Computational protocol: Sex‐Specific Mechanisms of Resistance Vessel Endothelial Dysfunction Induced by Cardiometabolic Risk Factors

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Protocol publication

[…] Mesenteric resistance arteries were embedded in freezing medium (Tissue‐Tek OCT), and transverse sections (10 μm) were obtained with a cryostat (−20°C). Sections were incubated for 30 minutes at 37°C in a light‐protected humidified chamber with phosphate buffer (0.1 mmol/L, pH 7.4) containing 2 mmol/L CaCl2 and 8 μmol/L DAF‐2 DA, used as NO‐sensitive dye. DHE (dihydroethidium [hydroethidine]; 5 μmol/L), a superoxide indicator dye, was topically applied to a second set of tissue sections in a light‐protected humidified chamber with phosphate buffer for 30 minutes for quantification of reactive oxygen species (ROS). Images were obtained with a fluorescence microscope (Eclipse Ti; Nikon) using a ×20 objective. The fluorescence was quantified by a blinded investigator by calculating the integrated density using ImageJ software (National Institutes of Health). [...] The data, analytic methods, and study materials will be made available to other researchers by request to the corresponding author for purposes of reproducing the results or replicating the procedures.All values are presented as mean±SEM. For , the normally distributed data (blood pressure, body weight, glucose) were analyzed by 3‐factor ANOVA with risk factor, genotype, and sex as the variables, followed by the Holm–Sidak post hoc test using SigmaPlot v.12.5 (Systat Software). For the nonnormally distributed data (cholesterol, aldosterone, corticosterone, estradiol) the Kruskal–Wallis test was used in R (R Foundation for Statistical Computing) to independently assess for effects of sex, genotype, and risk factor. For Figures , , , , , , through , means were compared by 2‐factor ANOVA followed by the Bonferroni post hoc test with risk factor and genotype as the variables, using GraphPad Prism 5.0. For all data, a value of P<0.05 was considered significant. […]

Pipeline specifications

Software tools ImageJ, SigmaPlot
Applications Miscellaneous, Microscopic phenotype analysis
Organisms Homo sapiens, Mus musculus
Diseases Hyperlipidemias, Liver Diseases, Vascular Diseases
Chemicals Nitric Oxide, Superoxides