Acute Injuries In Multiple Tissues In A Rat Heatstroke Model Induced By Co-stress Of Lipopolysaccharide And Heat

Study background:Heat stroke is a life-threatening heat-related illness characterized by a rapidly increasing core body temperature and central nervous system disorder,can cause shock,acute lung injury(ALI)/ acute respiratory distress syndrome(ARDS),and multiple organ dysfunction syndrome(MODS).The mortality and morbidity of heatstroke may increase with global warming and the predicted worldwide increase in the frequency and intensity of heat waves.The mediation of metabolic changes and tissue damage is not fully understood.Our pilot study has shown that co-stress of exogenous lipopolysaccharides(LPS) and hyperthermia may advance and augment systemic inflammatory response syndrome and thus contributes to the acute lung injury seen in this illness.Objective:Use an experimental animal model for severe heatstroke induced by a combination of hyperthermia and LPS,to monitor tissue injuries of the heart,lung, brain,liver,kidney,and muscle in severe heatstroke,to characterize the systemic inflammatory response syndrome(SIRS) pathway in heatstroke and MODS,to allow further study of the intensive care and pathogenesis of severe heatstroke on targeting decrease the clinical course and improvement outcome.Materials and methods: Design:A 2(dry bulb temperature,Td)(2 levels:(35.0±0.5)℃,(26.0±0.5)℃)-by-2(drugs,Dr)(2 levels:LPS,NS)-by-4(time,Tin)(4 levels:0,40,80,and 120 min time point) factorial design,randomized controlled experiment.Setting:Animal chamber of mimic climate room in the institute of public hygiene and tropic medicine,Southern Medical University.Subjects:Total 96 male specific pathogen-free Wistar rats(body mass 190 to 210 g),obtained from the Medical Experimental Animal Center of Southern Medical University.Interventions:(1) Of total 96 rats,80 rats were randomly assigned to the following groups:saline-injected normothermic control(C-Group),saline-injected heat exposed(H-Group),LPS-injected normothermic control(L-Group),and LPS-injected heat exposed(HL-Group).Each group was divided into 4 subgroups: 0min,40min,80min,and 120min point subgroups(n=5).All animals were anesthetized by intraperitoneal injection of 3%pentobarbital(1ml/kg).Rats in L-/HL-Group were given an intravenous injection of LPS(Escherichia coli,0111:B4, 10 mg/kg) via the tail vein.Rats in C-/H-Group were given an intravenous injection of 0.9%NaCl(10 ml/kg) via the tail vein.Rats in C-/L-Group were exposed in a chamber at an ambient Td of(26±0.5) degrees,wet bulb temperature(Tw) of(21±0.5) degrees.Rats in H-/HL-Group were exposed in a chamber at an ambient Td of (35.0±0.5) degrees,Tw of(27±0.5) degrees.The relative humidity in the chamber was controlled at(40±5)%.In H/HL-group,the moment in which systolic arterial pressure dropped to a value of 60 mm Hg from the peak level was about 100 mins after the initiation of heat stress;this time point was arbitrarily taken as the onset of heat stroke.(2) 16 of tatal 96 rats were randomly assigned to H and HL-Group.Rats had heat exposure withdrawn at 100 mins and were allowed to recover at room temperature(26℃).The experiments were performed in a blinded and randomized fashion.End points:(1) Heat stress response:①Vital signs:rectal temperature(Tr), heart rate(HR),systolic arterial pressure(SAP),and respiratory rate(RR) were continually monitored;②Heat load and heat rate,stroke time(Tst,interval between the start of heat and the onset of heatstroke) survival time(Tsr,interval between the onset of heatstroke and animal death) during heatstroke were also monitored.(2) Biochemical measurements:①Plasma peripheral platelet count(PLT), activated partial thromboplastin(aPTT),prothrombin time(PT),hematocrit(Hct), and②serum alanine aminotrasferase(ALT),aspartate aminotransferase(AST), creatinine(Cr),and blood urea nitrogen(BUN) were detected at 1,40,80,and 120min time point;(3) Cytokine production level:①Plasma levels of Interleukin -10(IL-10), Interleukin -8(IL-8),Interleukin-1 receptor antagonist(IL-1ra),soluble Tumor Necrosis FactorⅠ(sTNFrⅠ),and②soluble Tumor Necrosis FactorⅡ(sTNFrⅡ), and tissue levels of Tumor Necrosis Factor-α(TNF-α),Interleukin -6(IL-6),and Interleukin -1β(IL-1β) in the liver,kidney,spleen,and adrenal gland were observed using enzyme linked immunosorbent assay(ELISA) kits at every time point;(4) Tissue damage of brains,heart,lungs,livers,kidneys,spleens,small intestine and muscle were evaluated histologically at 120 min after treatment,and the expression of TNF-αin tissue were detected in situ by immunohistochemical SABC method.Statistical analyses:Statistical analyses were performed using SPSS for Windows(version 13.0.,offered