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Skeletal muscle atrophy is a known risk factor for immunosuppressive conditions and for a poor prognosis in sepsis. However, its immunopathology has not been experimentally elucidated. This study investigated the effects of skeletal muscle atrophy on the immunopathology of sepsis. Male C57BL/6J mice were subjected to sciatic denervation (DN) and caecal ligation and puncture (CLP) to induce muscle atrophy or sepsis. The macrophages, myeloid-derived suppressor cells (MDSC), and T-cells in peritoneal and spleen were analysed using flow cytometry. DN-induced muscle atrophy did not affect macrophage and MDSC populations. In contrast, the percentage of cytotoxic T-lymphocyte-associated antigen (CTLA)-4 + CD4 + T-cells, programmed death (PD)-1 + CD8 + T-cells, regulatory T-cells, and the CTLA-4 + regulatory T-cells was statistically significantly higher in DN-CLP mice than in sham-CLP mice. Skeletal muscle atrophy before sepsis triggers excessive T cell immunosuppression, which may contribute to the exacerbation of sepsis under skeletal muscle atrophy., (© 2024 The Author(s). FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Introduction/aims: The precise relationship between molecular mimicry and tissue-specific autoimmunity is unknown. Major histocompatibility complex (MHC) class II antigen presenting cell-CD4+ T-cell receptor complex interactions are necessary for adaptive immunity. This study aimed to determine the role of endoneurial endothelial cell MHC class II in autoimmune polyneuropathy., Methods: Cryopreserved Guillain-Barré syndrome (GBS) patient sural nerve biopsies and sciatic nerves from the severe murine experimental autoimmune neuritis (sm-EAN) GBS model were studied. Cultured conditional ready MHC Class II antigen A-alpha chain (H2-Aa) embryonic stem cells were used to generate H2-Aa flox/+ C57BL/6 mice. Mice were backcrossed and intercrossed to the SJL background to generate H2-Aa flox/flox SJL mice, bred with hemizygous Tamoxifen-inducible von Willebrand factor Cre recombinase (vWF-iCre/+) SJL mice to generate H2-Aa flox/flox ; vWF-iCre/+ mice to study microvascular endothelial cell adaptive immune responses. Sm-EAN was induced in Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+, H2-Aa flox/flox ; +/+, H2-Aa +/+ ; vWF-iCre/+ and untreated H2-Aa flox/flox ; vWF-iCre/+ adult female SJL mice. Neurobehavioral, electrophysiological and histopathological assessments were performed at predefined time points., Results: Endoneurial endothelial cell MHC class II expression was observed in normal and inflamed human and mouse peripheral nerves. Tamoxifen-treated H2-Aa flox/flox ; vWF-iCre/+ mice were resistant to sm-EAN despite extensive MHC class II expression in lymphoid and non-lymphoid tissues., Discussion: A conditional MHC class II knockout mouse to study cell- and time-dependent adaptive immune responses in vivo was developed. Initial studies show microvascular endothelial cell MHC class II expression is necessary for peripheral nerve specific autoimmunity, as advocated by human in vitro adaptive immunity and ex vivo transplant rejection studies., (© 2024 The Author(s). Muscle & Nerve published by Wiley Periodicals LLC.)

Immune cells infiltrate the peripheral nervous system (PNS) after injury and with autoimmunity, but their net effect is divergent. After injury, immune cells are reparative, while in inflammatory neuropathies (e.g., Guillain Barré Syndrome and chronic inflammatory demyelinating polyneuropathy), immune cells are proinflammatory and promote autoimmune demyelination. An understanding of immune cell phenotypes that distinguish these conditions may, therefore, reveal new therapeutic targets for switching immune cells from an inflammatory role to a reparative state. In an autoimmune regulator (Aire)-deficient mouse model of inflammatory neuropathy, we used single-cell RNA sequencing of sciatic nerves to discover a transcriptionally heterogeneous cellular landscape, including multiple myeloid, innate lymphoid, and lymphoid cell types. Analysis of cell-cell ligand-receptor interactions uncovered a macrophage-mediated tumor necrosis factor-α (TNF-α) signaling axis that is induced by interferon-γ and required for initiation of autoimmune demyelination. Developmental trajectory visualization suggested that TNF-α signaling is associated with metabolic reprogramming of macrophages and polarization of macrophages from a reparative state in injury to a pathogenic, inflammatory state in autoimmunity. Autocrine TNF-α signaling induced macrophage expression of multiple genes ( Clec4e , Marcksl1 , Cxcl1 , and Cxcl10 ) important in immune cell activation and recruitment. Genetic and antibody-based blockade of TNF-α/TNF-α signaling ameliorated clinical neuropathy, peripheral nerve infiltration, and demyelination, which provides preclinical evidence that the TNF-α axis may be effectively targeted to resolve inflammatory neuropathies., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

Decellularized extra-cellular matrix (ECM) has been studied as an alternative to anti-adhesive biomaterials and cartilage acellular matrix (CAM) has been shown to inhibit postoperative adhesion in several organs. This study aimed to evaluate the suitability of glutaraldehyde (GA) crosslinked CAM-films as anti-adhesion barriers for peripheral nerve injury. The films were successfully fabricated and showed improved physical properties such as mechanical strength, swelling ratio, and lengthened degradation period while maintaining the microstructure and chemical composition after GA crosslinking. In the in vitro study of CAM-film, the dsDNA content met the recommended limit of decellularization and more than 70% of the major ECM components were preserved after decellularization. The adhesion and proliferation of seeded human umbilical vein endothelial cells and fibroblasts were significantly lower in CAM-film than in control, but similar with Seprafilm. However, the CAM-film extract did not show cytotoxicity. In the in vivo study, the peri-neural fibrosis was thicker, adhesion score higher, and peri-neural collagen fibers more abundant in the control group than in the CAM-film group. The total number of myelinated axons was significantly higher in the CAM-film group than in the control group. The inflammatory marker decreased with time in the CAM-film group compared to that in the control group, whereas the nerve regenerative marker expression was maintained. Moreover, the ankle angles at contracture and toe-off were higher in the CAM film-treated rats than in the control rats. GA-crosslinked CAM films may be used during peripheral nerve surgery to prevent peri-neural adhesion and enhance nerve functional recovery., (© 2021 John Wiley & Sons Ltd.)

Object: Ninjurin2 (nerve injury induced protein 2, NINJ2) is a molecule which mediates cell-to-cell and cell-to-extracellular matrix interactions in the nervous system. Clinical study shows NINJ2 is associated with the development of postherpetic neuralgia. However, it is lack of direct evidence that NINJ2 participated in neuropathic pain. In this study, we aim to investigate the role of NINJ2 in the development of neuropathic pain in spared sciatic nerve injury rats and the underlying mechanism., Method: Spared sciatic nerve injury (SNI) models were established. The level of NINJ2 and p-p65 (a NF-κB family member) were measured in SNI rats by western blots and immunofluorescent staining. Lentivirus encoding small interfering RNA targeting NINJ2 (RNAi) was intrathecally injected into rats. Then the change of pain behavior of rats induced by NINJ2 RNAi was tested by Von-Frey hairs. The change of p-p65 in the spinal cord in rats after NINJ2 RNAi treatment was also measured by western blots. inhibitor of p-p65-induced change of TNF-α, IL-1β, and IL-6 levels were measured by ELISA., Results: NINJ2 and p-p65 were increased in the spinal cord of SNI rats on the 3, 7, 14th days after modeling. NINJ2 were mainly expressed in neurons, and co-located with p-p65 in the spinal dorsal horn. When down regulating the level of NINJ2 by RNAi, the development of pain in SNI rats was partially blocked. Phosphorylation of p65 was also inhibited by NINJ2 RNAi. Blocking the phosphorylation of NF-κB pathway could inhibit the increase of TNF-α, IL-1β, and IL-6 in the spinal cord of SNI rats., Conclusion: NINJ2 protein was increased in the spinal cord of SNI rats. It participated in the development of nerve injury-induced neuropathic pain by activating neuroinflammation in the spinal cord via NF-κB pathway. This study provides a new target to investigate the mechanism of neuropathic pain., (Copyright © 2021 Elsevier B.V. All rights reserved.)

