| BackgroundComplications caused by septic are a threat for human health, although a lot of research about sepsis have been done, but this disease still has a mortality rate of up to 28%-47%, and the difficulty of its treatment is still great, so studying on sepsis is still very much. In recent years, the clinical studies and experiments for sepsis are continuing, and the septic animal model is a necessary platform for many experiments in sepsis pathogenesis and clinical prevention. A lot of experiments must be with the help of sepsis animal model. Therefore, the generation and application of sepsis animal model have very vital significance, and an ideal model can make the research work more effective.Myocardial injury, as a common complication of sepsis, is a key factor of septic shock and multiple organ failure, and is a major cause of death in sepsis. After myocardial injury, cardiac hemodynamic and cardiac contractile will have a great change. A typical hemodynamic characteristic of septic shock is the high output and low resistance, namely, the high cardiac output, low circulation resistance, the enlarged heart, the decreased left ventricular ejection fraction and the decreased contractile response of the capacity load. Myocardial injury also is observed that the levels of plasma troponin I and Caspase-3 activity have a great change.In recent years, despite the anti-infection treatment and organ function support technology have made a great progress, the treatment of sepsis is a complicated and arduous task. Esmolol as β-adrenergic receptor blocking drugs with high selectivity, it has used for prevention and treatment of serious arrhythmia in clinical treatment, and this drug can reduce the production of oxygen free radical and inhibit neutrophil infiltration mechanism to protect the myocardium.Purpose1. The sepsis rat model copied with cecal ligation and puncture method (CLP) can provide an ideal animal model for the study of sepsis, and can lay the foundation for the next step experiment, too.2. The effect of esmolol on myocardial apoptosis and cardiac function in rats with sepsis was investigated for saving the myocardial injury, delaying the progression of the disease, reducing the mortality rate and providing experimental basis and new ideas for the early clinical treatment of sepsis.3. The effect of esmolol on β-adrenal receptor and cardiac hemodynamic in rats with sepsis was investigated for studying on effect of esmolol in improvement of severe sepsis hemodynamic feasibility.Methods1. The sepsis rat model was copied with cecal ligation and puncture method (CLP), and the changes of living habits and mental state of sepsis rat model were observed before and after preparation.2. The ascites and blood were taken at 24 h after operation, and ascites and blood were cultured in culture medium. The result of bacterial contamination was observed. The appearance of spleen, lung, intestine, liver and other organs were observed, and the difference was got with normal rats. The tissue from liver, spleen and lung were taken, and put into 10% formalin for routine pathological examination.3. Eighteen SD rats were randomly divided into six groups, containing sham group (n=3, for sham operation),2 h group (n=3, sepsis model),6 h group (n=3, sepsis model),12 h group (n=3, sepsis model),24 h group (n=3, sepsis model) and 48 h group (n=3, sepsis model). The sepsis rat model was copied with cecal ligation and puncture method (CLP), and the heart blood was taken at 2 h,6 h,12 h,24 h and 48 h after operation. The plasma was separated from the heart blood and the LPS concentration was measured.4. Thirty SD rats were randomly divided into sham group, CLP1 group, CLP2 group, the different treatment was operated, respectively. The blood (1 mL) was taken from femoral vein at 24 h after operation, and the plasma was separated from the blood. The lactic acid levels and blood glucose levels of rat’s plasma for sepsis model were measured.5. Thirty SD rats were randomly divided into sham group, CLP1 group, CLP2 group, the different treatment was operated, respectively, and the survival rate of rats was counted in a week.6. Ninety-six SD rats (weight:250±20 g) were randomly divided into sham group, sepsis group and esmolol group. The rats of sham group didn’t copy by cecal ligation and puncture, and only cut the abdomen, and sepsis group and esmolol group copied by cecal ligation and puncture. After operation, sepsis group was injected into the normal saline, and esmolol group was injected into esmolol (1mg/kg/min) through tail vein.5 mL blood of three groups was