Studies On The Damages Of Endothelial Cells And Its Association With The Organ Damages And Severity Of Sepsis

The development of modern medicine helps many severe patients survive. Therefore the morbidities of critical illness like sepsis increase correspondingly. Despite the accumulating new advances about the pathogenesis and treatment of sepsis, this disorder continues to be associated with an unacceptably high mortality rate. Early monitoring and diagnosis are very essential for the prevention and therapy of sepsis. Endothelial cell is a key organ involved in the pathogenesis of sepsis as both a target cell and an effector cell. Besides, under physiologic conditions, endothelial cells exert a number of functions that are important for the normal homeostasis. We hypothesized that evaluation of endothelial cell damage could reflect the severity of sepsis. But the current available methods for assessing endothelial cells'damage are not sensitive and lack specificity.ObjectiveIn this experiment, the damage of endothelial cells will be evaluated by the number of circulating endothelial cells(CECs), the mRNA expression of the endothelial-specific genes, the vascular permeability and tissue water content in rats with sepsis resulting from cecal ligation and puncture (CLP). These changes will be analyzed to evaluate their relations with organs'histologic and function. Based on animal research, the correlation between damage of endothelial cells and severity of septic patients will be studied.Material and Methods1. The evaluation of endothelial cell damage in CLP model of rats: 90 rats were divided into 6 groups: normal control, sham operation, CLP3h, CLP6h, CLP12h and CLP24h, 15 per group. CLP was performed as ligating the cecum below the ileocecal valve and puncturing thrice with a 16-gauge needle. Sham surgery was performed as CLP without ligation or puncture of the cecum. Blood were sampled from tail vein of 36 rats to count the number of leucocytes before and after the operation. The serum IL-6 was measured by enzyme linked immunosorbent assay (ELISA) simultaneously.2. CECs were separated using density gradient separation and counted in 36 rats (6 per group). Total RNA of whole blood were extracted to measure the mRNA levels of tie2 and vegfr2 by real-time PCR in another 6 groups of 36 rats. 18 rats (6 per group) were killed at different time points to determine the vascular permeability and tissue water content of heart, liver, kidney, ileum and lung by Evans blue method and by desiccation respectively.3. Functional changes of the major organs and its relationship with endothelial cells dameges: the contents of K~+,Na~+,Cl~-,AST,ALT,BUN and Cr in each group were measured by automatic biochemistry analyzer. The histologic change of heart, liver, lung, kidney and ileum were observed with H.E. staining. The amount of whole blood tie2 mRNA, histologic change and related biochemistry parameters of heart, liver, kidney, ileum and lung were investigated respectively.4. The association analysis between endothelial cells'damages and severity of sepsis in patients: Blood of trauma patients divided into sepsis and non-sepsis group were sampled on day 1, 3, 7, 14, 21, 28 after ICU admission. Extracted RNA of whole blood were purified and measured for mRNA levels of tie2 by real-time PCR. The levels of plasma vWF were detected with ELISA. The relationship between the amount of whole blood tie2 mRNA and the APACHEâ…¡scores, contents of vWF were analyzed respectively. The WBC number,levels of AST,ALT,BUN and Cr were measured, and analyzed for their association with the levels of tie2 mRNA.Results1. General evaluation of CLP model of rats: The 24-hour-mortality is 31.82 %( 7/22). The amount of leukocyte is higher in CLP3h group than others'(P <0.05) and decreases significantly at CLP12h, 24h compare with CLP3h and 6h (P <0.01). Compared with the normal control, the levels of IL-6 increase after CLP operation (P <0.05).2. The numbers of CECs peak at 3h after CLP compared with normal control (P <0.05), and then decrease gradually. The CECs number in CLP24h is obviously smaller than that in CLP3h and 6h group (P <0.05). The mRNA level of tie2 in CLP3h group is also markedly higher than other groups'(P <0.05). But the amounts of vegfr2 mRNA have no significant change after CLP (P >0.05). The vasopermeability and tissue water contents increase in heart, liver and kidney after CLP operation (P <0.05), whereas they are not changed in ileum, pulmonary tissue water content.3. Engorgement, degeneration and even necrosis are observed gradually after CLP in hepatocytes. Focal atelectasis, erthrocyte exdudation in pulmonary alveoli are found first in CLP3h group and aggravated in CLP24h group. Hyperemia, necrosis and detachment are found in ileum mucosa. Myocardium bleeding, renal glomerulus engorgement and tuble epithelial cells cast are also found. Compared with the normal controls, the significant increase of AST, ALT and BUN are first observed in CLP6h and 12h respectively (P <0.05). Serum levels of K~+,Na~+,Cl~- increase after CLP(P >0.05).4. The circulating mRNA levels of tie2 increase obviously in sepsis patients compared with non-septic trauma patients (P <0.01). Circulating tie2 mRNA is highly correlated with APACHEâ…¡scores (P <0.01). The Peason's coefficient correlation, represented as"r", is 0.532(P <0.01). The filiate regression equation is Y=12.66+4.922X (R~2 =0.283). Contents of plasma vWF have no significant difference between groups. But there's a significant correlation between the amounts of tie2 mRNA and plasma levels of vWF (P <0.05), r =0.334(P <0.05). The filiate regression equation is Y=180.932+57.93X (R~2 =0.112). The number of WBC in sepsis group is higher than that in the non-sepsis group (P<0.01). The levels of BUN and Cr in sepsis group are also higher than that in non-sepsis group (P<0.01). There no significant difference of ALT and AST between these two groups (P >0.05).Conclusion1. The CLP model of sepsis performed in this study provides a reliable basement for the subsequent experiments.2. The endothelial cells are damaged after CLP as evidenced by increased CECs number, circulating mRNA levels of tie2 and vasopermeability, tissue water content. Quantitative measurement of tie2 mRNA using real-time PCR is the most sensitive method to evaluate impairements of endothelial cells among these methods.3. In rat CLP model of sepsis, the parameters reflecting damages of endothelial cells change earlier than those routinely used clinical functional parameters for the major organs.4. Real-time quantitative PCR of blood tie2 mRNA is an easy and reliable method to quantify CECs in peripheral blood samples. Monitoring the levels of blood tie2 mRNA and routine clinical parameters simultaneously could facilitate the determination of the severity of sepsis, thus make the protective therapy start as early as possible.