A Study On Atherosclerotic Plaque Eruption In Carotid Artery From Apoe^(-/-) Deficient Mouse

BACKGROUNDAtherosclerosis is the basic pathogenesis of a variety of cerebrovascular diseases. Stable plaques do not cause obvious ischemia, even when the luminal stenosis reaches 70%. Most of the clinical emergencies are caused by plaque rupture and thrombosis, which partially or completely obstruct the blood supply for the organs. How to identify vulnerable plaques and prevent plaque rupture have become the focus of researches. The unstable plaques have large patch of lipids, thin fibrous caps, less collagen and vascular smooth muscle cells in caps, inflammatory cells, angiogenesis, and high expression of matrix metalloproteinases. However, the mechanism underlying plaque rupture is missing.OBJECTIVEThe present study was designed to investigate the mechanism underlying plaque eruption in carotid artery from ApoE^(-/-) deficient mice.METHODS1. Establishment of plaque rupture in ApoE^(-/-) deficient mice ApoE^(-/-)-deficient mice and C57BL/6 mice at the age of 9 weeks were used in present study. After the mice were anesthetised, the carotid artery of the mouse was separated and was ligated just proximal to its bifurcation. Four weeks after the ligation, a polyethylene cuff was applied just proximal to the ligated site. On the day of 2, 4 and 7 after the cuff placement, the carotid arteries were removed. The vessel was fixed and undergone HE and Masson staining. Histologic change of the vessel was observed. 2. Investigation of the mechanism underlying plaque rupture of the miceSeven days after cuff placement, the mice were sacrificed. The carotid artery were seperated. The expressions of smooth muscleα-actin, vWF, COX-1, COX-2, MMP-2 and MMP-9 were detected by immunohistochemistry. The changes of T cells in peripheral blood were analysed by flow cytometry.3. Role of OX40 ligand in the plaque ruptureOn the day after cuff placement, the mice were adminstrated with 300ug monoclonal antibody against OX40 ligand by intraperitoneal injection. Seven days after later, the carotid arteries of the mice were removed and frozen sections were made. The sections underwent HE staining for the observation of morphologic changes. The expression of OX40 ligand of the artery were detected by immunohistochemistry. The changes of OX40 ligand positive T-cells in the blood were analysed by flow cytometry.RESULTS1. Neointima formation hardly occurred in the carotid artery of ApoE^(-/-) deficient mice and C57BL/6 mice after 4 weeks of standard diet. Marked plaques composed of collagen, foam cells and fibrous caps were induced in the lumen after 4 weeks of ligation the carotid artery of ApoE^(-/-) deficient mice. Small neointimas which had less collagen and no foam cell or fibrous cap were induced in the lumen after 4 weeks of ligation the carotid artery of C57BL/6 mice. No intraplaque hemorrhage or evidence of plaque rupture was detected at this time. There were plaques ruptured at 2, 4 and 7 days after cuff placement in ApoE^(-/-) deficient mice. At 7 days after the cuff placement all of the ApoE^(-/-) deficient mice suffered plaque rupture. There were marked neointimas emerged in the lumens of three C57BL/6 mice, while the neointimas of the other three C57BL/6 mice ruptured.2. There were masses of smooth muscleα-actin positive cells distributed diffusely in the plaques of ApoE^(-/-) deficient mice, especially at the fibrous caps. The smooth muscleα-actin positive cells also distributed diffusely in neointimas of C57BL/6 mice. VWF was observed in the normal arteries and plaques induced by ligation of ApoE^(-/-) deficient mice and C57BL/6 mice. The expression of COX-1, COX-2, MMP-2, MMP-9 was detected in cracks of plaques of ApoE^(-/-) deficient mice. Higher expressions of COX-1, COX-2, MMP-2, MMP-9 was also found after plaque rupture. The amount of T cells increased significantly 7 days after the cuff-placement compared with that of the mice with intact plaques.3. After the treatment with OX40 ligand, five of six ApoE^(-/-) deficient underwent plaque ruture. The amount of OX40 positive T cells increased significantly at 7 days after the cuff-placement. OX40L was also detected at the cracks of plaque rupture.CONCLUSIONOn the site of plaque eruption of the carotid artery from ApoE^(-/-) deficient mice, the high expression of pro-plaque eruption factors maybe the major cause for the plaque eruption in this animal model.