taken from aortic, and the plasma was gotten with centrifugalization, and was used for measuring the levels of cTnI. The heart tissue was taken for measuring the activity of caspase-3 and myocardial apoptosis index. The facter containing LVEDd, LVPP, FS, LVEF were measured with GE VIVID7 ultrasonic diagnostic apparatus (The frequency of the probe:5 MHz).7. Measurement of cTnI concentration:The plasma was divided from heart blood, and the cTnl concentration was measured with the kit.8. Measurement of IL-1β level:The plasma was divided from heart blood, and the IL-1 β level was measured with ELISA.9. Measurement of the activity of caspase-3:The supernatant (50 μL) was divided from myocardial tissue homogenate, and the activity of caspase-3 was measured with the kit and the software CurveExpert.10.Measurement of macrophage migration inhibitory factor in cardiac muscular tissue with Western blotting.11.Measurement of myocardial apoptosis index:Specimens were fixed in paraffin embedded and sliced, after routine HE staining, the specimens were observed with optical microscope. The Specimens were treated with routine formalin fixed and paraffin embedded after dehydration treatment and the kit, the specimens were observed with optical microscope. Five perspective were randomly selected (400 ×), and the proportion of apoptotic cells was calculated.12. Grouping and treatment. Sixty SD rats (weight:250±20 g) were randomly divided into sham group, sepsis group and esmolol group. The rats of sham group didn’t copy by cecal ligation and puncture, and only cut the abdomen, and sepsis group and esmolol group copied by cecal ligation and puncture. After operation, sepsis group was injected into the normal saline, and esmolol group was injected with esmolol (0.2 mg/kg) through tail vein. The cardiac hemodynamic index was observed at time point of 6 h,12 h, and 18 h after operation. Eighty SD rats (weight:250±20 g) were randomly divided into sham group, sepsis group, the high esmolol group and the low esmolol group in the measurement of activity of myocardial tissue SOD and the levels of RCR. The treatments of sham group and sepsis group were same with the former, and the high esmolol group and the low esmolol group were injected with the high dose of esmolol (0.1 mg/kg) and the high dose of esmolol (0.4 mg/kg) through tail vein, respectively. The related index was recorded at time point of 6 h,18 h after operation.13. The parameters of heart function. The rats in any group were anesthetized intraperitoneally, femoral artery catheterization was taken for getting the biological signals by the sensor linked with MedLab system, mean arterial pressure and the electrocardiogram at time point of 6 h,12 h, and 18 h after operation were recorded, respectively. Mean left ventricular end diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP),+dP/dt,-dP/dt and heart rate (HR) were calculated by the electrocardiogram. The β-adrenergic receptor activity of sham group, sepsis group and esmolol group were measured at the time points of 0.5 h,2 h,4 h, and 6 h after treatment.14. Measuring of the SOD activity. The SOD activity was measured with the kit for measuring the SOD activity.15. Measuring of RCR. The heart was put into the buffer and homogenated using homogenizer after cutting. After centrifugation with 1000 rpm 10 min, the supernatant was suspended into heavy medium. Centrifugation with 1000 rpm 10 min again for separating the sediment, and the sediment is mitochondria. The RCR was measured with the kit.16. Statistical analysis:The data was analysized with SPSS13.0 statistical software, measurement data were expressed with the mean and standard deviation (x±s), Count data were expressed using percentage. For comparison of measurement data between three groups, single factor analysis of variance was used, LSD test were used between the two groups. Chi-square test and Fisher exact probability method were used for count data. P<0.05 was considered statistically significant difference.Results1. The changes of mental status and living habits. Before the operation, rats were lively, fur were soft, and the claw showed a healthy rosy, the eating and drinking were more active and normal. After the operation, the rat spirit were dispirited, the rat lips and claws have blood stasis, the eating and drinking were significantly reduced, the stool is not normal. Fever appeared after 4h, and the body was low body temperature after 17h. Some animal breathed faster, and eyes varied double loop.2. The changes of peritoneal tissue. The congestion point and tissue inflammation were found