| Objective:Establish rabbit PR-MODS model by applying home-made pneumatic chest compression device of small animals as well as modified tracheal occlusion asphyxia method.and disscuss the changing law of the model in aspects of inflammatory response, apoptosis, ion channels as well as mitochondrial damage.Methods:66 healthy and clean Chinese adult rabbits, weight 2.0-2.5kg, either male or female, by the Animal Experimental Center of Jilin University Norman Bethune Medical Department, responsive, shiny coat, without abnormal discharge in their ears, noses and eyes. Rearing cage before the experiment, no water fasting before the operation. Randomly divided into 3 groups.The first group included 10 rabbits, aiming to explore the animal model's asphyxia time of improved asphyxia methods which induced Post resuscitation multiple organ dysfunction syndrome. Comparing the differences of 48-hour survival rate between non-intervention time 3 minutes and 4 minutes after cardiac arrest which was caused by rabbit asphyxia.The second group whose non-intervention time were 3 minutes included 28 rabbits, and were randomly grouped into 4 groups,7 rabbits in each group, respectively. Each group of rabbits were killed after surgery but before resuscitation (sham group),12h,24h,48h after resuscitation respectively.Then took specimens from heart, brain, lung, kidney, liver and other organs and tissues. Observed Bcl-2, Bax and caspase-3 protein expression of major organs by immunohistochemistry; using transmission electron microscopy and TUNEL method to observe the ultrastructural changes and apoptosis of myocardial cell after resuscitation. Arterial blood of the 7 rabbits in group would be killed after 48 hours was withdrawn after surgery and before resuscitation, immediately after resuscitation,3h,6h,12h,24h as well as 48h after resuscitation.Then detect IL-6 and IL-10 level according to ELISA kit instructions;determine the total activity of serum SOD activity by method of xanthine oxidase; determine MDA capacity by thiobarbituric acid method.The third group whose non-intervention time were 3 minutes also included 28 rabbits, and were randomly grouped into 4 groups,7 rabbits in each group, respectively.Remove the rabbits'hearts and went through heart perfusion,then isolated myocardial cells to prepare single cell suspension after surgery but before resuscitation (sham group),3h,12h,24h after resuscitation. Then detect mitochondrial membrane potential(ΔΨm),while at the same time observed the ventricular action potential (AP), L-type calcium channel current (ICa-L) as well as delayed rectifier potassium current (IK) changes of PR-MODS model rabbits at different time points using whole cell patch clamp techniqueResults:1. When reaching to the standards of cardiac arrest,1 rabbit in 10 had a ventricular fibrillation, occurring 10%,6 showed electromechanical dissociation (60%),3 performanced for ECG static (30%). Spontaneous circulation of 10 rabbits after cardiopulmonary resuscitation were all recovery, ROSC rate was 100%, which need 282.06±43.06s.Survival rate was 80% after 6h; Survival rate was 60% after 12h; Survival rate was 60% after 24h(4 rabbits received cardiopulmonary resuscitation 3 minutes after cardiac arrest,2 rabbits received cardiopulmonary resuscitation 4 minutes after cardiac arrest).Survival rate was 50% after 48h(4 rabbits received cardiopulmonary resuscitation 3 minutes after cardiac arrest,1 rabbits received cardiopulmonary resuscitation 4 minutes after cardiac arrest)2. Serum IL-6 levels began to increase after resuscitation, and reached to the peak at 6h, then decreased gradually, its'serum level turned to be lower than the sham group at 12h, the levels of IL-6 was lowest at 48 hours.Serum IL-10 levels increased after resuscitation, and reached to the peak at 3h, then gradually decreased, the levels closed to the sham operation at 12h, and lower than the sham group at 48h.After resuscitation, IL-6/IL-10 ratio was significantly lower compared with that before asphyxiation, lowest at 3h, and then gradually increased, the ratio reached to a peak and higher than that before asphyxiation af 24h,then decreased3. The serum total SOD activity began to decrease after resuscitation, reached to the minimum value at 6h, then increased gradually,while the serum total SOD activity was still lower than the sham group up to 48 hours.Serum MDA increased after resuscitation, reached to the peak at 12h,then gradually decreased, while the serum MDA was,up to 48 hours, still higher than the sham group.4. No significant expression of BC1-2 protein was observed in organs of the sham group. BCl-2 protein expression in organs of 12h group after resuscitation was significantly higher than sham group, and then decreased gradually. BCl-2 protein expression in organs of 48h group after resuscitation was significantly less than the 12h group, but still higher than sham group.No significant Bax protein expression was observed in organs of the sham group. Bax protein expression in organs of 12h group after resuscitation was significantly higher than sham group, and then increased gradually. Bax protein expression in organs of 48h group after resuscitation was maximum, and was statistically significant compared with other groups。