Enhancing Techniques for Determining Inflammatory Edema Formation and Neutrophil Accumulation in Murine Skin

Abstract

Inflammatory edema formation and polymorphonuclear leukocyte (neutrophil) accumulation are common components of cutaneous vascular inflammation, and their assessment is a powerful investigative and drug development tool but typically requires independent cohorts of animals to assess each. We have established the use of a mathematical formula to estimate the ellipsoidal-shaped volume of the edematous wheal or bleb after intradermal injections of substances in mice pretreated intravenously with Evans blue dye (which binds to plasma albumin) to act as an edema marker. Whereas previous extraction of Evans blue dye with formamide is suitable for all strains of mice, we report this quicker and more reliable assessment of edema volume in situ. This therefore allows neutrophil accumulation to be assessed from the same mouse using the myeloperoxidase assay. Importantly, we examined the influence of Evans blue dye on the spectrometry readout at the wavelength at which myeloperoxidase activity is measured. The results indicate that it is feasible to quantify edema formation and neutrophil accumulation in the same mouse skin site. Thus, we show techniques that can assess edema formation and neutrophil accumulation at the same site in the same mouse, allowing paired measurements and reducing the total use of mice by 50%.