| Objective: To explore an isolated cardiac preparation and perfusion technique that can achieve continuous perfusion,to fundamentally overcome the limitations of traditional preparation methods of rat heart,and to provide a more perfect research model for isolated cardiac studies.Methods: 80 male SD rats were randomly divided into traditional group(T,n =40)and modified operation group(M,n =40).The cardiac function in vitro was compared by using Langendorff traditional in vitro cardiac method and improved surgical method.The cardiac function in vitro was compared by observing cardiac resuscitation(P-RT),stabilization time(R-ST),coronary flow(CF),heart rate(HR),left ventricular pressure(LVP)and arrhythmia,coronary outflow fluid was used to detect CK-MB content,LDH activity level.compared myocardial histopathological changes and cardiomyocyte apoptosis levels at 2 h.then,another 10 rats in each group were randomly used to further detect myocardial histopathological changes and cardiomyocyte apoptosis levels at 4 h.finally,the last 10 in each group were used for continuous perfusion to record isolated cardiac survival time.Results:The results of cardioverting experiment showed that the longest time of isolated cardiac aortic perfusion in the traditional group was 5s,the shortest time was2 s,and the median time was 3s;the longest time of cardiac perfusion-steady beating time was 8.8 min,the shortest perfusion-steady beating time was 4.2 min,the median perfusion-steady beating time was 5.05min;all the hearts in the improved group were able to achieve sustained and stable beat after isolation,no cardiac perfusion interruption was observed,and no arrhythmia was observed,which was significantly different from the traditional group(P <0.001);heart rate changes: at 1 min,the heart rate of the traditional group was only260.3±29.3 beats/min,while that of the modified group was 317.4±25.8 beats/min,P<0.001;at 5min、10min、15min、30min、1h、2h;The heart rate of the traditional group was 268.0±29.2 beats/min,266.5±26.3 beats/min,264.5±30.2 beats/min,268.6±19.6beats/min,258.5±26.1 beats/min.1 beats/min,264.0±19.3 beats/min,respectively;modified heart rate at various time points in the group:329.2±21.7 beats/min,328.9±16.1 beats/min,331.2 ± 33.6 beats/min,319.3±20.2 beats/min beats/min,318.0±20.4 beats/min,311.1±12.7 beats/min,all P values<0.001;Results of arrhythmia: the common incidence of each type of arrhythmia in the traditional group 1 was: 100%,100%,80%,60%,respectively.The maximum duration of arrhythmia was 492.9s,the minimum duration was 47.9s,and the median duration was 167.4s.On the contrary,the hearts isolated by the modified method did not have arrhythmia in the whole process and kept beating steadily,which was significantly different from the traditional group,with all P values less than 0.001.;Results of coronary flow: at 1min,the coronary flow was 5.17±0.94 ml/min in the traditional group and 9.51±0.84 ml/min in the modified group,P<0.001.At 5min,10 min and 15 min,the coronary flow in the traditional group was 9.61±0.81 ml/min,9.21±0.82 ml/min,and 9.18±0.83 ml/min,respectively.The coronary flow of the modified group was 9.69±0.87ml/min,9.83±0.76ml/min,and 9.88±0.86 ml/min.There was no significant difference between the two groups at the above time points,and the P values were 0.834,0.097,and 0.080 respectively.The coronary flow at30 min,1h and 2h in the traditional group was 8.76±0.85 ml/min,7.99±0.67ml/min,and 7.14±0.80 ml/min,respectively.The coronary flow in the modified group was9.89±0.85ml/min,9.66±0.79ml/min,and 9.17±0.78ml/min,respectively,with statistically significant differences.The P values were 0.008,<0.001,and <0.001,respectively;Results of left ventricular pressure parameters: all left ventricular pressure parameters in the modified group