in multiple organs, and the tissue of cecal ligation department had partial necrosis, while the surrounding tissue also observed necrosis and inflammation response in different degree. The dark red effusion was found in abdominal cavity, meanwhile, multiple organs tissues, containing lung, liver, kidney, etc, showed inflammatory changes.3. Blood and peritoneal contamination. The Enterococcus faecium was found in the culture medium of the blood and peritoneal effusion for 24 h after sepsis model operation.4. The level of endotoxin (Lipopolysaccharide, LPS). The level of LPS of sham group,2 h group,6 h group,12 h group,24 h group and 48 h group were 0.17,11.53, 16.42,32.59,27.15 and 11.50. Obviously, the levels of LPS had a change or very significantly change (P<0.01) in different time after operation, and were higher than that of sham group. The date indicates the rats have formed different degrees of inflammation and sepsis.5. The lactic acid levels and blood glucose levels. The lactic acid levels of sham group is stable before operation and after operation, and the change is big for CLP1 group and CLP2 group, the lactic acid levels of two groups increased significantly. The blood glucose levels of sham group is stable before operation and after operation, and the change is big for CLP1 group and CLP2 group, the blood glucose levels of two groups decreased significantly. Whether the lactic acid levels or the blood glucose levels, the CLP2 group had a more change than CLP1 group.6. The survival rate. The sepsis rat model was copied with cecal ligation and puncture method (CLP) after one week, no death of found, five rats of CLP1 group survived, and only one rat of CLP2 group survived, so the survival rate of sham group was 100%, the survival rate of CLP1 group was 50%, the survival rate of CLP2 group was 10%. This date indicated that the sepsis model of CLP1 group was more ideal model for the research than CLP2 group.7. The result of optical microscope. The myocardial cell structure was observed under the light microscope, and the cell structure of sham group was close, no obvious edema, hyperemia and cytoplasm leakage; the massive necrosis of myocardial cells of sepsis group were showed, marked edema, swelling of the nuclei, myocardial fiber fault changes, cell stromal vascular congestion; myocardial cell edema and focal necrosis was not obvious in esmolol group, and changed myocardial fiber fault was less than sepsis group.8. The level of cTnI in plasma. The level of serum cTnI had no significant change after operation in sham group (P>0.05). The level of serum cTnI rose for sepsis group and esmolol group, and was higher than that of sham group at every time end (P<0.05). The time point of the most high level of serum cTnI was 12 h after operation in sepsis group, and the time point of the most high level of serum cTnI was 6 h after operation in esmolol group(P<0.05).The level of serum cTnI for sepsis group was higher than that of esmolol group, but the level of serum cTnI for esmolol group was higher than that of sham group(P<0.05).9. Apoptosis index of myocardial cell. Compared with sham group, the apoptosis index of myocardial cell in sepsis group increased significantly at every time (P<0.05).Compared with sepsis group, the apoptosis index of myocardial cell in esmolol group decreased significantly at every time (P<0.05).10.Measurement of plasma IL-1 β. The levels of IL-1 β in sham group remained stable(P>0.05). Compared with sham group, The levels of IL-1β in sepsis group increased significantly at 3h(P<0.05), and reached the highest level at 12h(P<0.05). Compared with sepsis group, the levels of IL-1β in esmolol group decreased significantly at every time (P<0.05).11. The activity of Caspase-3. The activity of Caspase-3 of sepsis group and esmolol group were higher than that of sham group (P<0.05), and the time point of the most high activity was 12 h after operation in sepsis group (P<0.05) and the time point of the most high activity was 6 h after operation in esmolol group(P<0.05). The activity of Caspase-3 in sepsis group was higher than that of esmolol group (P<0.05).12. The level of macrophage migration inhibitory factor. Compared with sham group, the levels of macrophage migration inhibitory factor in sepsis group didn’t increased significantly at 3h and 6h(P>0.05), and increased significantly at 12h and 24h(P<0.05). The level of macrophage migration inhibitory factor in sepsis group was higher than that of esmolol group at 12h and 24h (P<0.05).13. Parameters of myocardial function. The levels of LVEF, LVPP and FS of sepsis group