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Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) was used to detect neurodegenerations in aged monkeys. Our previous studies have shown that aging-related NADPH-d positive bodies (ANBs) and megaloneurites appeared in the lumbosacral spinal cord of aged rats and dogs, respectively. To determine the occurrence of megaloneurites and ANBs in non-human primates, we used NADPH-d histochemistry to perform an advanced study of aging-related alterations in aged male monkeys. We identified two distinct abnormal NADPH-d positive alterations, which were expressed as ANBs and megaloneurites, mainly distributed in the superficial dorsal horn, dorsal gray commissure, lateral collateral pathway (LCP) and sacral parasympathetic nucleus of the sacral spinal cord in aged monkeys. Meanwhile, large diameter punctate NADPH-d abnormalities occurred and scattered in the lateral white matter of the LCP and dorsal root entry zone at the same level of megaloneurites in the gray matter. Immunohistochemical results showed that megaloneurites and ANBs are two distinct abnormal alterations, with megaloneurites co-localizing with vasoactive intestinal peptide immunoreactivity, whereas ANBs were not co-localized. Both ANBs and megaloneurites provide consistent evidence that the anomalous NADPH-d alterations in the aged sacral spinal cord are referred to as a specialized aging marker in the pelvic visceral organs in non-human primates., (© 2024. The Author(s).)

Background: Dysfunction of the blood-spinal cord barrier (BSCB) contributes to the occurrence and development of neuropathic pain (NP). Previous studies revealed that the activation of cyclophilin A (CypA)-metalloproteinase-9 (MMP9) signaling pathway can disrupt the integrity of the blood-brain barrier (BBB) and aggravate neuroinflammatory responses. However, the roles of CypA-MMP9 signaling pathway on BSCB in NP have not been studied. This study aimed to investigate the effect of CypA on the structure and function of the BSCB and pain behaviors in mice with NP., Methods: We first created the mouse chronic constriction injury (CCI) model, and they were then intraperitoneally injected with the CypA inhibitor cyclosporine A (CsA) or vehicle. Pain behaviors, the structure and function of the BSCB, the involvement of the CypA-MMP9 signaling pathway, microglia activation, and expression levels of proinflammatory factors in mice were examined., Results: CCI mice presented mechanical allodynia and thermal hyperalgesia, impaired permeability of the BSCB, downregulated tight junction proteins, activated CypA-MMP9 signaling pathway, microglia activation, and upregulated proinflammatory factors, which were significantly alleviated by inhibition of CypA., Conclusions: Collectively, the CypA-MMP9 signaling pathway is responsible for CCI-induced NP in mice by impairing the structure and function of the BSCB, and activating microglia and inflammatory responses., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 International Anesthesia Research Society.)

In the present study it was hypothesized that 5-((4-methoxyphenyl)thio)benzo[c][1,2,5] thiodiazole (MTDZ), a new acetylcholinesterase inhibitor, exerts antinociceptive action and reduces the oxaliplatin (OXA)-induced peripheral neuropathy and its comorbidities (anxiety and cognitive deficits). Indeed, the acute antinociceptive activity of MTDZ (1 and 10 mg/kg; per oral route) was observed for the first time in male Swiss mice in formalin and hot plate tests and on mechanical withdrawal threshold induced by Complete Freund's Adjuvant (CFA). To evaluate the MTDZ effect on OXA-induced peripheral neuropathy and its comorbidities, male and female Swiss mice received OXA (10 mg/kg) or vehicle intraperitoneally, on days 0 and 2 of the experimental protocol. Oral administration of MTDZ (1 mg/kg) or vehicle was performed on days 2-14. OXA caused cognitive impairment, anxious-like behaviour, mechanical and thermal hypersensitivity in animals, with females more susceptible to thermal sensitivity. MTDZ reversed the hypersensitivity, cognitive impairment and anxious-like behaviour induced by OXA. Here, the negative correlation between the paw withdrawal threshold caused by OXA and acetylcholinesterase (AChE) activity was demonstrated in the cortex, hippocampus, and spinal cord. OXA inhibited the activity of total ATPase, Na + K + - ATPase, Ca 2+ - ATPase and altered Mg 2+ - ATPase in the cortex, hippocampus, and spinal cord. OXA exposure increased reactive species (RS) levels and superoxide dismutase (SOD) activity in the cortex, hippocampus, and spinal cord. MTDZ modulated ion pumps and reduced the oxidative stress induced by OXA. In conclusion, MTDZ is an antinociceptive molecule promising to treat OXA-induced neurotoxicity since it reduced nociceptive and anxious-like behaviours, and cognitive deficit in male and female mice., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Long non-coding RNAs (lncRNAs) play important roles in various diseases, but the effect of lncRNA CASC9 on spinal cord injury (SCI) remains unclear. Therefore, the present study was conducted to explore the role of this lncRNA in SCI. SCI model was established by laminectomy in rats in vivo or induced by LPS in PC12 cells in vitro. Methylprednisolone (MP) was used for treatment in vivo. Spinal cord tissues were stained with H&E, and the oxidative stress- and inflammation-related factors were detected using their commercial kits. Cell apoptosis was determined using flow cytometry assay. Relative expression of corresponding genes was measured using qRT-PCR and western blotting. Luciferase reporter assay was used to verify binding site between CASC9 and miR-383-5p, as well as miR-383-5p and LDHA. The results showed that lncRNA CASC9 was downregulated and miR-383-5p was upregulated in SCI rats and LPS-induced PC12 cells. Severe histological injury and increased water content were also found in SCI rats. Increased levels of LDH, MDA, lactic acid, TNF-α, and IL-1β were found in SCI rats and LPS-induced PC12 cells. These changes could be reversed by MP treatment in vivo or overexpression of CASC9 in vitro. Besides, overexpression of CASC9 decreased cell apoptosis and protein expression of LDHA and increased protein expression of Nrf2 and HO-1 in LPS-induced PC12 cells. Furthermore, miR-383-5p was a direct target of CASC9 and was negatively regulated by CASC9. LDHA was a direct target of miR-383-5p and was negatively regulated by CASC9. In conclusion, lncRNA CASC9 exerted a protective role against oxidative stress, inflammation, and cell apoptosis in SCI, providing a novel therapeutic target or prognostic factor for SCI.

Heterogeneity of glia in different CNS regions may contribute to the selective vulnerability of neuronal populations in neurodegenerative conditions such as amyotrophic lateral sclerosis (ALS). Here, we explored regional variations in the expression of heat shock protein 25 in glia under conditions of acute and chronic stress. Hsp27 (Hsp27; murine orthologue: Hsp25) fulfils a number of cytoprotective functions and may therefore be a possible therapeutic target in ALS. We identified a subpopulation of astrocytes in primary murine mixed glial cultures that expressed Hsp25. Under basal conditions, the proportion of Hsp25-positive astrocytes was twice as high in spinal cord cultures than in cortical cultures. To explore the physiological role of the elevated Hsp25 expression in spinal cord astrocytes, we exposed cortical and spinal cord glia to acute stress, using heat stress and pro-inflammatory stimuli. Surprisingly, we observed no stress-induced increase in Hsp25 expression in either cortical or spinal cord astrocytes. Similarly, exposure to endogenous stress, as modelled in glial cultures from SOD1 G93A -ALS mice, did not increase Hsp25 expression above that observed in astrocytes from wild-type mice. In vivo, Hsp25 expression was greater under conditions of chronic stress present in the spinal cord of SOD1 G93A mice than in wild-type mice, although this increase in expression is likely to be due to the extensive gliosis that occurs in this model. Together, these results show that there are differences in the expression of Hsp25 in astrocytes in different regions of the central nervous system, but Hsp25 expression is not upregulated under acute or chronic stress conditions.

