Effects Of Two Re-warming Methods On Oxidative Stress And Inflammatory Cytokines Of Lung Injury In Rats With Acute Severe Seawater Immersion Hypothermia

Objective:The purpose of this experiment is to establish the lung injury model in rats with acute severe seawater immersion hypothermia and to compare the effects of active re-warming and passive re-warming on oxygen free radicals?MDA,SOD,GSH-PX?and serum inflammatory cytokines?IL-6,TNF-a,IL-1??in hypothermia rats.Methods1.Establishment of lung injury model in rats with acute severe seawater immersion hypothermia?1?Exploration on the injury conditions of rats with acute severe seawater immersion hypothermia.Totally 100 healthy SD male rats were randomly divided into five groups which were immersed in 20??17??15??13?and 10?seawater respectively.Each group contained 20 rats.The changes of physiological indexes such as respiration,heart rate and muscle fibrillation and abdominal temperature within 2 hours were observed and the survival time and survival rate of rats in each group were counted in24 hours.Then based on the above results,the ideal soaking temperature and duration for establishing the injury model of rats with acute severe seawater immersion hypothermia were summarized.?2?Establishment of lung injury model in rats with acute severe seawater immersion hypothermia16 healthy SD male rats were randomly divided into normal temperature group and hypothermia immersion group?15?seawater immersion for 5 hours?.The coagulation,blood routine,biochemical indexes and lung histopathology were compared between the two groups.The changes of physiological state,gross lung specimens,lung histopathology,arterial blood gas,lung wet/dry ratio?W/D?,blood routine and coagulation after soaking at low temperature were observed.2.Effects of two re-warming methods on oxidative stress and inflammatory cytokines of the lungs in rats with acute severe seawater immersion hypothermiaA total of 64 healthy male Sprague-Dawley rats were randomly divided into normothermia group?hypothermia immersion group?hypothermia followed by active re-warming group and passive re-warming group.At the same time,active re-warming group and passive re-warming group further subdivided into three subgroups HAW2h,HAW6h and HAW12h according to the time of re-warming.Each group contained 8rats.In the hypothermia immersion group,the rats were soaked in seawater at 15°C for5 hours and the rats of passive re-warming group were passively re-warmed at room temperature after being soaked in seawater at 15°C for 5 hours.In the active re-warming group,the rats were soaked in seawater at 15°C for 5 hours and then re-warmed at 37°C for 1 hour.The purpose of this part of experiment was to observe the effects of two re-warming methods on lung pathology,abdominal temperature,wet/dry ratio?W/D?,lung oxygen free radicals?SOD,MDA,GSH-PX?and serum inflammatory factors?IL-6,IL-1?,TNF-a?.Results1?Establishment of lung injury model in rats with acute severe seawater immersion hypothermia?1?Exploration on the injury condition of rats with acute severe seawater immersion hypothermiaSoaking for 10 minutes,the respiration and heart rate of each group were significantly increased,but there was no significant difference among groups?P>0.05?.The respiration and heart rate decreased rapidly between 10 and 40 minutes and the decline was slower relatively between 40 and 80 minutes.Soaking for 80 minutes,there was a significant difference in respiration among the rats?P<0.05?.Immersing for100 minutes,the heart rates of rats in each group were significantly different?P<0.05?.Soaking for 20 minutes,the muscle fibrillation of 15?,13?and 10?group reached the peak and there were significant differences among groups?P<0.05?,then disappeared.However the rats in 20?and 17?group reached the peak at 30 minutes,but there was no significant difference between two groups?P>0.05?,hereafter the muscle fibrillation maintained at a certain level.The mean survival time of 20?group,17?group,15?group,13?group and 10?group in 24 hours were?23.6±1.23?hours,?15.0±4.16?hours,?7.7±3.21?hours,?2.4±0.91?hours and?1.1±0.39?hours respectively and the survival curve of each group was statistically significant?P<0.05?.The intraperitoneal temperature of rats showed a decline in the cliff,the lower the water temperature,the faster the descending.Soaking for 40 minutes,the difference of intraperitoneal temperature of each group was statistically significant?P<0.05?.According to the difference of temperature,seawater immersion in rats respiration,heart rate,fibrillation,consciousness,survival time and survival rate,we initially summarized that the ideal experimental conditions for establishing the injury model for rats with acute severe seawater immersion hypothermia was 15?seawater immersion for 5h.?2?Establishment of lung injury model in rats with acute severe seawater immersion hypothermiaCompared with the normal temperature group,the rats in the 15°C seawater immersion for 5h lost consciousness of activity and were in a coma and their