeneration was assessed using axonal injury models and CD11b-TK(mt-30) mice expressing a mutated HSV-1 thymidine kinase (TK) gene regulated by the myeloid-specific CD11b promoter. Continuous delivery of ganciclovir at a sciatic nerve lesion site greatly decreased the number of granulocytes/inflammatory monocytes and macrophages in the distal stump of CD11b-TK(mt-30) mice. Axonal regeneration and locomotor function recovery were severely compromised in ganciclovir-treated CD11b-TK(mt-30) mice. This was caused by an unsuitable growth environment rather than an altered regeneration capacity of neurons. In absence of CD11b+ cells, the clearance of inhibitory myelin debris was prevented, neurotrophin synthesis was abolished, and blood vessel formation/maintenance was severely compromised in the sciatic nerve distal stump. Spinal cord-injured axons also failed to regenerate through peripheral nerve grafts in the absence of CD11b+ cells. Therefore, myeloid cells support axonal regeneration and functional recovery by creating a growth-permissive milieu for injured axons.

Clinical and experimental evidence suggests that glucocorticoids may be effective in the treatment of neuropathic pain, but their mechanism of action is unknown. We gave triamcinolone (3 mg/kg) to rats with an experimental post-traumatic painful peripheral neuropathy, chronic constriction injury (CCI), five days after nerve injury, when the abnormal pain syndrome is known to be present; and pain sensitivity was measured on postoperative days 7 - 14, a period during which symptoms are known to be at approximately peak severity. Additional CCI rats were treated similarly; and then they were sacrificed five days after the injection for an immunocytochemical analysis of endoneurial tumor necrosis factor-alpha (TNFalpha), macrophages, and mast cells in the sciatic nerve proximal to the site of injury. Vehicle-injected CCI rats demonstrated the expected neuropathic pain symptoms. Triamcinolone-treated CCI rats had a statistically significant reduction in the magnitude of heat-hyperalgesia and mechano-allodynia, but there was no effect on cold-allodynia or mechano-hyperalgesia. On the nerve-injured side of vehicle-injected rats, TNFalpha was present in Schwann cells and mast cells. On the nerve-injured side of triamcinolone-treated rats, there was a significant (71.5%) reduction in the number of TNFalpha-positive mast cells. Our results suggest that glucocorticoid therapy for neuropathic pain may work via the reduced expression of TNFalpha in endoneurial mast cells.

Aim: Although the neuropathology of ischemic nerve fiber degeneration is relatively well known, its pathogenesis is poorly understood. Local cytokines, which have neuroprotective effects on inflammation and repair, participate in the process by undefined mechanisms. In this study, we evaluated the effects of ischemia and reperfusion on the sciatic nerve of the rat and investigated the probable effects of cytokines on this period., Material and Methods: In the current study, ischemia and reperfusion injury of sciatic nerve was rendered by clamping the femoral artery and vein of the rat for three hours and was followed by varying durations of reperfusion. Activin A, TGF Beta1 and TGF, Beta2 levels were measured in serum samples., Results: TGF Beta1 and Activin A were found to be increased in the ischemic groups compared with the control group (p < 0.05). A significant difference was found between the experimental groups after reperfusion (p < 0.05). There was no statistical significance for TGF Beta2 levels between the study groups (p > 0.05)., Conclusion: Ischemia causes some important changes in biochemical parameters, and nerve injury continues for a while according to the reperfusion time. Ischemia-reperfusion injury of peripheral nerves caused by various reasons therefore affects the levels of cytokines.