Several neurodegenerative disorders are characterized by proteasome dysfunctions leading to protein aggregations and pathogenesis. Since we showed that estrogen receptor alpha (ERα) activates the proteasome, drugs able to stimulate ERα in the central nervous system (CNS) could hold potential for therapeutic intervention. However, the transcriptional effects of selective estrogen receptor modulators (SERMs), such as tamoxifen and raloxifene, can be tissue specific. A direct comparison of the effects of different SERMs on gene transcription in the CNS has never been performed. Here, we report an RNA-seq analysis of the spinal cord treated with estrogen, tamoxifen, or raloxifene. We find stark SERM and sex-specific differences in gene expression profiles in the spinal cord. Notably, raloxifene, but not estrogen or tamoxifen, modulates numerous deubiquitinating enzymes, proteasome subunits and assembly factors, and these effects translate into decreased protein aggregates. In the SOD1-G93A mouse model of amyotrophic lateral sclerosis, we found that even a low dose of raloxifene causes a significant decrease in mutant SOD1 aggregates in the spinal cord, accompanied by a delay in the decline of muscle strength in females, but not in males. These results strongly indicate SERM-selective as well as sex-specific effects, and emphasize the importance of sex as a biological variable to be considered for the careful selection of specific SERM for use in clinical trials for neurodegenerative diseases., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society.)

Axonal demyelination is a consistent pathological characteristic of Spinal cord injury (SCI). Promoting differentiation of oligodendrocytes is of importance for remyelination. Conversion of reactive astrocytes with stem cell potential to oligodendrocytes is proposed as an innovative strategy for SCI repair. Neuregulin-1 (Nrg1) plays an essential role in the differentiation of oligodendrocytes. Therefore, it's a potential treatment for demyelination in SCI that using Nrg1 to drive reactive astrocytes toward oligodendrocyte lineage cells. In this study, tumor necrosis factor-α (TNF-α) was used to induce dedifferentiation of primary rat spinal cord astrocytes into reactive astrocytes and Nrg1 was used to induce astrocytes in vitro and in vivo. The results showed that astrocytes treated with TNF-α expressed immaturity markers CD44 and Musashi1 at mRNA and protein levels, indicating that TNF-α induced the stem cell state of astrocytes. Nrg1 induced reactive astrocytes to express oligodendrocyte markers PDGFR-α and O4 at mRNA and protein levels, indicating that Nrg1 directly converts reactive astrocytes toward oligodendrocyte lineage cells. Moreover, upregulation of PI3K-AKT-mTOR signaling activation in response to Nrg1 was observed. In rats with SCI, intrathecal treatment with Nrg1 converted reactive astrocytes to oligodendrocyte lineage cells, inhibited astrogliosis, promoted remyelination, protected axons and eventually improved BBB score. All the biological effects of Nrg1 were significantly reversed by the co-administration of Nrg1 and ErbB inhibitor, suggesting that Nrg1 functioned through the receptor ErbB. Our findings indicate that Nrg1 is sufficient to trans-differentiate reactive astrocytes to oligodendrocytes via the PI3K-AKT-mTOR signaling pathway and repair SCI. Delivery of Nrg1 for the remyelination processes could be a promising strategy for spinal cord repair., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Bupivacaine (Bup) has a certain research basis in pain-related diseases, but it has not been studied in painful diabetic neuropathy. In this study, we investigated the role of Bupivacaine in painful diabetic neuropathy. Mouse model with painful diabetic neuropathy was established, and then treated with different concentrations of Bupivacaine. The blood glucose level in the tail vein and the changes in body weight was measured. The mechanical allodynia, thermal hyperalgesia and thermal allodynia was assessed by pain behavioral tests. Microglia were treated with high glucose (HG) and different concentrations of Bupivacaine. The levels of inflammatory cytokines were detected by using Enzyme-linked immunosorbent assays. Dual luciferase reporter assay explored the relationship between miR-23a and phosphodiesterase 4 B (PDE4B). The results displayed that Bupivacaine ameliorated the mechanical allodynia, thermal hyperalgesia, and thermal allodynia in mice with painful diabetic neuropathy, and is more effective at low concentration. Moreover, low concentration of Bupivacaine inhibited inflammation and promoted miR-23a expression in mice with painful diabetic neuropathy and in microglia induced by HIGH GLUCOSE. Overexpression of miR-23a reduced the levels of inflammatory cytokines by down-regulating PDE4B expression. Knockdown of miR-23a reversed the inhibition effect of Bupivacaine on microglial inflammation. These results revealed that low concentration of Bupivacaine inhibited microglial inflammation through down-regulating PDE4B via miR-23a, thereby attenuated painful diabetic neuropathy., (Copyright © 2020. Published by Elsevier B.V.)

Background: Neuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury., Methods: A total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1β-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1β, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay., Results: We found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3'UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1., Conclusions: JHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis., (Copyright © 2020. Published by Elsevier B.V.)

In this study, the deposition of platinum in oxaliplatin (OXA)-exposed mice and the effects of the oxidative damage on the central nervous system were investigated. The relationship between the reactive species (RS) levels as well as the expression and activity of enzymes, such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD) and acetylcholinesterase (AChE), in the development of peripheral neuropathy after OXA exposure, was evidenced. The effects of 7-chloro-4-(phenylselanyl) quinoline (4-PSQ) on OXA-induced peripheral neuropathy was also investigated. Swiss mice received OXA (10 mg kg -1 ) or vehicle by intraperitoneal route (days 0 and 2). Oral administration of 4-PSQ (1 mg kg -1 ) or vehicle was performed on days 2 to 14. Behavioural tasks started on day 9, after the first OXA administration. It was observed that 4-PSQ reduced the mechanical and thermal hypersensitivity induced by OXA. 4-PSQ and OXA did not affect locomotor and exploratory activities. The results revealed, for the first time, a high concentration of platinum in the spinal cord of mice exposed to OXA. 4-PSQ reversed the increased levels of RS in the spinal cord, cerebral cortex and hippocampus of mice exposed to OXA. The alterations in the activity and expression of the GPx, SOD, CAT and AChE induced by OXA exposure were normalized by 4-PSQ. Therefore, the 4-PSQ might be a good prototype for the development of a more effective drug for the treatment of OXA-induced peripheral neuropathy. The results obtained by the present study expanded the knowledge about the mechanisms involved in the physiopathology of peripheral neuropathy. Graphical abstract.

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.)

Peroxiredoxins (PRXs) are intracellular anti-oxidative enzymes but work as inflammatory amplifiers under the extracellular condition. To date, the function of PRXs in the pathogenesis of multiple sclerosis (MS) and neuromyelitis optica spectrum disorder (NMOSD) is not fully understood. The aim of this study was to investigate whether PRXs play a role in the pathogenesis of MS and NMOSD. We analyzed levels of PRXs (PRX1, PRX5 and PRX6) in the cerebrospinal fluid (CSF) and serum of 16 patients with MS, 16 patients with NMOSD and 15 patients with other neurological disorders (ONDs). We identified potential correlations between significantly elevated PRXs levels and the clinical variables in patients with MS and NMOSD. Additionally, pathological analyses of PRXs (PRX1-6) in the central nervous system (CNS) were performed using the experimental autoimmune encephalomyelitis (EAE), animal model of MS. We found that serum levels of PRX5 and PRX6 in patients with MS and NMOSD were higher compared with those in patients with ONDs (P < 0·05). Furthermore, high levels of PRX5 and PRX6 were partly associated with blood-brain barrier dysfunction and disease duration in NMOSD patients. No significant elevation was found in CSF PRXs levels of MS and NMOSD. Spinal cords from EAE mice showed remarkable PRX5 staining, especially in CD45 + infiltrating cells. In conclusion, PRX5 and PRX6 may play a role in the pathogeneses of MS and NMOSD., (© 2020 British Society for Immunology.)

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease in adults. While it is primarily characterized by the death of upper and lower motor neurons, there is a significant metabolic component involved in the progression of the disease. Two-thirds of ALS patients have metabolic alterations that are associated with the severity of symptoms. In ALS, as in other neurodegenerative diseases, the metabolism of glycosphingolipids, a class of complex lipids, is strongly dysregulated. We therefore assume that this pathway constitutes an interesting avenue for therapeutic approaches. We have shown that the glucosylceramide degrading enzyme, glucocerebrosidase (GBA) 2 is abnormally increased in the spinal cord of the SOD1 G86R mouse model of ALS. Ambroxol, a chaperone molecule that inhibits GBA2, has been shown to have beneficial effects by slowing the development of the disease in SOD1 G86R mice. Currently used in clinical trials for Parkinson's and Gaucher disease, ambroxol could be considered as a promising therapeutic treatment for ALS., (Copyright © 2020. Published by Elsevier B.V.)

