过热复合脂多糖致热射病大鼠动物模型多器官急性损伤的实验研究
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摘要
研究背景:热射病为重症热损伤,主要临床表现为体核温度骤升,出现中枢神经系统异常,可导致休克、靶器官损伤以及多器官功能障碍综合征(multipleorgan dysfunction syndrome,MODS),其发病率及病死率可能随着全球气候变暖以及热浪袭击频率和强度的逐年增加而上升。热射病代谢改变及组织损伤的介导机制至今未明。我们的前期研究显示,过热与外源性内毒素(脂多糖,lipopolysaccharides,LPS)复合打击可促发和加重炎症反应,造成急性肺损伤。
目的:运用过热与脂多糖复合打击技术复制重度热射病大鼠的动物模型,监测该模型心、肺、脑、肝、肾、骨骼肌等组织的病理生理损伤特点,探讨重度热射病、MODS的全身炎症反应综合征(systemic inflammatory responsesyndrome,SIRS)启动途径的特征,为重度热射病严密监护及发病机制方面的深入研究提供依据,以期缩短病程、改善预后。
材料与方法:
实验设计2(干球温度(Td),2水平:(35.0±0.5)℃、(26.0±0.5)℃)×2(药物(Dr),2水平:LPS、注射用生理盐水(NS))×4(应激时间(Tm),4水平:0min、40min、80min、120min)析因设计,随机对照实验。
实验环境南方医科大学公共卫生与热带卫生学学院仿真气候室动物舱。
实验对象无特殊病原体动物(Special pathogen free animal,SPF动物)级雄性Wistar大鼠96只,体质量(190～210)g,购自南方医科大学实验动物中心。
处理(1)将80只大鼠随机分为4大组,即常温生理盐水应激组(C组)、高温NS应激组(H组)、常温LPS应激组(L组)、高温+LPS应激组(HL组),各大组下设应激后0min、40min、80min、120min亚组,每组5只动物(n=5)。以3%戊巴比妥钠1ml/kg腹腔注射麻醉动物,L组、HL组动物经尾静脉注射LPS10mg/kg(Escherichia coli,0111:B4,1mg/ml),C组、H组动物经尾静脉注射NS 10ml/kg。将动物置入仿真热气候室动物舱行热暴露。H组、HL组动物暴露条件:Td:(35.0±0.5)℃,湿球温度(Tw):(27.0±0.5)℃;C组、L组动物暴露条件:Td:(26.0±0.5)℃,Tw:(21.0±0.5)℃,各实验组动物暴露环境相对湿度(RH)均为(40±5)%;(2)16只接受热应激大鼠随机分2组(n=8):H组、HL组,麻醉、暴露温度及药物处理同前,暴露100min后置于Td:(26.0±0.5)℃,Tw:(21.0±0.5)℃环境自主恢复。
主要终点指标
(1)热应激反应的指标:①持续监测应激动物生命体征(直肠温度(rectaltemperature,Tr)),心率(heart rate,HR),动脉收缩压(systolic arterial pressure,SAP),呼吸频率(respiratory rate,RR)的动态变化;②16只接受热应激大鼠的热应激反应:热负荷(heat load)、热速率(heat rate)、SAP的最高值(SAP max)及最低值(SAPmix)、动物从接受热暴露至发生热射病的时间(stroke time,Tst)以及发生热射病后的存活时间(survival time,Tsr);
(2)生化指标:于0min、40min、80min、120min时相点,检测:①外周血血小板计数(peripheric platelet count,PLT),血浆活化部分凝血活酶时间(activatedpartial thromboplastin,aPTT),凝血酶原时间(prothrombin time,PT),红细胞比容(hematocrit,Hct)以及②血清丙氨酸氨基转移酶(alanine aminotrasferase,ALT)、天门冬酸转移酶(aspartate aminotransferase,AST)、肌酐(creatinine,Cr)、尿素氮(blood urea nitrogen,BUN)含量等??
