Biological Effects Of Methane On Spinal Cord Ischemia-reperfusion Injury And Its Mechanism In Rats

Spinal cord ischemia-reperfusion(IR)injury is a devastating complication of spinal trauma and thoracoabdominal aortic aneurysm surgery.The potential subsequent central nervous system injuries that give rise to paraplegia or paralysis has seriously affected the recovery and life quality of patients.It is reported that oxidative stress and subsquent neuroinflammation and neuronal apoptosis are the important pathogenic mechanisms of spinal cord IR injury.However,the commonly used antioxidants are not effective for spinal cord IR injury due to low lipid solubility and large molecular weight.Methane(CH4)is the main component of natural gas,and the simplest alkane organic gas with small molecule,high lipid solubility and abundant source.It is traditionally considered that methane is a“bioinertia”gas which does not affect the physiological function.But methane has been reported to have protective effects against IR-induced injury to the intestine,liver,heart,retina and abdominal skin flaps.Methane has also been shown to have beneficial effects on autoimmune hepatitis,bacterial sepsis,endotoxic shock,ulcerative colitis,diabetic retinopathy,carbon monoxide poisoning encephalopathy and one-time exhaustive exercise induced muscle damages via antioxidant,anti-inflammatory and anti-apoptotic activity.Nuclear factor erythroid 2-related factor 2(Nrf2)is a transcription factor regulating oxidative stress,which contributes to expression of antioxidant and phase II detoxifying genes.Nrf2 binds to Kelch-like ECH-associated protein 1(Keap1),its main negative regulator.It has been reported that pharmacological induction of the Nrf2 signaling might constitute a potent neuroprotective strategy.According to the current research progress,the study puts forward the following questions:Does methane have the protective effects on spinal cord IR damage?Is it safe the therapeutic dose of methane in vivo?Does methane exert the protective effects on spinal cord IR injury through antioxidant,anti-inflammatory and anti-apoptotic actions,and whether Nrf2 signaling is involved in them?The goal of the study is to answer the above questions.Methane is flammable and explosive in gas state,which is unconvenient to storage,transportation and utilization.But methane can be dissolved in physiological saline under high pressure without altering its chemical and biological quality.Thus,the methane-rich saline(MS)is prepared.Use MS,the study is carried out in the following two aspects in a well-established rat model of spinal cord IR injury.Part I:Biological effects of methane on spinal cord ischemia-reperfusion injury and its safety in ratsObjective:To investigate the biological effects of methane on spinal cord IR injury and its safety in rats.Methods:Forty male Sprague-Dawley rats were randomly divided into 5 groups(n=8 each)using a random number table:sham operation group(Sham group),spinal cord IR group(IR group)and three dose of MS groups(MS-1,MS-5 and MS-10 groups).Sham animals were subjected to surgery but spinal cord ischemia was not induced.Spinal cord ischemia was induced by occlusion of the thoracic aorta combined with controlled hypotension for 9 min followed by reperfusion in spinal cord IR group.MS 1,5 and 10 ml/kg were administered intraperitoneally immediately after onset of reperfusion in MS-1,MS-5and MS-10 groups,respectively.The arterial oxygen tension(PaO2),arterial carbon dioxide tension(PaCO2),pH,plasma glucose and hematocrit were analyzed at pre-ischemia,5 min after ischemia,10 and 30 min after reperfusion onset.Methane concentration in spinal cord tissues and motor sensory deficit index(MSDI)were measured at 12,24,48 and 72 h of reperfusion.At 72 h after reperfusion,neuronal survival and apoptotic numbers were calculated.The blood-spinal cord barrier(BSCB)permeability,such as evans blue(EB)fluorescence density and content,and spinal cord water content were also measured.Results:9 min spinal cord ischemia induced hind-limb motor sensory dysfunction,and increase neuronal apoptosis,BSCB permeability and spinal cord edema in rats.Intraperitoneal injection of 5 and 10 ml/kg MS produced high concentrations of methane in spinal cords during reperfusion,attenuated MSDI and neuronal apoptosis,and promoted neuronal survival in the ventral,intermediate and dorsal gray matter of lumbar segments.The MS treatment also reduced BSCB permeability and spinal cord edema.The beneficial effects of MS were gradually enhanced with the increase of dose.But 1 ml/kg MS(i.p.)caused trace methane to appear in spinal cord tissues within reperfusion and had no beneficial effects on spinal cord IR injury.The three dose of MS had no adverse effects on PaO2,PaCO2,pH,plasma glucose and hematocrit.Conclusion:Our data indicate that methane can reduce spinal cord IR damage in rats,and its beneficial effects are