Mechanisms Of Pulmonary Source Lipopolysaccharide On Ventilator-induced Lung Injury

Party I The model of pulmonary source lipopolysaccharide induced lung injury in rats in vivoObjective To observe the effects of various dose lipopolysaccharide-instilled lungs on lung in rats in vivo and to investigate the pathophysiological changes and the mechanisms of the injurious lungs induced by pulmonary source lipopolysaccharide.Method 36 male, adult SD rats were randomly divided into 6 groups:control group(groupC,n=6);intratracheal instillation of 50 ug/kg body weight LPS group(group L1,n=6); intratracheal instillation of 100μg/kg body weight LPS group( group L2, n=6); intratracheal instillation of 200μg/kg body weight LPS group group(group L3,n=6); intratracheal instillation of 1000μg/kg body weight LPS group ( group L4,n=6).intratracheal instillation of 5000μg/kg body weight LPS group(group L5,n=6). The spontaneously breathing was keeped and the air was inhaled in the 6 groups'rats. Three hours after experiment, The rats were killed by exsanguination via arteria carotis interna. Then the rats'pulmonary pathomorphology scores,pulmonary tissue wet/dry weight ratio, white blood cell(WBC) count in bronchial lavage fluid(BALF), lung permeability index (LPI)were determined. The tumor necrosis factor-α(TNF-α)and macrophage inflammatory protein -2(MIP-2) concentrations in BALF were detected with a enzyme-linked immuno assay(ELISA method).Results pulmonary pathomorphology scores ,W/D and LPI: there was no significant difference in groupL1 , group L2and group L3 as compared to group C,but the elevation of groupL4 and groupL5 was significantly differen(tP<0.05or P<0.01);the elevation of group L5 had significant difference vs. group L4(P<0.05or P<0.01). BALF WBC: there was no significant difference in groupL1 and group L2 as compared to group C,but the group L3 was increased(P<0.05),and the group L4and group L5 were significant increased(P<0.01). TNF-αwas not detected in group C, there were a small quantity TNF-αin the groupL1 , group L2and group L3, but it was elevated in group L4 and group L5 vs. group L1(P<0.01). MIP-2: there was no significant difference in groupL1 , group L2and group L3 as compared to group C, but the elevation of groupL4 and groupL5 was significantly differen(tP<0.05or P<0.01);the elevation of group L5 had significant difference vs. group L4(P<0.05).Conclusions 50-100μg/kg body weight LPS instilled lung don't induce lung structural injury and inflammatory cell infiltrate, but there are some slight elevation of TNF-αconcentration . 200μg/kg body weight LPS instilled lung don't induce lung structural injury, but it induce a slight inflammatory cell infiltrate and elevation of TNF-αconcentration. 1000 -5000μg/kg body weight LPS instilled lung induce lung structural injury, inflammatory cell infiltrate, elevation of TNF-αconcentration, and decrease of PO2. Party II Effect of various ventilation manier on lung tissue and the expression of CD14 receptor in rats in vivoObjective To investigate the effects of mechanical ventilation (MV)on rat's lung and the expression changes of lung endotoxin receptor CD14.Methods 24 male Sprague-Dawley rats weighing 330-360g were randomly divided into 4 groups (n=6):group R received no mechanical ventilation, group P received small MV (VT=6ml/kg, PEEP=8mmHg) , group M received conventional MV (VT=12ml/kg, PEEP=0mmHg) , and group N received large tidal volume mechanical ventilation(VT =40ml/kg, PEEP=0mmHg). The animals were anesthetized with intraperitoneal pentobarbital 35-40mg.kg-1, tracheotomized and mechanically ventilated (I:E=1:1,FiO2=21%). The respiratory rate (RR) of MV was adjusted to maintain the end-tidal carbon dioxide in the rang of 35-45mm Hg throughout the procedure. Right carotid artery and left femoral vein were cannulated for BP monitoring and fluid and drug administration.. The experiment was culminated in 3 hours, then the rats were killed by by exsanguination via arteria carotis interna. Morphologic change scores of the rats'lungs, wet/dry weight ratio of lung tissue(W/D),bronchial lavage fluid(BALF)inflammtory cell population, and lung permeability index(LPI) were evaluated. The concentration of TNF-αand MIP-2 in the plasma were determined by enzyme immunoassay method(ELISA). The expressions of lung tissue endotoxin receptor CD14 were detected by RT-PCR, macrophage CD14 in BALF was also detected by immunohistochemistry.Results pulmonary pathomorphology scores:there were no alteration in R group and P group,but it were slightly increased in M group,there was significantly elevated in N group as compare to M group(P<0.01). pulmonary tissue wet/dry weight ratio(W/D):Compare with R group,There was no statistically significant difference in P group and M group; the elevation in N group(,P<0.05),LPI: Compare with R group, There was no statistically significant difference in P group and M group; the obviously elevation in N group (P<0.01).WBC in BALF:Compare with R group,there was no change in P group, the elevation in M group ( P<0.05 ) ,there was significantly elevated in N group (P<0.01).TNF-αhad no manifest variation in 4 groups.MIP-2:compare with R grroup, There was no statistically significant difference in P group, the elevation in M group, there was significantly elevated in N group(P<0.01)。