<i>CSH</i> is a powerful tool for a variety of stains.

<p>Common histological stains, displaying the fidelity of <i>CSH</i>. (Top panels): A mouse aorta stained with alkaline phosphatase (ALP, red) for detection of early calcification, with Gill's hematoxylin as counterstaining (purple), which depicts advanced calcification. ALP stain is scarlet red (denoted “A” in the top left panel), while hematoxylin is a shade of purple (denoted “H” in the top left panel). Visually, the hematoxylin interferes with the ALP, making it difficult to see where the ALP stain begins and ends. We analyzed the section for ALP-positive area using both CSH and an RGB-based method. (Bottom panels): A mouse liver stained with picrosirius red staining visualized using polarized light microscopy for detection of fibrosis. We analyzed the section using both CSH and an RGB-based method. The RGB method was unable to register the brightest parts of the stain as positive (gray), and falsely interpreted stain artifacts as positive areas (green in both “Merge” images).</p

Analytic performance across diverse section thicknesses.

<p>A) A section of infarcted mouse heart cut to 4 µm and stained with Masson's trichrome. Descending from the original image, we see the RGB binary image, the HSV binary image, a density map of the pixels mapped to the RGB color space, and a density map of the pixels mapped to the HSV color space. B) A section of infarcted mouse heart cut to 6 µm and stained with Masson's trichrome. C) A section of infarcted mouse heart cut to 8 µm and stained with Masson's trichrome. D) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the RGB method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. As the section thickness increases, RGB analysis decreases the perceived collagen area, despite analyzing adjacent sections of heart. E) For each of four experimental hearts and each of the three section thicknesses, the area identified as muscle is plotted next to the area identified as collagen using the HSV method. Because each heart has a different size infarction, these results for each heart are normalized as a percentage of the measured area in the 6 µm sample. There is no discernible change in perceived muscle or collagen area as the section thickness increases when using the HSV method.</p

<i>CSH</i> processing on an infarcted mouse heart.

<p>A) An infarcted mouse heart section cut 8 µm thick. Muscle tissue is scarlet red, while collagen fibers appear blue, and necrotic regions are purple-black. Insets show enlarged areas of muscle, collagen and necrotic region. B) The same mouse heart, post-processing by <i>CSH</i>. The areas that <i>CSH</i> determined as collagen are blue, and the areas that <i>CSH</i> determined as muscle are red. The background is yellow. C) A plot of the pixels from the original heart image mapped to HSV space. The gray arrows indicate the direction from which this 3-D graph will be displayed in the following 2-D images. D) A plot of the pixels from the original image in the Hue-Saturation plane. The borders collagen and the muscle rectangular thresholds are visible at Hue = {200, 300, 385}. E) A plot of the pixels from the original image in the Hue-Value plane. F) A plot of the pixels from the original image in the Value-Saturation plane. This graph most clearly shows the different shapes of the collagen peak (blue) and the muscle peak (red).</p

<i>CSH</i> is consistent between individuals.

<p>Apoe−/− mouse innominate arteries stained with MAC3 antibody for detection of macrophages. RGB1(threshold1) was optimized for Cross Section 1, and overestimates the positive area when applied to Cross Section 2. RGB2(threshold2) was optimized for Cross Section 2, and underestimates the positive area when applied to Cross Section 1. <i>CSH</i> was able to effectively use a single HSV threshold on both cross sections. In the overlays between the HSV and RGB1 and RGB2, yellow area shows where there is agreement between the HSV method and the RGB method. Green area in the overlays may indicate false positive area reported by the RGB method, while red area in the overlays may represent false negative area reported by the RGB method.</p

Data_Sheet_1_The effect of plaque morphology, material composition and microcalcifications on the risk of cap rupture: A structural analysis of vulnerable atherosclerotic plaques.pdf