(3)细胞因子水平检测:采用固相三明治夹心、酶联免疫吸附实验(enzymelinked immunosorbent assay,ELISA)法检测0min、40min、80min、120min时相点①血浆白细胞介素-10(Interleukin -10,IL-10),白细胞介素-8(Interleukin-8,IL-8),白细胞介素-1受体拮抗剂(Interleukin-1 receplor antagonist,IL-1ra)、可溶性TNF受体Ⅰ、Ⅱ(soluble Tumor Necrosis FactorⅠandⅡ)及②肝、肾、脾、肾上腺组织炎症介质肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)、白细胞介素-1β(Interleukin-1β,IL-1β)含量;
(4)组织学检查:120min时各组动物心、肺、脑、肝、肾、脾、骨骼肌等组织HE染色光镜观察(HL组各时相点均查);免疫组织化学法观察应激120min时各组动物心、肺、脑、肝、肾、脾、骨骼肌等组织TNF-α染色。
统计学分析计量资料数据用(?)±s表示,运用SPSS for Windows软件包(13.0版本,南方医科大学公共卫生与热带医学学院生物统计系提供),采用析因分析(analysis of variance,ANOVA)进行不同温度、时间、药物水平各监测指标间差异的主效应分析,固定时间水平,进行各指标的单因素方差分析(ony-way ANOVA),均数间多重比较用最小有意义差异(least significant difference,LSD)t检验。H/HL组间Tst以及Tsr的均数比较采用独立样本,检验(independent-sample t test)。认为P≤0.05为显著性水准。
结果:
主效应的方差分析表明,不同时间水平动物生命体征及血浆PLT、aPTT、PT、血清ALT、AST、BUN、血浆IL-1ra、sTNFrⅠ/Ⅱ、IL-8、IL-10、肝及脾组织TNF-α、IL-6、IL-1β、肾组织TNF-α、肾上腺组织TNF-α、IL-6含量均存在统计学差异(P＜0.05);不同温度水平存在显著性差异的指标包括生命体征及血浆PLT、aPTT、PT、血清ALT、AST、BUN、血浆IL-1ra、sTNFrⅠ、IL-8、脾组织TNF-α、IL-6、IL-1β、肝及肾上腺组织TNF-α、IL-6、肾组织TNF-α含量(P＜0.05);不同药物水平存在显著性差异的指标包括生命体征中的HR、SAP及血浆PLT、aPTT、PT、血清AST、BUN、血浆IL-1ra、sTNFrⅠ/Ⅱ、IL-8、IL-10、脾组织TNF-α、IL-6、IL-1β、肝及肾上腺组织TNF-α、IL-6含量。固定温度、药物水平,不同时相点各指标单向方差分析(one-way ANOVA)表明(以LSD做后续检验):
(1)热应激反应
实验30min起,热应激动物Tr即显著升高(P＜0.01,vs.C-/L-Group),但H/HL组间无显著性差异(P＞0.05)。H组、HL组动物的Tr波动范围分别为(34.2℃-43.3℃)和(34.3℃-43.5℃)。热应激组动物SAP值缓慢升高,于应激50min起高于常温组(P＜0.01,vs.C-/L-Group),并于100min-110min达峰值,随后急剧下降,此时HL组动物SAP值显著低于H组SAP水平。H组、HL组动物的SAP波动范围分别为(118mmHg-155mmHg)和(75mmHg-155mmHg)。120min时相点时HL组动物均发生休克。L组动物在应激50min-60min时SAP值显著降低,此外SAP稳定,与C组比较差异无统计学意义(P＞0.05)。
Tr:应激40min-120min,H组、HL组动物Tr水平显著升高,与C组、L组比较差异有统计学意义(P＜0.01),但H组、HL组组间差异无统计学意义(P＞0.05)。L组动物Tr水平较平稳,与C组比较差异无统计学意义(P＞0.05);
HR:应激80min～120min时相点,H组、HL组动物HR水平显著升高,与C组、L组比较差异有统计学意义(P＜0.01),HL组HR显著快于H组,其差异有统计学意义(P＜0.01);L组动物HR较平稳,与C组比较差异无统计学意义(P＞0.05);
SAP:H组、HL组SAP于应激80min时显著上升,于应激120min时显著下降,与C组、L组比较差异有统计学意义(P＜0.01);HL组动物SAP显著低于H组SAP水平,其差异有统计学意义(P＜0.01)。上述时相点L组动物SAP与C组比较差异无统计学意义(P＞0.05);
RR:H组、HL组于应激80min是RR显著加快,与C组比较差异有统计学意义(P=0.000),应激120min时H组、HL组RR约为正常对照值的2倍,与C组比较差异有统计学意义(P=0.000),但两者之间差异无统计学意义(P=0.940);L组与C组比较差异无统计学意义(P=0.304);应激80min起,L组RR与H组、HL组比较,差异有统计学意义(P=0.000)。
