Evaluation Of Endothelial Function Using Ultrasonography In Atherosclerotic Rabbits

IntroductionEndothelial dysfunction developed at the early stage of atherosclerosis. It is important to detect endothelial dysfunction, found risk factors and observe the therapeutic effects of medicine. In this study the vascular endothelium function and pathological change of iliac artery in atherosclerotic rabbits during different feeding time were observed to provide morphological foundation for evaluation of endothelial function using ultrasonography, and assess the effect of simvastatin on vascular endothelial function and the relation with VCAM -1 by high resolution sonography and explore the evaluation of therapeutic effect using ultrasonography . To explore the possibility of evaluating microvascular endothelial function using variables of time - intensity curve. Myocardial contrast echocardiography ( MCE) was used to investigate the changes of myocardial microvascular endothelial function in atherosclerotic rabbits.Methods1. Twenty four Japanese white big ear rabbits, weighting 1. 75 -2. 25kg, were randomly divided into four groups (6 rabbits one group) : control group were fed with normal feed;cholesterol diet 4 weeks group ,cholesterol diet 8 weeks group and cholesterol diet 12 weeks group were fed with 2g cholesterol a day. EDD (endothelial dependent dilation) and NEDD(non -endothelial dependent dilation) were measured by linear array transducer with frequency of 3 -11MHz. EDD = (D₁ -D₀)/ D₀ × 100%;NEDD = (D₂ -D₀)/ D₀×100%.Do is internal diameter of LEIA at base condition. Dj is internal diameter of LEIA at hyperemia after pressure. D2is internal diameter of LEIA after injection of NTG. Venous blood of all the rabbits was drawed before execute. Serum TC, HDL - C^TG^LDL - C were measured with enzymic method by automatic biochemistry analyzer. At the same time some LEIA were fixed with formalin^ sliced^HE stained and observed with light microscope;another LEIA were fixed with glutaral^sliced >, stained and observed with electron microscope. Reproduc-ibility test is carried out for the measurement of vascular endothelial function u-sing ultrasonography.2. Twenty six Japanese white big ear rabbits, weighting 2. 05 - 2. 40kg, were randomly divided into four groups (6 rabbits one group) : A group (control group,6) were fed with normal feed;B group (atherosclerosis group, 10) were fed with 2g cholesterol a day;C group (simvastatin group, 10) were fed with 2g cholesterol for 4 weeks and was fed with 2g cholesterol and simvastatin lOmg/kg a day for another 8 weeks. All the rabbits were fed 12 weeks. EDD and NEDD were measured with high resolution sonography at different time ( ditto methods). At the same time blood fat and pathology were detected. The expression of VCAM - 1 was also analyzed by immuno histochemistry with polyclone antibody of anti - rabbit VCAM - 1 using ABC method. It was defined positive cell if there are brown granas on cell membrane or in endochylema. The percentage of positive cells was calculated from the average value of random selected 5 fields under the light microscope (10 x40).3. Twenty Japanese white big ear rabbits, weighting 1. 80 -2. 50kg, were fed with 2g cholesterol for 12 weeks. Albumin and dextran -40 were mixed pro rate and vibrated with supersonic cell crusher at same time C3F8 was influxed then the acoustic contrast was prepared. The microbubble concentration is 2. 9x 10 /ml,the size is 2. 8 ±0.76(xm. MCE was implemented via injection of self- made contrast media using transducer S4 at different feeding time. Then time- intensity curve was fitted automatically. Peak intensity (PI) >, area under curve (AUC) ^half-time of descent ( HT) and mean transit time(MTT) were derived from time - intensity curve. After aortic root injection of acetylcholine ( ACH) and nitroglycerin(NG) and then PI ratio (PIR) and AUC ratio ( AUCR) of post- to pre - ACH or post - to pre - NG were calculated. EDD and NEDD of myocardial microcirculation were analyzed. At the same time blood fat was detected and aorta was stained with HE and left coronary artery was stained with sudan UhlV.Results1. Serum TC NHDL - C ^LDL - C stepped up with the progress of atherosclerosis , internal diameter of LEIA increased, EDD dysfunction occurred at different time and became worse progressively, and there is significant deviation inter-group(P<0. 01). NEDD dysfunction occurred at 12 weeks. Endothelial function impired and pathologic changes occured simultaneously.2. In atherosclerosis group blood fat increased gradually, EDD decreased gradually, NEDD damaged at 12 weeks and VCAM - 1 expressed in endomem-brane and smooth muscle cells and percentage of positive cells is (30. 12 ±9. 95) %. After intervention of Simvastatin, the blood lipid decreased and EDD function of was obviously improved. NEDD at 12 weeks was improved too. There was significant difference between the two groups (P<0.01,P<0.05). VCAM- 1 expressed at low level in simvastatin group. Pathological changes of macroscopic observation and light microscope lessened compared with AS group. VCAM -1 only presented in endomembrane and endothelial cells and percentage of positive cells is (19.93 ±6. 69) %. In control group there are small quantity brown granas on the surface of the endothelial cells.3. PI increased 2. 41 ±0. 06 times and AUC increased 1. 89 ±0. 07 times after ACH injection at 0 week. The increase degree of 4 week decreased obviously (P<0.01). PI and AUC of 8 week and 12 week decreased after ACH injection in and decreased more obviously with time ( all P <0.01). PI and AUC increased after injection of NG at all feeding time. Significant difference of PIR and AUCR was not found between 4 week,,8 week and 0 week, and significant difference was found between 12 week and 0 week(P<0.05,P<0.01). EDD of atherosclerotic myocardial microvascular endothelial function decreased following atherosclerotic progress and there is significant difference intergroup(P <0.01), NEDD decreased at 12 weeks.Conclusions1. AS is a process that morphological and functional changes occurred simultaneously. Endothelium dysfunction has occurred at the early stage of AS with corresponding pathological finding. EDD damage occurred before NEDD damage. High resolution sonography is a good method to evaluate vascular endotheli-al function.2. Simvastatin can improve impaired EDD function and suppress the express of VCAM - 1. High resolution sonography maybe used to assess the effect of drug therapy on endothelial function.3. Satisfactory MCE imaging can be gained by transthoraco echocardio-graphy with self - made acoustic contrast in rabbits. Myocardial microvascular endothelial function can be quantitative analysised with MCE technique on atherosclerotic rabbit model. EDD and NEDD of myocardial microvessel can be quantitatively evaluated using MCE. EDD of atherosclerotic myocardial microvessel decreased with time. NEDD of atherosclerotic myocardial microvessel did not change obviously.