The cholecystokinin B (2) receptor knockout (Cckbr KO) protects against allodynia induced by chronic constriction injury (CCI). The mechanism of this phenomenon is unknown, but must involve persistent changes in pain modulation and/or inflammatory pathways. We performed a gene expression study in two brain areas (midbrain and medulla) after surgical induction of CCI in Cckbr KO and wild-type (wt) control mice. The patterns of gene expression differences suggest that the immune system is activated in higher brain structures following CCI in the wt mice. The strongest differences include genes related to the MAPK pathway activation and cytokine production. In Cckbr KO mice this expressional pattern was absent. In addition, we found significant elevation of the Toll-like receptor 4 (Tlr4) in the supraspinal structures of the mice with deleted Cckbr compared to wt control mice. This up-regulation is most likely induced by the deletion of Cckbr. We suggest that there is a functional deficiency in the Tlr4 pathway which disables the development of neuropathic pain in Cckbr KO mice. Indeed, real time PCR analysis detected a CCI-induced upregulation of Tlr4 and Il1b expression in the lumbar region of wt but not Cckbr KO mice. Gene expression profiling indicates that elements of the immune response are not activated in Cckbr KO mice following CCI. Our findings suggest that there may be a role for CCK in the regulation of innate immunity.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Perineural alpha2-adrenoceptor activation relieves hypersensitivity induced by peripheral nerve injury or sciatic inflammatory neuritis. This effect is associated with a reduction in pro-inflammatory cytokines, as well as a reduction in local leukocyte number and their capacity to produce pro-inflammatory cytokines. Curiously, clonidine's antinociceptive effect appears with a 2-3-day delay after injection. Previous observations have shown that alpha-adrenoceptor activation induces apoptosis in leukocytes, which would reduce leukocyte number. Additionally, macrophage scavenging of apoptotic cells results in a shift to an anti-inflammatory phenotype, with expression of transforming growth factor (TGF)-beta1. We therefore examined the effects of perineural clonidine 24 h and 3 days after its injection on apoptosis, TGF-beta1 expression and lymphocyte and macrophage phenotype in acute sciatic inflammatory neuritis. Perineural clonidine reduced ipsilateral neuritis-induced hypersensitivity in a delayed manner (3 days after treatment), along with a reduction at this time in lymphocyte number and an increase in caspase-3 and TGF-beta1 expressing cells and macrophages co-expressing TGF-beta1 in the sciatic nerve. One day after injection clonidine treatment was associated with a reduction in lymphocytes and pro-inflammatory Th-1 cells as well as increased numbers of caspase-3 and TGF-beta1 expressing cells and macrophages co-expressing TGF-beta1 in sciatic nerve. Clonidine's effects were prevented by co-administration of an alpha2-adrenoceptor antagonist. These data suggest that alpha2-adrenoceptor activation in sciatic inflammatory neuritis increases local apoptosis and anti-inflammatory products early after treatment. This early effect likely underlies the delayed anti-inflammatory and anti-hypersensitivity effects of perineural clonidine in this setting.

Neuropathic pain, arising from nerve injury or secondary to other diseases, occurs in young children as well as adults but little is known about its postnatal development. Neonatal rat pups do not display mechanical allodynia following nerve injury and young rats recover faster from spinal nerve damage. Since both spinal microglia and astrocytes are strongly implicated in the maintenance of persistent pain, we hypothesized that the magnitude and time course of spinal cord glial activation following nerve injury change throughout postnatal development. To test this, we have compared the time course and intensity of the microglial and astrocytic response in the spinal cord dorsal horn at various times following spared nerve injury in postnatal day 3, 10, 21 and adult rats. The levels of the microglial markers OX-42 and IBA-1 and of the astrocytic marker GFAP were analysed using immunohistochemistry and Western blots. We show that in the adult SNI evokes clear dorsal horn microglial activation at 5 days and astrocytic activation at 7 days post surgery. In contrast, SNI in young animals evokes a weak microglial response but a robust astrocytic response with an early onset at day 1 that is not observed in adults, followed by a second activation at day 7. These results highlight the differential development of the glial response to nerve injury which may explain the lack of neuropathic allodynia in young animals.

Matrix metalloproteinase-9 (MMP-9) is an extracellular protease that is induced in Schwann cells hours after peripheral nerve injury and controls axonal degeneration and macrophage recruitment to the lesion. Here, we report a robust (90-fold) increase in MMP-9 mRNA within 24 h after rat sciatic nerve crush (1 to 60 days time-course). Using direct injection into a normal sciatic nerve, we identify the proinflammatory cytokines TNF-alpha and IL-1beta as potent regulators of MMP-9 expression (Taqman qPCR, zymography). Myelinating Schwann cells produced MMP-9 in response to cytokine injection and crush nerve injury. MMP-9 gene deletion reduced unstimulated neuropathic nociceptive behavior after one week post-crush and preserved myelin thickness by protecting myelin basic protein (MBP) from degradation, tested by Western blot and immunofluorescence. These data suggest that MMP-9 expression in peripheral nerve is controlled by key proinflammatory cytokine pathways, and that its removal protects nerve fibers from demyelination and reduces neuropathic pain after injury.

Nerve injury initiates Wallerian degeneration with subsequent alterations of cytokine expression contributing to neuropathic pain. To investigate the very early temporal pattern of cytokine regulation we studied 140 mice of C57Bl/6J background after chronic constriction injury (CCI) of the right sciatic nerve and measured the relative mRNA expression of the pro-inflammatory cytokines tumor necrosis factor-alpha (TNF) and interleukin-1beta (IL-1beta) and of the anti-inflammatory cytokines IL-4 and IL-10 with quantitative real-time polymerase chain reaction (qRT-PCR). The measurements were performed in ipsi- and contralateral sciatic nerves and dorsal root ganglia (DRG) 1, 3, 6, 9, 12, 24 h, and 3 and 7 days after CCI. We found an ipsilateral upregulation of TNF, IL-1beta and IL-10 mRNA levels as early as one hour after CCI. To investigate upstream regulatory mechanisms, we used inhibitors to the N-methyl-d-aspartate (NMDA) receptor ((+)-MK-801) and to calpain (MDL-28170). MDL-28170, but not (+)-MK-801 inhibited TNF and IL-1beta upregulation one hour after CCI. This leads us to suggest that calpain is one of the earliest mediators of cytokine upregulation in injured peripheral nerves.

