Evaluation of the time course of plasma extravasation in the skin by digital image analysis

Plasma extravasation (PE) can be triggered by neurotransmitters as part of a neuroinflammatory response. We present a technique based on video digital image processing that provides a simple, noninvasive, reliable, and quantitative method for measuring the time course and extent of PE in the skin. After intravenous infusion of Evans Blue dye, stimulation of the saphenous nerve caused the skin on the dorsomedial region of the hind paw to become dark blue. The change in reflectance of the skin was recorded with a monochrome video camera. Images were digitized and analyzed with inexpensive or public domain software. The change in pixel intensity was determined in a selected region. Stimulation at 4 Hz caused greater darkening of the skin than at 1 Hz, and this was confirmed with spectrophotometric measurements of Evans Blue content. The NK1 receptor antagonist CP-99, 994 blocked saphenous nerve and substance P-induced darkening of the skin. The results indicate that our measurement gives results similar to those obtained with classic methods that are widely accepted as an indication of PE. This simple and quick method reveals the extent, time course, and location of PE, is cheap to implement and easy to learn, and thus represents a useful and alternative tool for studies of PE and its modulation.