The Effects Of Hyperbaric Oxygen On The Intestinal After Resuscitation From Traumatic Shock

ObjectiveHBO has been used therapeutically in intestinal ischemia/reperfusion(I/R)injury models established by clamping superior mesenteric artery,with beneficial outcome. However,there have been few studies about the effect of HBO on I/R injury of gut after resuscitation from traumatic shock in animals.This study was designed to investigate whether HBO could have protective effect intestinal tissue and mucosal barrier in I/R animal model after resuscitation from traumatic shock,and to elucidate the underlying mechanisms of these protective effects.Materials and Methods1.Experimental animalsNinety-six male Wistar rats,weighing 260-300 grams each,were randomly divided into four groups with 24 rats each group:shock group(operation performed with induced traumatic shock);sham group(operation performed without induced traumatic shock);one HBO treatment(HBOT)group(traumatic shock with one HBOT);three HBOT group(traumatic shock with three HBOT).2.Traumatic shock modelMale Wistar rats were anesthetized with amobarbital sodium(80 mg/kg,i.p.)at room temperature(25℃).Then blood pressure was monitored via polyethylene tubing inserted into the right femoral artery,using pressure analyzer(Multiparameter Patient Monitor,M3046A,Boebingen,Germany).The left jugular vein was cannulated to administer normal saline and shed blood for resuscitation.The right carotid artery was cannulated with polyethylene tubing for withdrawal of shed blood.After above operation was done,an iron mass weighted 2.5kg from a 20 cm height smashed the middle part of left thigh of rat vertically,causing the compound fracture of femur and crush injury of muscular tissue,then the damaged thigh was bandaged and fixed.At the same time,the blood was withdrawn through the tube inserted into right carotid artery, until the mean artery pressure was reduced to 30-35mmHg within 5 minutes.Constant hypotension was maintained for 60 minutes by further withdrawal of small amounts of blood as necessary.After 60 minutes of hypotension,the rats were resuscitated by transfusing the shed blood over 5 minutes,followed by 4-6ml normal saline for 55 minutes with mean artery pressure above 70-80mmHg after the resuscitation.3.Scheme of HBO treatmentThe resuscitated rats were put into the hyperbaric chamber(10N-750,monoplace chamber,Ningbo,China)and the door was closed.The chamber was washed three times with pure oxygen in 1.5 atmosphere absolute(ATA)of pressure.Then the pressure inside the chamber arose to 2.5 ATA with a constant speed over 15 minutes and the oxygen concentration was higher than 95%.The pressure and oxygen concentration would be maintained for 60 minutes,HBO is given intermixed with one air-breathing period of 10 minutes.Finally,the pressure was decreased with a constant speed over 15 minutes.When the pressure in chamber was 1 ATA,the door was opened and rats were taken out.The animals in one HBOT group were given one-time HBO treatment immediately after resuscitation,and the animals in three HBOT group were given one-time HBO treatment immediately after resuscitation plus additional two-times HBO treatments within 24 hours(×q8h)after resuscitation.4.Measurements of levels of iNOS,NO,TNF-αand lactate in intestinal tissueThe tissue of small intestine at end of ileum was excised from each rat before and at end of shock,at the end of resuscitation,and 24 hours after resuscitation.The tissue was rinsed in ice normal saline(0-4℃),eliminating blood,drying out,weighting,then putting into a beaker with ice normal saline(0-4℃),and cut into small pieces.The tissue pieces was pulverized by using supersonic generator(Snoiprep 150)with amplitude of 14μm,10%of intestinal tissue homogenate was prepared,and centrifuged, and supernatant was ready for measurement of levels of iNOS,NO and lactate with using colorimetric method,and level of TNF-αby using radioimmunity method.5.Histological procedures24 hours after resuscitation,the terminal ileum was excised from each rat,and fixed with 10%paraformaldehyde.Fixed tissues were embedded in paraffin and processed for light microscopy.Sections were stained with hematoxylin and eosin. Finally,the slices were examined via a light microscopy and the severity of tissue injury was