Development Of A New Zealand Rabbit Model With Microcalcified Atherosclerotic Plaques And Comparison Of Staining Methods Of Microcalcification

BackgroundDespite recent advances in diagnosis and management, coronary heart disease(CHD) continues to be a major cause of morbidity and mortality throughout the world. In2010, nearly3.6million people died of cardiovascular disease in our country, namely more than9800people died of cardiovascular disease every day. Acute coronary syndrome(ACS) is the emergent syndrome in CHD,which covers a range of clinical pathophysiological condition,including unstable angina pectoris(UAP),acute myocardial infarction(AMl)and sudden cardiac death. Available data suggest that vulnerable plaque disruption and acute thrombus formation represents the principal pathophysiology underlying ACS. Autopsy studies suggest that these atherosclerotic lesions, which are prone to rupture, typically consist of a necrotic/lipid core, covered by a thin fibrous cap(<65μm) with severe infiltration of macrophages in the shoulder regions. The underlying mechanisms of plaque vulnerability include the expansion of lipid core, active inflammation, tissue proteolysis and local stress increased. Despite the above observations, the mechanism of vulnerable plaque rupture has remained unclear. So it is very important to identify other fators which play a pivotal role in the process of plaque rupture and search for the methods for the reversal and stabilization of vulnerable plaque.Vascular calcification is a prominent feature of atherosclerosis. Recently researchers found minute(10-μm-diameter) cellular-level microcalcifications in the fibrous cap and lipid core using high-resolution imaging modalities which include confocal microscopy with calcium-specific staining and micro-computed tomography imaging. Much interest has been focused on the role of microcalcifications in the vulnerable plaque rupture mechanics. Vengrenyuk etal first demonstrated that microcalcifications in the cap cause local stress concentrations that lead to interfacial debonding and rupture of thin fibrous cap atheroma (<65u m) by Goodier’s theoretical model. And then scholars confirmed the localization and geometry of microcalcification influence its effect on the stability of plaque by numerical modeling. A elongated microcalcification increased peak circumferential stress more than a spherical microcalcification. Microcalcification can explain why some plaques ruptured. But there are still some problems. The researches of microcalcification were based on3D numerical modeling and theoretical model. It is necessary to verify the accuracy in vivo experiments. Therefore development a microcalcified atherosclerotic plaques model and identification of the microcalcification are urgent.Atherosclerosis is closely related to local hemodynamic factors especially fluid shear stress, the frictional force generated by blood flow over the vascular endothelium. Low shear stress and osillatory shear stress are responsible for the localized nature of atherosclerosis. Mechanoreceptors on the endothelial cell surface sense changes in flow patterns converting biomechanical forces to biochemical responses, hence low shear stress and osillatory shear stress can up-regulate adhesion molecules promoting monocyte recruitment and stimulate the production of reactive oxygen species which oxidatively modify LDL into oxidized LDL (oxLDL).Shear stress are also associated with plaque calcification. Stefan found segments on the contralateral wall of the bifurcation which have previously been identified as regions with low shear stress not only exhibited a higher plaque burden, but also a higher degree of calcification using intravascular ultrasound (IVUS) and virtual histology (IVUS-VH) in coronary artery bifurcations. A perivascular shear stress modifier (referred to as a cast) can induce changes in shear stress patterns in vivo in a straight vessel and in a defined manner. New Zealand rabbit model in which rabbits were fed with high-cholesterol diets and undergone aortic endothelium denudation is the traditional atherosclerotic plaques model. In this model, calcification occurred rarely. We aim to modify the model to develop a New Zealand rabbit model with microcalcified atherosclerotic plaques.Alizarin red s staining and von kossa staining are routinely used in histopathology to stain calcium. The von kossa method indirectly localizes calcium in tissue by detecting phosphate or carbonate ions. Calcium cations are replaced by silver, with transformation of Ca3(PO4)2to Ag3PO4and of CaCO>3to Ag2CO3. Both silver salts are easily reduced to the metal, a reaction most easily accomplished by placing the staining dish under a100W light bulb. Calcified material is blackened and is sharply delineated. Alizarin red s is an anionic anthraquinone dye that forms sparingly soluble salts with calcium ions. Both the sulphonate group and the ionized hydroxy group participate in salt formation with calcium. Alizarin red s has fluorescence activity which faciliates result observation under laser scanning confocal microscope. We aim to compare of staining methods of microcalcification.Objectives(1) To develop a New Zealand rabbit model with microcalcified atherosclerotic plaques.(2)To compare the alizarin red s staining with von kossa staining on identifying microcalcification.MethodsThirty New Zealand rabbits were randomly divided into cast-placement group and non-cast-placement group. All animals were fed with high-cholesterol diets and undergone aortic endothelium denudation. For animals in cast-placement group, double-wedge-shaped casts were implanted around the aorta. At the end of12th week, rabbits were sacrificed and arterial samples were collected to undergo pathological examination,and immunohistochemistry. Ultrasound was performed to exam average blood flow, end-diastolic diameter of preoperative and postoperative abdominal aorta.Results(1)Atherosclerotic plaques could be found in abdominal aorta in both groups. (2) Microcalcification was evidenced more frequently in cast-placement group, and the calcification area index was also much larger.(3) The alizarin red s staining and von kossa staining had similar sensitivity for identifying microcalcification. The von kossa staining was superior to illuminate the accurate localization of the microcalcification, while the Alizarin red s staining was ready for imaging by laser scanning confocal microscope.Conclusions(1) A new animal model with microcalcified atherosclerotic plaques was developed successfully.(2)Both the alizarin red s staining and the von kossa staining could be used to identify microcalcification sensitively.