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these Naaa CD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in Naaa CD11b+ than in control mice. Naaa CD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Paclitaxel-induced neuropathic pain (PINP) is a dose-limiting side effect and is refractory to widely used analgesic drugs. Previous studies have demonstrated a protective role of peroxisome proliferator-activated receptor gama (PPARγ) in neuropathic pain. However, whether PPARγ activation could alleviate PINP remains to be elucidated. Our previous study has validated the analgesic effect of oltipraz, an nuclear factor erythroid-2 related factor 2 (Nrf2) activator, in a rat model of PINP. In this study, we tested the hypothesis that rosiglitazone, a selective agonist of PPARγ, could attenuate PINP through induction of Nrf2/heme oxygenase-1 (HO-1) signaling pathway. Paclitaxel was injected intraperitoneally on four alternate days to induce neuropathic pain. Paw withdrawal threshold was used to evaluate mechanical allodynia. Western blot and immunofluorescence were used to examine the expression and distribution of PPARγ, Nrf2 and HO-1 in the spinal cord. Our results showed that rosiglitazone attenuated established PINP and delayed the onset of PINP via activation of PPARγ, which were reversed by PPARγ antagonist GW9662. Moreover, rosiglitazone inhibited downregulation of PPARγ in the spinal cord of PINP rats. Furthermore, the analgesic effect of rosiglitazone against PINP was abolished by trigonelline, an Nrf2 inhibitor. Finally, rosiglitazone significantly increased expression of Nrf2 and HO-1 in the spinal cord of PINP rats. Collectively, these results indicated that PPARγ activation might mitigate PINP through activating spinal Nrf2/HO-1 signaling pathway. Our results may provide an alternative option for PINP patients., (Copyright © 2020. Published by Elsevier Masson SAS.)

Brachial plexus axotomy is a common peripheral nerve trauma. Artemisinin, an FDA-approved antimalarial drug, has been described to possess neuroprotective properties. However, the specific mechanisms by which artemisinin protects neurons from axotomy-induced neurotoxicity remain to be elucidated. In this study, we assessed the neuroprotective effects of artemisinin on an experimental animal model of brachial plexus injury and explored the possible mechanisms involved. Artemisinin treatment restored both athletic ability and sensation of the affected upper limb, rescued motoneurons and attenuated the inflammatory response in the ventral horn of the spinal cord. Additionally, artemisinin inhibited the molecular signals of apoptosis, activated signaling pathways related to cell survival and induced NSCPs differentiation into NeuN-positive neurons. Further validation of the involved key signaling molecules, using an in vitro model of hydrogen peroxide-induced neurotoxicity, revealed that both the inhibition of PKA signaling pathway or the silencing of Akt reversed the neuroprotective action of artemisinin on motoneurons. Our results indicate that artemisinin provides neuroprotection against axotomy and hydrogen peroxide-induced neurotoxicity, an effect that might be mediated by the PKA-Akt signaling pathway., (Copyright © 2020. Published by Elsevier Ltd.)

Oxidative stress induced post-translational protein modifications are associated with the development of inflammatory hypersensitivities. At least 90% of cellular reactive oxygen species (ROS) are produced in the mitochondria, where the mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), is located. MnSOD's ability to reduce ROS is enhanced by the mitochondrial NAD + -dependent deacetylase sirtuin (SIRT3). SIRT3 can reduce ROS levels by deacetylating MnSOD and enhancing its ability to neutralize ROS or by enhancing the transcription of MnSOD and other oxidative stress-responsive genes. SIRT3 can be post-translationally modified through carbonylation which results in loss of activity. The contribution of post-translational SIRT3 modifications in central sensitization is largely unexplored. Our results reveal that SIRT3 carbonylation contributes to spinal MnSOD inactivation during carrageenan-induced thermal hyperalgesia in rats. Moreover, inhibiting ROS with natural and synthetic antioxidants, prevented SIRT3 carbonylation, restored the enzymatic activity of MnSOD, and blocked the development of thermal hyperalgesia. These results suggest that therapeutic strategies aimed at inhibiting post-translational modifications of SIRT3 may provide beneficial outcomes in pain states where ROS have been documented to play an important role in the development of central sensitization., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Long noncoding RNAs (lncRNAs) have been involved in the development of multiple pathological processes including neuropathic pain. The aim of the present study is to investigate the role of lncRNA down-regulated in liver cancer stem cells (DILC) in the progression of neuropathic pain and its underlying mechanism. Neuropathic pain rat model was established with the bilateral chronic constriction injury (bCCI) method. The results from quantitative PCR analysis in the spinal cord showed that DILC was significantly up-regulated in rats with bCCI compared with the sham group. DILC down-regulation mediated by intrathecal administration of DILC siRNA significantly increased the mechanical shrinkage threshold (MWT) and paw withdrawal threshold latency (PWTL), decreased the positive frequency for nerve sensitivity to cold and suppressed the expression of inflammatory genes in bCCI rats. Down-regulation of DILC induced suppressor of cytokine signaling (SOCS3) expression and inhibited the phosphorylation of signal transducer and activator of transcription 3 (p-STAT3) in spinal cord tissues. Western blotting showed that down-regulation of DILC by DILC siRNA transfection induced SOCS3 expression and inhibited the expression of p-Janus kinase 2 (p-JAK2) and p-STAT3 and their downstream genes in primary microglia. Furthermore, down-regulation of DILC increased the viability of primary microglia, suppressed apoptosis, and inhibited the production of interleukin (IL)-6 and IL-1β in microglia. In contrast, overexpression of DILC showed the opposite functions to those of DILC knockdown. In conclusion, silence of lncRNA DILC attenuates neuropathic pain via SOCS3-induced suppression of the JAK2/STAT3 pathway., (© 2020 The Author(s).)

Spinal cord injury (SCI) is a neurological disease commonly caused by traumatic events on spinal cords. MiRNA-92a-3p is reported to be down-regulated after SCI. Our study investigated the effects of up-regulated miR-92a-3p on SCI and the underlying mechanisms. SCI mice model was established to evaluate the functional recovery of hindlimbs of mice through open-field locomotion and scored by Basso, Beattie, and Bresnahan (BBB) locomotion scale. Apoptosis of spinal cord cells was determined by flow cytometry. The effects of miR-92a-3p on SCI were detected by intrathecally injecting miR-92a-3p agomiR (agomiR-92) into the mice prior to the establishment of SCI. Phosphatase and tensin homolog (PTEN) was predicted as a target of miR-29a-3p by TargetScan. We further assessed the effects of agomiR-92 or/and overexpressed PTEN on apoptosis rates and apoptotic protein expressions in SCI mice. Moreover, the activation of protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling was determined by Western blot. The results showed that compared with the sham-operated mice, SCI mice had much lower BBB scores, and theapoptosis rate of spinal cord cells was significantly increased. After SCI, the expression of miR-92a-3p was down-regulated, and increased expression of miR-92a-3p induced by agomiR-92 further significantly increased the BBB score and decreased apoptosis. PTEN was specifically targeted by miR-92a-3p. In addition, the phosphorylation levels of Akt and mTOR were up-regulated under the treatment of agomiR-92. Our data demonstrated that the neuroprotective effects of miR-92a-3p on spinal cord safter SCI were highly associated with the activation of the PTEN/AKT/mTOR pathway., (© 2020 The Author(s).)