H组热负荷值显著低于HL组(t=-2.613,P=0.020),但2热应激组热速率不存在统计学差异(t=-1.768,P=0.096)。H、HL组间SAP max及SAP mix存在统计学差异,HL组SAP的波动幅度加大。
Tst及Tsr:H组、HL组动物Tst、Tsr存在显著性差异,复合应激组动物热应激后发生热射病的时间显著提前,热射病后存活时间显著缩短(P＜0.05,vs.H-Group)。
(2)生化指标
PLT:应激80min起H组动物PLT计数显著升高,与C组比较差异具有统计学意义(P＜0.05),L组动物PLT计数显著下降,与H组、HL组比较差异具有统计学意义(P＜0.05)。应激120min时,各组间PLT计数水平均存在统计学差异(P＜0.01),以LSD法做后续检验,LPT由高至低组别依次为H组、C组、HL组、L组:
aPTT:应激80min起时热应激组动物aPTT差异具有统计学意义(P＜0.05),以LSD法做后续检验,H/HL组与C/L组动物血清aPTT水平存在显著性差异(P＜0.05);
PT:应激80min起时热应激组动物PT差异具有统计学意义(P＜0.05),以LSD法做后续检验,H/HL组与C/L组动物血清PT水平存在显著性差异(P＜0.05);
ALT:应激120min时ALT差异具有统计学意义(P＜0.05),以LSD法做后续检验,HL组与C组动物血清ALT水平存在显著性差异(P＜0.01);
AST:应激40min起各组间AST差异具有统计学意义(P＜0.05),应激40min、80min时相点L组、H组、HL组血清AST水平显著升高(P＜0.05,vs.C-Group),应激120min时,HL组AST水平显著高于其余3组(P＜0.01);
Urea:应激40min起L组、H组、HL组动物血清Urea显著升高,与C组比较差异具有统计学意义(P＜0.05)。应激40min起,各组间Urea水平均存在统计学差异(P＜0.05),后续检验血Urea由高至低组别依次为HL组、H组、L组、C组。
(3)炎症介质水平
循环炎症介质
IL-10:HL组动物血浆IL-10水平于应激40min时显著上升,于应激80min时达峰值(约10倍对照组水平),与C、L、H组比较差异有统计学意义(P值分别小于0.001,0.05,0.001);120min时IL-10水平下降,仍显著高于C、H组(P值分别小于0.001,0.05);
IL-8:HL组动物血浆IL-8水平于应激40min时显著上升,于应激120min时达峰值(约3倍对照组水平),与C组比较差异有统计学意义(P＜0.001);
IL-1ra:HL组动物血浆IL-1ra水平于应激40min时显著上升,于应激120min时达峰值(约300倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);
sTNFrⅠ:HL组动物血浆sTNFrⅠ水平于应激40min时显著上升,于应激80min时达峰值(约8-10倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001),120min时sTNFrⅠ水平下降,仍显著高于C、H组(P＜0.001);
sTNFrⅡ:HL组动物血浆sTNFrⅡ水平于应激40min时显著上升,于应激120min时达峰值(约3-4倍对照组水平),与C、H组比较差异有统计学意义(P值分别小于0.01、0.05)。
组织炎症介质
120min时相点时,病理检查结果显示,复合打击动物的心、脑、肺、肝,肾、脾、骨骼肌及小肠组织的病理损伤较单因素打击组为重。
肝组织炎症介质:
TNF-α:HL组动物肝TNF-α水平于应激80min时显著上升并达峰值(约3-4倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,仍显著高于C、H组(P＜0.05);
IL-6:HL组动物肝IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:L、H组动物肝IL-1β水平较稳定,尽管HL组于120min时有所升高,但与C组比较差异无统计学意义(P＞0.05)。
肾组织炎症介质
NF-α:HL组动物肾TNF-α水平于应激80min时显著上升并达峰值(约3-4倍对照组水平),与C、L、H组比较差异有统计学意义(P值分别为0.01,0.05,0.01);120min时TNF-α水平下降,各组间均不存在统计学差异(P＞0.05);
肾上腺炎症介质
TNF-α:HL组动物。肾上腺TNF-α水平于应激80min时显著上升并达峰值(约5.6倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,与C、H组不存在显著性差异(P＞0.05);