BackgroundThe mechanical rupture of an atheroma cap may initiate a thrombus formation, followed by an acute coronary event and death. Several morphology and tissue composition factors have been identified to play a role on the mechanical stability of an atheroma, including cap thickness, lipid core stiffness, remodeling index, and blood pressure. More recently, the presence of microcalcifications (μCalcs) in the atheroma cap has been demonstrated, but their combined effect with other vulnerability factors has not been fully investigated.Materials and methodsWe performed numerical simulations on 3D idealized lesions and a microCT-derived human coronary atheroma, to quantitatively analyze the atheroma cap rupture. From the predicted cap stresses, we defined a biomechanics-based vulnerability index (VI) to classify the impact of each risk factor on plaque stability, and developed a predictive model based on their synergistic effect.ResultsPlaques with low remodeling index and soft lipid cores exhibit higher VI and can shift the location of maximal wall stresses. The VI exponentially rises as the cap becomes thinner, while the presence of a μCalc causes an additional 2.5-fold increase in vulnerability for a spherical inclusion. The human coronary atheroma model had a stable phenotype, but it was transformed into a vulnerable plaque after introducing a single spherical μCalc in its cap. Overall, cap thickness and μCalcs are the two most influential factors of mechanical rupture risk.ConclusionsOur findings provide supporting evidence that high risk lesions are non-obstructive plaques with softer (lipid-rich) cores and a thin cap with μCalcs. However, stable plaques may still rupture in the presence of μCalcs.</p

Blood biochemistry and plasma levels of osteopontin and pitavastatin.

<p>Mouse plasma was prepared from 32-weeks old apoE^-/- mice fed with high-fat diet. Levels of phosphate (A), calcium (B), creatinine (C), cystatin C (D), urea (E), total cholesterol (F) and osteopontin (G) were measured in plasma from apoE^-/- mice (n = 10), CRD apoE^-/- mice (n = 14) and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 18). Data are shown as mean ± SEM. H: Plasma concentration of pitavastatin given as food admixture in mice. ApoE^-/- mice were fed a chow supplemented with pitavastatin at doses of 30, 100 and 300 mg/kg diet (0.003, 0.01 and 0.03% wt/wt) for 2 weeks. These doses were equivalent to 3, 10 and 30 mg pitavastatin/kg body weight, respectively. Mice treated with pitavastatin at a dose of 100 mg/kg diet had plasma concentration of 5.3 ± 1.0 ng/mL. Data are shown as mean ± SEM (n = 5).</p

A: Pitavastatin has no significant effect on calcification in vascular smooth muscle cells. Mouse vascular smooth muscle cells were treated with or without 50 nM pitavastatin (PTV) in the presence of calcium/phosphate (Ca/P, 3 mM calcium and 2 mM phosphate) for 7 days. Calcium deposition was determined by o-cresolphthalein complexone method and normalized by cellular protein content. Data are shown as mean ± SEM (n = 3 each group). B and C: Pitavastatin reduces osteopontin mRNA expression in peritoneal macrophages. Macrophages were preincubated with either DMSO control or pitavastatin (100 nM or 300 nM) and followed by stimulation with calcium/phosphate (Ca/P, 3 mM calcium and 2 mM phosphate or 5 mM phosphate). mRNA levels of osteopontin (B,C) were determined by real-time PCR and normalized by mRNA levels of GAPDH. Data are shown as mean ± SEM (n = 6 each group).

<p>A: Pitavastatin has no significant effect on calcification in vascular smooth muscle cells. Mouse vascular smooth muscle cells were treated with or without 50 nM pitavastatin (PTV) in the presence of calcium/phosphate (Ca/P, 3 mM calcium and 2 mM phosphate) for 7 days. Calcium deposition was determined by o-cresolphthalein complexone method and normalized by cellular protein content. Data are shown as mean ± SEM (n = 3 each group). B and C: Pitavastatin reduces osteopontin mRNA expression in peritoneal macrophages. Macrophages were preincubated with either DMSO control or pitavastatin (100 nM or 300 nM) and followed by stimulation with calcium/phosphate (Ca/P, 3 mM calcium and 2 mM phosphate or 5 mM phosphate). mRNA levels of osteopontin (B,C) were determined by real-time PCR and normalized by mRNA levels of GAPDH. Data are shown as mean ± SEM (n = 6 each group).</p

Pitavastatin reduces macrophage accumulation in brachiocephalic arteries of CRD mice.