by medical biostatistical institute(institute of public hygiene and tropic medicine,Southern Medical University).Data were presented as mean and standard deviation of the mean.To test statistical differences,analysis of variance(ANOVA) was used,followed by least significant difference(LSD) t-test for evaluation of multiple comparisons of means where appropriate.The independent sample t test was used for quantitative variables(to compare Tst and Tsr between H and HL-groups).The level of significance was set at 0.05.Results:Statistical result:Main effects.There were significant main effects①of Tm in vital signs,plasma PLT,aPTT,PT,serum ALT,AST,BUN,plasma IL-10,IL-8, IL-1ra,sTNFrⅠ,sTNFrⅡ,and TNF-α,IL-6,and IL-1βin livers and spleens,TNF-α in kidneys,and TNF-αand IL-6 in adrenal gland(P＜0.05),②of Td in vital signs, plasma PLT,aPTT,PT,serum ALT,AST,BUN,plasma IL-8,IL-1ra,sTNFrⅠ,and TNF-α,IL-6,and IL-1βin spleens,TNF-αand IL-6 in livers and adrenal glands, TNF-αin kidneys(P＜0.05);③of Dr in HR,SAP,plasma PLT,aPTT,PT,serum AST, BUN,plasma IL-10,IL-8,IL-1ra,sTNFrⅠ,sTNFrⅡ,and TNF-α,IL-6,and IL-1βin spleens,TNF-αand IL-6 in livers and adrenal glands(P＜0.05).Fix the level of Td and Dr,one-way ANOVA of these variables at 4 time points were shown(followed by LSD) as follows:(1) Heat stress responseVital signsHyperthermia stressed rats displayed significantly high values of Tr from 30 min time point on(P＜0.01,vs.C-/L-Group) and there were no significant differences between HL-Group and H-Group(P＞0.05).The range of body temperature in H-Group and HL-Group was from 34.2℃to 43.3℃and 34.3℃to 43.5℃respectively.SAP in H-/HL-Group increased gradually but significantly(P＜0.01,vs. C-/L-Group) and peaked at 110-120 min time point,then decreased dramatically.The values of SAP in HL-Group were markedly lower than those in H-Group(P＜0.01). The range of SAP in H-Group and HL-Group was from 110 mmHg to 155 mmHg and 69 mmHg to 159 mmHg respectively.All rat in HL-Group were in shock(SAP＜90 mm Hg) at 120 min time point.The Tr kept steady in L-Group and there was no significant difference between L-Group and C-Group(P＞0.05).At 50～60 min time point,the values of SAP in L-Group decreased temporarily(P＜0.01,vs. C-/H-/HL-Group),then returned at baseline and kept steady.Tr.The rats in H-/HL-Group displayed significantly high values of Tr at 40min～120min time point(P＜0.01,vs.C-/L-Group) and there were no significant differences in the values of Tr between HL-Group and H-Group(P＞0.05).The Tr kept steady in L-Group and there was no significant difference in the values of Tr between L-Group and C-Group(P＞0.05).HR.The rats in H-/HL-Group displayed significantly high values of HR at 80min～120min time point(P＜0.01,vs.C-/L-Group) and there was a significant difference in the values of HR between HL-Group and H-Group at 120min time point (P＜0.01).The HR kept steady in L-Group and there was no significant difference in the values of HR between L-Group and C-Group(P＞0.05).SAP.The rats in H-/HL-Group displayed significantly high values of SAP at 80min and significantly low values of SAP at 120min time point(P＜0.01,vs. C-/L-Group) and there was a significant difference in the values of SAP between HL-Group and H-Group at 120min time point(P＜0.01).The SAP kept steady in L-Group and there was no significant difference in the values of SAP between L-Group and C-Group at these time points(P＞0.05).RR.The rats in H-/HL-Group displayed significantly high values of RR at 80min～120min time points and there were no significant difference in the values of RR between HL-Group and H-Group(P＞0.05).The RR kept steady in L-Group and there was no significant difference in the values of RR between L-Group and C-Group at these time points(P＞0.05).Heat stress responseHeat load and heat rate.The values of heat load in HL-Group were significantly higher than those in H-Group(t=-2.613,P=0.020).There was no significant difference in the values of heat rate between hyperthnic rats(t=-1.768,P=0.096).Tst and Tsr.There was significant differece in the values of Tst and Tsr between H/HL-Group.Tst and Tsr in HL-Group were significantky shorter than those in H-Group.