The Experimental Study Of Lung Protective Ventilation Strategy In Treatment Of Acute Respiratory Distress Syndrome

BackgroudAcute respiratory distress syndrome(ARDS) is a common life-threatening condition with a high mortality.In order to improve the prognosis of ARDS,many new treatment methods have been proposed,but large-scale clinical trials all showed negative results.Positive pressure mechanical ventilation(MV) is still the main means for ARDS treatment.However,improper applications of MV easily lead to mechanical ventilator-associated lung injure(VALI) and increase mortality,for example traditional ventilation with conventional tidal volume(VT) and low positive-end expiratory pressure(PEEP).Then Lung protective ventilation strategy(LPVS) was proposed,of which the core content is small VT and best PEEP.The proposal of LPVS results in the change of MV therapeutic target from maintaining nomal arterial blood gas to reduce mortality.Studies showed LPVS can improve the prognosis of ARDS via a variety of mechanisms,but there were significant differences in mortality in the reports of different areas,and domestic mortality is higher.Thus,there are many controversies on the measures of LPVS and its mechanism improving prognosis of ARDS:Is RM a measure of LPVS? Does proper buffer of respiratory acidosis influence treatment effectiveness of LPVS? Dose prevention to excessive apoptosis of cells in pulmonary tissue impact the improvement of LPVS on ARDS prognosis? Dose LPVS increase expression and activity of glucocorticoid receptor(GR),compared with traditional ventilation? In this study,we use canines as a model to investigate these questions.Objective1.To investigate the relationship of RM and LPVS by observing pulmonary pathological changes and inflammation-related indicators of oleic acid-induced ARDS canines.2.To investigate the effect of LPVS on excessive apoptosis of cells in pulmonary tissue of oleic acid-induced ARDS canines. 3.To investigate tolerance of oleic acid-induced ARDS canines to respiratory acidosis and the impact of the properly buffer of respiratory acidosis on treatment effectiveness of LPVS.4.To investigate the effect of LPVS on the expression and activities of NF-κB and GR of oleic acid-induced ARDS canines.MethodsTwenty five canines were randomly divided into five groups(n=5).ARDS canine model was established by intravenous oleic acid.ARDS canines were ventilated with the following VT and PEEP for six hours:â‘ Group A:ARDS canines were ventilated with 12ml/kg VT and 0 PEEP;â‘¡Group B:ARDS canines were ventilated with 12ml/kg VT and 5 cmH₂O PEEP;â‘¢Group C:ARDS canines were ventilated with 6ml/kg VT and best PEEP,and respiratory acidosis was bufferred;â‘£Group D:ARDS canines were ventilated with 6ml/kg VT and best PEEP,but respiratory acidosis was not bufferred;⑤Group E:RM(5OcmH₂O for 2 minutes)was performed firstly,then ARDS were ventilated with 6ml/kg VT and best PEEP.Except for group D,blood pH of other groups maintaned at the range of 7.35 to 7.45.1.Part OneThe indicators of hemodynamics,respiratory mechanics and arterial blood gas were monitored.Oxygenation index(OI),alveolar-artery oxygen pressure gradient[P_A-aO₂],ratio of shunted blood to total perfusion((Qs/Qt) and dynamic lung compliance(CLdyn) were calculated.2.Part TwoLung tissue was obtained by biopsy.Some lung sections were stained with hematoxylin and eosin(HE).Chest CT scans were performed at the sixth hour after diagnosis of ARDS(T6) and estimate the situation of lung ventilation by CT images.Wet-to-dry weight ratio(W/D) of lung and protein concentration of bronchoalveolar lavage fluid(BALF) were measured.3.Part ThreeApoptosis cells in pulmonary tissue were detected by TUNEL assay.4.Part FourBALF was collected for total and differential counts.TNF-αand IL-10 concentration of serum and BALF were measured by enzyme-linked immunoabsorbent assay(ELISA).rhe activity of NF-κB in peripheral blood leukocytes(PBL) were measured by ELISA.Other lung sections were prepared for immunohistochemistry using two polyclonal antibodies against NF-κB p65 and GR-α,respectively.Results1.Part OneCompared with Group B,OI and P_A-aO₂ of Group C were obviously higher than those in Group A and Group B at T6(P＜0.05),and Qs/Qt of Group C was obviously lower than that in GroupA and Group B at T6(P＜0.05).Oxygen delivery in arterial blood(DaO₂) of Group C was higher than that of Group A and Group B at T6,but there was significant difference only between Group A and Group C.There was no significant difference in C_Ldyn between Group B,Group A and Group C at T6.Compared with Group A and Group B,the inhibitory effect of LPVS on hemodynamics of Group C was not obvious.Mean blood pressure(mBP) of Group C was lower(P＜0.05 between Group C and Group A) at T6,but still within the normal physiological range.While mean pulmonary artery pressure(mPAP),central venous pressure(CVP) and cardiac output(CO) of Group C is higher than those of Group A and Group B at T6,but there were significant differences only between Group C and Group A.Pulmonary capillary wedge pressure(PCWP) and cardiac index(CI) in Group C were obviously higher than those in GroupA and Group B at T6(P＜0.05).PCWP in Group C at T6 was still within the normal physiological range.There were significant hemodynamics abnormalities in Group D at T4 and T6,for example,obvious decrease of mBP.CO,CI,mBP and DaO₂in Group D were obviously lower than those of Group C at T6(P＜0.05).There were no significant differences in CVP and PCWP between Group D and GroupC at T6.There were no significant differences in C_Ldyn,OI,P₍A-a_O2 and Qs/Qt between Group D and Group C at T2,T4 and T6.OI,P_A-aO₂ in Group E was obviously higher than those in Group C at T2(P＜0.05),while Qs/Qt in Group E was obviously lower than that in Group C at T2(P＜0.05).There were no