IL-6:HL组动物肾上腺IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:L、H组动物肾上腺IL-1β水平较稳定,尽管HL组于120min时有所升高,但与C组比较差异无统计学意义(P＞0.05)。
脾组织炎症介质
TNF-α:HL组动物脾TNF-α水平于应激40min时显著上升并于80min达峰值(约15倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,仍显著高于C、H组(P＜0.05);
IL-6:HL组动物脾IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:HL组动物脾IL-1β水平于应激80min时显著上升,于120min达峰值约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
(4)组织病理学改变
120min时,HE染色光镜观察发现,LPS与高温复合打击动物的心、肺、脑、肝、肾、脾、骨骼肌等组织出现病理损伤,程度重于比L/H组:
心组织学改变:HL组心肌细胞明显水肿、变性,肌间血管充血、水肿、炎细胞浸润。
脑组织学改变:HL组动物脑组织充血、水肿,神经元胞体及神经胶质细胞明显肿胀,部分神经尼氏小体消失,局部神经细胞变性、坏死,脑组织充血、水肿。
肺组织学改变:HL组动物120min时肺组织出现急性微血管损伤改变,表现为肺泡及间质水肿,淋巴细胞、PMN浸润,肺泡壁厚薄不均、透明膜形成、肺出血等。
肝组织学改变:HL组动物肝中央静脉及肝血窦明显淤血,主要在肝小叶中间区。部分肝细胞浆高度疏松,呈稀网状或空泡。中央静脉及其临近肝窦扩张、充血。汇管区血管亦见扩张、充血及炎细胞浸润。
肾组织学改变:HE染色观察可见肾间质及肾小管炎性浸润、细胞肿胀,并出现出血与坏死。
脾组织学改变:HL组动物脾血管及血窦明显扩张、淤血,红髓及白髓分界不清,脾小体结构消失。
骨骼肌组织学改变:HL组骨骼肌细胞明显水肿、变性,部分骨骼肌细胞核固缩及凝固性坏死。
小肠组织学改变:HL组粘膜层明显充血水肿,肠粘膜糜烂,肠绒毛结构破坏,粘膜上皮坏死、脱落。
结论:
1.本研究首次发现,高温与LPS同时打击可以复制重度热射病大鼠多器官急性损伤的动物模型。从炎症反应角度讲,动物的反应与人由中度热射病发展为重度热射病的反应具有相似性、重复性。动物热应激反应(如生命体征、热负荷及热速率)、循环衰竭及多器官急性损伤等表现与人重度热射病的临床表现相近。该模型可用于重度热射病炎症反应途径启动机制、评价治疗及重症监护措施对重度热射病病程及预后影响的进一步研究。
2.内毒素血症在从热射病向重度热射病发展进程中起重要作用。热应激下内毒素血症可通过SIRS途径造成生命体征、凝血.抗凝血平衡紊乱,使炎症介质表达异常,促使热应激个体出现循环衰竭、重度热射病甚至多器官急性损伤。
3.复合打击可促发和加重全身炎症反应综合征,表现为血浆细胞因了IL-1ra、sTNFrⅠ/Ⅱ)、IL-8、IL-10及组织细胞因子TNF-α、IL-1α、IL-6含量显著升高。这些细胞因子在调节局部及全身急性炎症反应中至关重要,所介导的炎症反应过程与人中度热射病及重度热射病的发展进程相一致。
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 response
Vital signs
Hyperthermia 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 response
Heat 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 measurements
PLT: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) Cytokines
Plasma 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-1βconcentration
Liver TNF-α,IL-6,and IL-1βconcentration
TNF-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-αconcentration
TNF-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-1βconcentration
TNF-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-1βconcentration
TNF-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.