respiration and heart rate were not noticeable.Arterial blood gas showed a decrease in blood pH?P<0.05?,partial pressure of oxygen?PaO₂?and partial pressure of carbon dioxide?PaCO₂?increased significantly?P<0.05?.The lung specimens were dark red with slightly dark texture,congestion,edema,and hemorrhagic foci on the surface.Pathological light microscopy of the lungs revealed alveolar septum thickening with rupture,local atelectasis,alveolar collapse and partially visible hyaline membrane formation within the alveolar space including edema fluid,extensive pulmonary capillary congestion and pulmonary interstitial scattered bleeding with inflammatory cell infiltration.Lung pathology scores and W/D increased?P<0.05?.Blood tests showed that the percentages of red blood cells,hemoglobin and neutrophils increased?P<0.05?,platelet and white blood cell counts decreased?P<0.05?,partial activated thromboplastin time,prothrombin time and international standardized rate increased?P<0.05?.The pathophysiological parameters of rats showed that the lung injury model in rats with acute severe seawater immersion hypothermia could be successfully established at 15°C seawater immersion for 5 hours.2?Effects of two re-warming methods on oxidative stress and inflammatory cytokines of the lungs in rats with acute severe seawater immersion hypothermia?1?Comparisons of two re-warming methods on abdominal temperature and lung pathology in ratsAt the time of active re-warming for 1 hour,the temperature of the abdominal cavity of the rat rapidly increased from about 16°C to about 38°C,followed by a short-term down-effect to about 35°C and it basically returned to normal at 2 hours.After passive re-warming for 8 hours,the temperature of the rat's abdominal cavity slowly increased from about 16°C to normal.Pulmonary pathological scores and W/D in the passive re-warming 2h and 6h groups were higher than those in the active re-warming group at the corresponding time points?P<0.05?.Pulmonary pathological scores of passive re-warming for 12h were slightly higher than those of active re-warming 12h groups?P>0.05?.?2?Comparisons of two re-warming methods on pulmonary oxygen free radicals in ratsCompared with the normal group,the MDA content in the lungs of the low temperature soaking group increased significantly?P<0.05?,the activities of the SOD and GSH-PX decreased significantly?P<0.05?.Compared with the low temperature soaking group,the content of MDA in the lungs of the passive re-warming 2h group was further increased?P<0.05?.Compared with active re-warming for 2h group,the content of MDA in lungs of passive re-warming for 2h group was significantly higher?P<0.05?.The MDA of passive re-warming 6h and 12h groups were slightly higher than those in active re-warming 6h and 12h groups?P>0.05?.Compared with passive re-warming 2h group,the activities of SOD and GSH-PX in the active re-warming 2h group were slightly higher?P>0.05?.Compared with passive re-warming 6h and 12h groups,the activities of SOD and GSH-PX in the lungs of active re-warming for 6h and 12h groups increased significantly?P<0.05?.?3?Comparisons of two re-warming methods on serum inflammatory cytokines in rats.Compared with the normal group,the levels of serum IL-1?,IL-6 and TNF-a in the low temperature soaking group were significantly higher?P<0.05?.Compared with the low temperature soaking group,the levels of serum IL-1?and IL-6 in the passive re-warming 2h group were further increased?P<0.05?.Compared with active re-warming for 2h group,the levels of serum IL-1?and IL-6 in the passive re-warming for 2h group increased?P<0.05?.Compared with the active re-warming 6h and 12h groups,the levels of serum IL-1?and IL-6 in passive re-warming 6h and 12h groups increased slightly?P>0.05?.The levels of serum TNF-a in passive re-warming 2h,6h and 12 groups were slightly higher than those in the corresponding active re-warming groups?P>0.05?.Conclusion1?The model of acute severe hypothermia seawater immersion hypothermia in rats was successfully constructed by immersing in seawater at 15°C for 5 hours.The main pathophysiological manifestations of this model were loss of activity awareness in rats,coma,difficulty in noticing breathing and heart rates.Hematological indicators showed that thrombocytopenia and coagulation disorders such as prothrombin time and increased international standardization rate,at the same time pulmonary pathology showed thickened alveolar space,local atelectasis,alveolar collapse,edema fluid in the alveolar cavity,extensive pulmonary capillary congestion and pulmonary interstitial with inflammatory cell infiltrations,besides lung pathological injury scores and W/D increased.2?Compared with the passive re-warming,warm bath re-warming might reduce lung injury of rats with acute severe seawater immersion hypothermia by weakening oxidative stress and inflammatory responses.The main manifestations were to reduce content of MDA,increase the activities of antioxidant enzymes SOD and GSH-PX,reduce the levels of inflammatory cytokines IL-1?,IL-6 and TNF-a.