We have previously reported that the spinal angiotensin (Ang) system is involved in the modulation of streptozotocin (STZ)-induced diabetic neuropathic pain in mice. An important drawback of this model however is the fact that the neuropathic pain is independent of hyperglycemia and produced by the direct stimulation of peripheral nerves. Here, using the leptin deficient ob/ob mouse as a type 2 diabetic model, we examined whether the spinal Ang system was involved in naturally occuring diabetic neuropathic pain. Blood glucose levels were increased in ob/ob mice at 5-15 weeks of age. Following the hyperglycemia, persistent tactile and thermal hyperalgesia were observed at 11-14 and 9-15 weeks of age, respectively, which was ameliorated by insulin treatment. At 12 weeks of age, the expression of Ang-converting enzyme (ACE) 2 in the spinal plasma membrane fraction was decreased in ob/ob mice. Spinal ACE2 was expressed in neurons and microglia but the number of NeuN-positive neurons was decreased in ob/ob mice. In addition, the intrathecal administration of Ang (1-7) and SB203580, a p38 MAPK inhibitor, attenuated hyperalgesia in ob/ob mice. The phosphorylation of spinal p38 MAPK was also attenuated by Ang (1-7) in ob/ob mice. These inhibitory effects of Ang (1-7) were prevented by A779, a Mas receptor antagonist. In conclusion, we revealed that the Ang (1-7)-generating system is downregulated in ob/ob mice and is accompanied by a loss of ACE2-positive neurons. Furthermore, Ang (1-7) decreased the diabetic neuropathic pain through inhibition of p38 MAPK phosphorylation via spinal Mas receptors., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Neuromodulation therapies offer a treatment option that has minimal side effects and is relatively safe and potentially reversible. Spinal cord stimulation (SCS) has been used to treat various pain conditions for many decades. High-frequency SCS (HFSCS) involves the application of a single waveform at 10,000 Hz at a subthreshold level, therefore providing pain relief without any paresthesia., Methods: We tested whether early HFSCS treatment attenuated spared nerve injury (SNI)-induced neuropathic pain. The phosphorylation profile of mitogen-activated protein kinases (MAPKs), i.e., extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38, was evaluated to elucidate the potential underlying mechanism., Results: SNI of rat unilateral sciatic nerves induced mechanical hyperalgesia in the ipsilateral hind paws. Rats were assigned to SCS sessions with HFSCS (frequency 10 kHz; pulse width 30 μs; pulse shape of charge-balanced, current controlled; delivered continuously for 72 h), or sham stimulation immediately after SNI. Tissue samples were examined at 1, 3, 7, and 14 days after SNI. Behavioral studies showed that HFSCS applied to the T10/T11 spinal cord significantly attenuated SNI-induced mechanical hyperalgesia compared with the sham stimulation group. Moreover, western blotting revealed a significant attenuation of the activation of ERK1, ERK2, JNK1, and p38 in the dorsal root ganglia and the spinal dorsal horn., Conclusion: Application of HFSCS provides an effective treatment for SNI-induced persistent mechanical hyperalgesia by attenuating ERK, JNK, and p38 activation in the dorsal root ganglia and the spinal dorsal horn., (Copyright © 2020. Published by Elsevier B.V.)

Peripheral nerve injury elicits spinal microgliosis, contributing to neuropathic pain. The aurora kinases A (AURKA), B (AURKB), and C (AURKC) are potential therapeutic targets in proliferating cells. However, their role has not been clarified in microglia. The aim of this study was to examine the regulation of aurora kinases and their roles and druggability in spinal microgliosis and neuropathic pain. Sprague-Dawley rats received chronic constriction injury (CCI). Gene expression of aurora kinases A-C was evaluated by quantitative RT-PCR and western blot, respectively, in spinal cords at 1, 3, 7, and 14 days after CCI. AURKB gene and protein expression was up-regulated concomitantly with the development of spinal microgliosis and neuropathic pain. Using lentiviral over-expression and adeno-associated viral knockdown approaches, the function of AURKB was further investigated by western blot, immunohistochemistry, RNA sequencing, and pain behavior tests. We found that AURKB over-expression in naive rats caused spinal microgliosis and pain hypersensitivity, whereas AURKB knockdown reduced microgliosis and alleviated CCI-induced neuropathic pain. Accordingly, RNA sequencing data revealed down-regulation of genes critically involved in signaling pathways associated with spinal microgliosis and neuropathic pain after AURKB knockdown in CCI rats. To examine its therapeutic potential for treatment of neuropathic pain, animals were treated intrathecally with the pharmacological AURKB inhibitor AZD1152-HQPA resulting in the alleviation of CCI-induced pain. Taken together, our findings indicated that AURKB plays a critical role in spinal microgliosis and neuropathic pain. Targeting AURKB may be an efficient method for treatment of neuropathic pain subsequent to peripheral nerve injury., (© 2019 International Society for Neurochemistry.)

Age-related loss of skeletal muscle innervation by motor neurons leads to impaired neuromuscular function and is a well-established clinical phenomenon. However, the underlying pathogenesis remains unclear. Studying mice, we find that the number of motor units (MUs) can be maintained by counteracting neurotoxic microglia in the aged spinal cord. We observe that marked innervation changes, detected by motor unit number estimation (MUNE), occur prior to loss of muscle function in aged mice. This coincides with gene expression changes indicative of neuronal remodeling and microglial activation in aged spinal cord. Voluntary exercise prevents loss of MUs and reverses microglia activation. Depleting microglia by CSF1R inhibition also prevents the age-related decline in MUNE and neuromuscular junction disruption, implying a causal link. Our results suggest that age-related changes in spinal cord microglia contribute to neuromuscular decline in aged mice and demonstrate that removal of aged neurotoxic microglia can prevent or reverse MU loss., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Patients undergoing surgery often feel anxious. Accumulating evidence indicated that presurgical anxiety was related to the more severe postsurgical pain. An animal model was established that exposed Sprague-Dawley rats to a single-prolonged stress (SPS) procedure to induce presurgical anxiety-like behaviors. The experiment revealed that presurgical anxiety not only aggravated but also prolonged postsurgical pain. However, the underlying mechanisms were unknown., Methods: The rats in group C + Cort, group I + Cort, group A + Cort, and group AI + Cort were injected with corticosterone. The rats in group C + RU486, group I + RU486, group A + RU486, and group AI + RU486 were injected with mifepristone (RU486). The rats in group C + GSK650394 and group AI + GSK650394 were injected with GSK650394. The rats in group C + FC1 and group AI + FC1 were injected with fluorocitrate (FC) 30 minutes before SPS, 30 minutes before incision, and on postoperative days 1, 2, 3, 4, and 5. The rats in group C + FC2 and group AI + FC2 were injected with FC on postoperative days 7, 8, 9, 10, 11, 12, and 13. The paw withdrawal mechanical threshold was assessed 24 hours before SPS and from postoperative days 1 to 28. The level of corticosterone was determined by enzyme-linked immunosorbent assay. The expression of serum/glucocorticoid regulated kinase 1 (SGK1), interleukin-1β, and tumor necrosis factor-α was visualized by Western blot. The concentrations of adenosine triphosphate (ATP) were measured by ATP assay kit., Results: This study showed SPS elevated plasma glucocorticoids and ATP release from astrocytes, which meant the mechanical pain hypersensitivity in presurgical anxiety-induced postsurgical hyperalgesia was dependent on GCs-SGK1-ATP signaling pathway. SGK1 protein level in astrocytes was increased in response to the glucocorticoid stimuli and enhanced the extracellular release of ATP. Furthermore, spinal astrocytes played a key role in the maintenance. Targeting spinal astrocytes in maintenance phase prevented the pathological progression., Conclusions: These data suggested an important signaling pathway that affected the pain sensitivity after operation caused by presurgical anxiety.

Spinal cord injury (SCI) is terrible damage leading to the deficiencies and results in infinite inconvenience to sufferers. The effective treatment for SCI still meets a larger number of problems. Herein, the underlying molecular mechanism and novel therapy of SCI are urgently to investigate. Arachidonate 12-lipoxygenase (ALOX12) is widely expressed in various cell types and plays important role in modulating different cellular processes, such as platelet aggregation, cell migration and cancer cell proliferation. Nevertheless, the effects of ALOX12 on SCI are unclear. In the study, SCI model was established in wild type (WT) mice and ALOX12 knockout mice. First, ALOX12 expression was up-regulated in spinal cord tissues of WT mice after SCI. ALOX12-knockout mice exhibited improved behavior after SCI operation. Glial activation triggered by SCI was also alleviated in mice with the loss of ALOX12, as evidenced by the down-regulated expression of glial fibrillary acidic protein (GFAP) and Iba-1 in spinal cord samples. Further, SCI-induced inflammation was markedly prevented in ALOX12-knockout mice through blocking inhibitor of NF-κB α (IκBα)/nuclear factor-κB (NF-κB) pathway signaling. Additionally, reducing ALOX12 expression attenuated apoptosis in spinal cord tissues of SCI mice by decreasing Cyto-c, cleaved Caspase-3 and poly (ADP-ribose) polymerases (PARP) expression. The protective role of ALOX12-decrease against SCI was verified in LPS-incubated glial cells through repressing inflammatory response and apoptotic formation. Moreover, transgenic mice with ALOX12 over-expression showed accelerated SCI, associated with intensified inflammation and apoptosis. Based on these results, strategies for inhibiting ALOX12 could be used to prevent SCI development by repressing inflammation and apoptosis., (Copyright © 2019. Published by Elsevier Inc.)