目的:运用过热与脂多糖复合打击技术复制重度热射病大鼠的动物模型,监测该模型心、肺、脑、肝、肾、骨骼肌等组织的病理生理损伤特点,探讨重度热射病、MODS的全身炎症反应综合征(systemic inflammatory responsesyndrome,SIRS)启动途径的特征,为重度热射病严密监护及发病机制方面的深入研究提供依据,以期缩短病程、改善预后。
材料与方法:
实验设计2(干球温度(Td),2水平:(35.0±0.5)℃、(26.0±0.5)℃)×2(药物(Dr),2水平:LPS、注射用生理盐水(NS))×4(应激时间(Tm),4水平:0min、40min、80min、120min)析因设计,随机对照实验。
实验环境南方医科大学公共卫生与热带卫生学学院仿真气候室动物舱。
实验对象无特殊病原体动物(Special pathogen free animal,SPF动物)级雄性Wistar大鼠96只,体质量(190～210)g,购自南方医科大学实验动物中心。
处理(1)将80只大鼠随机分为4大组,即常温生理盐水应激组(C组)、高温NS应激组(H组)、常温LPS应激组(L组)、高温+LPS应激组(HL组),各大组下设应激后0min、40min、80min、120min亚组,每组5只动物(n=5)。以3%戊巴比妥钠1ml/kg腹腔注射麻醉动物,L组、HL组动物经尾静脉注射LPS10mg/kg(Escherichia coli,0111:B4,1mg/ml),C组、H组动物经尾静脉注射NS 10ml/kg。将动物置入仿真热气候室动物舱行热暴露。H组、HL组动物暴露条件:Td:(35.0±0.5)℃,湿球温度(Tw):(27.0±0.5)℃;C组、L组动物暴露条件:Td:(26.0±0.5)℃,Tw:(21.0±0.5)℃,各实验组动物暴露环境相对湿度(RH)均为(40±5)%;(2)16只接受热应激大鼠随机分2组(n=8):H组、HL组,麻醉、暴露温度及药物处理同前,暴露100min后置于Td:(26.0±0.5)℃,Tw:(21.0±0.5)℃环境自主恢复。
主要终点指标
(1)热应激反应的指标:①持续监测应激动物生命体征(直肠温度(rectaltemperature,Tr)),心率(heart rate,HR),动脉收缩压(systolic arterial pressure,SAP),呼吸频率(respiratory rate,RR)的动态变化;②16只接受热应激大鼠的热应激反应:热负荷(heat load)、热速率(heat rate)、SAP的最高值(SAP max)及最低值(SAPmix)、动物从接受热暴露至发生热射病的时间(stroke time,Tst)以及发生热射病后的存活时间(survival time,Tsr);
(2)生化指标:于0min、40min、80min、120min时相点,检测:①外周血血小板计数(peripheric platelet count,PLT),血浆活化部分凝血活酶时间(activatedpartial thromboplastin,aPTT),凝血酶原时间(prothrombin time,PT),红细胞比容(hematocrit,Hct)以及②血清丙氨酸氨基转移酶(alanine aminotrasferase,ALT)、天门冬酸转移酶(aspartate aminotransferase,AST)、肌酐(creatinine,Cr)、尿素氮(blood urea nitrogen,BUN)含量等??
