MicroRNA let-7b enhances spinal cord nociceptive synaptic transmission and induces acute and persistent pain through neuronal and microglial signaling.

ABSTRACT: Secreted microRNAs (miRNAs) have been detected in various body fluids including the cerebrospinal fluid, yet their direct role in regulating synaptic transmission remains uncertain. We found that intrathecal injection of low dose of let-7b (1 µg) induced short-term (<24 hours) mechanical allodynia and heat hyperalgesia, a response that is compromised in Tlr7-/- or Trpa1-/- mice. Ex vivo and in vivo calcium imaging in GCaMP6-report mice revealed increased calcium signal in spinal cord afferent terminals and doral root ganglion/dorsal root ganglia neurons following spinal perfusion and intraplantar injection of let-7b. Patch-clamp recordings also demonstrated enhanced excitatory synaptic transmission (miniature excitatory postsynaptic currents [EPSCs]) in spinal nociceptive neurons following let-7b perfusion or optogenetic activation of axonal terminals. The elevation in spinal calcium signaling and EPSCs was dependent on the presence of toll-like receptor-7 (TLR7) and transient receptor potential ion channel subtype A1 (TRPA1). In addition, endogenous let-7b is enriched in spinal cord synaptosome, and peripheral inflammation increased let-7b in doral root ganglion/dorsal root ganglia neurons, spinal cord tissue, and the cerebrospinal fluid. Notably, let-7b antagomir inhibited inflammatory pain and inflammation-induced synaptic plasticity (EPSC increase), suggesting an endogenous role of let-7b in regulating pain and synaptic transmission. Furthermore, intrathecal injection of let-7b, at a higher dose (10 µg), induced persistent mechanical allodynia for >2 weeks, which was abolished in Tlr7-/- mice. The high dose of let-7b also induced microgliosis in the spinal cord. Of interest, intrathecal minocycline only inhibited let-7b-induced mechanical allodynia in male but not female mice. Our findings indicate that the secreted microRNA let-7b has the capacity to provoke pain through both neuronal and glial signaling, thereby establishing miRNA as an emerging neuromodulator.

A mast cell-thermoregulatory neuron circuit axis regulates hypothermia in anaphylaxis.

IgE-mediated anaphylaxis is an acute life-threatening systemic reaction to allergens, including certain foods and venoms. Anaphylaxis is triggered when blood-borne allergens activate IgE-bound perivascular mast cells (MCs) throughout the body, causing an extensive systemic release of MC mediators. Through precipitating vasodilatation and vascular leakage, these mediators are believed to trigger a sharp drop in blood pressure in humans and in core body temperature in animals. We report that the IgE/MC-mediated drop in body temperature in mice associated with anaphylaxis also requires the body's thermoregulatory neural circuit. This circuit is activated when granule-borne chymase from MCs is deposited on proximal TRPV1+ sensory neurons and stimulates them via protease-activated receptor-1. This triggers the activation of the body's thermoregulatory neural network, which rapidly attenuates brown adipose tissue thermogenesis to cause hypothermia. Mice deficient in either chymase or TRPV1 exhibited limited IgE-mediated anaphylaxis, and, in wild-type mice, anaphylaxis could be recapitulated simply by systemically activating TRPV1+ sensory neurons. Thus, in addition to their well-known effects on the vasculature, MC products, especially chymase, promote IgE-mediated anaphylaxis by activating the thermoregulatory neural circuit.

Mechanisms and treatments of neuropathic itch in a mouse model of lymphoma.

Our understanding of neuropathic itch is limited due to a lack of relevant animal models. Patients with cutaneous T cell lymphoma (CTCL) experience severe itching. Here, we characterize a mouse model of chronic itch with remarkable lymphoma growth, immune cell accumulation, and persistent pruritus. Intradermal CTCL inoculation produced time-dependent changes in nerve innervations in lymphoma-bearing skin. In the early phase (20 days), CTCL caused hyperinnervations in the epidermis. However, chronic itch was associated with loss of epidermal nerve fibers in the late phases (40 and 60 days). CTCL was also characterized by marked nerve innervations in mouse lymphoma. Blockade of C-fibers reduced pruritus at early and late phases, whereas blockade of A-fibers only suppressed late-phase itch. Intrathecal (i.t.) gabapentin injection reduced late-phase, but not early-phase, pruritus. IL-31 was upregulated in mouse lymphoma, whereas its receptor Il31ra was persistently upregulated in Trpv1-expressing sensory neurons in mice with CTCL. Intratumoral anti-IL-31 treatment effectively suppressed CTCL-induced scratching and alloknesis (mechanical itch). Finally, i.t. administration of a TLR4 antagonist attenuated pruritus in early and late phases and in both sexes. Collectively, we have established a mouse model of neuropathic and cancer itch with relevance to human disease. Our findings also suggest distinct mechanisms underlying acute, chronic, and neuropathic itch.

