| BackgroundHeat stroke(HS)is a severe heat stress with high mortality and high core temperature that is usually higher than 40 ?,as well as the central nervous system(CNS)abnormalities,severe cases often appear systemic inflammatory response syndrome(SIRS)and multiple organ dysfunction syndrome(MODS).CNS injury is a major clinical syndrome of heat stroke,characterized by cognitive decline,ataxia,delirium,convulsio ns and coma.Brain damage occurred at the early stage of heat stroke,attracting various studies focus on HS harm prevention and treatment.About the mechanism of brain damage following HS,previous conclusive evidence has been proven that thermal stimulation can damage brain cells and its internal structure,leading to the apoptosis and/or necrosis of neuronal progenitor cells.In addition to the direct effect of the CNS injury,in recent years,various explanations including neuroinflammatory aspects were put forward to account for the pathological process of brain injury following HS.HS can cause SIRS,including the severe whole body inflammation,the serious local inflammation of CNS.The overwhelming inflammatory responses can impair the cellular function and accelerate apoptosis of neurons,leading to the poor prognosis in HS patients with cognitive dysfunction,memory loss,mental disorders,etc.However,it is not clear,whether the CNS inflammatory responses of HS are secondary to peripheral inflammation,or a primary CNS inflammatory responses directly caused by high fever.Studies regarding the problem mentioned above will provide potential clinical treatment of HS,at least in partial relieve neuronal damage.The high mortality of HS associated with MODS,which is the common course in the pathological process of HS.The pathophysiological mechanism of this process is still not completely clear.A recent study has reported that the pathophysiological mechanism of HS is somewhat familiar with endotoxemia.HS elevates serum endotoxin concentration,activates mononuclear macrophages and neutrophils,secrete TNF-?,IL-1?,and IL-6 with a waterfall cascade amplification effect,involving the development of SIRS and MODS.But it is still controversial that whether the endotoxin linkaged with the endogenous gut sources or the endogenous blood endotoxin.The blood-brain barrier(BBB)and intestinal barrier are the responsible structure that isolate brain tissue from the peripheral blood,and isolate the peripheral blood from gut,respetively.Under normal circumstances the BBB blocking invasion of harmful substances through the blood into the brain,oppositely,inflammtory infiltration occurred in the CNS when the BBB structure damaged and the permeability increased,causing secondary central inflammatory responses.Similarly,the intestinal barrier can prevent invasion of potential intestinal bacteria and harmful substances into the peripheral blood.Thus,observation the change of BBB and intestinal barriers,and determination of inflammatory effects of peripheral circulations will be the hopeful approach to reveal the pathophysiological mechanism of CNS inflammation and the peripheral inflammation,as well as their relationship during HS.Therefore,this thesis based on an establishment of a brain injury model of HS,investigated the inflammatory change of CNS and peripheral circulations,as well as the damage degree and tendency of regularity of BBB and intestinal barrier following HS.The corresponding results will offer new reference for clinical treatment of brain damage in HS patients.Methods1,Healthy BALB/c mice,12 weeks of age,male,weight(29.1 ± 0.6)g.To establish the HS model,mice underwent continuous heat exposures as temperature(41.2±0.5)?,relative humidity(60±2)%,making mice's rectal temperature reach(42.4±0.2)?.Mice were divided into normal control group(CON group)and the indicated time points of recovery group after HS(HS-1h,HS-6h,and HS-24 h group).To explore the damage effect of HS on mice and the stress responses of mice during or following HS,physical performance outcomes of mice were observed,rectal temperature and survival rate were recorded in the stimulation and recovery period of HS;the subtle ultrastructure changes of the damaged brain were observed by hematoxylin-eosin(HE)staining and transmission electron microscope(TEM).2,To explore the brain