(3)细胞因子水平检测:采用固相三明治夹心、酶联免疫吸附实验(enzymelinked immunosorbent assay,ELISA)法检测0min、40min、80min、120min时相点①血浆白细胞介素-10(Interleukin -10,IL-10),白细胞介素-8(Interleukin-8,IL-8),白细胞介素-1受体拮抗剂(Interleukin-1 receplor antagonist,IL-1ra)、可溶性TNF受体Ⅰ、Ⅱ(soluble Tumor Necrosis FactorⅠandⅡ)及②肝、肾、脾、肾上腺组织炎症介质肿瘤坏死因子-α(Tumor Necrosis Factor-α,TNF-α)、白细胞介素-6(Interleukin-6,IL-6)、白细胞介素-1β(Interleukin-1β,IL-1β)含量;
(4)组织学检查:120min时各组动物心、肺、脑、肝、肾、脾、骨骼肌等组织HE染色光镜观察(HL组各时相点均查);免疫组织化学法观察应激120min时各组动物心、肺、脑、肝、肾、脾、骨骼肌等组织TNF-α染色。
统计学分析计量资料数据用(?)±s表示,运用SPSS for Windows软件包(13.0版本,南方医科大学公共卫生与热带医学学院生物统计系提供),采用析因分析(analysis of variance,ANOVA)进行不同温度、时间、药物水平各监测指标间差异的主效应分析,固定时间水平,进行各指标的单因素方差分析(ony-way ANOVA),均数间多重比较用最小有意义差异(least significant difference,LSD)t检验。H/HL组间Tst以及Tsr的均数比较采用独立样本,检验(independent-sample t test)。认为P≤0.05为显著性水准。
结果:
主效应的方差分析表明,不同时间水平动物生命体征及血浆PLT、aPTT、PT、血清ALT、AST、BUN、血浆IL-1ra、sTNFrⅠ/Ⅱ、IL-8、IL-10、肝及脾组织TNF-α、IL-6、IL-1β、肾组织TNF-α、肾上腺组织TNF-α、IL-6含量均存在统计学差异(P＜0.05);不同温度水平存在显著性差异的指标包括生命体征及血浆PLT、aPTT、PT、血清ALT、AST、BUN、血浆IL-1ra、sTNFrⅠ、IL-8、脾组织TNF-α、IL-6、IL-1β、肝及肾上腺组织TNF-α、IL-6、肾组织TNF-α含量(P＜0.05);不同药物水平存在显著性差异的指标包括生命体征中的HR、SAP及血浆PLT、aPTT、PT、血清AST、BUN、血浆IL-1ra、sTNFrⅠ/Ⅱ、IL-8、IL-10、脾组织TNF-α、IL-6、IL-1β、肝及肾上腺组织TNF-α、IL-6含量。固定温度、药物水平,不同时相点各指标单向方差分析(one-way ANOVA)表明(以LSD做后续检验):
(1)热应激反应
实验30min起,热应激动物Tr即显著升高(P＜0.01,vs.C-/L-Group),但H/HL组间无显著性差异(P＞0.05)。H组、HL组动物的Tr波动范围分别为(34.2℃-43.3℃)和(34.3℃-43.5℃)。热应激组动物SAP值缓慢升高,于应激50min起高于常温组(P＜0.01,vs.C-/L-Group),并于100min-110min达峰值,随后急剧下降,此时HL组动物SAP值显著低于H组SAP水平。H组、HL组动物的SAP波动范围分别为(118mmHg-155mmHg)和(75mmHg-155mmHg)。120min时相点时HL组动物均发生休克。L组动物在应激50min-60min时SAP值显著降低,此外SAP稳定,与C组比较差异无统计学意义(P＞0.05)。
Tr:应激40min-120min,H组、HL组动物Tr水平显著升高,与C组、L组比较差异有统计学意义(P＜0.01),但H组、HL组组间差异无统计学意义(P＞0.05)。L组动物Tr水平较平稳,与C组比较差异无统计学意义(P＞0.05);
HR:应激80min～120min时相点,H组、HL组动物HR水平显著升高,与C组、L组比较差异有统计学意义(P＜0.01),HL组HR显著快于H组,其差异有统计学意义(P＜0.01);L组动物HR较平稳,与C组比较差异无统计学意义(P＞0.05);
SAP:H组、HL组SAP于应激80min时显著上升,于应激120min时显著下降,与C组、L组比较差异有统计学意义(P＜0.01);HL组动物SAP显著低于H组SAP水平,其差异有统计学意义(P＜0.01)。上述时相点L组动物SAP与C组比较差异无统计学意义(P＞0.05);
RR:H组、HL组于应激80min是RR显著加快,与C组比较差异有统计学意义(P=0.000),应激120min时H组、HL组RR约为正常对照值的2倍,与C组比较差异有统计学意义(P=0.000),但两者之间差异无统计学意义(P=0.940);L组与C组比较差异无统计学意义(P=0.304);应激80min起,L组RR与H组、HL组比较,差异有统计学意义(P=0.000)。
