Effects Of Isoflurane Pretreatment On Acute Lung Injury Induced By Endotoxin In Piglets And Its Possible Mechanisms

Partâ… Effects of the different concentrations of isoflurane pretreatment on acute lung injury induced by endotoxin in pigletsObjective:1.Establish an endotoxin-induced acute lung injury(ALI) animal model in piglets for ALI research.2.To delineate the three concentrations of isoflurane pretreatment effects on lung mechanics,gas exchange,hemodynamics,pathology of lung,and inflammation in the model of ALI.Methods:Twenty-four piglets of 5-6 weeks old,weighing 9-14 kg were anaesthetized, intratracheally intubated and subjected mechanical ventilation.After isolation of left carotid artery and left external jugular vein,catheters were placed respectively to monitor hemodynamic parameters and infusion fluid,meanwhile monitor the blood gas analysis of artery,life sign,and the related respiratory parameters.The animals were divided into 4 groups(n=6 each group):LPS control group(CON group,n=6), 0.5 MAC(minimum alveolar concentration) isoflurane pretreatment group(0.5 MAC group,n=6),1.0 MAC isoflurane pretreatment group(1.0 MAC group,n=6),1.3 MAC isoflurane pretreatment group(1.3 MAC group,n=6).Each animal in CON group was treated by vein infusion with lipopolysaccharide(LPS) 20μg·kg^-1 for 60min.After the establishment of ALI for 12 hours,the animals were sacrificed;in 0.5 MAC,1.0 MAC,1.3 MAC group,each animal was treated by three different concentrations of isoflurane for 30min respectively.Waited for 60min,each animal was treated by vein infusion with LPS 20μg·kg^-1 for 60min.The animals were sacrificed after the establishment of ALI for12 hours.The lung mechanism,gas exchange,hemodynamic parameters,and the blood gas analysis were measured at the time of baseline,LPS 1h,LPS 2h,ALI 0h,ALI 4h,ALI 8h,ALI 12h.At the end of the experiment,animal lungs were processed for measurement of biochemical and biophysical alterations in brochoalveolar lavage fluid(BALF) or lung tissue,the wet/dry(W/D) lung weight ratio,and lung histopathology.The proinflammatory cytokine(TNFα) in plasma was detected by radioimmunoassay.Result:1.Twenty-seven piglets,in which three piglets died accidently during the experimental time,were treated by vein infusion with LPS 20μg·kg^-1.After 3-6 h infusion of LPS,ALI was induced when PaO₂/FiO₂≤300 mmHg,a more than 30%reduction of dynamic compliance(Cdyn) compared to baseline.2.Hemodynamics:After LPS infusion,there was a greater change in 0.5 MAC isoflurane pretreatment group than 1.3 MAC isoflurane pretreatment group in heart rate(HR),mean arterial blood pressure(MABP),cardiac output(CO), systemic vascular resistance(SVR) and dp/dtmax.By monitoring pulmonary vascular permeability index(PVPI) using Picco device,there was a trend in increment of PVPI with the prolonged experimental time in 1.3MAC group compared to CON group(P＜0.05).3.Lung mechanics,gas change and blood gas analysis:After LPS infusion,there was a less change in 1.0 MAC group than other groups at mean airway pressure (MAP),values of Vd/Vt,pH,PaO₂/FiO₂ compared to baseline among all groups.4.Among all groups,no statistical difference was detected in lung inflammation and injury by measuring MDA and MPO in lung tissue.5.Among all groups,no statistical difference was detected in the number of peripheral WBC and hepatic function.6.The tendency of TNFαin plasma in each group:After LPS infusion,each group significantly increased compared to baseline(P＜0.01).After establishment of ALI,TNFαin plasma of 1.3MAC group increased lightly less than other three groups.7.Compare to CON group,1.0MAC group had lower total protein(TP) in BALF and decreased W/D lung weight ratio(P＜0.05) was detected.Among all groups, there was no statistical difference in total phospholipids(TPL),disaturated phosphatidylcholine and surface tension in BALF.8.Among all groups,no statistical difference was detected in histopathology.Conclusion:1.20μg·kg^-1 LPS(E.coli 0111:B4) could successfully establish endotoxin-induced ALI in piglet.2.Compare to other two concentrations of isoflurane,the pretreatment of 1.3MAC isoflurane had better protection to keep the stable circulation,meanwhile,the pretreatment of 1.3MAC isoflurane could lightly inhibit the increase of TNFαin plasma.3.Inhalation of 1.0MAC isoflurane pretreatment for 30 min may improve lung mechanism,gas exchange and oxygenation;decrease the permeability of alveolar capillary.Partâ…¡Effect of mitochondrial ATP-sensitive