Methods for analyzing acetylcholine-induced vasodilation in human skin

Intradermal microdialysis, combined with laser-Doppler flowmetry and local heating, is commonly used to investigate mechanisms of endothelial function and dysfunction in the cutaneous microcirculation. Local perfusion of acetylcholine (ACh), with and without N^G-nitro-l-arginine methyl ester [l-NAME; a nonselective nitric oxide (NO) synthase inhibitor], allows for direct assessment of pharmacologically induced endothelium- and NO-dependent vasodilation. However, variability in the analysis and reporting of cutaneous vascular conductance (CVC) responses to in vivo cholinergic stimulation limits reproducibility. The purpose of this investigation was to compare analytical approaches used to quantify in vivo cutaneous vasodilation by retrospectively analyzing ACh dose-response data and NO-dependent vasodilation using five methods. Curve-modeling approaches included nonlinear regression with: 1) no constraints, 2) Hill slope constraint, and 3) top and bottom constraints. Point-by-point analysis and area under the dose-response curve (AUC) were also evaluated following graded ACh perfusion with and without l-NAME coperfusion. Nonlinear regression without constraints exhibited the highest rate of data exclusion due to poor model fit. Although all analysis methods detected the expected differences in CVC responses between ACh with and without l-NAME, each approach had distinct limitations. Nonlinear regression provides detailed pharmacodynamic parameters, including maximal and minimal vasodilator responses, logEC₅₀, and Hill slope, whereas point-by-point analysis and AUC offer straightforward quantitative assessments of NO-dependent vasodilation. Based on this retrospective analysis, we recommend the unconstrained model (methods 1A/1B) for characterizing the ACh dose-response relation, as the curves best reflect the visualized data. Point-by-point analysis, AUC, and l-NAME-sensitive component calculations may be more appropriate for quantifying NO-dependent vasodilation.NEW & NOTEWORTHY Although dose-response curves are commonly used to characterize in vivo cutaneous cholinergic vasodilator responses, modeling parameters and analytical approaches vary widely across studies. Here, we compare five methods applied to graded acetylcholine perfusion with and without N^G-nitro-l-arginine methyl ester [l-NAME; a nonselective nitric oxide (NO) synthase inhibitor] coperfusion to quantify NO-dependent vasodilation. We highlight key advantages and limitations of each method and provide practical recommendations for their implementation.