One hallmark of Guillain-Barre syndrome (GBS), a prototypic autoimmune peripheral neuropathy (APN) is infiltration of leukocytes (macrophages and T cells) into peripheral nerves, where chemokines and their receptors play major roles. In this study, we aimed to understand the potential contribution of chemokine receptors CCR2 and CX3CR1 in APN by using a well-established mouse model, B7.2 transgenic (L31) mice, which possesses a predisposed inflammatory background. We crossbred respectively CCR2KO and CX3CR1KO mice with L31 mice. The disease was initiated by partial ligation on one of the sciatic nerves. APN pathology and neurological function were evaluated on the other non-ligated sciatic nerve/limb. Our results revealed that L31/CX3CR1KO but not L31/CCR2KO mice were resistant to APN. CX3CR1 is needed for maintaining circulating monocyte and CD8 + T cell survival. While migration of a significant number of activated CD8 + T cells to peripheral nerves is essential in autoimmune response in nerve, recruitment of monocytes into PNS seems optional. Disease onset is independent of CCR2 mediated blood-derived macrophage recruitment, which can be replaced by compensatory proliferation of resident macrophages in peripheral nerve. CX3CR1 could also contribute to APN via its critical involvement in maintaining nerve macrophage phagocytic ability. We conclude that blockade of CX3CR1 signaling may represent an interesting anti-inflammatory strategy to improve therapeutic management for GBS patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oladiran, Shi, Fournier and Zhang.)

Peripheral nerve injury can cause debilitating disease and immune cell-mediated destruction of the affected nerve. While the focus has been on the nerve-regenerative response, the effect of loss of innervation on lymph node function is unclear. Here, we show that the popliteal lymph node (popLN) receives direct neural input from the sciatic nerve and that sciatic denervation causes lymph node expansion. Loss of sympathetic, adrenergic tone induces the expression of IFN-γ in LN CD8 T cells, which is responsible for LN expansion. Surgery-induced IFN-γ expression and expansion can be rescued by β2 adrenergic receptor agonists but not sensory nerve agonists. These data demonstrate the mechanisms governing the pro-inflammatory effect of loss of direct adrenergic input on lymph node function., Competing Interests: Disclosures: B. Schraml reported that the Schraml lab received funding from the Deutsche Forschungsgemeinschaft Projektnummer 360372040 - SFB 1335 (project 8; to B. Schraml). No other disclosures were reported., (© 2021 Chen et al.)

Inflammation is a hallmark of several neurodegenerative disorders including hereditary amyloidogenic transthyretin amyloidosis (ATTRv). ATTRv is an autosomal dominant neurodegenerative disorder with extracellular deposition of mutant transthyretin (TTR) aggregates and fibrils, particularly in nerves and ganglia of the peripheral nervous system. Nerve biopsies from ATTRv patients show increased cytokine production, but interestingly no immune inflammatory cellular infiltrate is observed around TTR aggregates. Here we show that as compared to Wild Type (WT) animals, the expression of several chemokines is highly downregulated in the peripheral nervous system of a mouse model of the disease. Interestingly, we found that stimulation of mouse Schwann cells (SCs) with WT TTR results in the secretion of several chemokines, a process that is mediated by toll-like receptor 4 (TLR4). In contrast, the secretion of all tested chemokines is compromised upon stimulation of SCs with mutant TTR (V30M), suggesting that V30M TTR fails to activate TLR4 signaling. Altogether, our data shed light into a previously unappreciated mechanism linking TTR activation of SCs and possibly underlying the lack of inflammatory response observed in the peripheral nervous system of ATTRv patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moreira, Costelha, Saraiva and Saraiva.)

The selection of control group is crucial, as the use of an inadequate group may strongly affect the results. In this study we examine the effect on contralateral tissue protein levels, in a model of unilateral UVB irradiation, as the contralateral side is commonly used as a control. Previous studies have shown that UVB irradiation increases immunoreactivity for inflammatory regulated neuropeptides. Unilateral UVB irradiation of rat hind paw was performed and corresponding contralateral spinal cord and dorsal root ganglia (DRG) were collected 2-96 h after and investigated for changes in galanin, substance P and c-fos immunoreactivity. Control tissue was collected from naïve rats. Measurement of skin blood flow from contralateral heel hind paws (Doppler), revealed no change compared to naïve rats. However, UVB irradiation caused a significant reduction in the contralateral proportion of galanin immunopositive DRG neurons, at all-time points, as well as an increase in the contralateral spinal cord dorsal horn, around the central canal and in the lateral spinal nucleus (2-48 h). The contralateral proportion of SP positive DRG neurons and dorsal horn immunoreactivity was unchanged, whereas the lateral spinal nucleus area showed increased immunoreactivity (48 h). UVB irradiation also induced a slight contralateral upregulation of c-fos in the dorsal horn/central canal area (24 and 48 h). In summary, unilateral UVB irradiation induced contralateral changes in inflammatory/nociceptive neuropeptides in spinal cord and afferent pathways involved in pain signaling already within 24 h, a time point when also ipsilateral neurochemical/physiological changes have been reported for rats and humans., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sciatic nerve crush injury triggers sterile inflammation within the distal nerve and axotomized dorsal root ganglia (DRGs). Granulocytes and pro-inflammatory Ly6C high monocytes infiltrate the nerve first and rapidly give way to Ly6C negative inflammation-resolving macrophages. In axotomized DRGs, few hematogenous leukocytes are detected and resident macrophages acquire a ramified morphology. Single-cell RNA-sequencing of injured sciatic nerve identifies five macrophage subpopulations, repair Schwann cells, and mesenchymal precursor cells. Macrophages at the nerve crush site are molecularly distinct from macrophages associated with Wallerian degeneration. In the injured nerve, macrophages 'eat' apoptotic leukocytes, a process called efferocytosis, and thereby promote an anti-inflammatory milieu. Myeloid cells in the injured nerve, but not axotomized DRGs, strongly express receptors for the cytokine GM-CSF. In GM-CSF-deficient ( Csf2 -/- ) mice, inflammation resolution is delayed and conditioning-lesion-induced regeneration of DRG neuron central axons is abolished. Thus, carefully orchestrated inflammation resolution in the nerve is required for conditioning-lesion-induced neurorepair., Competing Interests: AK, CY, LH, PD, RK, RP, HH, CJ, RK, KC, JJ, EH, DG, BS, RG No competing interests declared, (© 2020, Kalinski et al.)

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.)

