In vivo perfusion staining of atherosclerotic lesions and a novel quantification method for lesion size in sequential aortic root sectioning in murine models

Objectives To develop a simple, accurate and reproducible method, which combines macro and histopathological techniques for determining the degree of lipid deposition in genetically modified mice. Method The entire aortas from C57BL/6, ldlr-/- and apoE-/- mice were stained with Sudan IV using either in vivo perfusion or traditional in vitro enface staining techniques. Histological sections of aortic root and hearts were embedded in tissue freezing medium and cut with a cryostat, then stained with Oil Red O. The calculated aortic root area based on the aortic root circumference was used to reduce measurement errors. Results The in vitro en face staining can stain all fat, which include the adventitial tissue around aorta. However the in vivo perfusion staining can specifically stain the fatty deposition inside of aorta. Both entire aorta and aortic root section staining showed that there was a highly significant increase in fatty deposition in the aortas of the genetic modified mice. Although all mice genetic background was same, the apoE-/- mice had larger atherosclerotic lesions than ldlr-/- mice. Conclusions The new in vivo peffusion method is more accurate than the in vitro en face method. The combination of these macro and microscopic techniques overcomes the shortcomings of the earlier published methods which are generally limited to the measurement of fatty red staining areas only, neglecting non-specific adventitial fat staining around aorta and aortic root section tissue distortion.

Chemokine SR-PSOX/CXCL16 expression in peripheral blood of patients with acute coronary syndrome

Background Scavenger receptor that binds phosphatidylserine and oxidized lipoprotein/CXC chemokine ligand 16 （SR-PSOX/CXCL16） promotes foam cell formation through the tumor necrosis factor （TNF）-α mediated mechanism. Because chemokine CXCL16 could be expressed in atherosclerotic lesions and induce smooth muscle cell （SMC） proliferation, we＇presume that the monocyte SR-PSOX/CXCL16 detection in the patients＇ peripheral blood will be important for early diagnosis and prognosis of atherosclerosis （AS）. Methods Enrolled in this study were 40 patients with acute coronary syndrome （ACS）, including 20 patients with acute myocardial infarction （AMI） and 20 patients with unstable angina pectoris （UAP）, and 20 normal controls. Monocytes in the peripheral blood were isolated, and the changes of expression of CXCL16/SR-PSOX mRNA were compared using reverse transcription-polymerase chain reaction （RT-PCR）, with β-actin as internal control. We compared the expression of CXCL16/SR-PSOX in the ACS subgroups, using Western-blot to analyze protein expression levels. Tissue sections were made from biopsy specimens taken from patients with infective endocarditis, liver cirrhosis, and lung cancer as well as normal controls. And the expression of CXCL16/SR- PSOX was analyzed with a confocal microscope. Results The expression of CXCL16/SR-PSOX mRNA and protein in the monocytes of peripheral blood was significantly higher in ACS patients than in normal controls （P〈0.05）; however, there was no significant difference in CXCL16/SR-PSOX expression between UAP group and AMI group （P〉0.05）. Immunofluorescence showed that there were low expression of SR-PSOX in normal vascular endothelial cells and enhanced expression in every layer of the infected vessels, while spreading from endothelial cells to surrounding tissues as infection worsens. Confocal microscopy showed that the expression of SR-PSOX was enhanced in the infiltrated lymphocytes in liver cirrhosis, and that the expression level was proportionate to the degree of inflammation in the portal hepatis and folia. Conclusions The expression of CXCL16/SR-PSOX in the monocytes of peripheral blood was significantly higher in ACS patients than in the controls. CXCL16/SR-PSOX-mediated inflammation may contribute to the pathogenesis of ACS, and CXCL16 may play an important role in the pathogenesis and development of AS in humans.