dose-dependent and safe.Thus,methane may serve as an effective and safe therapeutic agent for treating spinal cord IR injury.Part II:Mechanism for methane ameliorating spinal cord ischemia-reperfusion injury in ratsObjective:To investigate the underlying mechanism for methane ameliorating spinal cord IR injury in rats.Methods:Two hundred and twenty-eight male Sprague-Dawley rats were randomly divided into 6 groups(n=38 each)using a random number table:Sham,Methane,IR,IR-Methane,Si-Methane and Con-Methane groups.Sham animals were subjected to surgery but no spinal cord IR injury.Methane rats received sham operation and MS(10 ml/kg,i.p.).IR rats underwent 9 min of spinal cord ischemia and subsequent reperfusion but no additional treatment.IR-Methane rats had injured spinal cords plus MS(10 ml/kg,i.p.)when reperfusion was initiated.Si-Methane or Con-Methane rats were intrathecally injected once daily for three consecutive days with Nrf2 siRNA plasmid(300?g/kg;Jima Inc.,Shanghai,China)or control siRNA plasmid(300?g/kg)that had been pre-incubated with Lipofectamine 2000(Invitrogen,Carlsbad,CA),and then were subjected to 9 min of spinal cord ischemia 24 h after the last injection.Firstly,the study examined the role of Nrf2 signaling in the improvement of hind-limb neurological function and the reduction of neuronal damage,BSCB dysfunction and neutrophil infiltration in rats with spinal cord IR injury.The MSDI was measured at 12,24,48 and 72 h of reperfusion.The neuronal survival numbers,BSCB permeability(EB fluorescence density and content),water content and myeloperoxidase(MPO)activity in the lumbar spinal cords were measured at 72 h after reperfusion.Secondly,the study investigated the expression of Nrf2 signaling spinal cords and its antioxidant activity after and spinal cord IR injury and methane treatment.Nrf2 gene and protein expressions,as well as its nuclear translocation and DNA binding activity were assessed at 12,24,48 and 72 h.Nrf2 negative regulatory factor,Keap1 was also determined.The antioxidant enzymes and II detoxification enzymes including superoxide dismutase(SOD),catalase(CAT),glutathione(GSH),oxidized glutathione(GSSG)and heme oxygenase-1(HO-1)were also measured.As a consequence,intracellular superoxide anion(O2~-),hydrogen peroxide(H2O2),and oxidative products such as malondialdehyde(MDA),8-hydroxy-2'-deoxyguanosine(8-OHdG)and 3-nitrotyrosine(3-NT)were determined.Thirdly,the study detected the activation and localization of Nrf2 in neurons,microglia and astrocytes of the lumbar segments,and examined its inhibitory effects on neuronal apoptosis and neuroinflammation after methane treatment.The activation and localization of Nrf2 in neurons,microglia and astrocytes were analyzed using double immunofluorescence at 72 h after initiation of reperfusion.We also evaluated hind-limb MSDI,neuronal survival,cell apoptosis,mitochondrial cytochrome c(cyto c)release to cytoplasm,and activation of caspase-9 and caspase-3.At 72 h of reperfusion,the activation of microglia and astrocytes in L3~6 spinal cords was determined,and the phosphorylation and nuclear translocation of nuclear factor kappa B(NF-?B)p65,and DNA binding activity were assessed.The inflammatory factors,including tumor necrosis factor-alpha(TNF-?),interleukin-1?(IL-1?),chemokine(C-X-C motif)ligand 1(CXCL1),intercellular adhesion molecule-1(ICAM-1)and matrix metalloproteinase-9(MMP-9)were detected.And then,we determined the expression of the tight junction associated proteins(claudin-5,occludin and ZO-1).Results:MS treatment increased Nrf2 expression and transcriptional activity in neurons,microglia and astrocytes in the ventral,intermediate and dorsal gray matter of lumbar segments.HO-1,SOD,CAT and GSH were upregulated;and GSSG,superoxide,H2O2,MDA,8-OHdG and 3-NT were downregulated in MS-treated spinal cords.MS treatment reduced motor sensory deficits and neuronal apoptosis in gray matter zones,which was consistent with the suppression of cyto c release to the cytosol from the mitochondria and the activation of caspase-9 and caspase-3.Throughout the gray matter,the activation of microglia and astrocytes was inhibited;the nuclear accumulation of phosphorylated NF-?B p65 was reduced;and TNF-?,IL-1?,CXCL1,ICAM-1 and MPO were decreased.MS treatment attenuated BSCB dysfunction by preventing the expression and activity of MMP-9 and disrupting tight junction proteins(claudin-5,occludin,ZO-1).Consecutive intrathecal injection of specific siRNAs plasmid targeting Nrf2 at 24-h intervals 3 days before ischemia reduced the beneficial effects of MS.Conclusion:Our data indicate that methane ameliorates IR-induced spinal cord damage in rats via antioxidant,anti-inflammatory and anti-apoptotic activities that involve the activation of Nrf2 signaling.Thus,this work suggests that methane has antioxidant,anti-inflammatory and anti-apoptotic actions on spinal cord IR damage which may be mediated by Nrf2 pathway.