| The whole-body hyperthermia(WBH) is widely used in treatment of maligant tumors,when the therapy kills the tumor cells,they also do harm to the body.The study showed that advanced tumor patients were under treatment WBH appeared systemic vasodilator,vascular resistance decreased and cardiac output increased,the cardiovascular system showed "high-ranked and low resistance'characteristics.The evaporation of perspiration and respiratory tract can also lead to a loss of numerous liquids.In order to maintain hemodynamic stability,we must infuse a large amount of fluid into the body,the amount of liquid infusued is very difficult to meet the needs of the body.The fluid infused in clinical is mostly to be Ringer's solution and artificial colloid,which,at a high rate,can cause cerebral edema and pulmonary edema. Researchers pointed out that blood-cerebrospinal fluid barrier(BCSFB) in choroid plexus works with the blood-brain barrier(BBB) in cerebral capillaries to stabilize the fluid environment of neurons.Dysfunction of BCSFB or BBB transport interface causes augmented fluxes of ions,water and proteins into the central nervous systerm (CNS).These barrier disruptions lead to problems with edema and other compromised homeostatic mechanisms.New functional data in animal experiment indicate that the choroidal epithelium and the ependymal cells are vulnerable to WBH. This is evidenced from the fact that rats subjected to 4 h of heat stress(38℃) showed a significant increase in the translocation of Evans blue and131Iodine from plasma to cistemal CSF,and manifested blue staining of the dorsal surface of the hippocampus and caudate nucleus.Degeneration of choroidal epithelial cells and underlying ependyma,a dilated ventricular space and damage to the underlying neuropil were frequent.A disrupted BCSFB is associated with a marked increase in edema formation in the hippocampus,candate nucleus,thalamus and hypothalamus.Some advanced tumor patients under treatment WBH at the end of anesthesia appear abnormal of CNS,it may probably related to heat stress and excessive fuid infusion, which,can cause the formation of the cerebral edema.Recent clinical studies show that advanced tumor patients who were under treatment WBH need less volume of fluid infusion and less application of cardiovascular drugs,in the condition of that they are infuse with hypertonic saline hydroxyethyl starch 40 injection(HSH). This,in turn,can relatively stabilize the hemodynamics,reduce heart failure in and post operation,and lower the posibility of pulmonary edema.However,It is still unsure that if the infusion of HSH during the WBH can reduce the occurrence of cerebral edema.In the experiment,by simulating tumor patients with WBH process. Putting rats exposed to WBH in a biological oxygen supply heated container maintained at 36℃for 3h,Thereafter rats were moved out of the heating container to let the temperature recover down for 1h,then observe the rat cerebral water content, Evans blue extravasation,the pyramidal cells morphological change in hippocampus region.Explore the acting mechanism of HSH on WBH. Materials and methods1.Experimental animals75 adult male rats,body weight(220-250)g,were provided by the experiment anmimal center of southern medical university.2.Establish whole-body hyperthermic rats modelAnesthesia administered by muscle injection of pentobarbital sodium 3% solution(45mg/kg).After being anesthetized,rats were subjected to femoral artery and vein cannulation for monitoring of mean arterial pressure and for fluid administration.Rats were exposed to WBH in a biological oxygen supply heated container maintained at 36℃for 3h to achieve the rectal temperature at(41～42)℃, Thereafter rats were moved out of the heating container to let the temperature recover down for 1h,then treated them with correspoding ways.At the beginning of the exposure to WBH,different kinds of fluid were administered intravenously within 30 minutes.3.Experiment group and treatment factorsRats were randomly divided into five groups,each with fifteen,groups and factors are:①normal control group(group C):rats stay at a temperature controlled room with(25-26)℃②whole-body hyperthermia group(group HT):rats treat with WBH but without fluid infusion③whole-body hyperthermia Ringer's injection group(group RL):rats treat with WBH and infuse with 36ml/kg RL④whole-body hyperthermia Haes+Ringer's injection group(group HRL):rats treat with WBH and infuse with 9ml/kg HES and 18ml/kg RL⑤whole-body hyperthermia HSH injection group(group HSH):rats treat with WBH and infuse with 8ml/kg HSH.All fluids must be infused evenly within 30 