The aim of this study was to investigate neuroprotective effect of bradykinin postconditioning on the rabbit spinal cord after 20 min of ischemia and 3 days of reperfusion. Bradykinin was administered by single i.p. application at 1, 6, 12 or 24 h after ischemia. Assessment of neurological function of hind limbs (Tarlov score) was estimated. Quantitative analysis was evaluated by Fluoro Jade B method, NeuN and ubiquitin immunohistochemistry in anterior horn neurons of the spinal cord. Histomorphologically distribution of ubiquitin and endogenous antioxidant enzymes (SOD1, SOD2, catalase) immunoreaction was described. Bradykinin postconditioning showed decreased number of degenerated neurons, increased number of surviving neurons and increase in number of ubiquitin positive neurons in all bradykinin postconditioned groups versus ischemia/reperfusion group. According to our results bradykinin postconditioning applied 24 h after ischemia significantly decreased (p < 0.001) number of degenerated neurons versus ischemia/reperfusion group. The least effective time window for bradykinin postconditioning was at 12 h after ischemia. Tarlov score was significantly improved (p < 0.05) in groups with bradykinin postconditioning applied 1, 6 or 24 h after ischemia versus ischemia/reperfusion group. Tarlov score in group with bradykinin application 12 h after ischemia was significantly decreased (p < 0.05) versus sham control group. Neuronal immunoreaction of ubiquitin, SOD1, SOD2 and catalase influenced by bradykinin postconditioning was dependent on neuronal survival or degeneration. In conclusion, bradykinin postconditioning showed protective effect on neurons in anterior horns of the rabbit spinal cord and improved motor function of hind limbs., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Generation of glial cell diversity in the developing spinal cord is known to depend on spatio-temporal patterning programs. In particular, expression of the transcription factor Olig2 in neural progenitors of the pMN domain is recognized as critical to their fate choice decision to form oligodendrocyte precursor cells (OPCs) instead of astrocyte precursors (APs). However, generating some confusion, lineage-tracing studies of Olig2 progenitors in the spinal cord provided evidence that these progenitors also generate some astrocytes. Here, we addressed the role of the heparan sulfate-editing enzyme Sulf2 in the control of gliogenesis and found an unanticipated function for this enzyme. At initiation of gliogenesis in mouse, Sulf2 is expressed in ventral neural progenitors of the embryonic spinal cord, including in Olig2-expressing cells of the pMN domain. We found that sulf2 deletion, while not affecting OPC production, impairs generation of a previously unknown Olig2-expressing pMN-derived cell subtype that, in contrast to OPCs, does not upregulate Sox10, PDGFRα or Olig1. Instead, these cells activate expression of AP identity genes, including aldh1L1 and fgfr3 and, of note, retain Olig2 expression as they populate the spinal parenchyma at embryonic stages but also as they differentiate into mature astrocytes at postnatal stages. Thus, our study, by revealing the existence of Olig2-expressing APs that segregate early from pMN cells under the influence of Sulf2, supports the existence of a common source of APs and OPCs in the ventral spinal cord and highlights divergent regulatory mechanism for the development of pMN-derived OPCs and APs., (© 2019 The Authors. Glia published by Wiley Periodicals, Inc.)

D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Gut microbiota has been proposed as an important environmental factor which can intervene and modulate central nervous system autoimmunity. Here, we altered the composition of gut flora with Clostridium butyricum and norfloxacin in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We found that appropriate C. butyricum (5.0 × 10 6 CFU/mL intragastrically daily, staring at weaning period of age) and norfloxacin (5 mg/kg intragastrically daily, 1 week prior to EAE induction) treatment could both ameliorate EAE although there are obvious differences in gut microbiota composition between these two interventions. C. butyricum increased while norfloxacin decreased the abundance and diversity of the gut microbiota in EAE mice, and both of the treatments decreased firmicutes / bacteroidetes ratio. In the genus level, C. butyricum treatment increased the abundance of Prevotella while Akkermansia and Allobaculum increased in norfloxacin treatment. Moreover, both interventions reduced Desulfovibroneceae and Ruminococcus species. Although there was discrepancy in the gut microbiota composition with the two interventions, C. butyricum and norfloxacin treatment both reduced Th17 response and increased Treg response in the gastrointestinal tract and extra-gastrointestinal organ systems in EAE mice. And the reduced activity of p38 mitogen-activated kinase and c-Jun N-terminal kinase signaling in spinal cord could be observed in the two interventions. The results suggested that manipulation of gut microbiota interventions should take factors such as timing, duration, and dosage into consideration. The discrepancy in the gut microbiota composition and the similar protective T cells response of C. butyricum and norfloxacin implies that achieving intestinal microecology balance by promoting and/or inhibiting the gut microbiota contribute to the well-being of immune response in EAE mice.

Synaptic abnormalities, perturbed endosomal recycling mediated by loss of the small GTPase RAB11, and neuroinflammatory signaling have been associated with multiple neurodegenerative diseases including the motor neuron disease, amyotrophic lateral sclerosis (ALS). This is consistent with the neuroprotective effect of RAB11 overexpression as well as of anti-inflammatory compounds. However, most studies were in animal models, and this phenomenon has not been demonstrated in human patients. Moreover, crosstalk between endosomal trafficking and inflammatory signaling pathways in ALS remains enigmatic. Here, we investigated RAB11 expression and MAPK/ERK/AKT signaling in 10 post-mortem spinal cord specimens from patients with sporadic ALS and age-matched controls. All 10 ALS patients showed TDP-43 pathology, whereas two specimens showed an overlapping FUS pathology and one had an acquired Q331K mutation in TDP-43. There was consistent RAB11 downregulation in all ALS cases, while p-AKT and phospho-ribosomal S6 kinase (p-p90RSK) were upregulated. Furthermore, competition between AKT and ERK pathways was observed in ALS, suggesting subtle differences among the TDP-43-ALS subtypes, which may influence patient therapeutic responses. Our findings demonstrate a complex regulation/perturbation pattern of signaling cascades involving MAPK/AKT/RAB11 in spinal cord tissue from ALS patients. These results underscore the relationships between ALS pathology, altered neuronal trafficking, and inflammation.

While evidence indicates that sigma-1 receptors (Sig-1Rs) play an important role in the induction of peripheral neuropathic pain, there is limited understanding of the role that the neurosteroidogenic enzymes, which produce Sig-1R endogenous ligands, play during the development of neuropathic pain. We examined whether sciatic nerve injury upregulates the neurosteroidogenic enzymes, cytochrome P450c17 and 3β-hydroxysteroid dehydrogenase (3β-HSD), which modulate the expression and/or activation of Sig-1Rs leading to the development of peripheral neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve induced a significant increase in the expression of P450c17, but not 3β-HSD, in the ipsilateral lumbar spinal cord dorsal horn at postoperative day 3. Intrathecal administration of the P450c17 inhibitor, ketoconazole during the induction phase of neuropathic pain (day 0 to day 3 post-surgery) significantly reduced the development of mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw. However, administration of the 3β-HSD inhibitor, trilostane had no effect on the development of neuropathic pain. Sciatic nerve injury increased astrocyte Sig-1R expression as well as dissociation of Sig-1Rs from BiP in the spinal cord. These increases were suppressed by administration of ketoconazole, but not by administration of trilostane. Co-administration of the Sig-1R agonist, PRE084 restored the development of mechanical allodynia originally suppressed by the ketoconazole administration. However, ketoconazole-induced inhibition of thermal hyperalgesia was not affected by co-administration of PRE084. Collectively these results demonstrate that early activation of P450c17 modulates the expression and activation of astrocyte Sig-1Rs, ultimately contributing to the development of mechanical allodynia induced by peripheral nerve injury., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.