Recent preclinical and clinical evidence suggest that immune system has a role in the progression and prognosis of Amyotrophic Lateral Sclerosis (ALS), but the identification of a clear mechanism and immune players remains to be elucidated. Here, we have investigated, in 30 and 60 days (presymptomatic) and 120 days (symptomatic) old SOD1-G93A mice, systemic, peripheral, and central innate and adaptive immune and inflammatory response, correlating it with the progression of the neurodegeneration in neuromuscular junction, sciatic nerves, and spinal cord. Surprisingly, we found a very initial (45-60 days) presence of IgG in sciatic nerves together with a gradual enhancement of A20/TNFAIP3 (protein controlling NF-κB signalling) and a concomitantly significant increase and activation of circulating mast cells (MCs) as well as MCs and macrophages in sciatic nerve and an enhancement of IL-6 and IL-10. This immunological frame coincided with a myelin aggregation. The 30-60 days old SOD1-G93A mice didn't show real elements of neuroinflammation and neurodegeneration in spinal cord. In 120 days old mice macrophages and monocytes are widely diffused in sciatic nerves, peripheral neurodegeneration reaches the tip, high circulating levels of TNFα and IL-2 were found and spinal cord exhibits clear signs of neural damage and infiltrating immune cells. Our results underpin a clear immunological disorder at the origin of ALS axonopathy, in which MCs are involved in the initiation and sustaining of inflammatory events. These data cannot be considered a mere epiphenomenon of motor neuron degeneration and reveal new potential selective immune targets in ALS therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Angelini, De Angelis, Vacca, Piras, Parisi, Nutini, Spalloni, Pagano, Longone, Battistini, Pavone and Marinelli.)

Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MPs) of the peripheral nervous system (PNS) are the main cellular agent controlling this process. Some evidence suggests that resident PNS-MPs along with MPs of hematogenous origin may be involved, but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporter mice and coherent anti-Stokes Raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatiotemporal choreography of resident and recently recruited MPs after injury and unveiled distinct functions of these subsets, with recruited MPs being responsible for efficient myelin stripping and clearance and resident MPs being involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Boissonnas et al.)

Background: Guillain-Barré syndrome (GBS), an autoimmune disease and an acute inflammation disorder, is currently the most frequent cause of acute flaccid paralysis worldwide. EAN, an animal model of GBS, is a CD4+ T cell-mediated autoimmune disease of the PNS. Wnt/β-catenin signals are critically important to several fundamental aspects of peripheral nerve development and play a crucial role in Schwann cell proliferation. Here, we investigate the role of Wnt/β-catenin signalling cascades in EAN rats. Methods: 28 male Lewis rats weighing 170 ± 10 g were randomly divided into control group ( n = 7) and EAN groups (Early group; Peak group and Recovery group. n  = 7 per group). EAN rats were immunized with P2 57-81 peptide; weighed daily, and the neurologic signs of EAN were evaluated every day. The sciatic nerve was taken on the days 10, 17, and 30 p.i. for H&E staining, transmission electron microscopy and immunohistochemical staining; blood samples were collected weekly from caudal vein to detect IFN-γ, IL-4, TGF-β1; and the sciatic nerve was taken to examinate the dynamics expression of Wnt/β-catenin pathway molecules. Results: In our study, we chose tail-root injection to better model GBS. Moreover, we observed that IFN-γ levels paralleled clinical EAN, and the levels of TGF-β1 and IL-4 gradually increased and peaked in the recovery phase. In addition, we have shown that canonical Wnt signalling is upregulated and reached a peak in the late recovery phase. Conclusion: Our findings suggest that Wnt/β-catenin signalling is associated with the promotion of remyelination in EAN rats.

Microglia and peripheral macrophages have both been implicated in amyotrophic lateral sclerosis (ALS), although their respective roles have yet to be determined. We now show that macrophages along peripheral motor neuron axons in mouse models and patients with ALS react to neurodegeneration. In ALS mice, peripheral myeloid cell infiltration into the spinal cord was limited and depended on disease duration. Targeted gene modulation of the reactive oxygen species pathway in peripheral myeloid cells of ALS mice, using cell replacement, reduced both peripheral macrophage and microglial activation, delayed symptoms and increased survival. Transcriptomics revealed that sciatic nerve macrophages and microglia reacted differently to neurodegeneration, with abrupt temporal changes in macrophages and progressive, unidirectional activation in microglia. Modifying peripheral macrophages suppressed proinflammatory microglial responses, with a shift toward neuronal support. Thus, modifying macrophages at the periphery has the capacity to influence disease progression and may be of therapeutic value for ALS.

The acceleration of peripheral nerve regeneration is crucial for functional nerve recovery. Our previous study demonstrated that human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSC) promote sciatic nerve recovery and regeneration via the direct upregulation and release of neurotrophic factors. However, the immunomodulatory role of hWJ-MSC in sciatic nerve recovery remains unclear. The effects of hWJ-MSC on innate immunity, represented by macrophages, natural killer cells, and dendritic cells, as well as on adaptive immunity, represented by CD4 + T, CD8 + T, B, and regulatory T cells (Tregs), were examined using flow cytometry. Interestingly, a significantly increased level of Tregs was detected in blood, lymph nodes (LNs), and nerve-infiltrating cells on POD7, 15, 21, and 35. Anti-inflammatory cytokines, such as IL-4 and IL-10, were significantly upregulated in the LNs and nerves of hWJ-MSC-treated mice. Treg depletion neutralized the improved effects of hWJ-MSC on sciatic nerve recovery. In contrast, Treg administration promoted the functional recovery of five-toe spread and gait stance. hWJ-MSC also expressed high levels of the anti-inflammatory cytokines TGF-β and IL-35. This study indicated that hWJ-MSC induce Treg development to modulate the balance between pro- and anti-inflammation at the injured sciatic nerve by secreting higher levels of anti-inflammatory cytokines.

This protocol describes a footpad inoculation model used to study the initiation and development of neuroinflammatory responses during alphaherpesvirus infection in mice. As alphaherpesviruses are main invaders of the peripheral nervous system (PNS), this model is suitable to characterize the kinetics of viral replication, its spread from the PNS to CNS, and associated neuroinflammatory responses. The footpad inoculation model allows virus particles to spread from a primary infection site in the footpad epidermis to sensory and sympathetic nerve fibers that innervate the epidermis, sweat glands, and dermis. The infection spreads via the sciatic nerve to the dorsal root ganglia (DRG) and ultimately through the spinal cord to the brain. Here, a mouse footpad is inoculated with pseudorabies virus (PRV), an alphaherpesvirus closely related to herpes simplex virus (HSV) and varicella-zoster virus (VZV). This model demonstrates that PRV infection induces severe inflammation, characterized by neutrophil infiltration in the footpad and DRG. High concentrations of inflammatory cytokines are subsequently detected in homogenized tissues by ELISA. In addition, a strong correlation is observed between PRV gene and protein expression (via qPCR and IF staining) in DRG and the production of pro-inflammatory cytokines. Therefore, the footpad inoculation model provides a better understanding of the processes underlying alphaherpesvirus-induced neuropathies and may lead to the development of innovative therapeutic strategies. In addition, the model can guide research on peripheral neuropathies, such as multiple sclerosis and associated viral-induced damage to the PNS. Ultimately, it can serve as a cost-effective in vivo tool for drug development.

