| ObjectiveTo investigate whether experimental hypercapnia is protective against acute lung injury (ALI) in an in vivo model of rabbits induced by oleic acid administration. Moreover, to observe the influence of hypercapnia on nuclear factor-kappaB and tumor necrosis factor-a ( TNF-a) , and to observe the effects of hypercapnia on nitric oxide-dependent pathway so as to investigate its potential mechanisms in ALI models.MethodsMaterialsTwenty-six New Zealand white rabbits (weighing 2.5 -3. Okg). Mixed gases ( 02: 0.75, balance N2 and 02: 0. 75, C02: 0. 08, balance N2). Bear Cub 750 ventilator. AVL OMNI V blood gas analyzer. RM 6000 physiological polygraph. TNF-a radio immuno assay (RIA) kit. Affinity-purified antibody against NF-kappaB p65. SABC kit. NO and iNOS kits. Anti-nitrotyrosine monoclonal antibody.Experiment procedureExperimental animals were anesthetized with 20% urethane (5ml kg-1 intraperitoneally). Atracheotomy was performed, and an endotracheal tube was inserted. The animals were ventilated using a Bear Cub 750 ventilator with fraction of inspired oxygen (Fi02) 0.75, rate 30 min-1, tidal volume ( VT) 7.5 ml kg'1, inspiratory time 0.66 seconds, inspiratory flow 2.0 L min"1 and 1 cm H2 0 positive end-expiratory pressure ( PEEP). These mechanical ventilation settings were continued for the duration of the experiment. The right carotid artery was cannulated for arterial pressure measurement and blood sampling. The left carotid vein was cannulated for infusion administration. Muscle relaxation was maintained with pancuronium ( 1 mg intravenously). Sterile technique wasutilized during all manipulations. Thereafter, baseline stability was confirmed for 20 minutes. Baseline arterial blood samples were then taken for blood gas measurement, and blood samples were taken for cytokine(TNF-a) assay, cen-trifuged, and the supernatant stored at - 70 C. Then the animals which met the criteria ( Pa02 >300mmHg, PaC0230 -40mmHg, HC03- > 20mmol L-1) were randomly allocated to control group (Group C) , nonnocapnia group (Group N) and hypercapnia group ( Group H). Immediately following randomization, the inspired gas concentrations were adjusted as follows: Group C and Group N ( FiC02 0.00, Fi02 0. 75, balance N2), Group H (FiC02 0.08, FiC02 0. 75, balance N2). 15 minutes later, oleic acid (0.1 ml kg-1) was injected intravenously in Group N and Group H. Sterile mormal saline (0.1 ml kg-1) was injected in Group C. Tidal volume (VT) , peak airway pressure (PIP) , positive end-expiratory pressure (PEEP) and lung dynamic compliance (Cdyn) were recorded throughout. Heart rate (HR) , arterial systolic pressure (BPsys) and arterial diastolic pressure (BPdia) were monitored throughout. Pa02, PaC02 and pH were determined at hourly intervals from the confirmation of baseline stability to the end of the experiment.Sample processingBlood samples were taken at baseline and stored at - 70 T!. The rabbits were ventilated for 3 hours after oleic acid administration. Then blood samples were taken and stored at - 70掳C. The rabbits were exsanguinated, and the lungs and heart were taken out from the thorax. Bronchoalveolar lavage ( BAL) was carried out by flushing the left lower lobe three times with 4 ml of saline (0.9% ). The BAL fluid was collected, and BALF cells were counted, then the fluid was centrifuged at 1200 r/min for 10 minutes and the supernatant stored at - 1Q掳C. The left upper lobe was fixed with 4% paraformaldehyde and embedded in paraffin, sectioned, and stained with hematoxylin-esoin for light microscopy. Immunohistochemical assay was also performed. A piece of core sample was extracted from the right middle lobe for wet/dry ratio measurement. A 1cm x 1cm xlcm core sample was extracted from the right lower lobe, and stored at -70C .MeasurementProtein content in HALF and plasma: Biuret Reaction method.Lung tissue myeloperoxidase: Biochemical method.TNF-a: The content of TNF-a in serum and BALF was measured with RIA kit according to the manufacture's introduction.Histopathol... |
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