| Objective1. Groping the percentage range of residual lumen cross-sectional area of rat IVC stenosis model in ensuring low mortality not only thrombus stability, but also to better mimic the clinical environmental.2. To reseach the establishment of DVT model and GSH intervention DVT model in rats by the IVC stenosis method, and observe their differ-ent manifestations of pathology.3. Using the TBA method and QPCR technology to observe the expression of HIF-1α and VEGF mRNA in oxidative stress promote rat DVT formation.Materials and MethodsThe first part:Pathology and established of rats DVT model. Section one:To determine the percentage range of the Rat IVC residual lumen cross-sectional area.1. Experimental grouping:45 rats were divided into group A (n=15), group B (n=15), group C (n=15) by randomly grouping principles, and each group was used by the inferior vena cava stenosis method to set up thrombosis models; A, B, C three groups were used for the syringe needle of 0.7mm (22G),0.9mm (20G),1.2mm (18G) outer diameter to control the percentage of residual lumen cross-sectional area after rats DVT modeling.2. Get intravenous tissue and IVC contents:in 24 hours after modeling, to open and observe the inferior vena cava and its contents is removed between the ligature to the bifurcation of the common iliac vein after the rats were anesthetized overdose and to see if there is thrombosis.3. Measurement indicators and statistical analysis:mortality, the rate of thrombosis, blood vessel diameter and percentage of residual lumen cross-sectional area of each group rats; Using statistical software SPSS 17.0 for statistical analysis, measurement data using mean ± standard deviation (x±s); Between the two groups were compared using ANOVA with LSD method (least squares method),*p<0.05 considered statistically significant.Section two:DVT rats model creation and pathology.1. Experimental grouping:80 rats were divided into normal group (n=20), sham group (n=20), DVT model group (n=20) and DVT model intervention group (n=20) by randomly grouping principles. To establish- ing thrombosis model through the inferior vena cava stenosis method. Model intervention group both of the before modeling 24 hours and after the completion of the modeling were injected glutathione 660 mg/Kg; Use a syringe needle of 0.9mm (20G) outer diameter to control the percentage range of residual lumen cross-sectional area with the 9.9% ± 1.7%.2. Get intravenous tissue and arterial blood:Abdominal aorta blooding after opening the abdominal cavity when the rats were overdose anesthetized in the 24 hours after modeling. The rats anticoagulated whole blood by centrifugation to obtained the plasma and placed -80℃cryopreservation; Divided the inferior vena cava and its contents into two parts which is removed between the ligature to the bifurcation of the common iliac vein, one part in 4% paraformaldehyde to save prepared the pathology and the other placed in-80℃ cryopreservation.3. Measurement indicators and statistical analysis:the rate of thrombosis, rats thrombus wet weight, length, and thrombus wet weight/length ratio; Using statistical software SPSS 17.0 for statistical analysis, measurement data using mean ± standard deviation (x±s); Between the two groups were compared using ANOVA with LSD method (least squares method),*p<0.05 considered statistically significant.The second part:Detection of rat inferior vena cava MDA content, the expression of HIF-1α and VEGF mRNA1. Detection of MDA content of each group intravenous tissue by TBA method and the MDA content can indirect response to oxidative stress levels.2. Total RNA was extracted by Trizol method from each group of the rats inferior vena cava. Using RT-PCR technology to make rats intrave- nous tissue RNA into cDNA; with Real-time PCR was used to detect the expression of HIF-1α and VEGF mRNA with GAPDH as an internal reference.3. Statistical analysis:Using statistical software SPSS 17.0 for statistical analysis, measurement data using mean ± standard deviation (x± s); Between the two groups were compared using ANOVA with LSD method (least squares method),*p<0.05 considered statistically significant.ResultsThe first part:Pathology and established of rats DVT model. Section one:To determine the percentage range of the Rat IVC residual lumen cross-sectional area.1. Three groups of rats mortality rates were 6.67%,6.67%,0%; thrombosis rates were 92.9%,92.9%,57.1%.2. Three groups of rats vascular outer diameter measurements were 2.84 ±0.38mm, 2.90±0.28mm,2.86±0.29mm (*p<0.05); after modeling the corresponding percentage of residual lumen cross-sectional area were 6.4%±1.6%,9.9%±1.7%,18.1%±3.9%.Section two:DVT rats model creation and pathology.1. HE staining:Both the normal group and the sham group showed the intact of the whole intimal surface, endothelial cells arranged in neat, uniform size, the lumen without thrombosis; DVT model group complete thrombosis in microscope, in part with the vessel wall adhesions, the main component of platelet beams, cellulose and red blood cells, and showed obvious inflammatory cell infiltration of the vessel wall and thrombus organization; Part of thrombosis of DVT model GSH interve-ntion group which complete thrombosis in microscope, in part with the vessel wall adhesions, the main component of platelet beams, cellulose and red blood cells, and showed obvious inflammatory cell infiltration of the vessel wall and thrombus organization; No thrombosis of DVT model GSH intervention group showed the intact of the whole intimal surface, endothelial cells arranged in neat, uniform size, the lumen without thrombosis but visible inflammatory cell infiltration of the vessel wall.2. The rate of thrombosis:Both the model group and the model intervention group were differences than the normal group and sham group(*p<0.05); and the model intervention group compared with the model group thrombosis rate decreased significantly (*p<0.05).3. Thrombus length:Eexcept between the normal control group and the sham group was no statistically significant (*p<0.05), the rest between each two have statistically significant (*p<0.05), the thrombus length of which DVT model group maximum, the model intervention group followed.4. Thrombus wet weight:Eexcept between the normal control group and the sham group was no statistically significant (*p<0.05), the rest between each two have statistically significant (*p<0.05), which of the model intervention group increased compared with the model group.5. Thrombus wet weight/length ratio:The model intervention group increased compared with the model group (*p<0.05).The second part:Detection of rat inferior vena cava MDA content, the expression of HIF-la and VEGF mRNA1. The content of MDA in model group and the model intervention group are increased(*p<0.05), the highest is model group, followed by the intervention group, but no statistical significance (*p> 0.05).2. Expression of HIF-1α mRNA in model group were higher than normal group and the sham group (*p<0.05), whereas the expression of the model intervention group is lower than the model group (*p<0.05); Expression of HIF-1α mRNA in model group were higher than normal group and the sham group (*p<0.05), whereas the expression of the model intervention group is lower than the model group (*p<0.05).Conclusion1. Use the rat IVC stenosis method successfully established the rats model of DVT.2. Control the percentage of residual lumen cross-sectional area for inferior vena cava is 9.9%±1.7% after rats modeling, rats thrombosis stable, can be used as animal modeling. 3. Glutathione may influenced the formation of the deep vein throm-bosis.4. HIF-1α and VEGF have relevance in the oxidative stress promote DVT formation. |
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