While CNS microglia have been extensively studied, relatively little is known about macrophages populating the peripheral nervous system. Here we performed ontogenic, transcriptomic and spatial characterization of sciatic nerve macrophages (snMacs). Using multiple fate-mapping systems, we show that snMacs do not derive from the early embryonic precursors colonizing the CNS, but originate primarily from late embryonic precursors and become replaced by bone-marrow-derived macrophages over time. Using single-cell transcriptomics, we identified a tissue-specific core signature of snMacs and two spatially separated snMacs: Relmα + Mgl1 + snMacs in the epineurium and Relmα - Mgl1 - snMacs in the endoneurium. Globally, snMacs lack most of the core signature genes of microglia, with only the endoneurial subset expressing a restricted number of these genes. In response to nerve injury, the two resident snMac populations respond differently. Moreover, and unlike in the CNS, monocyte-derived macrophages that develop during injury can engraft efficiently in the pool of resident peripheral nervous system macrophages.

Our study objective was testing for anti-neuronal autoantibodies within commercially available intravenous immunoglobulin (IVIg) preparations. Sixteen samples from 5 different commercially available IVIg preparations were tested with cell-based assays (CBA) and enzyme-linked immunosorbent assay (ELISA) to detect and characterize common neuronal autoantibodies, and with immunohistochemistry on teased fibers from mouse sciatic nerve and on mouse brain sections to screen for nodal and not yet identified neuronal antigens. In 15/16 IVIg preparations, anti-GAD antibodies were detected in titers ranging from 40 to 1507 IU/mL, as typically seen in type 1 diabetes, but not in the range (> 2000 IU/mL) seen in GAD-positive neurological patients. None of the preparations was however positive with anti-GAD CBA. Antibodies to AQP4 were also detected by ELISA in 15/16 IVIg preparations with titers comparable to those seen in AQP4-seropositive NMO patients; with CBA, however, all IVIg samples were AQP4-negative. IVIg preparations contained IgG-anti-MAG antibodies by ELISA at statistically significant higher titers compared to controls. Two of the 16 IVIg samples were positive for human 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies. All IVIg preparations were negative for antibodies to MOG, NMDAR, anti-nodal, and other neuronal-specific proteins. IVIg preparations contain antibodies against GAD and AQP4 in titers comparable to those seen in autoimmune patients when tested by ELISA, but not by CBA or tissue immunohistochemistry, suggesting that the autoantibodies within the IVIg are against linear rather than structural epitopes, as part of the natural antibody immune repertoire. The information is clinically important for diagnosis when testing patients' sera after they have received therapy with IVIg to avoid false interpretation.

Guillain-Barré syndrome (GBS) is characterized by acute immune-mediated peripheral neuropathy, which may result in rapidly progressive paralysis and fatal respiratory failure. As the underlying pathological mechanisms of GBS are unclear, we surveyed the transcriptome of rats with experimental autoimmune neuritis (EAN), a model of GBS. Briefly, sciatic nerves on both sides were collected from 8-10-week-old Lewis rats during early (10 days post-induction), peak (19 days) and late neuritis (30 days). Total RNA was sequenced to identify differentially expressed genes. Compared to control rats without induced neuritis, 33 genes were differentially expressed in the early phase (14 up-regulated and 19 down-regulated), with an adjusted P-value < 0·05 and |log 2 fold-change| >1, as were 137 genes in the peak phase (126 up-regulated and 11 down-regulated) and 60 genes in the late phase (58 up-regulated and two down-regulated). Eleven of these genes were common to all stages, suggesting their crucial roles throughout the disease course. Analysis of protein-protein interactions revealed Fos, Ccl2, Itgax and C3 as node genes at different stages. Functional analysis of differentially expressed genes identified biological processes and pathways that are activated as neuritis progresses. This is the first genomewide gene expression study of peripheral nerves in experimental autoimmune neuritis model. Dynamic gene expression and significantly altered biological functions were detected in different phases of the disease, increasing our understanding of the molecular mechanisms underlying EAN and highlighting potential targets for its diagnosis and treatment., (© 2019 British Society for Immunology.)

Mutations in the GDAP1 mitochondrial outer membrane gene cause Charcot-Marie-Tooth (CMT) neuropathy. Reduction or absence of GDAP1 has been associated with abnormal changes in the mitochondrial morphology and dynamics, oxidative stress and changes in calcium homeostasis. Neuroinflammation has been described in rodent models of genetic demyelinating CMT neuropathies but not in CMT primarily associated with axonopathy. Inflammatory processes have also been related to mitochondrial changes and oxidative stress in central neurodegenerative disorders. Here we investigated the presence of neuroinflammation in the axonal neuropathy of the Gdap1 -/- mice. We showed by transcriptome profile of spinal cord and the in vivo detection of activated phagocytes that the absence of GDAP1 is associated with upregulation of inflammatory pathways. We observed reactive gliosis in spinal cord with increase of the astroglia markers GFAP and S100B, and the microglia marker IBA1. Additionally, we found significant increase of inflammatory mediators such as TNF-α and pERK, and C1qa and C1qb proteins of the complement system. Importantly, we observed an increased expression of CD206 and CD86 as M2 and M1 microglia and macrophage response markers, respectively, in Gdap1 -/- mice. These inflammatory changes were also associated with abnormal molecular changes in synapses. In summary, we demonstrate that inflammation in spinal cord and sciatic nerve, but not in brain and cerebellum, is part of the pathophysiology of axonal GDAP1-related CMT., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: Dimethyl fumarate (DMF) exerts immunomodulatory and neuroprotective effects in the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat. DMF has been shown to modulate gut microbiota in veterinary medicine, however the effects of oral DMF on the gut-associated lymphoid tissue (GALT) remain unknown. Methods: Lewis rats were treated orally twice daily with DMF up to day 10 after immunization with immunogenic P2 peptide. Histological, flow cytometric and RT-PCR analyses of the GALT (intraepithelial layer, lamina propria, and Peyer patches) in duodenum, jejunum, and ileum were performed ex vivo . Moreover, cell transfer experiments were used to examine the protective effects of GALT regulatory T cells of the Peyer patches. Results: In the upper layers of duodenum, DMF induced a reduction of the toll-like receptor 4 (TLR4) mRNA expression. This was combined by a decrease of the pro-inflammatory lamina propria IFN-γ mRNA expression. In the ileum, we detected an immunoregulatory phenotype characterized by an increase of FoxP3 mRNA expression and of the nuclear factor (erythroid-derived-2)- like 2 (Nrf2) downstream molecule heme oxygenase-1 (HO-1) mRNA. Finally, CD4 + CD25 + regulatory T cells were increased in the Peyer patches. In vivo , the protective effect of these regulatory cells was verified by cell transfer into recipient EAN rats. Conclusions: Our results identified a novel immunomodulatory effect of DMF through the different regions and layers of the small intestine, which led to an increase of regulatory T cells, exerting a protective role in experimental neuritis.