showed significant differences at 30 min,1h and 2h.At 30 min,the maximum rate of increase of left ventricular systolic pressure,left ventricular developmental pressure,left ventricular developmental pressure,and the minimum rate of decrease of left ventricular developmental pressure in the conventional group were respectively 109.3 ± 5.1mmhg,104.3 ± 6.1mmhg,2427.8 ± 143.1mmhg /s,and-1874.1 ± 177.3mmhg /s.At 1h,the above indexes in the traditional group were respectively 104.7 ± 5.3 mm Hg,99.2 ± 5.7 mm Hg,2195.1 ± 190.1 mm Hg/s,and-1868.0 ± 256.6 mm Hg/s.At 2h,the above indexes in the traditional group were respectively 96.1 ± 6.2 mm Hg,91.1 ± 5.7 mm Hg,1968.6 ± 166.6 mm Hg/s,and-1653.3 ± 255.5 mm Hg/s.Correspondingly,at 30 min,the above indexes of theimprovement group were 122.5 ± 11.1mmhg,117.1 ± 11.0 mm Hg,2794.1 ± 170.6mm Hg/s,and-2615.2 ± 298.6 mm Hg/s,respectively.At 1h,the above indicators in the improvement group were: 118.0± 10.4mmhg,112.8±9.0 mm Hg,2636.4±170.4mm Hg/s,-2509.3±302.9 mm Hg/s.At 2h,the above indexes in the improvement group were respectively 111.0±11.5 mm Hg,106.1±10.1 mm Hg,2502.9±132.9 mm Hg/s,and-2323.9 ± 245.3 mm Hg/s.The specific P value is shown in the text;Results of ck-mb content and LDH activity in coronary efflux: the ck-mb content in the traditional group was 93.36±33.87ng/ml,while the ck-mb content in the modified group was only 53.31±17.88ng/ml,P=0.010,and the difference was statistically significant.The LDH activity of the traditional group was 2109.82±55.80 U/ml,while the LDH activity of the modified group was only 1944.46±125.66 U/ml,P=0.007,and the difference was statistically significant;Results of pathological structure changes of myocardial tissue: in the modified group,the size of myocardial cells was relatively normal,the cytoplasm and nucleus were uniformly stained,the arrangement of myocardial cells was regular,the density tended to be normal,and the leap disk structure was seen under the microscope.The myocardial fibers were clear and continuous,no fractures were observed,the myocardial space was slightly enlarged,and the myocardial interstitium was slightly edema.In contrast,we observed irregular arrangement of cardiomyocytes,extensive rupture and disorder of cardiac fibers in the conventional group,and severe enlargement of the myocardial interstitium and severe edema of the myocardial interstitium;Results of myocardial cell apoptosis: the incidence of myocardial cell apoptosis in 2h conventional group(27.25 ± 9.15%)was significantly higher than that in the modified group(0.37 ± 0.12%),P<0.001.The incidence of myocardial cell apoptosis in the 4h conventional group(40.90 ± 6.56%)was significantly higher than that in the modified group(0.42 ± 0.14%),P<0.001.The incidence of myocardial cell apoptosis at 4h(40.90 ± 6.56%)was significantly higher than that at 2h(27.25 ± 9.15%),and the difference was statistically significant(P=0.001).However,in the improvement group,this phenomenon was not found,P=0.380,the difference was not statistically significant.Results: median cardiac survival time in vitro in the traditional group was7.2h.Median cardiac survival time in vitro in the modified group was 10.55 h,P<0.001.The survival time in vitro in the modified group was significantly longer than that in the traditional group.Conclusion:We successfully developed an in vitro heart preparation and perfusion technique that can achieve continuous perfusion in rat in vitro.This technique can achieve no heparin application,no perfusion interruption,no hypothermia stimulation,no ischemia-reperfusion injury,no hypoxia-reoxygenation injury,no arrhythmia in the whole process.The rats with this technique had less heart injury and less apoptosis.Rat hearts made with this technique can survive longer. |
PDF Full Text Request