H组热负荷值显著低于HL组(t=-2.613,P=0.020),但2热应激组热速率不存在统计学差异(t=-1.768,P=0.096)。H、HL组间SAP max及SAP mix存在统计学差异,HL组SAP的波动幅度加大。
Tst及Tsr:H组、HL组动物Tst、Tsr存在显著性差异,复合应激组动物热应激后发生热射病的时间显著提前,热射病后存活时间显著缩短(P＜0.05,vs.H-Group)。
(2)生化指标
PLT:应激80min起H组动物PLT计数显著升高,与C组比较差异具有统计学意义(P＜0.05),L组动物PLT计数显著下降,与H组、HL组比较差异具有统计学意义(P＜0.05)。应激120min时,各组间PLT计数水平均存在统计学差异(P＜0.01),以LSD法做后续检验,LPT由高至低组别依次为H组、C组、HL组、L组:
aPTT:应激80min起时热应激组动物aPTT差异具有统计学意义(P＜0.05),以LSD法做后续检验,H/HL组与C/L组动物血清aPTT水平存在显著性差异(P＜0.05);
PT:应激80min起时热应激组动物PT差异具有统计学意义(P＜0.05),以LSD法做后续检验,H/HL组与C/L组动物血清PT水平存在显著性差异(P＜0.05);
ALT:应激120min时ALT差异具有统计学意义(P＜0.05),以LSD法做后续检验,HL组与C组动物血清ALT水平存在显著性差异(P＜0.01);
AST:应激40min起各组间AST差异具有统计学意义(P＜0.05),应激40min、80min时相点L组、H组、HL组血清AST水平显著升高(P＜0.05,vs.C-Group),应激120min时,HL组AST水平显著高于其余3组(P＜0.01);
Urea:应激40min起L组、H组、HL组动物血清Urea显著升高,与C组比较差异具有统计学意义(P＜0.05)。应激40min起,各组间Urea水平均存在统计学差异(P＜0.05),后续检验血Urea由高至低组别依次为HL组、H组、L组、C组。
(3)炎症介质水平
循环炎症介质
IL-10:HL组动物血浆IL-10水平于应激40min时显著上升,于应激80min时达峰值(约10倍对照组水平),与C、L、H组比较差异有统计学意义(P值分别小于0.001,0.05,0.001);120min时IL-10水平下降,仍显著高于C、H组(P值分别小于0.001,0.05);
IL-8:HL组动物血浆IL-8水平于应激40min时显著上升,于应激120min时达峰值(约3倍对照组水平),与C组比较差异有统计学意义(P＜0.001);
IL-1ra:HL组动物血浆IL-1ra水平于应激40min时显著上升,于应激120min时达峰值(约300倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);
sTNFrⅠ:HL组动物血浆sTNFrⅠ水平于应激40min时显著上升,于应激80min时达峰值(约8-10倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001),120min时sTNFrⅠ水平下降,仍显著高于C、H组(P＜0.001);
sTNFrⅡ:HL组动物血浆sTNFrⅡ水平于应激40min时显著上升,于应激120min时达峰值(约3-4倍对照组水平),与C、H组比较差异有统计学意义(P值分别小于0.01、0.05)。
组织炎症介质
120min时相点时,病理检查结果显示,复合打击动物的心、脑、肺、肝,肾、脾、骨骼肌及小肠组织的病理损伤较单因素打击组为重。
肝组织炎症介质:
TNF-α:HL组动物肝TNF-α水平于应激80min时显著上升并达峰值(约3-4倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,仍显著高于C、H组(P＜0.05);
IL-6:HL组动物肝IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:L、H组动物肝IL-1β水平较稳定,尽管HL组于120min时有所升高,但与C组比较差异无统计学意义(P＞0.05)。
肾组织炎症介质
NF-α:HL组动物肾TNF-α水平于应激80min时显著上升并达峰值(约3-4倍对照组水平),与C、L、H组比较差异有统计学意义(P值分别为0.01,0.05,0.01);120min时TNF-α水平下降,各组间均不存在统计学差异(P＞0.05);
肾上腺炎症介质
TNF-α:HL组动物。肾上腺TNF-α水平于应激80min时显著上升并达峰值(约5.6倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,与C、H组不存在显著性差异(P＞0.05);
