Experimental Study On The Effect Of The Cardiopulmonary Bypass On Cerebral Vascular Changes And Associated Cerebral Protective Strategy

Introduction With the improvement of techniques of cardiac surgery and cardiopulmonary bypass(CPB),attention is increasingly focused on the multi-organ dysfunction caused by the CPB.The early and late neurologic morbidities are still high.The interruption of cerebral blood flow and the regional low-flow cerebral perfusion are the vast mechanisms of the brain injury,and the cerebral perfusion is totally based on the functional status of the cerebral vessels.There are few studies on effect of the CPB on cerebral vascular changes nowadays;especially the field is almost empty in domestic.This research was assigned to experimentally study on the effect of the CPB on cerebral vascular changes and mechanisms,and raised associated cerebral protective strategy.PartⅠThe Study of the Animal Model of Closed Chest Cardiopulmonary Bypass in Sprague-Dawley RatsObjectives To establish the animal model of closed chest cardiopulmonary bypass in the Sprague-Dawley rats and provide a economical animal model for the basic research of multi-organ dysfunction induced by CPB.Methods The S.D.rats were assigned to undergo CPB via right carotid and jugular cannulation,and self-designed and improved the elements of CPB circuit.The cardiac arrest was induced by the hypothermal ventricular fibrillation.Mean body weight of the 30 rats was(467.9±44.5)g.The 30 rats were equally divided into five groups which were underwent CPB for 60 minutes(Group A),CPB for 120 minutes(Group B), deep hypothermic low flow(DHLF) bypass 60 minutes(Group C),deep hypothermic circulatory arrest(DHCA) 60 minutes(Group D) and sham(Group E) individually. During the bypass,temperature,invasive arterial blood pressure,electrocardiograph and arterial blood gas analysis were monitored and recorded at the following time point:after the anesthesia(T1),before CPB(T2),30min(T3),60min(T4),90min(T5), 120min(T6) during CPB and end of CPB(T7).Theα-stat strategy was used as blood gas analysis management.Results The CPB were performed successfully in all the rats.The blood gas analysis results were all maintained in acceptable range and the mean arterial pressure were over 60 mmHg when off-pump.The cardiac function recovered after the rat weaned from the CPB.Conclusions The animal model of closed chest CPB can be established successfully in rats with excellent survive.This model is a stable platform for the study of the pathophysiology of CPB and post-CPB multiple organ dysfunctions.PartⅡThe Study on Effect of the Cardiopulmonary Bypass on Cerebral Vascular Changes in Rats and Associated MechanismsObjectives 1.To study on the cerebral vasomotor changes during the CPB and cerebral vascular reactivity changes after different bypass time and modes and to investigate the associated mechanisms.2.To test the cerebral injuries and metabolism in protein and cell levels to correlate the cerebral injuries and cerebral vasomotor changes.Methods1.The Study on the Cerebral Vasomotor Changes During the CPB and Cerebral Vascular Reactivity Changes after Different Bypass Time and Modes in Rats. The 30 rats were divided into 5 groups as PartⅠ.The internal jugular vein blood samples were taken before CPB(T2),30 min(T3),60 min(T4),90 min(T5) and 120 min(T6) during CPB to test the serologic endothelin-1(ET-1) and nitric oxide(NO) changes.The ET-1/NO ratio was calculated to investigate the cerebral vasomotor changes.The right middle cerebral artery(MCA) was harvested and prepared for assessment of the myogenic tone with different transmural pressure,the endothelial cell response induced by different concentration of acetylcholine and the vascular smooth muscle cell response induced by the sodium nitroprusside and serotonin.The hippocampus tissue was harvested to observe the ultramicrostructure of endothelial cell in capillary with electron microscope. 