| Objectives:Severe acute pancreatitis (SAP), a dangerous clinical process with high mortality, will develop acute lung injury (ALI) as demonstrated by the current studies; and finally it will develop acute respiratory distress syndrome (ARDS), the outcome of ALI and one of the leading causes of SAP, with over 65% patients died associated with ARDS. Therefore, it is significantly important to clarify the mechanism of ALI during SAP and to determine the early-stage prevention and the treatment in order to decrease the mortality and to improve the diagnosis of SAP.During SAP, over inflammatory reaction of the body causes pulmonary vessel endothelia and alveolus epithelia extensively destroyed, resulting in ALI. Polymorphonuclear neutrophil leukocytes (PMN) can trigger and amplify inflammatory reaction, so it plays a key role in and is the important mark of inflammatory reaction. As documented by some studies, during SAP, PMN coagulate and assemble in the lungs, leading to pulmonary vessel endothelia and alveolus epithelia extensively destroyed and accordingly permeability increased, pneumonedema and microthrombus developed, and finally ALI. Coagulation and activation of PMN in the lungs is the initial process of ALI during SAP. Therefore, if the coagulation and activation can be intervened, the early prevention of ALI can be performed. The mechanism of it, however, is not clear, wanting further study. Currently, mesentery-thoracic duct lymph is highly valued in the severe burns, infectious toxic shock and other diseases, and is regarded as the mediator of lung injury in severe infection/trauma, finally leading to inflammatory reaction in the lungs or systemically. Clinical and animal experiments showed that ARDS will first occur in the multiple organs dysfunction syndromes (MODS) caused by multiple reasons, for lungs are the first organ to accept mesentery lymph, the one which possibly mediates lung injury. Other studies displayed that mesentery lymph collected after shock caused endothelia injured or died, finally increasing the permeability of the endothelia, and that ligating the mesentery lymph ducts can decrease lung injury after heamorrhagic shock and burn. All these studies suggest that mesentery lymph contains some substances that can mediate lung injury during systemic inflammation. Furthermore, no bacteria or toxins were found in the portal vein blood of the animal models or patients with traumatic shock. Meanwhile, portal vein plasma only causes light injury to the vessel endothelia. All this shows that the portal vein blood coming back through enteric canal capillaries plays a minor role in the organ injury caused by shock. As Louis reported, lung injury and increased permeability of endothelia caused by heamorrhagic shock was caused by enteric toxic substances in lymph not in portal vein blood. Based on this, we presume that this kind of function also exists in SAP caused by burn and severe infection, and may mediate SAP by inducing PMN coagulation and activation in the lung.SD rats were used in this experiment to establish cervical thoracic duct drainage models, and then to prepare models of SAP accompanied by ALI, with the aim to observe the pathogenesis of the lung, to detect the changes of blood gas analysis, thus to find out the effect of thoracic duct ligation /conduction on ALI. Meanwhile, to detect the changed content of anti-inflammatory and proinflammatory factors, pancreatin, and endotoxin, and to understand the prognosis effect of these factors on PMN functions in the arterial blood, and finally to observe the changes of the indicators mentioned above during ALI secondary to SAP. In short, this study is to explore the functions of the changed substances in the thoracic duct lymph in the activation of PMN in the lung, in the mediating of ALI during SAP, to find out the feasible interventions, and to explain the mechanism of AVI secondary to SAP, finally to provide theoretical and experimental supports for clinical practice.Methods:Part I: Establishing models of cervical thoracic duct drainage in ratsSixty clean SD adult male rats were randomized into 3 groups, with 20 in each. Group 1: cervical thoracic duct direct group; group 2: cervical thoracic duct indirect group; group 3: abdominal thoracic duct group. After being anaesthetized with napental and being fixed, the models were performed with microsurgery instruments to dissect cervical thoracic duct, left jugular trunk, and abdominal thoracic duct; 24G remaining needles then were used to puncture the above parts to drain lymph. Meanwhile, the drained lymph volumes in 3 groups and model making condition were recorded.Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI and on the changed functions of PMN in arterial bloodNinety-six healthy male SD rats were randomized into 12 groups, with 8 in each, and the following operations were performed respectively: cervical sham-operation + abdominal sham-operation 2 hours (SO2h), cervical sham-operation + abdominal sham-operation 6 hours (SO6h), cervical sham-operation + abdominal sham-operation 12 hours (SO12h); cervical sham-operation + SAP2 hours (SAP2h), cervical sham-operation + SAP6 hours (SAP6h), cervical sham-operation + SAP12 hours (SAP12h); cervical thoracic duct ligation + SAP2 hours (LC2h), cervical thoracic duct ligation + SAP6 hours (LC6h), cervical thoracic duct ligation + SAP12 hours (LC12h); cervical thoracic duct drainage + SAP2 hours (DC2h), cervical thoracic duct drainage + SAP6 hours (DC6h), cervical thoracic duct drainage + SAP12 hours (DC12h). According to the grouping requirement, SOA, SAP, LC, and DC models were prepared. Histopathological sections of pancreas and lung were observed, and the lung underwent light microscope scan; serum amylopsin (AMY) and pancreatic lipase were detected bio-chemically; and automatic blood gas system was employed to make arterial blood gas analysis. Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial bloodThe sample was collected from abdominal aorta blood (when the blood for blood gas analysis was collected). Flow Cytometry (FCM) was employed to detect the changed functions of PMN in the arteries.Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALITwenty healthy SD rats were randomized into 2 groups, with 10 in each, with the operations of cervical thoracic duct drainage + abdominal sham operation (Sham) and cervical thoracic duct drainage + SAP (DC) respectively. After the model making, the conduction was performed continually, the drained lymph being collected for a certain period and then kept in low tempreture in time. AMY and LIP in the lymph and blood were detected bio-chemically; ELISA was employed to detect TNF-αand IL-10 in the lymph and blood; the method of reaction time was used to detect endotoxin content in the lymph.Results:Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats1 Light milky white fluid was successfully conducted from the models of all the groups, no differences in appearance and properties, and all lymph.2 The success rate of model establishing in cervical thoracic duct direct group was apparently lower than those in cervical thoracic duct indirect group and abdominal thoracic duct direct group (P<0.05).3 The amount of lymph in abdominal thoracic duct group was greater than that in cervical thoracic duct direct and indirect groups, with significant difference (P<0.05).Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI1 Compared with SO groups at the same time point, the amount of ascites, pancreatic pathogenesis score, lung pathogenesis score, and serum AMY and LIP all increased significantly (P<0.01);compared with SO groups at the same time point, PaO2 in SAP2h and SAP6h also increased significantly (P<0.01), with PaO2 in SAP2h obviously higher than that in SAP6h (P<0.01), and PaO2 in SAP12h obviously higher than that in SO12h (P<0.01); compared with SO groups at the same time point, PaCO2 in SAP2h and SAP6h decreased significantly (P<0.01), while PaCO2 in SAP12h obviously higher than that in SO12h (P<0.01).2 Compared with SAP groups at the same time point, the amount of ascites in LC6h and LC12h, lung pathogenesis score in LC6h, and serum AMY and LIP in LC2h all decreased significantly (P<0.01 or P<0.05);the ascites in LC2h , lung pathogenesis scores in LC2h and LC12h tended to decrease but with no significant difference; pancreatic pathogenesis scores in LC2h and LC6h, and PaO2 in LC12h tended to increase but with no significant difference, and with pancreatic pathogenesis scores in LC12h increased significantly (P<0.01), and likely PaO2 in LC6h (P<0.01).3 Compared with SAP groups at the same time point, the amount of ascites in DC12h, lung pathogenesis score in DC6h and DC12h, and serum AMY and LIP in DC2h, DC6h and DC12h all decreased significantly (P<0.01 or P<0.05);lung pathogenesis score in DC2h tended to decrease but with no significant difference; PaO2 in DC6h and DC12h increased significantly (P<0.01).Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood1 Compared with SO groups at the same time point, the proportion of PMN with CD11b expression positive, the average fluorescence intensity of PMN respiratory burst in SAP groups all increased significantly (P<0.01); compared with SO groups at the same time point, average fluorescence intensity of CD11b in SAP2h tended to increase, with no significant difference (P>0.05), while that in SAP6h and SAP12h increased significantly (P<0.01 or P<0.05), with that in SAP12h apparently higher than that in SAP6h.2 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst, the proportion of PMN with CD11b expression positive, average fluorescence intensity of CD11b in LC all tended to decrease, with no significant difference.3 Compared with SAP groups at the same time point, the average fluorescence intensity of PMN respiratory burst and that of CD11b in DC, and the proportion of PMN with CD11b expression positive in DC6h and DC12h all decreased significantly (P<0.01或P<0.05).Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI1 TNF-α,IL-101.1 TNF-αin lymph and serumTNF-αlevel in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph TNF-αin SAP groups at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.TNF-αlevel in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum TNF-αin SAP at all time points compared with the abdominal sham operation groups at the same time point(P<0.01). Comparison among SAP groups: TNF-αlevel in SAP6h and 12h was apparently higher than that in SAP2h (P<0.01), while no significant difference between the former two.There is no significant difference between the lymth and serum TNF-αconcentration; and likely the lymth and serum TNF-αconcentration in DC groups.1.2 IL-10 in lymph and serumIL-10 level in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h.IL-10 level in