(2) Biochemical measurementsPLT:The rats in H-Group displayed significantly high values of PLT from 80min time points(P＜0.01 vs.C-Group).The rats in L-Group displayed significantly low values of PLT from 80min time point on compared with H-/HL-Group(P＜0.05). According to LSD,there was a significant difference in PLT in every 2 groups at 120min time point(P＜0.05).The highest values were shown in H-Group.The values in C-Group were in the next place,and those in HL-Group next them;aPTT:There was a significant difference in aPTT between Groups(P＜0.05) from 80min time point on.According to LSD,there was a significant difference in aPTT in H-/HL-Group(P＜0.05 vs.C/L-Group);PT:There was a significant difference in PT between Groups from 80min time point on(P＜0.05).According to LSD,there was a significant difference in PT in H-/HL-Group(P＜0.05 vs.C/L-Group);ALT:There was a significant difference in ALT between Groups(P＜0.05) at 120min time point.According to LSD,there was a significant difference in ALT in HL-Group(P＜0.01 vs.C-Group);AST:There was a significant difference in AST between Groups from 40min time point on(P＜0.05).According to LSD,the rats in L/H/HL-Group displayed significantly high values of AST at 40 to 80min time points(P＜0.05 vs.C-Group). At 120min time point,the values of AST in HL-Group were significantly higher than those in other 3 Groups(P＜0.05);BUN:There was a significant difference in BUN between Groups from 40min time point on(P＜0.05).According to LSD,there was a significant difference in BUN in every 2 groups at 120min time point(P＜0.05).The highest values were shown in HL-Group.The values in H-Group were in the next place,and those in L-Group next them.(3) CytokinesPlasma IL-8,IL-10,IL-1ra and sTNFrⅠ/Ⅰconcentration:The baseline levels for cytokines and cytokine receptors were similar between the 4 groups and remained unchanged in the control group throughout the experiment.IL-10:According to LSD,the rats in HL-Group displayed significantly high values of IL-10 from their baseline levels from 40min time points on(P＜0.01 vs. C-Group) and peaked at 80min time point(10-fold of those in C -Group,P＜0.01 vs. C/L/H-Group).The value of IL-10 deceased at 120min point in co-stressed rats,but it was still significantly higher than those in C/H-Group(P＜0.05);IL-8:According to LSD,the rats in HL-Group displayed significantly high values of IL-8 from their baseline levels from 40min time points on(P＜0.01 vs. C-Group) and peaked at 120min time point(3-fold of those in C -Group,P＜0.01 vs. C-Group);IL-1ra:According to LSD,the rats in HL-Group displayed significantly high values of IL-1ra from their baseline levels from 40min time points on(P＜0.01 vs. C-Group) and peaked at 120min time point(300-fold of those in C -Group,P＜0.01 vs.C/L/H-Group);STNFrⅠ:According to LSD,the rats in HL-Group displayed significantly high values of STNFrⅠfrom their baseline levels from 40min time points on(P＜0.01 vs. C-Group) and peaked at 80min time point(8-10-fold of those in C -Group,P＜0.01 vs.C/L/H -Group).The value of STNFrⅠdeceased at 120min point in co-stressed rats, but it was still significantly higher than those in C/H-Group(P＜0.05);STNFrⅡ:According to LSD,the rats in HL-Group displayed significantly high values of STNFrⅠfrom their baseline levels from 40min time points on(P＜0.01 vs. C-Group) and peaked at 120min time point(3-4-fold of those in C -Group,P＜0.01 vs.C/H-Group).Tissue TNF-α,IL-6,and IL-1βconcentrationLiver TNF-α,IL-6,and IL-1βconcentrationTNF-α:According to LSD,the livers in HL-Group displayed significantly high values of TNF-αfrom their baseline levels from 80min time points on(P＜0.01 vs. C/L/H-Group) and peaked at 120min time point(3-4 times higher than those in C-Group,P＜0.01 vs.C/H -Group).The value of TNF-αdeceased at 120min point in livers of co-stressed rats,but it was still significantly higher than those in C/H-Group (P＜0.05);IL-6:According to LSD,the rats in HL-Group displayed significantly high values of IL-6 in livers from their baseline levels at 120min time points(3-4 times higher than those in C-Groups,P＜0.01 vs.C/L/H -Group);IL-1β:The value of IL-1βkept stable in L/H-Group.The value of IL-1βinceased at 120min point in livers of co-stressed rats,but it was still not significant;Kidneyr TNF-αconcentrationTNF-α:According to LSD,the kidney in HL-Group displayed significantly high values of TNF-αfrom their baseline levels from 80min time points on(3-4 times higher than those in C-Group,P＜0.01 vs.C/L/H -Group).The value of TNF-αin co-stressed rats decreased and there was no significant diference between any 2 groups;Adrenal gland TNF-α,IL-6,and IL-1βconcentrationTNF-α:According to LSD,the livers in HL-Group displayed significantly high values of TNF-αfrom their baseline levels from 80min time points on(5-6 times higher than those in C-Group,P＜0.01 vs.C/L/H -Group).The value of TNF-αin co-stressed rats decreased and there was no significant diference between any 2 groups;IL-6:According to LSD,the IL-6 value in adrenal gland in HL-Group was significantly higher at 120min time points(3-4 times higher than those in C-Group,P＜0.01 vs.C/L/H -Group);IL-1β:The value of IL-1βkept stable in L/H-Group.The value of IL- 1βinceased at 120min point in adrenal gland of co-stressed rats,but it was still not significant;Spleen TNF-α,IL-6,and