Novel proresolving lipid mediator mimetic 3-oxa-PD1n-3 docosapentaenoic acid reduces acute and chronic itch by modulating excitatory and inhibitory synaptic transmission and astroglial secretion of lipocalin-2 in mice.

ABSTRACT: Specialized proresolving mediators (SPMs) have demonstrated potent analgesic actions in animal models of pathological pain. The actions of SPMs in acute and chronic itch are currently unknown. Recently, n-3 docosapentaenoic acid (DPA) was found to be a substrate for the biosynthesis of several novel families of SPMs and 3-oxa-PD1 n-3 DPA (3-oxa-PD1) is an oxidation-resistant metabolic stable analogue of the n-3 DPA-derived protectin D1 (PD1). In this article, we demonstrate that 3-oxa-PD1 effectively reduces both acute and chronic itch in mouse models. Intrathecal injection of 3-oxa-PD1 (100 ng) reduced acute itch induced by histamine, chloroquine, or morphine. Furthermore, intrathecal 3-oxa-PD1 effectively reduced chronic itch, induced by cutaneous T-cell lymphoma (CTCL), allergic contact dermatitis with dinitrofluorobenzene, and psoriasis by imiquimod. Intratumoral injection of 3-oxa-PD1 also suppressed CTCL-induced chronic itch. Strikingly, the antipruritic effect lasted for several weeks after 1-week intrathecal 3-oxa-PD1 treatment. Whole-cell recordings revealed significant increase in excitatory postsynaptic currents in spinal dorsal horn (SDH) neurons of CTCL mice, but this increase was blocked by 3-oxa-PD1. 3-oxa-PD1 further increased inhibitory postsynaptic currents in SDH neurons of CTCL mice. Cutaneous T-cell lymphoma increased the spinal levels of lipocalin-2 (LCN2), an itch mediator produced by astrocytes. 3-oxa-PD1 suppressed LCN2 production in CTCL mice and LCN2 secretion in astrocytes. Finally, CTCL-induced anxiety was alleviated by intrathecal 3-oxa-PD1. Our findings suggest that 3-oxa-PD1 potently inhibits acute and chronic itch through the regulation of excitatory or inhibitory synaptic transmission and astroglial LCN2 production. Therefore, stable SPM analogs such as 3-oxa-PD1 could be useful to treat pruritus associated with different skin injuries.

Macrophages and microglia in inflammation and neuroinflammation underlying different pain states.

Pain is a main symptom in inflammation, and inflammation induces pain via inflammatory mediators acting on nociceptive neurons. Macrophages and microglia are distinct cell types, representing immune cells and glial cells, respectively, but they share similar roles in pain regulation. Macrophages are key regulators of inflammation and pain. Macrophage polarization plays different roles in inducing and resolving pain. Notably, macrophage polarization and phagocytosis can be induced by specialized pro-resolution mediators (SPMs). SPMs also potently inhibit inflammatory and neuropathic pain via immunomodulation and neuromodulation. In this review, we discuss macrophage signaling involved in pain induction and resolution, as well as in maintaining physiological pain. Microglia are macrophage-like cells in the central nervous system (CNS) and drive neuroinflammation and pathological pain in various inflammatory and neurological disorders. Microglia-produced inflammatory cytokines can potently regulate excitatory and inhibitory synaptic transmission as neuromodulators. We also highlight sex differences in macrophage and microglial signaling in inflammatory and neuropathic pain. Thus, targeting macrophage and microglial signaling in distinct locations via pharmacological approaches, including immunotherapies, and non-pharmacological approaches will help to control chronic inflammation and chronic pain.

IL-23/IL-17A/TRPV1 axis produces mechanical pain via macrophage-sensory neuron crosstalk in female mice.