blood: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood IL-10 in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the level of IL-10 in SAP6h was significantly higher than that in SAP2h, and IL-10 in SAP12h higher than those in SAP2h and SAP6h. There is no significant difference between the lymth and serum IL-10 concentration; and likely the lymth and serum IL-10 concentration in DC groups.2 AMY and LIP2.1 AMY in lymph:Level of AMY in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.2.2 AMY in serum:No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased blood AMY in SAP at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among SAP groups: the levels of AMY in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.In DC groups, AMY concentration in lymph was apparently higher than that in serum (P<0.01).2.3 LIP in lymph:Level of LIP in lymph: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased lymph LIP in DC groups at all times compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.2.4 LIP in serum:Level of LIP in serum: No significant difference among abdominal sham operation 2h, 6h and 12h groups; significantly increased serum LIP in DC groups at all time points compared with the abdominal sham operation groups at the same time point (P<0.01). Comparison among DC groups: the levels of serum LIP in SAP6h and SAP12h were significantly higher than that in SAP2h (P<0.05), with no significant difference between those in SAP12h and SAP6h.In DC groups, LIP concentration in lymph at certain time point was apparently higher than that in serum (P<0.01).3 Changed levels of endotoxins in lymphNo significant difference among the same time point sham groups; compared with sham groups, DC2h showed no significant difference, while both DC6h and DC12h showed apparently significant increase (P<0.01), with the increase in the latter apparently higher than that in the former (P<0.01), and the increase in DC6h and DC12h higher than that in DC2h. Conclusions:Part I: Model establishing of cervical thoracic duct lymphatic fistula in rats1 The success rate of the cervical thoracic duct direct group is lower than that of the other two groups, with no significant difference in the cervical thoracic duct indirect group and abdominal thoracic duct group.2 There is no significant difference in properties about the drained lymph by the three methods. Compared with the cervical thoracic duct direct and indirect groups, there is more drained lymph in abdominal thoracic duct group.3 Cervical thoracic duct indirect fistula in rats, which can drain lymph stably and does not disturb abdominal cavity, is a stable and reliable method to make animal models of thoracic duct lymphatic fistula, and can be used in the experimental study of SAP accompanied by ALI associated with lymphatic circulation.Part II: Experimental study on the early stage intervention of thoracic duct ligation /conduction in the SAP accompanied by ALI1 Early stage SAP will cause damaging and pathomorphological changes of the lung, followed by functional disorder.2 Cervical thoracic duct conduction can relieve pathomorphological changes and dysfunction of the lung caused by ALI, and cervical thoracic duct ligation tends to have the same effect as conduction: at the early phase (2h, 6h), ligation and conduction show no significant difference in improving the function of the lung; while at the middle or late phase (12), conduction can apparently relieve pathomorphological changes and dysfunction of the lung caused by ALI secondary to SAP.3 Cervical thoracic duct conduction cannot relieve pathomorphological changes of the pancreas secondary to SAP, while cervical thoracic duct ligation will aggravate pathomorphological changes.Part III: Experimental study on the effect of thoracic duct ligation /conduction in SAP on PMN functions in the arterial blood1 Cervical thoracic duct conduction can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b; while cervical thoracic duct ligation tends to have the same effects as conduction.2 Cervical thoracic duct conduction/ligation can lower the effect of respiratory burst of arterial PMN in SAP, the proportion of PMN with CD11b expression positive, and MFI of CD11b, which may be attributed to the fact that cervical thoracic duct conduction can relieve the pathomorphological change and dysfunctions of ALI secondary to SAP.Part IV: Experimental study on the concentration changes of TNF-α, IL-10, AMY, LIP, and endotoxins in thoracic duct lymph in SAP accompanied by ALI1 The concentrations of endotoxin, AMY, LIP, TNF-α, and IL-10 in the lymph increase apparently during SAP.2 The lymph components, complicated in the thoracic duct during SAP, may activate PMN directly or indirectly through the activated pancreatin, endotoxin, TNF-α, IL-10, etc., and finally injure the lung tissue.3 During SAP, the concentrations of AMY and LIP in the lymph and in the serum show significant difference, thereby indicating that AMY and LIP is one of the origins causing the concentration of AMY and LIP increase in the serum.4 During SAP, the concentrations of TNF-αin the lymph and that in the serum show no significant difference, so as IL-10, thereby indicating that TNF-αand IL-10 is not the origin causing the concentration of those increase in the serum.5 Reaction time method (one of the Limulus Amebocyte Lysate assay) is a reliable method to determine the endotoxin content in the lymph.
|
PDF Full Text Request