Glutamate Neurotransmitter System Mediated Heat Stroke-Induced Brain Injury And Hyperbaric Oxygen Intervention

ObjectiveHeat stroke(HS)causes neurological sequelae,even the death.Currently,the excitotoxic neurotransmitters was viewed as one of the important mechanisms of brain injury.However,the brain features of the detailed roles glutamate system in HS-induced brain injury was still unknown,such as the mGluR5 and glutamate transporters.Herein,we observed the clinical characteristics and prognosis of HS in patients and screen the possible transmitters in HS-induced brain injury and the mechanisms underlying HS-induced brain injury.Methods1.Patients with HS admitted to the Third Affiliated Hospital of Wenzhou Medical University from January 2019 to September 2022 were enrolled.According to the consciousness,they were divided into "good" or "poor" outcome group.Logistic Analysis was used to evaluate the Glasgow Coma Scale(GCS)score,lactate level and their combination and Receiver Operating Characteristic(ROC)curves to predict the outcome of consciousness of patients with HS.Meanwhile,the positive locations and characteristics of cerebral CT and/or Magnetic Resonance Imaging(MRI)were also observed.2.Animal experiments:Rat HS model was established,and the neurobehavioral was evaluated with water maze and pathological alterations were detected with H&E and immunohistochemistry.With metabolomics technology(1H-nuclear magnetic resonance,NMR),the possible biomarkers of rat brain transmitters and metabolites was screened.Further,with targeted metabolomics technology(liquid chromatography-mass spectrometry,LC-MS/MS),the specific biomarkers for HS-induced hippocampal and cerebellar injury,such as glutamate was evidenced and screened for further studies.3.Microglia experiments:Microglial HS model was established by heating the culture at 40 degrees.The glutamate release was tested using colorimetric assay.IL-1β and IL-18 was detected with ELISA and Western blot.RT-PCR was used to detect mRNA of mGLuR2,mGLuR3,mGLuR5(metabolic receptors),EAAT1,EAAT2,EAAT3(glutamate transporters)and ionotropic receptors(NR1 and NR2A).Moreover,The effects of hyperbaric oxygen,Riluzole(EAAT3 inhibitor)and CHPG(mGluR5 agonist)on Heat-induced microglial activation were evaluated.Results1.Clinical characteristics of patients with HS and MRI features of the brainComparison between "good" and "poor" patients,in good outcome group patients with clear consciousness had short-term adverse outcome(including death),indicators,including the time of first medical contact,lactate,APTT,D-dimer,GCS were significantly altered.The AUC of GCS score combined lactate for predicting adverse outcome is at 0.887.Aspartate transaminase,myoglobin and creatine kinase of Exertional group were different from Classic group.2.The metabolites characteristics of rat brain after HS using 1H-NMR techniqueHS induced rat nerve dysfunction,such as the movement,balance,and muscle tension.HS impaired rat memory and learning with water maze test,HS enhanced the latency and the navigation distance of the target platform and reduced the percentage of navigation time in the target quadrant.Metabolomics of indicated HS increased glutamate(Glu),glutamine(Gln),lactate(Lac),and succinate(Suc),while HS decreased aspartate(Asp),glycine(Gly),glutathione(GSH),and pyruvate(Py).It indicated that these brain transmitters and metabolites involved in HS-induced brain injury.3.HS altered hippocampal and cerebellar neurotransmitters and metabolitesTargeted metabolomics indicated HS altered of hippocampal and cerebellar neurotransmitters.L-methionine,L-valine,gamma-aminobutyric acid(GABA),5-hydroxyindoleacetic acid,L-glutamine,taurine,L-tryptophan,and L-glutamate were significantly elevated,while L-aspartylglutamine,acetylcholine,serotonin,levodopa,5-hydroxytryptophan,melatonin,and vanillylmandelic acid were significantly decreased.Similarly,cerebellar L-methionine and L-tryptophan were significantly increased,while 5-hydroxytryptophan,L-alanine,L-aspartylgl utamine,L-aspartate,L-cysteine,dopamine hydrochloride,norepinephrine,L-serine,arginine,ornithine,L-tyrosine,and 5-hydroxytryptophan were significantly decreased.4.Heat-treated changed microglial expression of glutamate transmitter system,IL-1β and IL-18In heat-stimulated microglia,glutamate release increased significantly;mRNA of mGLuR2,mGLuR3,mGLuR5,EAAT1,EAAT2,EAAT3 increased sharply,especially mRNA of EAAT3 and mGLuR5,while no alteration of NR1 and NR2A.Riluzole(EAAT 3 inhibitor)and CHPG(mGLuR5 agonist)were selected to evaluated the roles of both in microglia activation.Both decreased the glutamate release from Heat-treatment microglia.Riluzole downregulated the mRNA of EAAT3 and CHPG upregulated the mRNA of mGLuR5 receptor,respectively.With the heat-treatment with or without glutamate stimulation,microglia expressed IL-1βand IL-18 and released both into culture fluid significantly;Riluzole and CHPG reduced the elevation of IL-1β and IL-18.5.Hyperbaric oxygen improved microglia expression of glutamate transmitter system and IL-1β and IL-18With in heat-treated microglia,hyperbaric oxygen reduced the glutamate release.hyperbaric oxygen significantly changed mRNA of mGLuR2,mGLuR3,mGLuR5,EAAT 1,EAAT 2 and EAAT 3;Hyperbaric oxygen downregulated IL-1β and IL-18.It indicated the possible inhibition of microglia activation.Conclusions1.The combination of GCS score and lactate levels predict short-term consciousness recovery in patients after heat stroke;Multiple sites of ischemia and a few bleeding lesions were seen on MRI imaging.2.HS induced brain injury including cortical neuron loss,hippocampal and cerebellar astrocyte and microglia activation.HS induced metabolic disorders of neurotransmitters,and there is specific biomarkers for hippocampal and cerebellar injury,such as glutamate in the early stages of HS.3.The glutamate transporters and receptor system involved the inflammatory response of heat-treated microglia.Riluzole and CHPG significantly improved the microglia activation by reducing glutamate release.4.Hyperbaric oxygen down-regulated mRNA of glutamate transporters and receptors in heat-treated microglia,and reduced glutamate release and IL-18,indicating the possible alleviation of microglia activation.