Although sex dimorphism is increasingly recognized as an important factor in pain, female-specific pain signaling is not well studied. Here we report that administration of IL-23 produces mechanical pain (mechanical allodynia) in female but not male mice, and chemotherapy-induced mechanical pain is selectively impaired in female mice lacking Il23 or Il23r. IL-23-induced pain is promoted by estrogen but suppressed by androgen, suggesting an involvement of sex hormones. IL-23 requires C-fiber nociceptors and TRPV1 to produce pain but does not directly activate nociceptor neurons. Notably, IL-23 requires IL-17A release from macrophages to evoke mechanical pain in females. Low-dose IL-17A directly activates nociceptors and induces mechanical pain only in females. Finally, deletion of estrogen receptor subunit α (ERα) in TRPV1+ nociceptors abolishes IL-23- and IL-17-induced pain in females. These findings demonstrate that the IL-23/IL-17A/TRPV1 axis regulates female-specific mechanical pain via neuro-immune interactions. Our study also reveals sex dimorphism at both immune and neuronal levels.

A new synthetic protectin D1 analog 3-oxa-PD1n-3 DPA reduces neuropathic pain and chronic itch in mice.

The resolution of inflammation is a biosynthetically active process controlled by the interplay between oxygenated polyunsaturated mediators and G-protein coupled receptor-signaling pathways. These enzymatically oxygenated polyunsaturated fatty acids belong to distinct families of specialized pro-resolving autacoids. The protectin family of mediators has attracted an interest because of their potent pro-resolving and anti-inflammatory actions verified in several in vivo disease models. Herein, we present the stereoselective synthesis and biological evaluations of 3-oxa-PD1n-3 DPA, a protectin D1 analog. Results from mouse models indicate that the mediators protectin D1, PD1n-3 DPA and the new analog 3-oxa-PD1n-3 DPA all relieved streptozotocin-induced diabetic neuropathic pain at doses of 90 and 300 pmol, equivalent to 30 and 100 ng, respectively, following intrathecal (I.T.) injection. Of interest, at a low dose of only 30 pmol (10 ng; I.T.) only 3-oxa PD1n-3 DPA was able to alleviate neuropathic pain, directly compared to vehicle controls. Moreover, using a chronic itch model of cutaneous T-cell lymphoma (CTCL), all three compounds at 300 pmol (100 ng) showed a significant reduction in itching for several hours. The biomolecular information on the structure-functions of the protectins and the new synthetic analog 3-oxa-PD1n-3 DPA is of interest towards developing new immunoresolvents.

Central opioid receptors mediate morphine-induced itch and chronic itch via disinhibition.

Opioids such as morphine are mainstay treatments for clinical pain conditions. Itch is a common side effect of opioids, particularly as a result of epidural or intrathecal administration. Recent progress has advanced our understanding of itch circuits in the spinal cord. However, the mechanisms underlying opioid-induced itch are not fully understood, although an interaction between µ-opioid receptor (MOR) and gastrin-releasing peptide receptor (GRPR) in spinal GRPR-expressing neurons has been implicated. In this study we investigated the cellular mechanisms of intrathecal opioid-induced itch by conditional deletion of MOR-encoding Oprm1 in distinct populations of interneurons and sensory neurons. We found that intrathecal injection of the MOR agonists morphine or DAMGO elicited dose-dependent scratching as well as licking and biting, but this pruritus was totally abolished in mice with a specific Oprm1 deletion in Vgat+ neurons [Oprm1-Vgat (Slc32a1)]. Loss of MOR in somatostatin+ interneurons and TRPV1+ sensory neurons did not affect morphine-induced itch but impaired morphine-induced antinociception. In situ hybridization revealed Oprm1 expression in 30% of inhibitory and 20% of excitatory interneurons in the spinal dorsal horn. Whole-cell recordings from spinal cord slices showed that DAMGO induced outward currents in 9 of 19 Vgat+ interneurons examined. Morphine also inhibited action potentials in Vgat+ interneurons. Furthermore, morphine suppressed evoked inhibitory postsynaptic currents in postsynaptic Vgat- excitatory neurons, suggesting a mechanism of disinhibition by MOR agonists. Notably, morphine-elicited itch was suppressed by intrathecal administration of NPY and abolished by spinal ablation of GRPR+ neurons with intrathecal injection of bombesin-saporin, whereas intrathecal GRP-induced itch response remained intact in mice lacking Oprm1-Vgat. Intrathecal bombesin-saporin treatment reduced the number of GRPR+ neurons by 97% in the lumber spinal cord and 91% in the cervical spinal cord, without changing the number of Oprm1+ neurons. Additionally, chronic itch from DNFB-induced allergic contact dermatitis was decreased by Oprm1-Vgat deletion. Finally, naloxone, but not peripherally restricted naloxone methiodide, inhibited chronic itch in the DNFB model and the CTCL model, indicating a contribution of central MOR signalling to chronic itch. Our findings demonstrate that intrathecal morphine elicits itch via acting on MOR on spinal inhibitory interneurons, leading to disinhibition of the spinal itch circuit. Our data also provide mechanistic insights into the current treatment of chronic itch with opioid receptor antagonist such as naloxone.