and esmolol group were lower than that of sham group (P<0.05), and the levels of LVEDd of sepsis group and esmolol group were higher than that of sham group (P<0.05). The levels of LVEF of esmolol group was higher than that of sepsis group (P<0.05), and the levels of LVPP of sepsis group was no difference with that of esmolol group. This results suggested that the effect of esmolol on LVEF was achieved by reducing LVEDd and increasing FS (P<0.05).14. Mean arterial pressure (MAP). Compared with the sham group, MAP of sepsis group significantly decreased at 6 h after operation (P<0.01), and the MAP at 18 h after operation was nearly half of MAP of sepsis group at 6 h after operation. Compared with the sham group, MAP of esmolol group significantly decreased at 6 h after operation (P<0.05), but the MAP significantly increased compared to the sepsis group (P<0.05).15. Heart rate (HR). The HR of sepsis group at 6 h after operation significantly increased compared to sham group, but HR of sepsis group significantly decreased at 12 h and 18 h after operation (P<0.05). Compared to sepsis group,the HR of esmolol group significantly decreased at 6, was no significantly difference at 12 h, significantly increased at 18 h after operation(P<0.05). The HR of esmolol group was more stable than the sepsis group.16. Mean left ventricular end diastolic pressure (LVEDP). LVEDP of sepsis group had a significant drop with sham group at 6 h after operation (P<0.01) and with the time increasing, the decrease of LVEDP was more obvious. LVEDP of esmolol group had a significant drop with sham group at 6 h after operation (P<0.05) and the LVEDP was increasingly reducing with the time increasing, and it was more stable than sepsis group. After treatment, LVEDP of esmolol group had significantly difference with sham group and sepsis group (P<0.01).17. Left ventricular systolic pressure (LVSP). LVSP of sepsis group had no significantly difference with sham group at the time point of 6 h,12 h, and 18 h after operation, and LVSP of esmolol group had no significantly difference with sham group at the time point of 6 h,12 h, and 18 h after operation, too.18.+dP/dt.+dP/dt of sepsis group was significantly lower than that of sham group at 6 h,12 h,18 h after operation (P<0.05).+dP/dt of esmolol group was significantly lower than that of sham group at 6 h,12 h,18 h after operation (P<0.05), and was higher than that of sepsis group (P<0.05).19.-dP/dt.-dP/dt of sepsis group was significantly lower than that of sham group at 6 h,12 h,18 h after operation (P<0.05).-dP/dt of esmolol group was significantly lower than that of sham group at 6 h,12 h,18 h after operation (P<0.05), and was higher than that of sepsis group (P<0.05).20. The P-adrenergic receptor activity of myocardial cell. In the three groups, the β-adrenergic receptor activity of sham group was the most high and the β-adrenergic receptor activity of sepsis group was the lowest at the time points of 6 h,12 h,18 h after treatment.21. The activity of myocardial tissue SOD. The activity of SOD of sepsis group, the high esmolol group and the low esmolol group were lower than that of sham group (P<0.05). The activity of SOD of the high esmolol group and the low esmolol group were higher than that of sepsis group (P<0.05). The activity of SOD of the high esmolol group was higher than that of the low esmolol group (P<0.05).22. The levels of RCR. The levels of RCR of sepsis group, the high esmolol group and the low esmolol group were lower than that of sham group (P<0.05). The levels of RCR of the high esmolol group and the low esmolol group were higher than that of sepsis group (P<0.05). The levels of RCR of the high esmolol group was higher than that of the low esmolol group (P<0.05). In addition, except sham group, the levels of RCR of sepsis group, the high esmolol group and the low esmolol group at time point of 18 h after operation were lower than that of this three groups at time point of 6 h after operation (P<0.05).Conclusion1. In this part, we have successfully copied the sepsis model with CLP, and the different ways were used for verification the success of the sepsis model. The changes of mental status and living habits containing eating, drinking, etc, were observed and these habits have significantly change, after the operation, the rat spirit were dispirited, the rat lips and claws have blood stasis, the eating and drinking were significantly reduced, the stool is not normal. The changes of peritoneal tissue were observed with microscope, the congestion point and tissue inflammation were observed in multiple organs, while