IL-1βconcentrationTNF-α:According to LSD,the spleens in HL-Group displayed significantly high values of TNF-αfrom their baseline levels from 40min time points on(P＜0.01 vs. C/L/H-Group) and peaked at 80min time point(15 times higher than those in C-Group,P＜0.01 vs.C/H -Group).The value of TNF-αdeceased at 120min point in spleen of co-stressed rats,but it was still significantly higher than those in C/H-Group (P＜0.05);IL-6:According to LSD,the IL-6 value in spleen in HL-Group was significantly higher at 120min time points(3-4 times higher than those in C-Group,P＜0.01 vs. C/L/H -Group);IL-1β:According to LSD,the spleens in HL-Group displayed significantly high values of IL-1βfrom their baseline levels from 80min time points on(P＜0.01 vs. C/L/H-Group) and peaked at 120min time point(3-4 times higher than those in C-Group,P＜0.01 vs.C/L/H -Group).(4) Acute injuries in multiple tissues (Histological examination by hematoxylin and eosin staining)At 120min time point,histological examination revealed that the heart,the brain, the lung,the liver,the kidney,the spleen,the muscle,and the small intestine of the rats co-treated with hyperthermia and LPS(HL) had more intensive morphologic abnormalities than those of rats in H/L Group.Histological examination of the heart:Acute injury of myocardial cell was observed in co-stressed rats,such as edema and degeneration,and congestion,edema and inflammatory neutrophil infiltration were also noted in myocytes;Histological examination of the brain:Acute injury of brain cell was observed in co-stressed rats,such as edema and degeneration in brain cell,glial cell and neuron swelling,karyopyknosis,karyorrhexis,thinning or loss of Nissl's body and local congestion and necrosis of neural cells were also observed;Histological examination of the lung:The histological changes in the HL-Group at 120min time point characterized by interstitial edema and neutrophil infiltration, perivascular edema,intra-alveolar hemorrhage,attenuation of bronchial epithelium, the appearance of lymphocytes and PMN,and diffuse hyaline membranes of the lung;Histological examination of the liver:Congestion in liver central venous and sinus were seen,mainly in the area of hepatic lobules.Parts of the plasma in liver cell were slakend,net-shaped or emptied.Infiltration and erythrocytes and neutrophils infiltration were observed in the liver in rats under co-stress.Histological examination of the kidney:Inflammatory infiltrates as well as cellular swelling hemorrhage and necrosis were noted in the renal interstitium and renal tubulointerstitial in the kidney of co-stressed rats.Histological examination of the spleen:In the spleen,boundaries between red pulp and white pulp were unclear,some structure of splenic corpuscle disappeared.Histological examination of the muscle:Acute injury of musle cell was observed in co-stressed rats,such as edema and degeneration in brain cell,karyopyknosis and karyorrhexis in musle cells were also observed;Histological examination of the small intestine:There were edemas and mucosal erosion in the intestinals.The desquamation of intestine villi and infiltration of inflammatory cells were also observed.Conclusion:1.The present study shows for the first time that the experimental rat model for severe heatstroke can be replicated by a combination of hyperthermia and LPS. The rats displayed a uniform heat stress response and reacted similarly to humans with moderate to severe heatstroke,in terms of inflammatory responses.Heat stess response(such as vital signs,heat load and heat rate) injuries in multiple tissues and circular failure reproduced closely the clinical manifestations in human disease.The rats could therefore be a suitable model for further study on the inflammatory pathway in heatstroke and for testing whether therapeutic intervention and intensive care targeting this pathway can alter the clinical course and improve outcome.2.Endoxemia plays a critical role in the process of moderate to severe heatstroke. Under heat stress,endoxemia may though SIRS pathway advance and augment disorders in vital signs,break balance between coagulation and anticoagulation, produce excessive inflammatory mediators,render the host at risk from heat stress to circulatory failure,severe heatsroke or even acute injuries in multiple organs.3.Co-stress of LPS and heat primes the rat to advance and augment systemic inflammatory response syndrome,as indicated by increased production of IL-1ra, sTNFrⅠ/Ⅱ,IL-8,IL-10 in plasma,and TNF-α,IL-6,and IL-1βin tissue,key mediators that modulate local and systemic acute inflammatory response.As in human,the inflammatory response was suitable during the course of moderate and was exacerbated when h??troke was more severe.