The ratio of Bcl-2/Bax in experimental group reached a peak at 12h after resuscitation,and then decreased gradually. The Bcl-2/Bax ratio of 24h and 48h group after resuscitation was minimum.No significant caspase-3 protein expression was observed in organs of the sham group, caspase-3 protein expression in organs of 12h group after resuscitation was significantly higher than sham group, and then increased gradually. caspase-3 protein expression in organs of 24h group after resuscitation was significantly increased,and was statistically significant compared with 12h group. caspase-3 protein expression in organs of 48h group after resuscitation was further increased, however,there was no statistically significant compared with the 24h group.5. TUNEL positive cells were not found in sham group organs by TUNEL detection, while it increased significantly 12h after resuscitation, and continued to increase at 24h as well as 48h after resuscitation. Positive labeled cells of every part in 48h group after resuscitation reached to the highest level during the study.6. Detected Rabbits mitochondrial membrane potential in each group using laser scanning confocal microscope,ΔΨm of the sham group was 129.42±29.29 (RFU),ΔΨm of 3h group after resuscitation sharply decreased to 13.67±2.88 (RFU),ΔΨm of 12h group after resuscitation was 15.79±3.73 (RFU), and then increased gradually.ΔΨm of 24h group after resuscitation was 43.50±2.55(RFU).The data between 3h and 12h did not have significant difference,while data among other groups all had statistical significance.7. The action potentials(AP) of 3h group after resuscitation decreased compared with that of the sham group, but it did not have statistical significance; the AP change of the 12h group, whose AP was obviously lower than the control group,was most significant; compared with the 12h group.to a degree, the AP of the 24h group restored a little, but still had significant difference compared with the control group.The ICa-L of the 3h group after resuscitation decreased compared with the sham group, but the difference was not statistically significant; ICa-L got the most prominent change at the 12th hour in our study, and was significantly lower than the sham group; ICa-L of the 24h group was higher than that of the 12h group, while was still lower than the sham group, but the differences were not statistically significant. the calcium channel was restrained early after resuscitation, and the ICa-L current density (pA/pF) turned to be lower than that of normal myocardial cells.IK gradually decreased till 24h after resuscitation.IK of 3h and 12h group had no statistically significance compared with the sham operation group. IK decreased more sharply than before,and had statistically significance compared with the sham operation group. 8. In the Electron microscope we observed that a great part of the mitochondrial of 12h group after resuscitation turned to be vacuoles and the remaining were swollen apparently, the mitochondriale crista were sparseness and nubilous; myocardial fiber of 24h after resuscitation was derangement and the texture was not clear,yet myocardial mitochondria were unabroken, but the mitochondriale crista collapsed, and was nubilous; the myocardial cells of 48h after resuscitation become focal necrotic, mitochondrial cavitation apparently, while the myofilaments arrangement were still neat, the light zone and the dark zone were clear, Z line became thicker, edema seemed to disappeared in the myocardial cytes.Conclusion:(1) the cardiac arrest PR-MODS model induced by asphyxia is reliable, and the pathological changes, inflammation, mitochondrial damage, apoptosis and apoptosis-related proteins expression in model organs all reached the multiple organ dysfunction standards. The model would have a higher success rate if we set the non-intervention time set for 3 minutes, and could be easily repeated.(2) IL-6/IL-10 gradually increased after resuscitation, together with the destroy of promoting/anti-inflammatory cytokine imbalance,which lead to MODS.(3) apoptosis plays an important role in organ dysfunction after resuscitation, which is caused by ischemia and reperfusion;(4) myocytes ICa-L began to decline 3 hours after resuscitation, and was significantly inhibited at 12th hour, while IK was not; ICa-L was gradually less inhibited as time extended, the inhibition of IK was gradually greater and became the most significant at 24th hour.(5)ΔΨm, SOD, MDA changed significantly, and the mitochondrial function was significantly impaired after resuscitation.
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