the surrounding tissue also observed necrosis and inflammation in different degree. In the experiment of blood and peritoneal contamination, the Enterococcus faecium was found in culture medium of the blood and peritoneal effusion for 24 h after sepsis model operation. The level of LPS were detected for verficating the success of the sepsis model, and the levels of LPS had change or very significantly change (P<0.01) in different time after operation, and were significantly higher than that of sham group. Moreover, the lactic acid levels and blood glucose levels were analysized, the great change was observed in CLP1 group and CLP2 group, the lactic acid levels of two groups increased significantly, while the blood glucose levels of two groups decreased significantly. The survival rate was counted, the survival rate of sham group was 100%, the survival rate of CLP1 group was 50%, and the survival rate of CLP2 group was 10%. This date indicated that the sepsis model of CLP1 group was more ideal model for the research than CLP2 group. The above results indicated that the sepsis model has been successfully copied with rat in this chapter, and the model in CLP1 can be suitable to use in the next experiments.2. In this work, the high level of cTn1 is a biomarker of myocardial damage in sepsis, and this indicates that myocardial cell injury severely. Meanwhile, the massive necrosis of myocardial cells was observed in sepsis group with the light microscope, and the result of esmolol group suggested esmolol could depress the myocardial damage in sepsis. Although the heart rate of rats is lower after treatment than before treatment, the myocardial damage is lighter than before treatment, and this result indicated cardiac output was compensated by increasing heart rate in sepsis. In addition, esmolol could ease reduce the inflammation response, and inhibited the myocardial apoptosis. So this drug can improve the myocardial function, and get relatively stable hemodynamic. In this work, the activity of Caspase-3 of sepsis group and esmolol group were higher than that of sham group (P<0.05), and the time point of the most high activity was 12 h after operation in sepsis group (P>0.05) and the time point of the most high activity was 6 h after operation in esmolol group (P>0.05). These results indicated that the increasing activity of Caspase-3 was coincident with the change of the myocardial apoptosis, and maybe, esmolol can inhibit the myocardial apoptosis by reducing the activity of Caspase-3. In sum, esmolol can inhibit the myocardial apoptosis in early sepsis, ease the myocardial damage, and get stable hemodynamic.3. In this work, MAP of sepsis group significantly decreased after operation, but the MAP of esmolol group significantly increased compared to the sepsis group (P<0.05), this indicated that esmolol has the effect of improving the MAP. At time point of 6 h after CLP operation, the increasing the HR in every group was considered as the result of stress reaction, and after 12 h after CLP operation the myocardial function was inhibited and the HR reduced. At the same time, esmolol can regulate the change of HR at the different phase. Another important research contents in our experiments is the significant effect on the improving the myocardial function. LVSP mainly reflects myocardial contractility, and LVEDP is on behalf of the left ventricular preload and indirectly reflects the ventricular diastolic compliance.+dP/dt and -dP/dt reflect the change of velocity of ventricular wall tension, so these indexes are the sensitive indexes to reflect myocardial systolic and diastolic function of left ventricle. In our experiments, the results suggested that esmolol can improve the heart systolic and diastolic function arrhythmia in the sepsis, and have significant effect on prevention and treatment of myocardial dysfunction inducing by sepsis. The levels of SOD activity and RCR of sepsis group, the high esmolol group and the low esmolol group were lower than that of sham group (P<0.05), and this indicated mitochondria function is affected with sepsis. The levels of SOD activity and RCR of the high esmolol group and the low esmolol group were higher than that of sepsis group (P<0.05), and this indicated mitochondria function can be improved with esmolol. The levels of RCR of the high esmolol group was higher than that of the low esmolol group (P<0.05), and this indicated the effect of esmolol is related with the dose. In sum, esmolol can stabilize the heart rate in sepsis, improve the hemodynamic, and the myocardial function. |
PDF Full Text Request