scored.The degree of intestinal tissue injury was evaluated by using Park's grading scale.2.5%of glutaric dialdehyde was dropped in the terminal ileum,the intestinal tissue with 0.5cm long in the part fixed with glutaric dialdehyde was excised,and put on ice cubes,cleaned with phosphate buffered solution(PBS,PH 7.4),the intestine sample of 1 mm³ was made,then put into and rinsed in PBS,then fixed in 1%osmium tetroxide (OsO₄)with PBS,dehydrated through graded ethanols and acetones,embedded in epoxide resin(EPON812),thin sections(with thickness of approximately 60nm)were cut by using LKB ultramicrotome,stained with uranyl acetate and lead citrate. Subcellular structure of mucosal epithelium was observed by using transmission electron microscope(JEM-1200EX,JEOL Ltd,Japan).6.Statistical analysisThe data were analyzed using SPSS 13.0 for Windows software.Data are represented as Mean±Standard Deviation((?)±s);the groups were compared with SNK-q test of One-Way analysis of variance(One-Way ANOVA);Correlation coefficient was calculated on the basis of Pearson's Bivariate method.Differences were considered significant when P＜0.05.Results1.The effects of HBO on the levels of the lactate,TNF-α,iNOS and NO in intestinal tissueThe levels of lactate,TNF-α,iNOS and NO were not altered significantly in sham group,but increased significantly after TRAUMATIC SHOCK in other three groups (P＜0.05).HBO significantly decreased the levels of lactate,TNF-α,iNOS and NO 24 hours after resuscitation from shock,and the differences of levels of lactate,TNF-α, iNOS and NO in intestinal tissue between shock and one or three HBOT group were significant(P＜0.05).Although these parameters in three HBOT group were better than those in the one HBOT group,the differences were not significant(P＞0.05 by one-way ANOVA and SNK)2.Analysis of correlation between the levels of iNOS and NO in intestinal tissueThere was positive correlation between the levels of iNOS and NO in intestinal tissue(r=0.796,P＜0.001,by Pearson's Bivariate method)3.Effect of HBO on histological change of intestinal tissue after resuscitation from traumatic shock.(1)Histological injury severity scoringThe histological injury grading score of small intestine in sham group were significantly lower than those in three other groups(P＜0.05).The injury grading score of small intestine in one or three HBOT group were significantly lower than that in shock group(P＜0.05).There was no significant difference of injury scores between two HBOT groups(P＞0.05,by One-Way ANOVA)(2)The changes of intestinal epithelium at transmission electron microscope Intestinal epithelia from sham-operated animals in electron micrograph showed there was no swelling in epithelia with integrated cellular membrane,well-arranged microvilli without ablating and rarefying,normal desmosome and tight junction,no reduction of cytoplasm and cellular organelle,no breakage of mitochondrial crista,no expansion of endoplasmic reticulum,cellular nucleus with normal chromatin.In contrast,intestinal epithelia in shock group exhibited the swelling,rarefaction of desmosomes,tight junction clearly opened,microvilli ablating,rarefying and often blown up,mitochondrial swelling,mitochondrial crista disappearing and widespread mitochondrial dissolution with vacuole formation,endoplasmic reticulum swelling and expansion,vague of perimeter space of nuclei,nuclei swelling with increased heterochromatin increasing.By contrast,the changes described above were significantly less severe in one and three HBOT groups:tight junction and desmosomes were preserved and partial mitochondrial dissolution.ConclusionHBO can decrease the production of inflammatory factor after traumatic shock, inhibit the excessive inflammatory reaction.HBO can decrease the intestinal pathological change under the light microscope.HBO can decrease the change of pathology on intestinal epithelia after resuscitation from traumatic shock that happened in microvillus,mitochondrial,endoplasmic reticulum and cell nucleus,especially,can decrease the pathological damage of cell juction.Those may provide the expedient evidents on intestinal permeability raising and the theory of bacterium displace.Those may give the reasonable explanations on mechanism of I/R after resuscitation from traumatic shock and HBO treatment.