Glutathione peroxidase 4 (GPx4) is the only enzyme capable of reducing toxic lipid hydroperoxides in biological membranes to the corresponding alcohols using glutathione as the electron donor. GPx4 is the major inhibitor of ferroptosis, a non-apoptotic and iron-dependent programmed cell death pathway, which has been shown to occur in various neurological disorders with severe oxidative stress. In this study, we investigate whether GPx4 expression is altered in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). The results clearly show that mRNA expression for all three GPx4 isoforms (cytoplasmic, mitochondrial and nuclear) decline in multiple sclerosis gray matter and in the spinal cord of MOG 35-55 peptide-induced EAE. The amount of GPx4 protein is also reduced in EAE, albeit not in all cells. Neuronal GPx4 immunostaining, mostly cytoplasmic, is lower in EAE spinal cords than in control spinal cords, while oligodendrocyte GPx4 immunostaining, mainly nuclear, is unaltered. Neither control nor EAE astrocytes and microglia cells show GPx4 labeling. In addition to GPx4, two other negative modulators of ferroptosis (γ-glutamylcysteine ligase and cysteine/glutamate antiporter), which are critical to maintain physiological levels of glutathione, are diminished in EAE. The decrease in the ability to eliminate hydroperoxides was also evidenced by the accumulation of lipid peroxidation products and the reduction in the proportion of the docosahexaenoic acid in non-myelin lipids. These findings, along with presence of abnormal neuronal mitochondria morphology, which includes an irregular matrix, disrupted outer membrane and reduced/absent cristae, are consistent with the occurrence of ferroptotic damage in inflammatory demyelinating disorders., (© 2018 International Society for Neurochemistry.)

Background: Chronic pain has been shown to depend on nociceptive sensitization in the spinal cord, and while multiple mechanisms involved in the initiation of plastic changes have been established, the molecular targets which maintain spinal nociceptive sensitization are still largely unknown. Building upon the established neurobiology underlying the maintenance of long-term potentiation in the hippocampus, this present study investigated the contributions of spinal atypical protein kinase C (PKC) isoforms PKCι/λ and PKMζ and their downstream targets (p62/GluA1 and NSF/GluA2 interactions, respectively) to the maintenance of spinal nociceptive sensitization in male and female rats., Results: Pharmacological inhibition of atypical PKCs by ZIP reversed established allodynia produced by repeated intramuscular acidic saline injections in male animals only, replicating previously demonstrated sex differences. Inhibition of both PKCι/λ and downstream substrates p62/GluA1 resulted in male-specific reversals of intramuscular acidic saline-induced allodynia, while female animals continued to display allodynia. Inhibition of NSF/GluA2, the downstream target to PKMζ, reversed allodynia induced by intramuscular acidic saline in both sexes. Neither PKCι/λ, p62/GluA1 or NSF/GluA2 inhibition had any effect on formalin response for either sex., Conclusion: This study provides novel behavioural evidence for the male-specific role of PKCι/λ and downstream target p62/GluA1, highlighting the potential influence of ongoing afferent input. The sexually divergent pathways underlying persistent pain are shown here to converge at the interaction between NSF and the GluA2 subunit of the AMPA receptor. Although this interaction is thought to be downstream of PKMζ in males, these findings and previous work suggest that females may rely on a factor independent of atypical PKCs for the maintenance of spinal nociceptive sensitization.

The pathophysiological mechanism of central post-stroke pain (CPSP) is complicated and not well understood. Recently, it has been reported that an increase in the levels of spinal nitric oxide synthetase (NOS) occurs in cerebral ischemia, and spinal NOS is involved in the development of neuropathic pain. The aim of this study was to elucidate the mechanism of spinal NOS signaling in the development of CPSP. Male ddY mice were subjected to 30-min long bilateral carotid artery occlusion (BCAO). The withdrawal responses to mechanical stimuli were significantly increased as determined with von Frey test on days 1 and 3 after BCAO. Protein expression of spinal N (G) ,N (G) -dimethylarginine dimethylaminohydralase 1 (DDAH1), a key enzyme involved in the metabolism of the endogenous NOS, increased on day 1 after BCAO, but not on day 3. Intrathecal (i.t.) injection of PD404182, a DDAH1 inhibitor, significantly suppressed mechanical allodynia on day 1, but not on day 3 after BCAO. In addition, i.t. administration of N G -nitro-L-arginine methyl ester (L-NAME), a non-selective NOS inhibitor, significantly blocked mechanical allodynia on days 1 and 3 after BCAO. Furthermore, BCAO-induced increment of spinal NOS activity was inhibited by the pretreatment with PD404182. These results suggest that mechanical allodynia in the early stage of CPSP is caused by increment of NOS activity through upregulated DDAH1 in the spinal cord.

Phosphodiesterase-5 inhibitors (PDE5Is) have been shown to modulate cell death/cell survival in different in vivo and in vitro models of disease by activating many signaling pathways. This review aimed at elucidating how PDE5Is can inhibit apoptosis. In this study, we describe many signaling pathways involved with the mechanism of action of PDE5Is that ultimately inhibit apoptosis and thus promote cell survival.

Objective: To investigate the effect of microRNA-210 on the spinal cord injury (SCI) and its underlying mechanism., Materials and Methods: The mouse SCI model was established. Mice were randomly assigned into 4 groups, namely the sham operation group (sham group), surgery group (SCI group), surgery+NC group (SCI+NC group) and surgery+microRNA-210 overexpression group (SCI+microRNA-210 mimics group). The mRNA levels of microRNA-210 and the key genes in the JAK-STAT pathway of the four groups were detected by Real-Time Polymerase Chain Reaction (RT-PCR) at different time points. Protein levels of JAK2 and STAT3 in mice of the four groups were detected by Western blot. To investigate the role of microRNA-210 in SCI recovery, changes in the motor function of mice were detected., Results: Grip strengths of right and left forelimbs in mice from the sham group were temporarily decreased at the early stage after surgery, which were gradually recovered to the preoperative levels on the 3rd postoperative day. However, mice in SCI group were unable to complete the grip strength determination at the early stage after surgery. Mice in SCI group were capable of grasping on the 7th postoperative day. Besides, grip strengths of mice in SCI group were remarkably lower than those of sham group until the end-point (on the 50th day). Furthermore, mRNA levels of microRNA-210 in mice of SCI group were decreased in a time-dependent manner (p<0.05). Higher grip strengths were observed in mice of SCI+microRNA-210 mimics group in comparison with those of SCI group and SCI+NC group (p<0.05). In addition, Western blot showed that protein levels of JAK2 and STAT3 in mice of SCI group were increased in a time-dependent manner (p<0.05). Moreover, protein levels of JAK2, STAT3, and MCP-1 in mice of SCI+NC group were remarkably higher than those in the sham group and SCI+microRNA-210 mimics group (p<0.05)., Conclusions: MicroRNA-210 is down-regulated in SCI mice. Grip strengths of SCI mice can be recovered after microRNA-210 overexpression via inhibiting inflammatory response by the JAK-STAT pathway.

The protein kinase C family regulates many cellular functions, including multiple forms of neuroplasticity. The novel PKCθ and atypical PKCζ isoforms have been implicated in distinct forms of spinal, respiratory motor plasticity, including phrenic motor facilitation (pMF) following acute intermittent hypoxia or inactivity, respectively. Although these PKC isoforms are critical in regulating spinal motor plasticity, other isoforms may be important for phrenic motor plasticity. We tested the impact of conventional/novel PKC activator, phorbol 12-myristate 13-acetate (PMA) on pMF. Rats given cervical intrathecal injections of PMA exhibited pMF, which was abolished by pretreatment of broad-spectrum PKC inhibitors bisindolymalemide 1 (BIS) or NPC-15437 (NPC). Because PMA fails to activate atypical PKC isoforms, and NPC does not block PKCθ, this finding demonstrates that classical/novel PKC isoforms besides PKCθ are sufficient to elicit pMF. These results advance our understanding of mechanisms producing respiratory motor plasticity, and may inspire new treatments for disorders that compromise breathing, such as ALS, spinal injury and obstructive sleep apnea., (Copyright © 2017. Published by Elsevier B.V.)