<p>A: Ex vivo fluorescence reflectance imaging (FRI) analysis. Representative images of the fluorescence intensity in the entire aorta were shown as red-green-blue (RGB) readout. Quantitative assessment of the signal intensity in the brachiocephalic artery (ROI) was shown as mean ± SEM. B: Mac3 immunostaining of brachiocephalic arteries. Representative images of macrophage accumulation within atherosclerotic lesions in brachiocephalic arteries of control apoE^-/- mice (n = 9), CRD apoE^-/- mice (n = 9), and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 15). L indicates lumen. Quantitative assessment of Mac3-postive area was shown as mean ± SEM.</p

A: Pitavastatin reduces osteopontin expression in brachiocephalic arteries of CRD mice. Representative images of osteopontin immunostaining within atherosclerotic plaques in brachiocephalic arteries of control apoE^-/- mice (n = 8), CRD apoE^-/- mice (n = 12), and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 14). L indicates lumen. Quantitative assessment of OPN-positive area was shown as mean ± SEM. B: Pitavastatin has no significant effect on calcification in brachiocephalic arteries of CRD mice. Representative images of advanced calcification within atherosclerotic lesions in brachiocephalic arteries of control apoE^-/- mice (n = 6), CRD apoE^-/- mice (n = 11), and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 15). Quantitative assessment of von Kossa-positive area was shown as mean ± SEM. L indicates lumen.

<p>A: Pitavastatin reduces osteopontin expression in brachiocephalic arteries of CRD mice. Representative images of osteopontin immunostaining within atherosclerotic plaques in brachiocephalic arteries of control apoE^-/- mice (n = 8), CRD apoE^-/- mice (n = 12), and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 14). L indicates lumen. Quantitative assessment of OPN-positive area was shown as mean ± SEM. B: Pitavastatin has no significant effect on calcification in brachiocephalic arteries of CRD mice. Representative images of advanced calcification within atherosclerotic lesions in brachiocephalic arteries of control apoE^-/- mice (n = 6), CRD apoE^-/- mice (n = 11), and CRD apoE^-/- mice treated with pitavastatin (CRD apoE^-/- PTV, n = 15). Quantitative assessment of von Kossa-positive area was shown as mean ± SEM. L indicates lumen.</p

A: Study design. High-cholesterol-fed apoE^-/- mice at 19 weeks of age were randomized into control mice (n = 10) and CRD mice treated or untreated with pitavastatin (n = 20 per group). Pitavastatin was administered as a food admixture for 10 weeks starting at 22 weeks. Development of luminal stenosis in brachiocephalic arteries was monitored by ultrasonography at 19 weeks (before nephrectomy) and at 31 weeks. Ex vivo near infrared fluorescence molecular imaging and tissue harvesting for histology were performed at 32 weeks. B: Histological evidence of kidney insufficiency in CRD mice. Hematoxylin and eosin staining demonstrates normal kidney morphology in control apoE^-/- mice and enlarged glomeruli in CRD apoE^-/- mice treated with or without pitavastatin (Black bar = 50 μm).

<p>A: Study design. High-cholesterol-fed apoE^-/- mice at 19 weeks of age were randomized into control mice (n = 10) and CRD mice treated or untreated with pitavastatin (n = 20 per group). Pitavastatin was administered as a food admixture for 10 weeks starting at 22 weeks. Development of luminal stenosis in brachiocephalic arteries was monitored by ultrasonography at 19 weeks (before nephrectomy) and at 31 weeks. Ex vivo near infrared fluorescence molecular imaging and tissue harvesting for histology were performed at 32 weeks. B: Histological evidence of kidney insufficiency in CRD mice. Hematoxylin and eosin staining demonstrates normal kidney morphology in control apoE^-/- mice and enlarged glomeruli in CRD apoE^-/- mice treated with or without pitavastatin (Black bar = 50 μm).</p