2.The Mechanism of Cerebral Vascular Dysfunction Induced by the Cardiopulmonary Bypass in Rats.The internal jugular vein blood samples were taken before CPB(T2),30 min(T3),60 min(T4),90 min(T5) and 120 min(T6) during CPB to test the tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6) in serum.The statistical analysis was used to test correlation between the cytokines and the cerebral vasomotor changes.The eNOS protein expression in hippocampus was determined by the Western immunoblotting and the correlation between the eNOS expression and NO content was accessed.3.The Study on the Correlation Between the Cerebral Vascular Changes and Cerebral Injuries Induced by CPB in Rats.The internal jugular vein blood samples were taken 30 min(T3),60 min(T4),90 min(T5) and 120 min(T6) during CPB to test the S100βprotein levels and assess the correlation between S100βlevels and ET-1/NO ratio.The apoptosis cell percentage in hippocampus was determined by Annexin V/PI exam and the correlation between the apoptosis cell ratio and ET-1/NO ratio on T6 was accessed.The neuron injury was scored by the histopathology.The ATP content in frontal and apical lobe of the brain was tested by the electrophoresis and correlated with the ET-1/NO ratio on T6.The lactic acid content and internal jugular vein saturation were tested before CPB(T2),30 min(T3),60 min(T4),90 min(T5) and 120 min(T6) during CPB.The correlation were analyzed between the lactic acid content/ internal jugular vein saturation and the ET-1/NO ratio.Results1.The Study on the Cerebral Vasomotor Changes During the CPB and Cerebral Vascular Reactivity Changes after Different Bypass Time and Modes in Rats. All the rats were successfully established and weaned from the bypass.The ET-1 content increased with the bypass time and in DHLF and DHCA group,the ET-1 content increased in rewarming period.The NO content reached summit at T3 but decreased gradually after that.The ET-1/NO ratio was lower than T2 in T3 but increased to summit at the end of bypass.The bypass did not alter the results of myogenic tone test with different transmural pressure.Furthermore,the vascular smooth muscle cell response induced by the sodium nitroprusside and serotonin were also intact in all the rats.Acetylcholine caused a dose-dependent vasodilation in the sham group that was absent in animals undergoing CPB.Significantly,this was apparent after only 60 minutes of CPB and the endothelial cell vasodilation responses were lower in DHLF and DHCA groups compared with group B.The ultramicrostructure of endothelial cell and capillary in hippocampus observed by electron microscope found normal structure in group A and B,but the V-R space widened in group C and structural injury in endothelial cell and cerebral edema in group D.2.The Mechanism of Cerebral Vascular Dysfunction Induced by the Cardiopulmonary Bypass in Rats.CPB led to an early and marked increase in TNF-αand IL-6.The correlation coefficient between TNF-αand ET-1/NO ratio was 0.637(P＜0.001).The correlation coefficient between IL-6 and ET-1/NO ratio was 0.521(P＜0.001).The ICAM-1 were overexpressed in all the CPB groups and the intensity of ICAM-1 expression increased by turns in group A,B,C and D.The correlation coefficient between ICAM-1 and ET-1/NO ratio was 0.872(P＜0.001).The CPB was correlated with the significantly reduction in eNOS expression.The ranks comparison based on Wilcoxson tests indicated that the eNOS protein level was lower in group B than group A(P=0.002) and group D than group C(P=0.002).The correlation coefficient between eNOS protein expression and NO content in internal jugular vein was 0.866(P＜0.001),and the regression equation was NO content= 1.91+1.73×eNOS expression.3.The Study on the Relationship Between the Cerebral Vascular Changes and Cerebral Injuries Induced by CPB in Rats.The CPB was correlated with the significantly increased in S100βprotein.DHCA caused highest S100βprotein levels compared with group B and C.The ranks comparison indicated that the S100βprotein level was higher in DHLF group compared with group B.The correlation coefficient between S100βprotein and ET-1/NO ratio was 0.753(P＜0.001).The CPB was correlated with the significantly increased in apoptosis cell percentage in hippocampus.DHCA caused highest percentage of apoptosis cells compared with group B and C.The correlation coefficient between percentage of apoptosis cells and ET-1/NO ratio was 0.793(P＜0.001).No neuron injury was found pathologically and scored 0 in all the groups.CPB did not alter the level of ATP compared with sham group,but the level reduced significantly in group C and D.The ranks comparison based on Wilcoxson tests indicated that the ATP level was lower in group D than group C(P=0.002).The correlation coefficient between ATP level and ET-1/NO ratio in T6 was -0.808 (P＜0.001).The lactic acid were higher in group C and D compared with group A and B in T4,T5,T6,but weak correlation was found with ET-1/NO ratio.Conclusions1.The Study on the Cerebral Vasomotor Changes During the CPB and Cerebral Vascular Reactivity Changes after Different Bypass Time and Modes in Rats.a.The cerebral vasoconstriction effect is greater than vasodilatation effect during the CPB,which lead to poor cerebral perfusion.b.The specific loss of acetylcholine- induced vasodilation suggests endothelial cell dysfunction,which appeares 60 min after the onset of CPB and are worsen in DHLF and DHCA group.Adversely,the vascular smooth muscle functions are not impaired after CPB.c.The observation of ultramicrostructure of endothelial cell and capillary in hippocampus finds structural injury in endothelial cell and cerebral edema in DHCA group.2.The Mechanism of Cerebral Vascular Dysfunction Induced by the Cardiopulmonary Bypass in Rats.a.The increase of TNF-αand IL-6 induced by CPB is correlated with the cerebral vasomotor changes,which demonstrates that the impair cerebrovascular function is caused by the systemic inflammatory response.b.The eNOS protein expression is reduced by CPB,especially in DHLF and DHCA group,which suggests the systemic inflammatory response and ischemia-reperfusion injury are the vast factors that affect the expression of eNOS protein.c.The cerebrovascular eNOS protein expression is correlated with the NO content in internal jugular vein,which suggests the expression level of eNOS is a main factor of cerebral vasomotor changes. 3.The Study on the Relationship Between the Cerebral Vascular Changes and Cerebral Injuries Induced by CPB in Rats.a.CPB,especially DHCA and DHLF leads to the increase of serum S100βprotein, which is a marker of the cerebral damage.And the serum S100βprotein level is correlated with the cerebral vasomotor changes.b.Apoptosis is induced by CPB in cells of hippocampus,which suggests that apoptosis is a vast mechanism of cerebral damage.The apoptosis cell percentage is correlated with the cerebral vasomotor changes.c.No evidence of neuron damage can be found pathologically in 2 hours after CPB.d.DHLF and DHCA can lead to the reduction of ATP level in brain and increase of the lactic acid level in internal jugular vein.The ATP level is correlated with the cerebral vasomotor changes.PartⅢThe Study of Protective Effect of NO Donor on Cerebral Damage Induced by CPB in RatsObjectivs To investigate the cerebral protective effect and mechanism of NO donor reducing the cerebral damage induced by CPB.Methods 36 rats were equally divided into six groups which were underwent CPB for 120 minutes(Group B),DHLF 60 minutes(Group C),DHCA 60 min(Group D),CPB for 120 minutes with SNP(Group F),DHLF 60 minutes with SNP(Group G),DHCA 60 min with SNP(Group H).The protective strategy was to infuse SNP 3mg/kg/h intravenously with deoxyepinephrine to maintain the MAP over 60mmHg till end of CPB.The serum S100βprotein level,the apoptosis cell percentage,the ATP levels, the lactic acid and internal jugular vein saturation and the microstructure were compared between the corresponding groups with and without SNP.The blood samples were taken at T2 and T6 to evaluate the TNF-αand IL-6 levels. The levels were compared between the corresponding groups with and without SNP. The eNOS protein expression were determined to assess the changes affected by the NO donor.Results 36 rats were successfully established the bypass.The serum S100βprotein levels and apoptosis cell percentages were lower at T5 and T6 in groups with SNP compared with the control groups.The ATP levels were higher in groups with SNP compared with the control groups while the lactic acid levels were lower in DHCA/DHLF groups with SNP compared with corresponding control groups.The IL-6 levels were lower in groups with SNP compared with corresponding control groups at T6.The SNP altered the eNOS protein expression in group F(P=0.037) compared with group B,while the expression levels were similar in DHLF/DHCA groups with or without SNP.Conclusions1.As an exogenous NO donor,SNP can significantly reduce the cerebral damage and cellular apoptosis induced by CPB.2.The mechanism of cerebral protection of SNP is contributed to the vasodilatation to increase the blood flow and oxygen and inhibition of systemic inflammatory response induced by CPB.