Despite the requirement for effective medication against neuropathic pain associated with type 2 diabetes mellitus (T2DM), mechanism-based pharmacotherapy has yet to be established. Given that long-lasting neuroinflammation, driven by inflammatory macrophages in the peripheral nerves, plays a pivotal role in intractable pain, it is important to determine whether inflammatory macrophages contribute to neuropathic pain associated with T2DM. To generate an experimental model of T2DM, C57BL/6J mice were fed a high-fat diet (HFD) ad libitum. Compared with control diet feeding, obesity and hyperglycemia were observed after HFD feeding, and the mechanical pain threshold evaluated using the von Frey test was found to be decreased, indicating the development of mechanical allodynia. The expression of mRNA markers for macrophages, inflammatory cytokines, and chemokines were significantly upregulated in the sciatic nerve (SCN) after HFD feeding. Perineural administration of saporin-conjugated anti-Mac1 antibody (Mac1-Sap) improved HFD-induced mechanical allodynia. Moreover, treatment of Mac1-Sap decreased the accumulation of F4/80 + macrophages and the upregulation of inflammatory mediators in the SCN after HFD feeding. Inoculation of lipopolysaccharide-activated peritoneal macrophages in tissue surrounding the SCN elicited mechanical allodynia. Furthermore, pharmacological inhibition of inflammatory macrophages by either perineural or systemic administration of TC-2559 [4-(5-ethoxy-3-pyridinyl)- N -methyl-(3E)-3-buten-1-amine difumarate], a α 4 β 2 nicotinic acetylcholine receptor-selective agonist, relieved HFD-induced mechanical allodynia. Taken together, inflammatory macrophages that accumulate in the SCN mediate the pathophysiology of neuropathic pain associated with T2DM. Inhibitory agents for macrophage-driven neuroinflammation could be potential candidates for novel pharmacotherapy against intractable neuropathic pain., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that leads to paralysis and death within 3-5 years. Although the vast majority of studies have focused on vulnerable neurons, growing evidence has shown that non-neuronal cells contribute to the pathogenesis and disease progression. Here, we showed that intrathecal injection of scAAV9-VEGF at 60 days of age significantly reduced the number of microglia and inhibited the neuroinflammatory response in the CNS. Meanwhile, we found that administration of VEGF inhibited the invasion of macrophages into the PNS, including ventral nerve roots, sciatic nerves and muscles. Overall, our study indicated the anti-inflammation effect of VEGF in the CNS and PNS of ALS mice when delivered by intrathecal injection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The present study aimed to investigate the mechanism of glucagon‑like peptide‑1 receptor (GLP‑1R) agonists in the progression of diabetic peripheral neuropathy (DPN) in streptozotocin (STZ)‑induced diabetic rats, through inflammatory signaling pathways. The DPN rat model was generated by intraperitoneal injection of STZ and then treated with the GLP‑1R agonist liraglutide or saline for 8 weeks. These animals were randomly divided into 4 groups (10 rats in each): The normal control + saline group, the normal control + liraglutide group, the diabetic + saline (DM) group and the diabetic + liraglutide (DML) group. The nerve conduction velocity (NCV) in the sciatic nerves of the rats was monitored over a period of 8 weeks. Peripheral serum was obtained for the measurement of blood glucose, tumor necrosis factor‑α (TNF‑α), interleukin‑6 (IL‑6) and IL‑1β level. The protein levels of phosphorylated (p‑) and total extracellular signal‑regulated kinase, c‑Jun NH2‑terminal kinases, p38 mitogen‑activated protein kinases (MAPK), and nuclear and cytoplasmic nuclear factor‑κB (NF‑κB) were measured through western blot analysis. Sciatic nerve mRNA expression levels of proinflammatory chemokines (TNF‑α, IL‑6 and IL‑1β), chemokines [monocyte chemoattractant protein‑1 (MCP‑1)], adhesion molecules [intercellular adhesion molecule 1 (ICAM‑1)], neurotrophic factors [neuritin, nerve growth factor (NGF) and neuron‑specific enolase (NSE)] and NADPH oxidase 4 (NOX4) were evaluated by reverse transcription-quantitative polymerase chain reaction. Subsequent to 8 weeks of treatment with liraglutide, the density of myelin nerve fibers was partially restored in the DML group. The delayed motor NCV and sensory NCV in the DML group were improved. The IOD value of NOX4 staining in the DML group (24.43±9.01) was reduced compared with that in the DM group (56.60±6.91). The levels of TNF‑α, IL‑1β and IL‑6 in the peripheral serum of the DML group were significantly suppressed compared with those of the DM group. It was also observed that the mRNA expression levels of TNF‑α, IL‑6, IL‑1β, MCP‑1, ICAM‑1 and NOX4 in the sciatic nerve were attenuated in the DML group. The mRNA expression of neuritin and NGF was significantly increased in the DML group compared with that of the DM group; NSE was reduced in the sciatic nerves of the DML group compared with that of the DM group. Additionally, the protein expression of p‑p38 MAPK and NF‑κB in the DML group was significantly suppressed. These data demonstrated that GLP‑1R agonists may prevent nerve dysfunction in the sciatic nerves of diabetic rats via p38 MAPK/NF‑κB signaling pathways independent of glycemic control. GLP‑1R agonists may be a useful therapeutic strategy for slowing the progression of DPN.

Aluminum (Al) is a neurotoxic associated with a number of chronic human diseases. We investigated the effects of Al exposure at doses similar to human dietary levels and at a high level exposure to Al on the peripheral nervous system. Wistar male rats were divided into two major groups and received orally: 1) First group - Low level - rats were subdivided and treated for 60days: a) Control - received ultrapure water; b) AlCl 3 - received Al at 8.3mg/kg body weight (bw) for 60days; and 2) Second group - High level - rats were subdivided and treated for 42days: C) Control - received ultrapure water through oral gavage; d) AlCl 3 - received Al at 100mg/kg bw for 42days. Von Frey hair test, plantar test, the presence of catalepsy and the spontaneous motor activity were investigated. Reactive oxygen species, lipid peroxidation and total antioxidant capacity, immunohistochemistry to investigate the nerve inflammation and, the specific presence of Al in the sciatic nerve fibers were investigated. Al exposure at a representative human dietary level promotes the development of mechanical allodynia, catalepsy, increased inflammation in the sciatic nerve, systemic oxidative stress and, is able to be retained in the sciatic nerve. The effects of low-dose Al were similar to those found in rats exposed to Al at a dose much higher (100mg/kg). Our findings suggest that Al may be considered toxic for the peripheral nervous system, thus inducing peripheral dysfunction., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease leading to paralysis and death within 3-5 years of its diagnosis. The SOD1G93A mouse is used extensively as an ALS animal model. Increasing evidence shows that non-neuronal cellscontribute to the pathogenesis and progression of ALS. Among them, many studies focus on microgliosis in the spinal cord (SC); while few on macrophage activation in the sciatic nerves. Substantial evidence shows that insulin-like growth factor 1 (IGF1) delays disease progression and increases the lifespan of SOD1G93A mice, and some studies indicate that IGF1 reduces inflammation in the SC of ALS mice. However, no studies have focused on the effect of IGF on sciatic nerve inflammation. Here, we find that ALS progression is characterized by increasing macrophage invasion and activation accompanied by significant TNF-α production in the sciatic nerve. Furthermore, IGF1 treatment and knockdown alleviate and aggravate these responses, respectively. Collectively, our findings show the first time that IGF1 has an anti-inflammatory effect in the sciatic nerves of SOD1G93A mice., (Copyright © 2017. Published by Elsevier B.V.)

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.)

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated ≥ 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

It is known that transient receptor potential ankyrin 1 (TRPA1) channels, expressed by nociceptors, contribute to neuropathic pain. Here we show that TRPA1 is also expressed in Schwann cells. We found that in mice with partial sciatic nerve ligation, TRPA1 silencing in nociceptors attenuated mechanical allodynia, without affecting macrophage infiltration and oxidative stress, whereas TRPA1 silencing in Schwann cells reduced both allodynia and neuroinflammation. Activation of Schwann cell TRPA1 evoked NADPH oxidase 1 (NOX1)-dependent H 2 O 2 release, and silencing or blocking Schwann cell NOX1 attenuated nerve injury-induced macrophage infiltration, oxidative stress and allodynia. Furthermore, the NOX2-dependent oxidative burst, produced by macrophages recruited to the perineural space activated the TRPA1-NOX1 pathway in Schwann cells, but not TRPA1 in nociceptors. Schwann cell TRPA1 generates a spatially constrained gradient of oxidative stress, which maintains macrophage infiltration to the injured nerve, and sends paracrine signals to activate TRPA1 of ensheathed nociceptors to sustain mechanical allodynia.