IL-6:HL组动物肾上腺IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:L、H组动物肾上腺IL-1β水平较稳定,尽管HL组于120min时有所升高,但与C组比较差异无统计学意义(P＞0.05)。
脾组织炎症介质
TNF-α:HL组动物脾TNF-α水平于应激40min时显著上升并于80min达峰值(约15倍对照组水平),与C、L、H组比较差异有统计学意义(P＜0.001);120min时TNF-α水平下降,仍显著高于C、H组(P＜0.05);
IL-6:HL组动物脾IL-6水平于应激120min时显著上升并达约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
IL-1β:HL组动物脾IL-1β水平于应激80min时显著上升,于120min达峰值约3-4倍对照组水平,与C、L、H组比较差异有统计学意义(P＜0.001);
(4)组织病理学改变
120min时,HE染色光镜观察发现,LPS与高温复合打击动物的心、肺、脑、肝、肾、脾、骨骼肌等组织出现病理损伤,程度重于比L/H组:
心组织学改变:HL组心肌细胞明显水肿、变性,肌间血管充血、水肿、炎细胞浸润。
脑组织学改变:HL组动物脑组织充血、水肿,神经元胞体及神经胶质细胞明显肿胀,部分神经尼氏小体消失,局部神经细胞变性、坏死,脑组织充血、水肿。
肺组织学改变:HL组动物120min时肺组织出现急性微血管损伤改变,表现为肺泡及间质水肿,淋巴细胞、PMN浸润,肺泡壁厚薄不均、透明膜形成、肺出血等。
肝组织学改变:HL组动物肝中央静脉及肝血窦明显淤血,主要在肝小叶中间区。部分肝细胞浆高度疏松,呈稀网状或空泡。中央静脉及其临近肝窦扩张、充血。汇管区血管亦见扩张、充血及炎细胞浸润。
肾组织学改变:HE染色观察可见肾间质及肾小管炎性浸润、细胞肿胀,并出现出血与坏死。
脾组织学改变:HL组动物脾血管及血窦明显扩张、淤血,红髓及白髓分界不清,脾小体结构消失。
骨骼肌组织学改变:HL组骨骼肌细胞明显水肿、变性,部分骨骼肌细胞核固缩及凝固性坏死。
小肠组织学改变:HL组粘膜层明显充血水肿,肠粘膜糜烂,肠绒毛结构破坏,粘膜上皮坏死、脱落。
结论:
1.本研究首次发现,高温与LPS同时打击可以复制重度热射病大鼠多器官急性损伤的动物模型。从炎症反应角度讲,动物的反应与人由中度热射病发展为重度热射病的反应具有相似性、重复性。动物热应激反应(如生命体征、热负荷及热速率)、循环衰竭及多器官急性损伤等表现与人重度热射病的临床表现相近。该模型可用于重度热射病炎症反应途径启动机制、评价治疗及重症监护措施对重度热射病病程及预后影响的进一步研究。
2.内毒素血症在从热射病向重度热射病发展进程中起重要作用。热应激下内毒素血症可通过SIRS途径造成生命体征、凝血.抗凝血平衡紊乱,使炎症介质表达异常,促使热应激个体出现循环衰竭、重度热射病甚至多器官急性损伤。
3.复合打击可促发和加重全身炎症反应综合征,表现为血浆细胞因了IL-1ra、sTNFrⅠ/Ⅱ)、IL-8、IL-10及组织细胞因子TNF-α、IL-1α、IL-6含量显著升高。这些细胞因子在调节局部及全身急性炎症反应中至关重要,所介导的炎症反应过程与人中度热射病及重度热射病的发展进程相一致。
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 response
Vital signs
Hyperthermia 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 response
Heat 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 measurements
PLT: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) Cytokines
Plasma 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-1βconcentration
Liver TNF-α,IL-6,and IL-1βconcentration
TNF-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-αconcentration
TNF-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-1βconcentration
TNF-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-1βconcentration
TNF-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.
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