How Do Sensory Neurons Sense Danger Signals?

Sensory neurons are activated by physical and chemical stimuli, eliciting sensations such as temperature, touch, pain, and itch. From an evolutionary perspective, sensing danger is essential for organismal survival. Upon infection and injury, immune cells respond to pathogen/damage-associated molecular patterns (PAMPs/DAMPs) through pattern recognition receptors (PRRs) such as Toll-like receptors (TLRs), and produce inflammatory mediators that activate sensory neurons through neuro-immune interactions. Sensory neurons also express TLRs and other PRRs that directly sense danger signals after injury or during infection, leading to pain, itch, or analgesia. In addition to slow-acting canonical TLR signaling, TLRs function uniquely in sensory neurons through non-canonical coupling to ion channels, enabling rapid modulation of neuronal activity. We discuss how sensory neurons utilize TLRs and other PRR pathways to detect danger signals in their environment.

Computer-aided Discovery of a New Nav1.7 Inhibitor for Treatment of Pain and Itch.

BACKGROUND: Voltage-gated sodium channel Nav1.7 has been validated as a perspective target for selective inhibitors with analgesic and anti-itch activity. The objective of this study was to discover new candidate compounds with Nav1.7 inhibitor properties. The authors hypothesized that their approach would yield at least one new compound that inhibits sodium currents in vitro and exerts analgesic and anti-itch effects in mice. METHODS: In silico structure-based similarity search of 1.5 million compounds followed by docking to the Nav1.7 voltage sensor of Domain 4 and molecular dynamics simulation was performed. Patch clamp experiments in Nav1.7-expressing human embryonic kidney 293 cells and in mouse and human dorsal root ganglion neurons were conducted to test sodium current inhibition. Formalin-induced inflammatory pain model, paclitaxel-induced neuropathic pain model, histamine-induced itch model, and mouse lymphoma model of chronic itch were used to confirm in vivo activity of the selected compound. RESULTS: After in silico screening, nine compounds were selected for experimental assessment in vitro. Of those, four compounds inhibited sodium currents in Nav1.7-expressing human embryonic kidney 293 cells by 29% or greater (P < 0.05). Compound 9 (3-(1-benzyl-1H-indol-3-yl)-3-(3-phenoxyphenyl)-N-(2-(pyrrolidin-1-yl)ethyl)propanamide, referred to as DA-0218) reduced sodium current by 80% with a 50% inhibition concentration of 0.74 µM (95% CI, 0.35 to 1.56 µM), but had no effects on Nav1.5-expressing human embryonic kidney 293 cells. In mouse and human dorsal root ganglion neurons, DA-0218 reduced sodium currents by 17% (95% CI, 6 to 28%) and 22% (95% CI, 9 to 35%), respectively. The inhibition was greatly potentiated in paclitaxel-treated mouse neurons. Intraperitoneal and intrathecal administration of the compound reduced formalin-induced phase II inflammatory pain behavior in mice by 76% (95% CI, 48 to 100%) and 80% (95% CI, 68 to 92%), respectively. Intrathecal administration of DA-0218 produced acute reduction in paclitaxel-induced mechanical allodynia, and inhibited histamine-induced acute itch and lymphoma-induced chronic itch. CONCLUSIONS: This study's computer-aided drug discovery approach yielded a new Nav1.7 inhibitor that shows analgesic and anti-pruritic activity in mouse models.

PD-1 blockade inhibits osteoclast formation and murine bone cancer pain.

