| Cardiopulmonary bypass (CPB) is a powerful systemic stimulation to the body, generating ischemia, anoxemia and reperfusion injury and intense stress reaction. Excessive inflammatory reaction induced by stress is one of the main causes of death in cardiac surgery, leading to tissue damage, systemic inflammation response syndrome (SIRS), and even multiple organs dysfunction syndrome (MODS). Organ protection during CPB is one of the hot areas in both basic and clinical research of cardiovascular surgery. Many in-depth studies have long focused on the function of important organs like heart, brain, lung and kidney in the CPB. It was not until recently that researchers have realized that intestine is not only one of the organs that initiate stress but also one of the paramount target organs of stress. The key point is that the dysfunction of intestinal mucosal barrier results in the bacteria/endotoxin translocation. The impairment of intestinal mucosal barrier after CPB is a marginal area of study with little attention, and the studies on its occurrence and mechanism are fairly few. The incidence of serious complications of digestive system after clinical CPB is low (0.41～3.7%), while the mortality rate is rather high(13.9～52%). Therefore, it is clinically important to study the mechanism of impairment of intestine barrier in CPB, and to discuss the corresponding protective measures.The maintenance of normal intestine barrier depends on the intact intestinal mucosal epithelia, normal flora and secretion in intestine, peristalsis and immune function of intestine, with intestinal mucosal barrier, the most critical part. Vigorous metabolic ability of intestinal mucosa along with severe traumatic ischemia, anoxemia and reperfusion thereafter contributes to apoptosis of large amount of excessive mucosal epithelial cells leading to the damage of mucosal barrier. Necrosis of intestinal mucosa was believed to be the pathological basis of intestinal barrier dysfunction, yet increasing studies have suggested that the main cause is apoptosis of mucosal epithelial cells. Recently, the production of specially made micro membrane lung of small animals and the closed-chest puncture technique have enabled the establishment of full-flow CPB rat model in common temperature, which is economic, practical, minimally invasive, easy to operate, reliable, and closer to clinical process, providing ideal model for the research of early stage systemic inflammation reaction, multiple organs dysfunction syndrome and tactics of prevention and care.Ulinastatin is a kind of broad spectrum enzyme inhibitor with multidunious biological functions. It inhibits the activity of various proteins, glucose and lipidic hydrolase, hampers excessive secretion of inflammatory mediators, improves microcirculation and pharmacological action of tissue perfusion. It is effective in the rescue of critical patients, protecting important organs.The research is to discuss the form of intestinal mucosal barrier of rats that went through CPB and its dysfunction, as well as the role of inflammatory reaction and apoptosis of intestinal mucosal epithelial cells in the impairment of intestinal mucosa, and to study preventive function and mechanism of ulinastatin by treating the rats with it. The research is discussed in three parts with methods, results and conclusion as follows.1. Establishment of CPB animal model in common temperature on rats with intubation.1.1 ObjectiveTo set up the full-flow CPB experiment model on rats in common temperature with intubation, which is economic, practical, minimally invasive, easy to operate, reliable, and closer to clinical process.1.2 Methods14 male adult SD rats (350～500g) were anesthetized and given tracheal intubation and assisted ventilation by breathing machine. The tube placed in the femoral artery was connected to the monitor for real-time blood pressure monitoring, and arterial blood gas was collected on time. Blood was transmitted through peristaltic pump to specially made micro membrane lung for oxidation before its infusion into right cervical artery. The model was pre-inflated with Ringer's solution, middle molecular hydroxyethly starch and small amount of rat blood. The total amount was 22ml, the ratio of crystal and colloid 1:1, infusion flow 120～160ml·kg-1·min-1, and mean bypass time 60 minutes.1.3 ResultsTwo out of fourteen rats died of hemorrhage during the establishment of CPB, another two failed to establish the model because of the obstructed drainage as a result of resistance in jugular vein intubation and inadequate length of the catheter in the vein. Success rate of model establishment was 71% with mean bypass time 60 minutes and bypass flow approaching 120～160ml·kg-1·min-1, approximate to rats'cardiac output and in accordance with the requirements of complete bypass. During the experiment, hemodynamics was stable, and oxygenation of oxygenator fully met CPB standard. Blood gas test during CPB indicated frequent acidosis, while other indices of blood gas and electrolytes were normal. During the discontinuance of CPB, blood pressure and heart rate began to drop, the level was remarkably lower than before the bypass even with the help of vasoactive agents, and serious dilution of blood worsened the condition, rats did not live long and generally died of circulation and respiratory failure in 7 hours.1.4 ConclusionFull-flow CPB experimental animal model could be established by intubation, drainage through right jugular vein cavity and right jugular artery infusion. The surgery was simple, easy to manage, economic and practical. Miniaturized circuit design provided guarantee for the establishment of stable CPB, and micro membrane lung and peristaltic pump met the requirements of oxygenation and hemodynamics of CPB rats. The model resulted in high short-term survival rate, exhibiting relevant detection capability for pathological, biological, biochemical and molecular biological index, and enabled the serialization and formularization of CPB related basic research, promising comprehensive application perspective. 