Myelinating glial cells (MGCs), oligodendrocytes (OLs) in the central nervous system (CNS) and Schwann cells (SCs) in the peripheral nervous system (PNS), generate myelin sheaths that insulate axons. After myelination is completed in adulthood, MGC functions independent from myelin are required to support axon survival, but the underlying mechanisms are still unclear. Dicer is a key enzyme that is responsible for generating functional micro-RNAs (miRNAs). Despite the importance of Dicer in initiating myelination, the role of Dicer in mature MGCs is still unclear. Here, Dicer was specifically deleted in mature MGCs in 2-month old mice (PLP-CreERT; Dicer fl/fl) by tamoxifen administration. Progressive motor dysfunction was observed in the Dicer conditional knockout mice, which displayed hind limb ataxia at 3 months post recombination that deteriorated into paralysis within 5 months. Massive axonal degeneration/atrophy in peripheral nerves was responsible for this phenomenon, but overt demyelination was not observed in either the CNS or PNS. In contrast to the PNS, signs of axonal degeneration were not observed in the CNS of these animals. We induced a Dicer deletion in oligodendroglia at postnatal day 5 in NG2-CreERT; Dicer fl/fl mice to evaluate whether Dicer expression in OLs is essential for axonal survival. Dicer deletion in oligodendroglia did not cause motor dysfunction at the age of 7 months. Neither axonal atrophy nor demyelination was observed in the CNS. Based on our results, Dicer expression in SCs is required to maintain axon integrity in adult PNS, and Dicer is dispensable for maintaining myelin sheaths in MGCs., (© 2018 Wiley Periodicals, Inc.)

Background: Over-expression of spinal protein kinase Cγ(PKCγ) contributes to the induction of persistent bilateral hyperalgesia following inflammatory injury, yet the role of spinal PKCγ in short- and long-lasting pain behavior is poorly understood., Objective: This study aimed to characterize the contribution of spinal PKCγ to spontaneous pain and long-lasting bilateral hyperalgesia in formalin-induced inflamed mice using pharmacological inhibition., Study Design: Laboratory animal study., Setting: The study was performed in the Department of Human Anatomy and K.K. Leung Brain Research Centre, Preclinical School of Medicine, the Fourth Military Medical University (Xi'an, China) and the Department of Anesthesiology, Fuzhou General Hospital (Fuzhou, China)., Methods: Male mice were unilaterally intraplantarly injected with formalin to induce inflammatory pain. Spontaneous pain behaviors, including flinches and lickings, were recorded by off-line video during the first hour post-injection and counted. Using von Frey tests, long-lasting bilateral mechanical paw withdrawal thresholds were determined before injection and at indicated time points thereafter. Temporal expression of spinal PKCγ was observed by immunohistochemical staining. For pharmacological inhibition, mice were treated daily with intrathecal Tat carrier or selective PKCγ inhibitor KIG31-1, from 1 hour prior to 10 days after formalin injection. Spontaneous pain behaviors and long-lasting bilateral mechanical hyperalgesia were assessed. Spinal PKCγ expression was also observed by using immunohistochemical staining and western blot., Results: The number of PKCγ-immunoreactive (ir) spinal neurons was significantly higher at 10 days, but not 2 hours, after formalin intraplantar injection, and accompanied by long-lasting bilateral hyperalgesia. Furthermore, long-lasting bilateral hyperalgesia could be reversed by pharmacological inhibition of over-expressed spinal PKCγ; however, pretreating with intrathecal KIG31-1 showed no antinociceptive effects on short-term spontaneous pain behaviors., Limitations: All results were obtained from the mice and no PKCγ inhibitors were available through clinical practice. Therefore, it remains difficult to draw definitive connections between animal research and human application., Conclusion: Our findings suggest that spinal PKCγ plays a predominant role in long-lasting bilateral hyperalgesia, but not in the spontaneous pain behaviors induced by formalin., Key Words: Formalin, spontaneous pain, mechanical hyperalgesia, protein kinase C gamma, KIG31-1, mice.

We examined whether chondroitin sulfate (CS), a compound used to treat osteoarthritis and joint pain, is effective against partial sciatic nerve ligation (PSNL)-induced neuropathic pain. Repeated oral administration of CS (300 mg/kg, b.i.d. for 20 days) resulted in inhibition of tactile allodynia observed 21 days after PSNL. On day 21, phosphorylation of spinal p38 mitogen-activated protein kinase (MAPK) was attenuated by CS. CS also inhibited c-Fos upregulation in ipsilateral deep dorsal horn (laminae III-IV) neurons, which receive Aβ-fiber afferent inputs. These findings suggest that CS attenuates PSNL-induced tactile allodynia by inhibiting spinal p38 MAPK phosphorylation and Aβ-fiber activation., (Copyright © 2018 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

It is well known that remifentanil, a widely used intravenous anesthesia drug, can paradoxically induce hyperalgesia. The underlying mechanisms are still not clear despite the wide investigations. The present study demonstrated that withdrawal from spinal application of remifentanil could dose-dependently induce long term potentiation (LTP) of C-fiber evoked field potentials. Remifentanil withdrawal could activate Src family kinases (SFKs) in microglia, and upregulate the expression of tumor necrosis factor alpha (TNFα) in spinal dorsal horn. Furthermore, pretreatment with either microglia inhibitor Minocycline, SFKs inhibitor PP2 or TNF αneutralization antibody could block remifentanil withdrawal induced spinal LTP, whereas supplement of recombinant rat TNFα to the spinal cord could reverse the inhibitory effect of Minocycline or PP2 on remifentanil withdrawal induced LTP. Our results suggested that TNFαrelease following SFKs activation in microglia is involved in the induction of LTP induced by remifentanil withdrawal.

Upregulation of Ca 2+ -permeable AMPA receptors (CP-AMPARs) in dorsal horn (DH) neurons has been causally linked to persistent inflammatory pain. This upregulation, demonstrated for both synaptic and extrasynaptic AMPARs, depends on the protein kinase C alpha (PKCα) activation; hence, spinal PKC inhibition has alleviated peripheral nociceptive hypersensitivity. However, whether targeting the spinal PKCα would alleviate both pain development and maintenance has not been explored yet (essential to pharmacological translation). Similarly, if it could balance the upregulated postsynaptic CP-AMPARs also remains unknown. Here, we utilized pharmacological and genetic inhibition of spinal PKCα in various schemes of pain treatment in an animal model of long-lasting peripheral inflammation. Pharmacological inhibition (pre- or post-treatment) reduced the peripheral nociceptive hypersensitivity and accompanying locomotive deficit and anxiety in rats with induced inflammation. These effects were dose-dependent and observed for both pain development and maintenance. Gene-therapy (knockdown of PKCα) was also found to relieve inflammatory pain when applied as pre- or post-treatment. Moreover, the revealed therapeutic effects were accompanied with the declined upregulation of CP-AMPARs at the DH synapses between primary afferents and sensory interneurons. Our results provide a new focus on the mechanism-based pain treatment through interference with molecular mechanisms of AMPAR trafficking in central pain pathways.

A fundamental issue in developmental biology and in organ homeostasis is understanding the molecular mechanisms governing the balance between stem cell maintenance and differentiation into a specific lineage. Accumulating data suggest that cell cycle dynamics play a major role in the regulation of this balance. Here we show that the G2/M cell cycle regulator CDC25B phosphatase is required in mammals to finely tune neuronal production in the neural tube. We show that in chick neural progenitors, CDC25B activity favors fast nuclei departure from the apical surface in early G1, stimulates neurogenic divisions and promotes neuronal differentiation. We design a mathematical model showing that within a limited period of time, cell cycle length modifications cannot account for changes in the ratio of the mode of division. Using a CDC25B point mutation that cannot interact with CDK, we show that part of CDC25B activity is independent of its action on the cell cycle., Competing Interests: FB, AM, MR, MA, SB, JG, FP, EA No competing interests declared, (© 2018, Bonnet et al.)