The expressions of macrophage CD14 protein in BALF and lung tissue CD14 mRNA were fundamentally coincident in R group and P group; the expressions of CD14 mRNA were elevated, but the expressions of CD14 protein were no change in M group; the expressions of CD14 in N group manifestly elevated (P<0.01).Conclusions conventional MV induces minor injury in rat's lung and can up regulate the expression of CD14 mRNA in the lung, but no up regulate the expression of CD14 protein;large tidal volume MV induces injury of rat's lung and evidently up regulates CD14 expression in the lung. Protective MV can avoid the above mentioned variations in rat's lung.Party III Effect of mechanical ventilation with different tidal volumes on alveolar macrophage and CD14 receptor expression in alveolar macrophage in ratsObjective To investigate the effect of mechanical ventilation with different tidal volumes on alveolar macrophage and the expression changes of alveolar macrophage endotoxin receptor CD14 in rats.Methods 18 male, adult SD rats weighing 230-240g were randomly divided into 3 groups (n=6 each): groupC (control) received no mechanical ventilation; group P received mechanical ventilation with small tidal volume (VT =8 ml·kg-1,PEEP=8mmHg) and group N received mechanical ventilation with large tidal volume (VT =40 ml·kg-1,PEEP=0 ). The animals were anesthetized with intraperitoneal 3% pentobarbital 35-40 mg·kg-1 , tracheostomized and mechanically ventilated (I:E=1:1, FiO2 =21%). The respiratory rate (RR) was adjusted to maintain the PET CO2 at 35-45 mmHg. The rats were killed by exsanguination after 3h mechanical ventilation. Morphologic change scores of the rats'lungs, wet/dry weight ratio of lung tissue(W/D),and lung permeability index(LPI) were evaluated. The left lung was lavaged, the number of inflammatory cells and alveolar macrophage in broncho-alveolar lavage fluid (BALF) were counted in light microscope, and the AM separated from BALF was cultivanted in the incubator for 2h. Then the expression of CD14 protein on the AM collected from BALF was determined by immuno-histochemistry. The CD14 receptor mRNA expression in AM was determined by RT-PCR.The concentration of MIP-2 and MCP-1 in culture fluid were determined by enzyme immunoassay method(ELISA).Results The pathological scores of lung, W/D lung weight ratio, the number of inflammatory cells and AM in BALF, the expression of CD14 receptor mRNA in AM and CD14 receptor protein on macrophage were significantly higher in large tidal volume group than in control group while there was no significant difference between small tidal volume and control group.Conclusion Mechanical ventilation with large tidal volume for 3h induces lung injury and up-regulation of CD14 receptor expression on alveolar macrophage. Protective MV can avoid the above mentioned variations .Party IV Effects and mechanisms of pulmonary source lipopolysaccharide on ventilator-induced lung injuryObjective To investigate the effects and mechanisms of lipopolysaccharide-instilled lungs on ventilator-induced lung injury.Method 24 male, adult SD rats were randomly divided into 4 groups:a spontaneously breathing group(groupC,n=6);intratracheal instillation of LPS+ spontaneously breathing group(group C1,n=6); mechanical ventilation group( group M, n=6); intratracheal instillation of LPS +ventilation group(group M1,n=6); group M and M1 group: Vt=20ml/kg, PEEP=0mmHg; group C1 and group M1: 100μg/kg of LPS was dropped in the trachea. The air was inhaled in the 4 groups'rats. Three hours after experiment, The rats were killed by exsanguination via arteria carotis interna. Then the rats'pulmonary pathomorphology scores,pulmonary tissue wet/dry weight ratio, bronchial lavage fluid(BALF)white blood cell(WBC) count and AM count, lung permeability index, LPI were determined. The tumor necrosis factor-α(TNF-α)and macrophage inflammatory protein -2(MIP-2) concentrations in BALF were detected with a enzyme-linked immuno assay(ELISA method).Lung tissue endotoxin receptor CD14 mRNA expression was determined with RT-PCR, BALF macrophage CD14 expression was determined with immunohistochemistry.Results W/D and LPI: there was no significant difference in group C1 and group M as compared to group C,but the elevation of group M1 was significantly different(P<0.01);the elevation of group M1 had significant difference vs. group M(P<0.01). Pulmonary pathomorphology score, BALF WBC,AM, and MIP-2: there was no significant difference between group C and group C1,the above values of group M and group C1 significantly increased than those of group(P<0.01);the above values of group M1 significantly increased than those of group M(P<0.01).TNF-αwas not detected in C group and M group,but it was elevated in group C1 and group M1,the increase in M1 group was more predominant than that of C1 group(P<0.01).Pulmonary tissue endotoxin receptor CD14 mRNA and BALF macrophage CD14 expressions were all elevated(P<0.01)in group M as compared to group C.Conclusions Microamount pulmonary source endotoxin can aggravate ventilator induced lung injury ,the possible cause is that mechanical ventilation of a large Vt sensitizes the lung to LPS stimulation, a phenomenon that may occur via the up regulation of CD14 and the increase of AM.