It has been proposed that complex regional pain syndrome (CRPS) is a posttraumatic autoimmune disease, and we previously observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture CRPS model. The current study used the mouse model to evaluate the progression of postfracture CRPS-like changes in wild-type (WT) and muMT fracture mice lacking B cells and antibodies. The pronociceptive effects of injecting WT fracture mouse serum antibodies into muMT fracture mice were also evaluated. Postfracture pain behaviors transitioned from being initially dependent on both innate and autoimmune inflammatory mechanisms at 3 weeks after fracture to being entirely mediated by antibody responses at 12 weeks after fracture and spontaneously resolving by 21 weeks after fracture. Furthermore, serum IgM antibodies from WT fracture mice had pronociceptive effects in the fracture limb when injected into muMT fracture mice. IgM antibody levels gradually increased in the fracture limb hind paw skin, sciatic nerve, and corresponding lumbar cord, peaking at 12 to 18 weeks after fracture and then declining. Immunohistochemistry localized postfracture IgM antibody binding to antigens in the fracture limb hind paw dermal cell nuclei. We postulate that fracture induces expression of neoantigens in the fracture limb skin, sciatic nerve, and cord, which trigger B cells to secret IgM antibodies that bind those antigens and initiate a pronociceptive antibody response. Autoimmunity plays a key role in the progression of nociceptive and vascular changes in the mouse fracture model and potentially contributes to the CRPS disease process.

Brown adipose tissues (BAT) burn lipids to generate heat through uncoupled respiration, thus representing a powerful target to counteract lipid accumulation and obesity. The tumor suppressor liver kinase b1 (Lkb1) is a key regulator of cellular energy metabolism; and adipocyte-specific knockout of Lkb1 (Ad-Lkb1 KO) leads to the expansion of BAT, improvements in systemic metabolism and resistance to obesity in young mice. Here we report the unexpected finding that the Ad-Lkb1 KO mice develop hindlimb paralysis at mid-age. Gene expression analyses indicate that Lkb1 KO upregulates the expression of inflammatory cytokines in interscapular BAT and epineurial brown adipocytes surrounding the sciatic nerve. This is followed by peripheral neuropathy characterized by infiltration of macrophages into the sciatic nerve, axon degeneration, reduced nerve conductance, and hindlimb paralysis. Mechanistically, Lkb1 KO reduces AMPK phosphorylation and amplifies mammalian target-of-rapamycin (mTOR)-dependent inflammatory signaling specifically in BAT but not WAT. Importantly, pharmacological or genetic inhibition of mTOR ameliorates inflammation and prevents paralysis. These results demonstrate that BAT inflammation is linked to peripheral neuropathy., (Copyright © 2017. Published by Elsevier B.V.)

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 present study was designed to investigate the effect of ferulic acid (FA) in vincristine-induced neuropathic pain in rats. Vincristine (50 µg/kg, i.p. for 10 consecutive days) was administered to induce painful neuropathy in rats. Various pain sensitive tests, viz., pinprick, hot plate, paint-brush, and acetone test were performed on different days (1, 6, 14, and 21) to assess the degree of mechanical hyperalgesia, heat hyperalgesia, mechanical dynamic allodynia, and cold allodynia, respectively. The electrophysiological and histopathological evaluations were also investigated. The tissue thiobarbituric acid reactive species (TBARS), reduced glutathione (GSH), myeloperoxidase (MPO), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-10 (IL-10), and total calcium were measured as the markers of inflammation and oxidative stress. FA (50 and 100 mg/kg, i.p.) and gabapentin (10 mg/kg, p.o.) were administered for 11 days. Administration of FA attenuated the vincristine-induced behavioral alteration along with electrophysiological and histopathological changes significantly (P < 0.05). FA also attenuated the vincristine-induced oxidative stress (TBARS, GSH, and total calcium levels) and inflammation (MPO, TNF-alpha, IL-6, and IL-10). It may be concluded that FA has ameliorative potential in mitigation of the painful states associated with vincristine-induced painful neuropathy that may further be attributed to anti-inflammatory actions with subsequent reduction in oxidative stress.

Chronic pain is commonly and closely correlated with inflammation. Both cannabinoid signaling and mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammatory pain. Although cannabinoid signaling is essential for mesenchymal stem cell survival and differentiation, little is known about its role in modulatory effect of MSCs on inflammation and pain sensitivity. Here we showed that mouse bone-marrow derived MSCs (BM-MSCs) expressed both cannabinoid receptor type 1 and 2 (CB1 and CB2). CB2 expression level in BM-MSCs increased with their maturation. In addition, we found that tetrahydrocannabinol (THC) activated CB2 receptor and ERK signaling, consequently enhancing the modulation of MSCs on inflammation-associated cytokine release from lipopolysaccharides-stimulated microglia. Consistent with in vitro data, THC pretreatment enhanced the immunomodulatory effects of BM-MSC on thermal hyperalgesia and mechanical allodynia in chronic constriction injury model, by decreasing the release of pro-inflammation cytokines. Our study revealed the crucial role of THC in promoting the immunomodulatory effects of MSCs and proposed a new strategy to alleviate pain based on stem cells therapy.

Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Macrophages are the major immune cells in peripheral nerves and may exert tissue-damage or tissue-protective activity during EAN. While considered to define a subpopulation of T lymphocytes, CD8 expression has been found on certain macrophages that show cytotoxic effects. Here we have studied the spatiotemporal accumulation of CD8(+) cells in sciatic nerves of EAN rats. A robust accumulation of CD8(+) cells was observed in the sciatic nerves of EAN rats, which was positively correlated with the severity of neurological signs in EAN. Moreover, double-labelling experiments showed that the major cellular sources of CD8 were reactive macrophages. Therefore, our data here suggest a pathological role of CD8(+) macrophages in EAN, which makes CD8(+) macrophage a potential therapeutic target for EAN.