inflammatory changes of HS mice,the expression of TNF-?,IL-1?,and IL-6 in cortex and hippocampus were detected by quantitative real-time polymerase chain reaction(qPCR)and enzyme linked immunosorbent assay(ELISA).3,To explore the peripheral inflammatory changes of HS mice,the secretion of TNF-?,IL-1?,and IL-6 in the blood serum were detected by ELISA,endotoxin levels in the blood serum were detected by the limulusamebocyte lysate assay.The correlation between serum endotoxin and circulating inflammation was compared;also,the inflammatory cytokines in local spleen were detected by qPCR and HE.4,Pathological changes of BBB was evaluated by Evans blue(EB)dye,HE staining and TEM.The expression of matrix metalloproteinases 2 and matrix metalloproteinases 9 were determinated by Western Blotting.The intestinal barrier permeability was detected by 4-kDa FITC-dextran,HE staining and TEM.Results1,The HS model in mice was established by setting environment temperature(41.2 ± 0.5)?,relative humidity(60 ± 2)%,rectal temperature of continuous exposure to mice(42.4 ± 0.2)?.During or after HS,the mice appeared moving restlessly,dehydration,delirium,less activity or even coma,HS-1h group mice's rectal temperature significantly decreased,HS-24 h group mice's rectal temperature is slight higher than CON group;HE staining and TEM show that HS can damage cells and cause endoplasmic reticulum dysfunction,suggesting an obvious pathological changes of CNS.Thus,a HS mouse model with significant CNS injury is successfully established.2,At 1 and 6 h after HS,m RNA transcription and protein synthesis of TNF-?,IL-1?,and IL-6 in cortex and hippocampus were significantly increased compared to the CON group(P<0.01).At 24 h after HS,mRNA transcription and protein synthesis of TNF-?,IL-1?,and IL-6 in cortex and hippocampus slightly increased,but no significant change compared to the CON group(P>0.05).These results suggest that an obvious inflammatory effect was occurred 6 h after HS,and returned to normal levels at approximately 24 h.3,Compared to the CON group,at 1 h,6 h and 24 h after HS,the levels of TNF-?,IL-1?,IL-6,and endotoxin in peripheral circulations were significantly increased(P<0.01),the inflammatory factors peaked at 1 h,and the endotoxin concentration peaked at 6 h,and were sustained up to 24 h after HS.A similar pattern was observed in spleen.The results showed that there was a significant peripheral inflammatory response in the recovery stage of HS.4,HE staining and TEM show that brain capillaries surrounding edema and oppression of the vascular lumen,vascular endothelial cells connected closely,and swelling and endothelial disappeared,in the small intestine mucosa in shorter,sparse,loss of villi,indicating the damage of BBB and intestinal barrier structure after HS.At 1 h and 6 h after HS,brain MMP-2 and MMP-9 protein synthesis were significantly increased compared to the CON group(P<0.01),and were sustained up to 24 h after HS(P<0.01).At 1 h and 6 h after HS,EB content in the brain was increased significantly compared to the CON group(P<0.01),and EB content was still higher than the CON group 24 h after HS(P<0.01),indicating that HS can damage BBB and increase BBB permeability.Similarly,serum FD4 concentration was increased significantly 1 h and 6 h after HS compared to the CON group(P<0.01),and were sustained up to 24 h after HS,suggesting that HS can also damage the intestinal barrier.Conclusion1.During heat stroke recovery,obvious inflammation have been implicated within 6 h in the CNS,which returned to normal level approximately at 24 h.CNS inflammation may be closely associated with central nerve damage during HS.2.HS can damage the structure of intestinal barrier and increase its permeability,and then the gut endotoxin may act through the intestinal barrier into the blood circulation,causing significant peripheral inflammation within 24 h after HS.3.HS can damage the structure and permeability of the BBB,within 6 h after HS,peripheral inflammation may aggravate the permeability of BBB and the inflammatory responses of CNS.But through 24 h of HS recoveryCNS inflammation may characterized by independent development of inflammatory process,which was not affected by the peripheral inflammation... |
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