Chronic nicotine exposure and the immune response to peripheral nerve injury has not been investigated thoroughly. Rats were exposed to chronic nicotine or saline followed by chronic constriction injury (CCI) of the sciatic nerve. Mechanical sensitivity was measured at various time points and the immune response was investigated at 21 days post-CCI. Chronic nicotine increased mechanical hypersensitivity, microglia activation, and the production of IL-1beta, but not the number of immune cells at the site of injury. These results suggest that chronic nicotine increases mechanical hypersensitivity following peripheral nerve injury through a mechanism that may involve an increased production and release of central and peripheral cytokines.

The purpose of this study was to investigate quantitatively the longitudinal temporal, spatial changes of the tumor necrosis factor-alpha (TNF) and interleukin-10 (IL-10) immunopositive cells during Wallerian degeneration and the following regeneration after crush injury in rat sciatic nerve using immunohistochemistry and enzyme linked immunosorbent assay (ELISA). The number of TNF-immunopositive cells reached its peak and increased significantly in all the segments distal to the crush site 3 days after injury. On Day 7, TNF-immunopositive cells decreased in all the segments distal to the crush site, and a significant decrease was observed 14 days after injury. From Day 21 to Day 56, there were no significant differences in the numbers of TNF-immunopositive cells. The average size of TNF immunopositive cells became significantly larger with degeneration. The number of IL-10-immunopositive cells decreases significantly 1 day after crush injury. IL-10-immunopositive cells increased on Day 3, returning to control levels. Seven days after injury, a significant increase in the number of IL-10-immunopositive cells was observed. There was also no significant difference in the number of IL-10-immunopositive cells beyond Day 14 except for a part of distal segments. The number of IL-10-immunopositive cells showed no significant differences in all the segments on Day 56. The protein levels of IL-10 measured by ELISA were similar to the result of immunohistochemistry. These results suggest that the significant change in IL-10 occurred prior to the significant change in TNF and that IL-10 may be the key to the change in TNF.

Recent studies have proposed that neurite outgrowth is influenced by specific nerve cell surface gangliosides, which are sialic acid-containing glycosphingolipids highly enriched in the mammalian nervous system. For example, the endogenous lectin, myelin-associated glycoprotein (MAG), is reported to bind to axonal gangliosides (GD1a and GT1b) to inhibit neurite outgrowth. Clustering of gangliosides in the absence of inhibitors such as MAG is also shown to inhibit neurite outgrowth in culture. In some human autoimmune PNS and CNS disorders, autoantibodies against GD1a or other gangliosides are implicated in pathophysiology. Because of neurobiological and clinical relevance, we asked whether anti-GD1a antibodies inhibit regeneration of injured axons in vivo. Passive transfer of anti-GD1a antibody severely inhibited axon regeneration after PNS injury in mice. In mutant mice with altered ganglioside or complement expression, inhibition by antibodies was mediated directly through GD1a and was independent of complement-induced cytolytic injury. The impaired regenerative responses and ultrastructure of injured peripheral axons mimicked the abortive regeneration typically seen after CNS injury. These data demonstrate that inhibition of axon regeneration is induced directly by engaging cell surface gangliosides in vivo and imply that circulating autoimmune antibodies can inhibit axon regeneration through neuronal gangliosides independent of endogenous regeneration inhibitors such as MAG.

Interleukin (IL)-17A, a recently described novel T cell cytokine, orchestrates inflammation in a variety of immune-mediated diseases. In the present investigation, we analyzed the temporal gene expression pattern of IL-17A and its main regulators IL-23 and IL-15 after chronic constriction injury (CCI) of the sciatic nerve, a lesion paradigm inducing neuropathic pain, by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in mice. IL-17A displayed a monophasic expression in degenerating nerves at day 7 after CCI while transcripts for the IL-17A regulatory cytokines IL-23 and IL-15 peaked earlier. Accordingly, IL-17A positive T cells were detectable within the endoneurium of the injured nerves by immunocytochemistry. In support of a crucial role of T cell inflammation, RAG-1 knockout mice lacking functional T lymphocytes did not express IL-17A mRNA in distal nerve segments following CCI. Interestingly, T cell deficiency was associated with less thermal hyperalgesia and reduced mRNA levels for the macrophage marker molecule F4/80 and the chemokine macrophage chemoattractant protein-1 (MCP-1) after CCI. Our study supports the notion that T cells and T-cell-derived cytokines contribute to the inflammatory response after peripheral nerve injury.