significant differences in OI,P_A-aO₂ and Qs/Qt between Group E and Group C at T4 and T6.There were no significant differences in C_Ldyn and DaO₂ between Group E and Group C at T2,T4 and T6.There was a transient abnormalities of hemodynamics in Group E when RM was performed.There were no significant differences in the indicators of hemodynamics between Group E and Group C at T2,T4 and T6.2.Part TwoChest CT,the gross specimens of lung,HE stain of lung sections showed hyperventilation regions and hypoventilation regions of Group C were less than those of Group A and Group B.W/D of Group C was obviously lower than that of Group A Group B(P＜0.05).There was no significant difference in protein concentration of BALF between Group C,Group B and Group A at T0,T3 and T6.Imaging performance and the severity of pathological lesions in lung of Group D are similar with those of Group C.There were no significant differences in protein concentration of BALF and W/D between Group D and Group C.Chest CT and the gross specimens of lung showed diffuse lesions in the upper and middle lung in Group E.At the same time,HE stain of lung sections aslo showed alveolar exudative lesions in the upper and middle lung in Group E,but these lesions could not be found in Group C.There were no significant differences in protein concentration of BALF and W/D between Group E and Group C.3.Part ThreeThe ratio of apoptotic cells in lung(alveolar epithelial cells, bronchial endothelial cells,vascular endothelial cells) obviusly increased at T6.But there was no significant differences between groups.4.Part fourLevels of TNF-αin serum and BALF,the number of leukocyte in BALF, and the activity of NF-κB in PBL of Group C were lower than those of Group A and Group B.There were significant differences in all these indicators between Group A and Group C at r6,but there was significant difference only in the TNF-αlevel of BALF between Group B and Group C at T6.The number of polymorphonuclear neutrophile(PMN) in BALF and the ratio of TNF -αand IL-10 in serum of Group C was lower than those of Group A and Group B,but there was significant difference only in serum the ratio of TNF -αand IL-10 between Group A and Group C.Levels of IL-10 in serum and BALF of Group C higher than those of Group A and Group B.There were significant differences in all these indicators between Group A and Group C at T6,but there was significant difference only in the IL-10 level of serum between Group B and Group C at T6..Immunohistochemistry of lung sections for GR-αshowed cytoplasm +++ and nucleus +++ in Group C, cytoplasm +/++ and nucleus +/++ in Group A and Group B,and immunohistochemistry scores of Group C were obviously higher than those of Group A and Group B(P＜0.05).Immunohistochemistry of lung sections for NF-κB p65 showed cytoplasm +/++ and nucleus +/++ in Group C,cytoplasm ++ and nucleus ++ in Group B,cytoplasm +++ and nucleus +++ in Group A,and there were significant differences in immunohistochemistry scores only between Group C and Group A.Levels of TNF-αin serum and BALF,the number of leukocyte in BALF, the ratio of INF-αand IL-10 in serum,and the activity of NF-κB in PBL of Group D lower than those of Group C,and the number of PMN in BALF, levels of IL-10 in serum and BALF of Group D higher than those of Group C.But there were no significant differences in these indicators between Group D and Group C.Immunohistochemistry of lung sections for GR-αshowed cytoplasm +++ and nucleus +++ in Group C and Group D,and immunohistochemistry scores of Group D were higher than those of Group C(P＞0.05).Immunohistochemistry of lung sections for NF-κB p65 showed cytoplasm +/++ and nucleus +/++ in Group C and Group D,and immunohistochemistry scores of Group D were lower than those of Group C(P＞0.05).Levels of TNF-αin serum and BALF,the number of leukocyte and PMN in BALF,the ratio of TNF-αand IL-10 in serum,and the activity of NF-κB in PBL of Group E higher than those of Group C,while Levels of IL-10 in serum and BALF of Group E lower than those of Group C.But there were no significant differences in these indicators between Group E and Group C.Immunohistochemistry of lung sections for GR-αshowed cytoplasm+/++ and nucleus+/++ in Group E,and immunohistochemistry scores of Group E were lower than those of Group C(P＞0.05).Immunohistochemistry of lung sections for NF-κB p65 showed cytoplasm++ and nucleus++ in Group E,and immunohistochemistry scores of Group E were higher than those of Group C(P＞0.05). Conclusions1.Compared with traditional ventilation,LPVS was more beneficial for oleic acid-induced ARDS canines in aveolar recruitment,avoiding open alveolar excessive expansion,alleviating pulmonary lesions and pulmonary edema,improving inflammation and anti-inflammation,and ameliorating pulnomary oxygenation and cardiac function.2.Compared with not dealing with repiratory acidosis of PHC,proper buffer of respiratory acidosis can significantly improve circulation and tissue oxygen supply of oleic acid-induced ARDS canines,while has no significant impact on inflammation,anti-inflammation,imaging performance and the severity of pathological lesions in lung.3.Compared conventional LPVS,RM can further improve pulmonary oxygenation of oleic acid-induced ARDS canines,but the duration of improvement effect is relatively short,and the impact of RM on hemodynamics is not obvious.Too long time of RM maybe aggravate pulmonary lesions and inflammation,and have an adverse impact on anti-inflammation. Whether RM is a measure of LPVS maybe depend on time of RM.4.The excessive apoptosis in lung tissue maybe play a certain role in the development of ARDS.Obvious impacts of small VT and best PEEP, respiratory acidosis and RM on cell apoptosis in lung tissue were not observed in this study,which was related with not enough time of observation.