potassium channels on isoflurane pretreatment in endotoxin-induced acute lung injury in pigletsObjective:To investigate whether the mitochondrial ATP-sensitive potassium channels(mitoK_ATP) were involved in 1.0MAC isoflurane pretreatment in endotoxin-induced ALI of piglets.Methods:On the basis of the ALI model establishement in partâ… ,six piglets were infused 5mg·kg^-1 5-hydroxydecanoate(5-HD),it was the highly specific mitoK_ATP channel antagonist.After 30min,each animal was treated by the pretreatment of 1.0MAC isoflurane for 30min.After 1 hour,each animal was treated by 20μg·kg^-1 LPS in vein infusion for 60min.After the establishment of ALI for 12h,the animals were sacrificed.This group was defined as 5-HD group(n=6).5-HD group compared with the four groups in partâ… .During the experiment,the similar treatment in monitoring parameters and lung measurement were performed as partâ… .Results:Compared to its baseline,the hemodynamics of 1.0MAC and 5-HD group had the similar change during experimental time.And no statistical difference was detected within and between the two groups.Compared to baseline,pH,PaO₂/FiO₂ at LPS 1h in 5-HD group was significant lower(P＜0.01),whereas,no such statistical difference was detected in 1.0MAC group.Compared to CON group and 1.0MAC group,the number of peripheral WBC in 5-HD group at ALI 4h and ALI 8h decreased significantly(P＜0.01).With the prolonged experimental time,total bilirubin(TBIL) and conjugated bilirubin(CBIL) in 5-HD group increased.Compared to CON group,TBIL in 5-HD group increased at ALI 8h(P＜0.05);CBIL increased at ALI 0h and ALI 8h(P＜0.05).AST in 5-HD group also increased.However,no statistical differences were found in AST and ALT within and among the groups.Compared to 1.3MAC group,TNFαof plasma in 5-HD group increased at ALI 4h(P＜0.05).Compared to CON group and 1.0MAC group,Myeloperoxidase(MPO) activity in 5-HD group increased(P＜0.05).TP and TPL of BALF in 5-HD group decreased (P＜0.05),compare to CON group.There were no statistical differences in W/D lung tissue ratio,malondialdehyde(MDA) between the groups.Conclusion:1.Using the highly specific mitoK_ATP channel antagonist 5-HD,there were no effects on the permeability of alveolar capillary between 1.0MAC group and 5-HD group.MitoK_ATP channel may not affect the permeability of alveolar capillary in ALI.2.The inflammatory reatcion of endotoxin-induced lung injury deteriorated in 5-HD group.In 5-HD group the production of TPL in BALF was detected lower compared to CON group,which suggested inhibition of mitoK_ATP channel resulted in more injury in alveolar epithelial typeâ…¡cell(AECâ…¡).Partâ…¢Effects of isoflurane pretreatment on adhesive molecular and proinflammatory cytokine mRNA in endotoxin-induced ALIObjective:To study the expression of adhesive molecular(ICAM-1,VCAM-1) mRNA, proinflammatory cytokine(TNFα,IL-6,IL-8) mRNA and ICAM-1 protein in lung tissue after the pretreatment of the different concentrations of isoflurane or using mitoK_ATP channel antagonist,and research its possible molecular mechanism.MethodsOn the piglet ALI model and the same group with partâ… andâ…¡,the lung tissue of each group was harvested and preserved in liquid nitrogen.RNA of lung tissue was extracted.The adhesive molecular(ICAM-1,VCAM-1) and proinflammatory cytokine(TNFα,IL-6,IL-8) mRNA in lung tissue of five groups were tested by RT-PCR,and the protein of ICAM-1 was detected by immunohistochemistry.ResultsCompared to 1.0MAC group,the expression of ICAM-1mRNA in 5-HD group increased(P＜0.05).In five groups,no statistical difference was detected in the expression of VCAM- 1 mRNA.Compared to CON,0.5MAC,1.0MAC,1.3MAC group,the expression of IL-8 mRNA in 5-HD group increased(P＜0.05).However,there was no statistical difference in the expression of IL-6 mRNA,TNFαmRNA among the groups.The protein of ICAM-1 was expressed on pulmonary vascular endothelium,alveolar and bronchial epithelium.Conclusion:1.The highly expression of ICMA-1mRNA in lung tissue in 5-HD group and combining the increased result of MPO activity of partâ…¡,which indicated the more aggravated inflammation in 5-HD group.It was maybe suggested the mitoK_ATP channel was involved in the process of 1.0MAC isoflurane pretreatment and affected the inflammatory in ALI.2.The highly expression of IL-8 mRNA in lung tissue in 5-HD group suggested that the inhibited mitoK_ATP channel had synergistic effect in aggravating the lung inflammatory and the highly expression of proinflammatory cytokine.