Computational protocol: The Effect of a Nonpeptide Angiotensin II Type 2 Receptor Agonist, Compound 21, on Aortic Aneurysm Growth in a Mouse Model of Marfan Syndrome

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[…] Serial cross-sections (7 μm, 10 series) were made after paraffin embedding (Microm HM360; Thermo Scientific, Walldorf, Germany). Hematoxylin and eosin as well as Verhoeff–Van Gieson (VVG) staining were performed. Images of the ascending aorta were obtained at the level of the sinotubular junction at ×40 magnification using a Zeiss Axioplan microscope and an Axiocam MRc5 camera. Two blinded observers (D.V. and M.C.) performed measurements using ImageJ software and results were averaged. Media thickness, the average of 8 measurements (2 per quadrant), was obtained using hematoxylin and eosin–stained samples. VVG was used to evaluate wall architecture at 4 different locations, using an arbitrary scale based on the number of elastin breaks per cross-section. A “break” was defined as an isolated area where an elastic fiber was fragmented with interposed excessive connective tissue matrix. A scale of (1) indicated no breaks, (2) 1 to 2 breaks, (3) 3 to 4 breaks, (4) 5 to 6 breaks, and (5) 7 or more breaks. Dividing the area of elastic fibers by the total area of the media determined the elastin fiber content. [...] Statistical analysis was conducted using SPSS (IBM SPSS statistics; IBM Corporation, Armonk, NY). The Kolmogorov–Smirnov test evaluated normal distribution and the Levene's test evaluated the homogeneity of variance. One-way analysis of variance (ANOVA) with post hoc Scheffe's correction was used to compare mean values. A P-value of ≤0.05 was set for statistical significance. Data are presented as mean ± 2 SEM. Measurements were not corrected for weight of the mice because there were no differences between the groups after 3 and 6 months of treatment. […]

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