Inflammation is associated with peripheral neuropathy, however the interplay among cytokines, chemokines, and neurons is still unclear. We hypothesized that this neuroinflammatory interaction can be defined by computational modeling based on the dynamics of protein expression in the sciatic nerve of rats subjected to chronic constriction injury. Using Dynamic Bayesian Network inference, we identified interleukin (IL)-18 as a central node associated with neuropathic pain in this animal model. Immunofluorescence supported a role for inflammasome activation and induction of IL-18 at the site of injury. Combined in vivo and in silico approaches may thus highlight novel targets in peripheral neuropathy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

To shed light on the early immune response processes in severed peripheral nerves, we performed genome-wide transcriptional profiling and bioinformatics analyses of the proximal (P, regenerating) and distal (D, degenerating) nerve stumps on day 1 in the sciatic nerve axotomy model in rats. Multiple cell death-related pathways were activated in the degenerating D stump, whereas activation of the cytoskeletal motility and gluconeogenesis/glycolysis pathways was most prominent in the P stump of the axotomized nerve. Our bioinformatics analyses also identified the specific immunomodulatory genes of the chemokine, IL, TNF, MHC, immunoglobulin-binding Fc receptor, calcium-binding S100, matrix metalloproteinase, tissue inhibitor of metalloproteinase, and ion channel families affected in both the P and D segments. S100a8 and S100a9 were the top up-regulated genes in both the P and D segments. Stimulation of cultured Schwann cells using the purified S100A8/A9 heterodimer recapitulated activation of the myeloid cell and phagocyte chemotactic genes and pathways, which we initially observed in injured nerves. S100A8/A9 heterodimer injection into the intact nerve stimulated macrophage infiltration. We conclude that, following peripheral nerve injury, an immediate acute immune response occurs both distal and proximal to the lesion site and that the rapid transcriptional activation of the S100a8 and S100a9 genes results in S100A8/A9 hetero- and homodimers, which stimulate the release of chemokines and cytokines by activated Schwann cells and generate the initial chemotactic gradient that guides the transmigration of hematogenous immune cells into the injured nerve., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neuropathic pain resulting from peripheral nerve injury involves many persistent neuroinflammatory processes including inflammatory chemokines that control leukocyte trafficking and activate resident cells. Several studies have shown that CCL2 chemokine, a potent attractant of monocytes, and its cognate receptor, CCR2, play a critical role in regulating nociceptive processes during neuropathic pain. However, the role of CCL2 in peripheral leukocyte infiltration-associated neuropathic pain remains poorly understood. In particular, the contribution of individual CCL2-expressing cell populations (i.e. stromal and leukocytes) to immune cell recruitment into the injured nerve has not been established. Here, in preclinical model of peripheral neuropathic pain (i.e. chronic constriction injury of the sciatic nerve), we have demonstrated that, CCL2 content was increased specifically in nerve fibers. This upregulation of CCL2 correlated with local monocyte/macrophage infiltration and pain processing. Furthermore, sciatic intraneural microinjection of CCL2 in naïve animals triggered long-lasting pain behavior associated with local monocyte/macrophage recruitment. Using a specific CCR2 antagonist and mice with a CCL2 genetic deletion, we have also established that the CCL2/CCR2 axis drives monocyte/macrophage infiltration and pain hypersensitivity in the CCI model. Finally, specific deletion of CCL2 in stromal or immune cells respectively using irradiated bone marrow-chimeric CCI mice demonstrated that stromal cell-derived CCL2 (in contrast to CCL2 immune cell-derived) tightly controls monocyte/macrophage recruitment into the lesion and plays a major role in the development of neuropathic pain. These findings demonstrate that in chronic pain states, CCL2 expressed by sciatic nerve cells predominantly drove local neuro-immune interactions and pain-related behavior through CCR2 signaling., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neuropathic pain is a debilitating condition caused by damage to the somatosensory nervous system, such as peripheral nerve injury. The immune system, and in particular the adaptive T cell response, plays a key role in mediating such pain. Regulatory T (Treg) cells are a small subpopulation of inhibitory T cells that prevent autoimmunity, limit immunopathology and maintain immune homeostasis. Here, we investigated the effects of conditional depletion of Treg cells on mechanical allodynia and serum cytokines in mice with chronic constriction injury (CCI) of the sciatic nerve, an animal model of neuropathic pain. We demonstrate that CCI induced the infiltration of small numbers of Treg cells within effected neuronal tissue. Utilising the transgenic DEREG (DEpletion of REGulatory T cells) mice, we confirmed effective depletion of Foxp3+ Treg cells by diphtheria toxin injections. Following CCI we observed a transient, though significant, increase in pain hypersensitivity for Treg-depleted DEREG mice compared to non-Treg-depleted mice. Analysis of systemic cytokine levels demonstrated significant changes in serum cytokine expression profiles. In particular, we observed significant increases in systemic concentration of RANTES, IL-2 and IL-5, and significant decreases in IL-12 and IFN-γ in nerve-injured Treg-depleted DEREG mice. Further analysis indicated a substantial increase in the serum concentration of IL-12p40 as a direct result of Treg cell depletion. These results suggest that depletion of Foxp3+ Treg cells promote nerve injury-induced pain hypersensitivity, partially by inducing altered systemic concentrations of cytokines, which may act to regulate neuropathic pain., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Laquinimod is an immunomodulatory drug with neuroprotective potential. We used the animal model of experimental autoimmune neuritis (EAN) in the Lewis rat to study the effects of laquinimod treatment. After immunization with the neuritogenic peptide aa 53-78 of P2 myelin protein, preventive therapy with 12.5mg/kg laquinimod once daily inhibited neuritis in clinical and electrophysiological terms. Histology corroborated a lower degree of inflammatory lesions and demyelination in the sciatic nerve. The proportion of FoxP3-positive regulatory T cells in the peripheral lymph nodes of treated rats remained unchanged. We conclude that laquinimod may represent a therapeutic option in human autoimmune neuropathies., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Aims: To investigate the effect of locally slow-released rapamycin (RAPA) from bionic peripheral nerve stent to reduce the incidence of neuropathic pain or mitigate the degree of pain after nerve injury., Main Methods: We constructed a neural tissue engineering scaffold with sustained release of RAPA to repair 20mm defects in rat sciatic nerves. Four presurgical and postsurgical time windows were selected to monitor the changes in the expression of pain-related dorsal root ganglion (DRG) voltage-gated sodium channels 1.3 (Nav1.3), 1.7 (Nav1.7), and 1.8 (Nav1.8) through immunohistochemistry (IHC) and Western Blot, along with the observation of postsurgical pathological pain in rats by pain-related behavior approaches., Key Findings: Relatively small upregulation of DRG sodium channels was observed in the experimental group (RAPA+poly(lactic-co-glycolic acid) (PLGA)+stent) after surgery, along with low degrees of neuropathic pain and anxiety, which were similar to those in the Autologous nerve graft group., Significance: Autoimmune inflammatory response plays a leading role in the occurrence of post-traumatic neuropathic pain, and that RAPA significantly inhibits the abnormal upregulation of sodium channels to reduce pain by alleviating inflammatory response., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Inflammation is a key pathological hallmark of several neurodegenerative disorders including Alzheimer's disease, Parkinson's disease and familial amyloidotic polyneuropathy (FAP). Among all inflammatory cytokines associated with FAP, IL-1β, in particular, has been implicated in playing a key pathogenic role. In the present study, we sought to investigate whether blocking IL-1β signaling provides disease-modifying benefits in an FAP mouse model., Methods: We assessed the effect of chronic administration of Anakinra, an IL-1 antagonist, on FAP pathogenesis in vivo, using real-time polymerase chain reaction (qPCR), semi-quantitative immunohistochemistry (SQ-IHC), western blot and nerve morphometric analyses., Results: We found that treatment with Anakinra prevents transthyretin (TTR) extracellular deposition in sciatic nerve, protecting unmyelinated nerve fibers from aggregate-induced degeneration. Moreover, Anakinra administration significantly suppressed IL-1 signaling pathway and inhibited apoptosis and nitrative stress., Conclusions: The present work highlights the relevance of the IL-1 signaling pathway in the pathophysiology of FAP. Our results bring to light the importance of non-amyloid targets in the therapeutic strategies for this disorder. Thus, we propose the use of Anakinra as a potential therapeutic agent for TTR-related amyloidosis.

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.)