2. Research on the early stage dysfunction of intestinal mucosal barrier, inflammatory reaction and mechanism after CPB.2.1 ObjectiveTo discuss clinical significance of the intestinal mucosal barrier impairment after CPB by observing the ileal mucosa morphous, barrier impairment and the variance of serum inflammatory factor concentration. And by detecting the level of protein and gene, to approach the existence of excess apoptosis and generation of inflammatory factors as well as the prospective mechanism of intestinal mucosal barrier impairment.2.2 Methods30 clean healthy adult SD rats were divided randomly into normal control group (group N,n=10), sham-operated group(SH,n=10), CPB group(n=10). Rats in SH group received intubation in corresponding area while being injected 22ml of pre-inflated fluid slowly through femoral artery in 10 minutes. CPB was not performed on these rats which were killed 2 hours later for sample collection. Rats in CPB group were executed 2 hours after bypass for sample collection. The height of end-piece ileal mucosa villi was examined by light microscope and graded according to Chiu's score, ultramicrostructural change of intestinal mucosa was observed by electron microscope, and serum DAO activity, the variance of D-lactic acid and NO. The change of serum TNFα, IL-6 concentration was detected by ELISA. The serum values were corrected to eliminate the interference caused by the dilution of pre-inflation. Then, apoptotic index of small intestinal mucosa of rats was tested by TUNEL, the changes of TNFα, iNOS, Bcl-2, Bax, Caspas-3 protein expression of intestinal mucosa went through image analysis by immunohistochemical. The expression variance of TNFα, iNOS, Bcl-2, Bax, Caspase-3mRNA was tested by RT-PCR.2.3 ResultsAccording to the study, obvious mucous hyperemia and dilation of small vessels in interstitium were observed in rats in CPB group, as well as massive infiltration of neutrophils, interstitial edema, expansion of intestinal canal, shortening and deformity of villi, and frequent focal exfoliation of chorioepithelia. Besides, necrosis and exfoliation of mucosal epithelia exacerbated, and some parts of proper layer exposed, with notable decrease in the number of glands in proper layer. The condition of rats in N group and SH group was much better than that of CPB group with fairly normal villi of small intestine. Chiu's score demonstrated that the intestinal mucosal impairment was obvious in CPB group than that of N group and SH group(P<0.05). Electron microscope showed that in N group and SH group, microvilli of intestinal mucosal epithelial cells were lined up in order with the complete structure of cellula columnoepithelialis, and that no abnormality was detected in cell organs, and that tight junction, intermediate junction desmosome and gap junction were clear without being widened. in N group and SH group, microvilli of intestinal mucosal epithelial cells were lined up in order with the complete structure of cellula columnoepithelialis, and that no abnormality was detected in cell organs, and that tight junction, intermediate junction, desmosome and gap junction were clear without being widened. The structure of glands and various cells in proper layer was normal. In CPB group, two different ways of apoptosis and swelling death of small intestine epithelial cells were presented. Rarefaction of microvilli was observed whose arrangement was not in order but downward, and some of which was absent. Junctions between epithelial cells widened with opening of some tight junctions, mitochondria swollen, and endoplasmic reticulum expanded. Several apoptotic cells were seen with the cell nucleus gathering under the nuclear envelop and apoptotic bodies exfoliated into the enteric cavity. Edema of proper layer was obvious with increased lymphocytes and neutrophils. Some apoptotic cells resulted in further necrosis with disintegration, loss and incomplete membranes of intracytoplasm cell organs. Epithelial cells died of swelling were also observed with the destruction of nuclear membrane and cell membrane, and disintegration of intracytoplasm cell organs like mitochodria. Rats that went through CPB presented the impairment of intestinal barrier with serum DAO activity, D-lactic acid concentration higher than those of N group and SH group (P<0.05). DAO activity, D-lactic acid concentration in SH group was higher than those of N group, yet no significance of difference was detected. Rats that went through CPB were in systemic inflammatory state with serum TNFα, IL-6 and NO concentration remarkably higher than those of N group and SH group (P<0.05). Serum TNFαand NO concentration in SH group were higher than those of N group (P<0.05). Linear regression and relevant analysis indicated that height of intestinal mucosal villi and Chiu's score were positively correlated with serum DAO activity and D-lactic acid concentration, and that serum DAO activity and D-lactic acid concentration were positively correlated with serum TNFα, IL-6 and NO concentration. Apoptotic index of intestinal mucosal epithelial cells was significantly higher than that of N group and SH group (P<0.01). Apoptotic index was positively correlated with Chiu's score, serum DAO activity, D-lactic acid, serum TNFα, IL-6 and NO concentration. TNFα, iNOS, Caspase-3 protein and mRNA expression