minutes.4.Indicators observation and specimens collectionMean arterial pressure and rectal temperture were recorded before the experiment(TO),15min into WBH(T1),30min into WBH(T2),45min into WBH(T3),60min into WBH(T4),75min into WBH(T5),90min into WBH(T6),105min into WBH(T7),120min into WBH(T8),135min into WBH(T9),150min into WBH(T10),165min into WBH (T11),180min into WBH(T12).And collected blood sample to test arterial blood gases before the experiment,after the fluid infused and after the WBH.At the end of the experiment,Oberserved the rat cerebral water content,Evans blue extravasation and the pyramidal cells morphological change in hippocampus region.5.Cerebral water content measurementAt the end of experiment,six rats in each group were rapid craniotomy brain. Placed the brain tissue in petri dishes where have a qualitative 0.5ml saline moist filter paper to prevent water evaporation,stripping clean leptomeninges and blood clots,filter drain liquid surface,cut right brain which weight(0.2～0.3)g.Then the brain tissue was put in the electric incubators 105℃for a 72h drying to constant weight,weight the dry tissue.By Elliot formula cerebral water content(%),cerebral water content=(wet weight-dry weight)/wet weight×100%.6.Brain Evans blue extravastion determinationWhen the rats rectal temperature fell to 37℃,six rats in each group were injected with 2%Evans blue 3ml/kg from the femoral vein,1h later,we did the thoracotomy and exposed the heart,inserted needle from the apical of the heart and then went to the aorta to fix firmly.Cut the right atrial appendage,using 37℃heparin saline (100u/ml) lavage until the right atrial appendage outflow of transparent liquids,rats were rapid craniotomy brain,Place the brain tissue in petri vessels where have a qualitative 0.5ml saline moist filter paper to prevent water evaporation,stripping clean leptomeninges,filter drain liquid surface,cut right brain which weights (0.2～0.3)g,put the brains that are filled with 3ml formamide,then lidded them,put them into 37℃constant temperature water bath tank,48h later,1500g centrifugate them for 10min.The extravasation of EB dye in the brain was then measured by Molecular Devices M5 continuous spectrum multifunctional microporous Reader at 632nm wavelength,measured OD values.According to the standard curve, calculating the EB content(μg/g,wet brain weight).7.The pyramidal cells morphological change in hippocampus regionTake three rats from each group,time it when the rectal temperature dropped to 37℃,1h later,we did the thoracotomy and exposed the heart,inserted the needle from the apical of the heart and then went to the aorta to fix firmly.Cut the right atrial appendage,perfusd 37℃heparin saline(100u/ml) 150ml quickly,followed by rapid infusion of ice-cold 4%paraformaldehyde 200ml,then perfused 300ml slowly in 2h. Decapitated brain,immersed the right brain tissue in 4%paraformaldehyde untill the temperature rose to 4℃,then the tissue fixed for 48 hours,cut it into 4mm thick block,conventional dehydration,paraffin-embedded,and cut it into 5μm in coronal sections line,do it with HE staining,observe the thin piece under Nikon microscope, and take some pictures.8.Statistical analysisAll data are reported as the mean±standard deviation((?)±s).SPSS 13.0 were used to analyze the data.Analysis of one-way-anova was used to evaluate the body weight,baseline rectal temperature,baseline mean arterial pressure,cerebral water content and evans blue extravasation among groups.Post Hocmultiple comparisons among groups were analyzed by using LSD test.Analysis of variance of repeated measure data was used to analyze the repeated measures data such as mean arterial pressure,rectal temperature and arterial blood gases.Post Hocmultiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test.P values of＜0.05 were accepted as significant. Result:1.The baseline dataThere is no significanct difference between body weight,baseline mean arterial pressure and baseline rectal temperature(P＞0.05).2.Mean arterial pressureAnalysis of variance of repeated measure data shows that there is significance different among groups in the mean arterial pressure during the WBH process(F =31.923 P=0.000),there is significant difference among different time spots in each group(F=55.191 P=0.000),and there have crossover effect between different groups and different time spots(F=9.413 P=0.000).Further analysis of the separate effect,Post Hocmultiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test.It shows that on the time spots of T11 and T12,there is significant difference in mean arterial preesure in group HT and other