Emerging immune therapy, such as with the anti-programmed cell death-1 (anti-PD-1) monoclonal antibody nivolumab, has shown efficacy in tumor suppression. Patients with terminal cancer suffer from cancer pain as a result of bone metastasis and bone destruction, but how PD-1 blockade affects bone cancer pain remains unknown. Here, we report that mice lacking Pdcd1 (Pd1-/-) demonstrated remarkable protection against bone destruction induced by femoral inoculation of Lewis lung cancer cells. Compared with WT mice, Pd1-/- mice exhibited increased baseline pain sensitivity, but the development of bone cancer pain was compromised in Pd1-/- mice. Consistently, these beneficial effects in Pd1-/- mice were recapitulated by repeated i.v. applications of nivolumab in WT mice, even though nivolumab initially increased mechanical and thermal pain. Notably, PD-1 deficiency or nivolumab treatment inhibited osteoclastogenesis without altering tumor burden. PD-L1 and CCL2 are upregulated within the local tumor microenvironment, and PD-L1 promoted RANKL-induced osteoclastogenesis through JNK activation and CCL2 secretion. Bone cancer upregulated CCR2 in primary sensory neurons, and CCR2 antagonism effectively reduced bone cancer pain. Our findings suggest that, despite a transient increase in pain sensitivity following each treatment, anti-PD-1 immunotherapy could produce long-term benefits in preventing bone destruction and alleviating bone cancer pain by suppressing osteoclastogenesis.

Regulation of pain by neuro-immune interactions between macrophages and nociceptor sensory neurons.

Inflammation is the body's protective reaction to injury and infection. Pain is a hallmark of inflammation and can be either protective or detrimental during acute or chronic phase. Macrophages play a chief role in the pathogenesis of pain and have bilateral communications with nociceptors, the specialized primary sensory neurons that sense pain. Macrophages 'talk to' nociceptors by releasing pro-inflammatory mediators (e.g. pro-inflammatory cytokines) that induce pain via direct activation of nociceptors. Macrophages also 'listen to' nociceptors, by which nociceptors secrete neuropeptides and chemokines which act on macrophages. Activation of toll-like receptors (TLRs) in nociceptors releases CCL2, activating macrophages and potentiating pathological pain. Emerging evidence also points to a pro-resolution role of macrophages in inflammation and pain. Macrophage GPR37 is activated by neuroprotectin D1, a specialized pro-resolving mediator (SPM) and resolves inflammatory pain via phagocytosis and production of IL-10 that inhibits nociceptors. Macrophage-nociceptor interactions are also mediated by microRNAs and microRNA-containing exosomes in chronic pain. Notably, extracellular microRNAs (e.g. let-7b and miR-711) can directly bind and activate nociceptors. Targeting macrophage-nociceptor interactions will help to control inflammation and pain.

High-concentration hydrogen protects mouse heart against ischemia/reperfusion injury through activation of thePI3K/Akt1 pathway.

The study investigated the role of Akt1 through the cardioprotection of high-concentration hydrogen (HCH). C57BL/6 mice were randomly divided into the following groups: sham, I/R, I/R + HCH, I/R + HCH + LY294002 (PI3K inhibitor), I/R + HCH + wortmannin (PI3K inhibitor), I/R + LY294002, and I/R + wortmannin. After 45 min of ischemia, HCH (67% H2 and 33% O2) was administered to mice during a 90-min reperfusion. To investigate the role of Akt1 in the protective effects of HCH, mice were divided into the following groups: I/R + A-674563 (Akt1 selective inhibitor), I/R + HCH + A-674563, I/R + CCT128930 (Akt2 selective inhibitor), and I/R + HCH + CCT128930. After a 4-h reperfusion, serum biochemistry, histological, western blotting, and immunohistochemical analyses were performed to evaluate the role of the PI3K-Akt1 pathway in the protection of HCH. In vitro, 75% hydrogen was administered to cardiomyocytes during 4 h of reoxygenation after 3-h hypoxia. Several analyses were performed to evaluate the role of the Akt1 in the protective effects of hydrogen. HCH resulted in the phosphorylation of Akt1 but not Akt2, and Akt1 inhibition markedly abolished HCH-induced cardioprotection. Our findings reveal that HCH may exert cardioprotective effects through a PI3K-Akt1-dependent mechanism.

Comments and hypotheses on the mechanism of methane against ischemia/reperfusion injury.

As we all know, methane is a kind of fuel. Previous studies have shown that methanogens in the colon can react with carbon dioxide and hydrogen to produce methane. In a recent study, the anti-inflammatory effects of methane were shown in a dog model of small intestinal ischemia/reperfusion. The mechanism of this anti-inflammatory effect needs further investigation. Recently, studies have shown anti-inflammatory, anti-apoptotic and anti-oxidative effects of methane on different organic injuries. According to the results of these studies, we hypothesize that the initial effects of methane are to react with free radicals and enhance expression of antioxidase through forkhead box transcription factor class O pathway. The anti-inflammatory effect is following the anti-oxidative effect, and the anti-apoptotic effect relies on anti-inflammatory and anti-oxidative effects.