of intestinal mucosal epithelial cells in CPB group stepped up notably compared with those of N group and SH group. Yet no significance of difference was detected between N group and SH group. Decrease of Bcl-2 protein and mRNA expression, increase of Bax protein expression and mRNA expression were of no statistical significance, and yet the drop of Bcl-2/Bax was of significance compared with that of N group and SH group.2.4 ConclusionRats that went through CPB presented the morphous and barrier dysfunction of intestinal mucosa with systemic inflammatory state. Linear regression and relevant analysis indicated the important role of the morphous and barrier dysfunction of intestinal mucosa in inflammatory reaction after CPB. Apoptosis of intestinal mucosal epithelial cells is one of the main ways of death for epithelial cells after CPB. Ischemia, anoxemia and reperfusion after CPB and excessive secretion of inflammatory factors and increase of NO synthesis after stress promoted apoptosis, and the decrease of Bcl-2/Bax also contributed to the apoptosis of intestinal mucosa.3. Research on the mechanism of ulinastatin prevention of the early stage dysfunction of intestinal mucosal barrier3.1 ObjectiveTo discuss feasibility and mechanism of intestinal mucosal barrier after CPB by observing the pathological, physiological index and inflammatory factors and control of apoptosis pretreated with large and small dose groups of ulinastatin before the establishment of bypass.3.2 Methods40 clean healthy male adult Spranue-Dawley (SD) rats were divided randomly into sham-operated group (SH,n=10), CPB group(n=10), ulinastatin small dose group(U1 group,n=10), ulinastatin large dose group(U2 group,n=10). Before the bypass, U1 group was given 40,000U/kg of ulinastatin through femoral artery, while U2 group was given 100,000U/kg of ulinastatin. The bypass and sample collection methods were identical to those taken in CPB group. SH group was the same as CPB group. Mean blood pressure and heart rate of rats that went through CPB were monitored continuously. The height of end-piece ileal mucosa villi was examined by light microscope and graded according to Chiu's score, ultramicrostructural change of intestinal mucosa was observed by electron microscope, and serum DAO activity, the variance of D-lactic acid and NO concentration. The change of serum TNFα, IL-6 concentration was detected by ELISA. Apoptotic index of small intestinal mucosa of rats was tested by TUNEL, the changes of TNFα, iNOS, bcl-2, Bax, Caspas-3 protein expression of intestinal mucosa went through image analysis by immunohistochemical . The expression variance of TNFα, iNOS, Bcl-2, Bax, Caspase-3mRNA was tested by RT-PCR.3.3 ResultsThe drop of mean blood pressure and heart rate of rats that went through CPB in ulinastatin interference group improved compared with that of CPB, U2 group in particular (P<0.05). In U1, U2 groups, intestinal mucosal hyperemia and dilation of small vessels in interstitium were observed, as well as infiltration of neutrophils, interstitial edema, expansion of intestinal canal, shortening and deformity of villi, and focal exfoliation of chorioepithelia, all of which were better than those of CPB group, in accordance with the indication by villi height and Chiu's score that the improvement of intestinal mucosal damage in U1, U2 groups was more obvious than that of CPB group (P<0.05), yet there was no difference between U1 and U2 groups. Electron microscope showed that in U1 and U2 groups, microvilli of intestinal mucosal epithelial cells were lined up in order with most of cellula columnoepithelialis remained complete structure, and with opening of some tight junction. The swelling of mitochondria mitigated, and endoplasmic reticulum slightly expanded with few apoptotic cells. Rats that went through CPB and received ulinastatin treatment showed intestinal barrier impairment. Serum DAO activity, D-lactic acid concentration were remarkably higher than those of SH group (P<0.05), yet the drop of the values was significant compared with that of CPB group (P<0.05), and the drop in U2 group was greater than that of U1 group (P<0.05). Systemic inflammatory reaction of rats that went through CPB and received ulinastatin treatment alleviated. Serum TNFα,IL-6 and NO concentration in U1 and U2 groups extremely descended than those of CPB group(P<0.01); The drop in U2 group was greater than that of U1 group(P<0.05).The increase of serum TNFαand NO concentration in U2 group was not obvious than that of SH group(P>0.05). Apoptotic index of intestinal mucosal epithelial cells dropped notably compared with that of CPB group (P<0.01), higher than that of SH group, yet no difference between U1 and U2 groups was detected. TNFα, iNOS, Caspase-3 protein and mRNA expression of intestinal mucosal epithelial cells in U1 and U2 groups decreased remarkably compared with those of CPB group, while the drop of Bcl-2/Bax was significant in comparison with that of CPB group. TNFα, iNOS, Caspase-3 protein expression and mRNA expression of U2 group dropped remarkably compared with those of U1 group.3.4 ConclusionUlinastatin effectively enhanced the condition of rats that went through CPB, not only alleviating the morphological change and impairment of intestinal mucosa barrier, but also mitigating systemic inflammatory reaction. Besides, it downregulated the apoptosis of intestinal mucosal epithelial cells by inhibiting the secretion of inflammatory factors and lessening stress reaction which to some extent prevents the early stage dysfunction of intestinal mucosal barrier after CPB, indicating dose-effective relationship of ulinastatin prevention.
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