experimental groups(P＜0.05 ),There is no different significance between group HSH and group C(P=0.338),while the mean arterial preesure in group RL is lower than group C.3.Rectal temperatureAnalysis of variance of repeated measure data shows that there is significance different among groups in the mean arterial pressure during the WBH process(F =729.519 P=0.000),there is significant difference among different time spots in each group(F=3208.790 P=0.000),and there have crossover effect between different groups and different time spots(F=210.281 P=0.000).Further analysis of the separate effect,Post Hocmultiple comparisons among each time spot in different groups and different time spots in each group were analyzed by using LSD test.It shows that on each time spot in different groups and different time spots in each group,the rectal temperature is increased in the experimental groups,there is significant difference in rectal temperature in group HSH and other experimental groups(P＜0.05).4.Cerebral water contentCerebral water content of all groups are significantly different(F=81.886,P =0.000),cerebral water content of group C,group HT,group RL,group HRL,group HSH are as follows(%):78.11±0.58,82.83±0.54,81.86±0.57,80.22±0.60,79.15±0.26. Post Hocmultiple comparisons among groups were analyzed by using LSD test,it shows that the cerebral watet content in group HSH is higher than group C,there is significantly different in the cerebral water between group HSH and group HT(P＜0.05).5.EB dye extravasationThe extravasation of EB dye of all groups are significantly different(F=286.629 P=0.000),the results show that EB extravastion of the brain was(2.26±0.09) ug of EB/g of tissue in the C group,whereas it were(9.79±0.48) ug of EB/g of tissue in the HT group,(8.41±0.33) ug of EB/g of tissue in the RL group,(6.98±0.61) ug of EB/g of tissue in the HRL group,(5.87±0.38)ug of EB/g of tissue in the HSH group.The multiple comparsion in groups show that the extravasation of EB dye in group HSH in higher than group C,and there is significantly different in the extravasation of EB dye between the group HSH and group HT(P＜0.05).6.Aterial blood gasesAnalysis of variance of repeated measure data shows that there is significant difference in PH,PaO2,PaCO2,HCT,Na+ and K+ betwwen group HT and group C during the WBH(P＜0.05 ),mlutiple comparsion among each time spot in different groups and different time spots in each group shows that at the end of fluid infusion, there is significant difference in Na+ and K+ between group HSH and group C(P＜0.05),while there is no significant difference in PH,PaO2,PaCO2 and HCT between the two groups.There is no significantly different in PH,PaO2,PaCO2,HCT,Na+,K+ between group RL and group HRL.7.The pyramidal cells morphological change in hippocampus regionThe pyramidal cells in CA3 area in normal brain tissue were arranged regularly, there were appeared complete forma,full nucleus,clear nucleolus and rich in Nissl bodies.In HT group,the number of brain cells decreased,the residual neurons appeared large nucleus,some nucleus dissolved or disappeared,the cell body increases,the edge of ambiguity is not clear,the Nissl bodies is disappeared,the majority of cells become ghost cells.In RL group,the cell morphology is irregular, showing polyhedrosis or shuttle type,the membrane is shrink,nuclear pyknosis, concentrated dye,nuclear membrane is sag,nucleolus blurred or disappeared, cytoplasmic vacuolization.degeneration.In HRL group shows vacuolar degeneration cells significantly reduced and appeared lots of concentrated nuclear staining neurons. In HSH group,Numrous normal pyramidal cells scattered between the concentrated nuclear staining neurons,occasional seen some neurons with nuclear pyknosis, nuclear membrane depression,nuclear dense staining,nucleolus blurred,cell body and nucleus of irregular shape.Conclusion:1.Whole-body hyperthermia increase the blood brain barrier preability,cause cerebral edeama and damage the pyramidal cells morphological change in hippocampus region in rats.2.Fluid therapy can decrease the blood brain barrier preability,reduce the cerebral edema and ameliorate the pyramidal cells morphogical chang in hippocampus region in whole-body hyperthermic rats.3.Compared with Ringer's and hydroxyethyl Starch,Hypertonic sodium chloride hydroxyethl starch 40 injection may significantly decrease the blood brain barrier preablity,reduce the cerebral edema and ameliorate the pyramidal cells morphogical chang in hippocampus region,it's the suitable therapy fluid in the whole-body hyperthermic rats.
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