Inhalation of high concentrations of hydrogen ameliorates liver ischemia/reperfusion injury through A2A receptor mediated PI3K-Akt pathway.

BACKGROUND AND AIMS: This study explored the hepatoprotection of high concentrations of hydrogen (HCH) inhalation in a mouse hepatic ischemia/reperfusion (I/R) injury model and the potential mechanism. METHODS: To explore the role of the PI3K-Akt pathway in the hepatoprotection of HCH, C57BL/6 mice were randomly divided into five groups: Sham, I/R, I/R+HCH, LY294002 (PI3K inhibitor)+I/R+HCH, and LY+I/R groups. Mice received inhalation of 66.7% hydrogen and 33.3% oxygen for 1h immediately after surgery. LY294002 was intravenously injected at 10mol/kg. To explore whether PI3K-Akt pathway activation was mediated by the A2A receptor, additional four groups were included: ZM241385 (A2A receptor antagonist)+I/R+HCH, ZM241385+I/R, bpv(HOpic) (PTEN inhibitor)+I/R, and ZM241385+bpv+I/R+HCH. Six hours after I/R, serum biochemistry, histological examination, Western blotting, and immunohistochemistry were performed to evaluate the hepatoprotection of HCH and the role of the PI3K-Akt pathway and A2A receptor in this protection. RESULTS: Liver dysfunction, hepatic pathological injury, infiltration of inflammatory cytokines, and hepatocyte apoptosis were observed after hepatic I/R, accompanied by inhibition of the PI3K-Akt pathway. HCH significantly improved liver function, reduced serum inflammatory cytokines, and inhibited hepatocyte apoptosis, and also induced the PI3K-Akt pathway activation. In the presence of LY294002 or ZM241385, the protective effects of HCH were markedly attenuated, but the effects of ZM241385 were reversed by bpv(HOpic). CONCLUSION: Our findings indicate that HCH may protect the liver against I/R injury through the A2A dependent PI3K-Akt pathway.

Methane ameliorates spinal cord ischemia-reperfusion injury in rats: Antioxidant, anti-inflammatory and anti-apoptotic activity mediated by Nrf2 activation.

Methane is reported to have antioxidant, anti-inflammatory and anti-apoptotic properties. We investigated the potential neuroprotective effects of methane-rich saline (MS) on spinal cord ischemia-reperfusion injury and determined that its therapeutic benefits are associated with the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Rats received 9min of spinal cord ischemia induced by occlusion of the descending thoracic aorta plus systemic hypotension followed by a single MS treatment (10ml/kg, ip) and 72h reperfusion. MS treatment attenuated motor sensory deficits and produced high concentrations of methane in spinal cords during reperfusion, which increased Nrf2 expression and transcriptional activity in neurons, microglia and astrocytes in the ventral, intermediate and dorsal gray matter of lumbar segments. Heme oxygenase-1, superoxide dismutase, catalase and glutathione were upregulated; and glutathione disulfide, superoxide, hydrogen peroxide, malondialdehyde, 8-hydroxy-2-deoxyguanosine and 3-nitrotyrosine were downregulated in MS-treated spinal cords. MS treatment reduced neuronal apoptosis in gray matter zones, which was consistent with the suppression of cytochrome c release to the cytosol from the mitochondria and the activation of caspase-9 and -3. Throughout the gray matter, the activation of microglia and astrocytes was inhibited; the nuclear accumulation of phosphorylated nuclear factor-kappa B p65 was reduced; and tumor necrosis factor α, interleukin 1ß, chemokine (C-X-C motif) ligand 1, intercellular adhesion molecule 1 and myeloperoxidase were decreased. MS treatment attenuated blood-spinal cord barrier dysfunction by preventing the expression and activity of matrix metallopeptidase-9 and disrupting tight junction proteins. Consecutive intrathecal injection of specific siRNAs targeting Nrf2 at 24-h intervals 3 days before ischemia reduced the beneficial effects of MS. Our data indicate that MS treatment prevents IR-induced spinal cord damage via antioxidant, anti-inflammatory and anti-apoptotic activities that involve the activation of Nrf2 signaling. Thus, methane may serve as a novel promising therapeutic agent for treating ischemic spinal cord injury.