Guillain-Barré syndrome (GBS) is an acute, post-infectious, immune-mediated, demyelinating disease of peripheral nerves and nerve roots. Experimental autoimmune neuritis (EAN) is an animal model of GBS. Chrysin, which is a naturally occurring flavonoid, exhibits various biological activities. This study was designed to investigate the anti-inflammatory and neuroprotective properties of preventative and therapeutic chrysin treatment in EAN rats. For preventative treatment, chrysin was administered orally from day 1 to day 16 (50mg/kg once daily) while, for therapeutic treatment, rats received chrysin from day 7 to day 16 at the same dose once daily. Control animals received the same volume of the vehicle (phosphate-buffered saline/2% dimethylsulfoxide). Regardless of the treatment regimen, chrysin attenuated the severity and duration of the clinical course of EAN and reduced inflammatory cell infiltration and demyelination of sciatic nerves. In the sciatic nerves, the expression of inducible nitric oxide synthase, cyclooxygenase-2 and nuclear factor kappa B was reduced. Furthermore, chrysin inhibited the splenic mononuclear cell secretion of interleukin-1β, interleukin-2, interleukin-6, inteleukin-12, interferon γ and tumor necrosis factor α, and elevated the level of inteleukin-4. In summary, our data demonstrate that chrysin is a potentially useful agent for the treatment of EAN with its anti-inflammatory and neuroprotective effects., (Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.)

Experimental autoimmune neuritis (EAN) is an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system. Erythropoietin (EPO) has been known to promote EAN recovery but its haematopoiesis stimulating effects may limit its clinic application. Here we investigated the effects and potential mechanisms of an EPO-derived nonerythropoietic peptide, ARA 290, in EAN. Exogenous ARA 290 intervention greatly improved EAN recovery, improved nerve regeneration and remyelination, and suppressed nerve inflammation. Furthermore, haematopoiesis was not induced by ARA 290 during EAN treatment. ARA 290 intervention suppressed lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3+/CD4+ regulatory T cells and IL-4+/CD4+ Th2 cells and decrease of IFN-γ+/CD4+ Th1 cells in EAN. In addition, ARA 290 inhibited inflammatory macrophage activation and promoted its phagocytic activity. In vitro, ARA 290 was shown to promote Schwann cell proliferation and inhibit its inflammatory activation. In summary, our data demonstrated that ARA 290 could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that ARA 290 could be a potent candidate for treatment of autoimmune neuropathies.

Objective: Wistar Ottawa Karlsburg W (RT1(u)) rats (WOKW) develop obesity, dyslipidemia, moderate hypertension, hyperinsulinemia and impaired glucose tolerance prone to induce peripheral neuropathy (PN). Autophagy has been shown to prevent neurodegeneration in the central and peripheral nervous system. We analyzed the potential protective role of autophagy in an established rat model in preventing PN., Methods: We examined electrophysiology (motor-and sensory/mixed afferent conduction velocities and the minimal F-wave latency) and morphology, including ultrathin sections, myelin sheath thickness (g-ratio) and immunohistochemical markers of autophagy and inflammation in the sciatic nerve of five-month-old, male WOKW as compared to Wistar derived, congenic LEW.1W control rats, characterized by the same major histocompatibility complex as WOKW rats (RT1(u)). Moreover, the expression of axonal and synaptic proteins (NF68, GAP43, MP0), autophagy- (Atg5, Atg7, LC3), and apoptosis (cleaved caspase-3)-related markers was measured using Western blot., Results: No abnormalities in nerve electrophysiology and morphology were found in WOKW compared to LEW.1W rats. However, autophagosomes were more frequently apparent in sciatic nerves of WOKW rats. In Western blot analyses no significant differences in expression of neuronal structural proteins were found, but autophagy markers were up-regulated in WOKW compared to LEW.1W sciatic nerves. Immunostaining revealed a greater infiltration of Iba1/ED-1-positive macrophages, CD-3-positive T-cells and LC3-expression in sciatic nerves of WOKW rats., Conclusions: Our results indicate that WOKW rats show an up-regulated autophagy and a mild inflammatory response but do not develop overt neuropathy. We suggest that autophagy and inflammatory cells may exert a protective role in preventing neuropathy in this rat model of the metabolic syndrome but the mechanism of action is still unclear., (© 2013.)

Gabapentin (GBP) is an anti-convulsive drug often used as analgesic to control neuropathic pain. This study aimed at evaluating whether oral GBP treatment could improve nerve inflammation response after sciatic nerve constriction in association with selected pain and motor spontaneous behavior assessments in Wistar rats. We evaluated nerve myeloperoxidase (MPO) and inflammatory cytokines on the 5th day post-injury, time in which nerve inflammation is ongoing. In addition, the role of GBP on carrageenan-induced paw edema and peritoneal cell migration was analyzed. GBP was given by gavage at doses of 30, 60 and 120mg/kg, 60min prior to chronic constriction of the sciatic nerve (CCSN) and during 5 days post-injury, 12/12h. CCSN animals treated with saline were used as controls and for behavioral and inflammation assessments untreated sham-operated rats were also used. On the 5th day, GBP (60 and 120mg/kg) alleviated heat-induced hyperalgesia and significantly increased delta walking scores in CCSN animals, the latter suggesting excitatory effects rather than sedation. GBP (60mg/kg) significantly increased nerve MPO, TNF-α, and IL-1β levels, comparing with the saline group. GBP (120mg/kg) reduced the anti-inflammatory cytokine IL-10 nerve levels compared with the CCSN saline group. Furthermore, GBP (60 and 120mg/kg) increased carrageenan-induced paw edema and peritoneal macrophage migration compared with the CCSN saline group. Altogether our findings suggest that GBP accentuates nerve and peripheral inflammatory response, however confirmed its analgesic effect likely due to an independent CNS-mediated mechanism, and raise some concerns about potential GBP inflammatory side effects in widespread clinical use., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Experimental autoimmune neuritis (EAN), an autoantigen-specific T-cell-mediated disease model for human demyelinating inflammatory disease of the peripheral nervous system, is characterized by self-limitation. Here we investigated the regulation and contribution of erythropoietin (EPO) in EAN self-limitation. In EAN sciatic nerves, hypoxia, and protein and mRNA levels of hypoxia-inducible factor 1α (HIF-1α), HIF-2α, EPO and EPO receptor (EPOR) were induced in parallel at disease peak phase but reduced at recovery periods. Further, the deactivation of HIF reduced EAN-induced EPO/EPOR upregulation in EAN, suggesting the central contribution of HIF to EPO/EPOR induction. The deactivation of EPOR signalling exacerbated EAN progression, implying that endogenous EPO contributed to EAN recovery. Exogenous EPO treatment greatly improved EAN recovery. In addition, EPO was shown to promote Schwann cell survival and myelin production. In EAN, EPO treatment inhibited lymphocyte proliferation and altered helper T cell differentiation by inducing increase of Foxp3(+)/CD4(+) regulatory T cells and decrease of IFN-γ(+)/CD4(+) Th1 cells. Furthermore, EPO inhibited inflammatory macrophage activation and promoted its phagocytic activity. In summary, our data demonstrated that EPO was induced in EAN by HIF and contributed to EAN recovery, and endogenous and exogenous EPO could effectively suppress EAN by attenuating inflammation and exerting direct cell protection, indicating that EPO contributes to the self-recovery of EAN and could be a potent candidate for treatment of autoimmune neuropathies., (Copyright © 2013 Elsevier B.V. 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.