Neuropathic pain resulting from nerve injury can become persistent and difficult to treat but the molecular signaling responsible for its development remains poorly described. Here, we identify the neuronal stress sensor dual leucine zipper kinase (DLK; Map3k12 ) as a key molecule controlling the maladaptive pathways that lead to pain following injury. Genetic or pharmacological inhibition of DLK reduces mechanical hypersensitivity in a mouse model of neuropathic pain. Furthermore, DLK inhibition also prevents the spinal cord microgliosis that results from nerve injury and arises distant from the injury site. These striking phenotypes result from the control by DLK of a transcriptional program in somatosensory neurons regulating the expression of numerous genes implicated in pain pathogenesis, including the immune gene Csf1 . Thus, activation of DLK is an early event, or even the master regulator, controlling a wide variety of pathways downstream of nerve injury that ultimately lead to chronic pain., Competing Interests: JW, JG, EN, HS, JT, AC, CL No competing interests declared

Objective: To investigate the role of p38MAPK signal pathway in spinal cord and dorsal root ganglion (DRG) in rats with phantom limb pain and the effects of specific inhibitors.
 Methods: Healthy adult male SD rats (n=48) were cut off one side of the sciatic under anesthesia to establish a model of phantom limb pain. In addition, the healthy rats were taken as a sham group (group S, n=24). The animals were scored by observing the action of chewing (0=no chewing, 13=the worst chewing) after the operation and were sacrificed on the following day after the operation. The successful model of phantom limb pain were randomly divided into 2 groups: a phantom limb pain group (group P, n=24) and a phantom limb pain plus inhibitor group (group P+I, n=24). SB203580 was given to the rat at 0.8 mg/kg on every Monday until the rats were sacrificed, the rest of the rats received an equal amount of saline. Eight rats from each group were randomly taken for the determination of levels of P-p38MAPK in spinal cord and DRG before administration and on the 4th, 6th, 8th weekend following the administration, respectively.
 Results: In the sham group, no animal developed chewing. Meanwhile, rats in successful model of phantom limb pain group began chewing from the 2nd day after operation with scores at eight to eleven. The chewing scores in the P+I group were reduced after the treatment. Compared with group S, P-p38MAPK levels were elevated in groups of P and P+I (P<0.05 or P<0.01). Compared with group P, P-p38MAPK level was decreased in the group P+I (P<0.05 or P<0.01).
 Conclusion: P38MAPK signal pathway involves in the development of phantom limb pain.

Development of chemotherapy-induced neuropathic pain (CINP) compromises the use of chemotherapy and greatly impacts thousands of lives. Unfortunately, there are no Food and Drug Administration-approved drugs to prevent or treat CINP. Neuropathological changes within CNS, including neuroinflammation and increased neuronal excitability, are driven by alterations in neuro-glia communication; but, the molecular signaling pathways remain largely unexplored. Adenosine is a potent neuroprotective purine nucleoside released to counteract the consequences of these neuropathological changes. Adenosine signaling at its adenosine receptors (ARs) is dictated by adenosine kinase (ADK) in astrocytes, which provides a cellular sink for the removal of extracellular adenosine. We now demonstrate that chemotherapy (oxaliplatin) in rodents caused ADK overexpression in reactive astrocytes and reduced adenosine signaling at the A3AR subtype (A3AR) within the spinal cord. Dysregulation of ADK and A3AR signaling was associated with increased proinflammatory and neuroexcitatory interleukin-1β expression and activation of nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome, but not putative oxaliplatin-associated GSK3β transcriptional regulation. Intrathecal administration of the highly selective A3AR agonist MRS5698 attenuated IL-1β production and increased the expression of potent anti-inflammatory and neuroprotective IL-10. The effects of MRS5698 were blocked by attenuating IL-10 signaling in rats with intrathecal neutralizing IL-10 antibody and in IL-10 knockout mice. These findings provide new molecular insights implicating astrocyte-based ADK-adenosine axis and nucleotide-binding oligomerization domain-like receptor protein 3 in the development of CINP and IL-10 in the mechanism of action of A3AR agonists. These findings strengthen the pharmacological rationale for clinical evaluation of A3AR agonists already in advanced clinical trials as anticancer agents as an adjunct to chemotherapy.

The role of AMP-activated protein kinase (AMPK) in the regulation of energy metabolism and the control of skeletal muscle regeneration post injury has been described previously. It remains unknown whether this metabolic sensor plays a role in the mechanism of axonal regeneration post injury. In this study, we used a sciatic nerve crushed mouse model to detect the expression of AMPK in sciatic nerve and spinal motor neurons at 1 week, 2 weeks and 3 weeks after injury by immunofluorescence staining. Electrophysiological and histopathological studies were used to confirm the nerve injury and regeneration. Our results showed that frequency of AMPK-positive spinal motor neurons was significantly higher on day 7 after sciatic nerve crush (SNC) and peaked on day 14. No expression of AMPK was detected in axons of the sciatic nerve before and after the injury. Taken together, our study suggested a possible role of AMPK in the mechanism of motor nerve regeneration after injury., (Copyright © 2018 Kaohsiung Medical University. Published by Elsevier Taiwan. All rights reserved.)

Bone cancer pain (BCP) remains a serious complication of malignancy, which is an intractable clinical problem due to the gap in knowledge of its underlying mechanisms. Recent studies have demonstrated that the major involvement of neuroinflammation, particularly high-mobility group box 1 (HMGB1), which was identified as a late mediator of inflammation, in a number of pain conditions. However, the underlying mechanisms and functions of HMGB1 release in spinal cord, and its contributions to the development of BCP as well, are poorly understood. In the present study, we examined the theory that PKC activation lead to nuclear translocation and cytosolic HMGB1 secretion, which subsequently induces spinal neuro inflammatory responses (cytokine release) causing hyperalgesia. Our results showed that PKC activation and HMGB1 release in spinal neurons as well as mechanical allodynia in BCP rats, were all attenuated by intrathecal administration of the PKC inhibitor Gö6983 and aggravated by its activator PMA. Intrathecal administration of anti-HMGB1 antibody also alleviated hypersensitivity caused by BCP. Meanwhile, phospho-PKC and cellular HMGB1 were found co-localized in neurons, but not in microglia and astrocytes, of the spinal dorsal horns of tumor-bearing rats. Additionally, we found that HMGB1 translocation from nuclei to cytoplasm may be the consequence of PKC translocation into the nuclei, which occurred 9 days after tumor inoculation. Total p-HMGB1 as well as nuclear and cytoplasmic HMGB1 expression levels were tested in response to broad-spectrum PKC inhibitor Gö6983 or activator PMA in BCP rats. Together, these findings suggest that bone cancer related hyperalgesia is driven by PKC induced phosphorylation of HMGB1, which results in its translocation from the nucleus, and releasing from the cytosol of the dorsal horn, and the activation of spinal pro-inflammatory mediators., (Copyright © 2018. Published by Elsevier Inc.)

The central nervous system controls the innate immunity by modulating efferent neuronal networks. Recently, we have reported that central brain stimulation inhibits inflammatory responses. In the present study, we investigate whether spinal p38 mitogen-activated protein kinase (MAPK) affects joint inflammation in experimental arthritis. Firstly, we observed that intra-articular administration of zymosan in mice induces the phosphorylation of the spinal cord p38 MAPK. In addition, we demonstrated that spinal p38 MAPK inhibition with intrathecal injection of SB203580, a conventional and well-characterized inhibitor, prevents knee joint neutrophil recruitment, edema formation, experimental score and cytokine production. This local anti-inflammatory effect was completely abolished with chemical sympathectomy (guanethidine) and beta-adrenergic receptors blockade (nadolol). In conclusion, our results suggest that pharmacological strategies involving the modulation of spinal p38 MAPK circuit can prevent joint inflammation via sympathetic networks and beta-adrenoceptors activation., (© 2017 Société Française de Pharmacologie et de Thérapeutique.)