Image analysis comparison of nerve staining with food dye, methylene blue or tissue marker

Objective Novel locoregional techniques use dye studies to confirm successful nerve targeting. The goal was to objectively quantify and compare nerve staining characteristics of dye mixtures commonly reported in the literature using image analysis software.Study design Prospective, randomized cadaveric study.Methods Thirty-six brachial plexus nerves from unpreserved pig cadavers were randomized into three groups of 12: FD (1:10 mixture of blue food dye and bupivacaine 0.5%), MB (methylene blue 1%) and TM (0.1:10 mixture of blue tissue marker and lidocaine 2%). Nerves were immersed in dye for 1, 15, 30 or 60 minutes (n = 3 each). Images of nerves before immersion (baseline) and at each time point with epineurium intact (superficial staining) and after longitudinal bisection (deep staining) were processed using image analysis software. Color saturation values were divided into quartiles (dark, medium-dark, medium-light or light). Percentage of stained nerve area in each quartile was calculated and compared using two-way ANOVA.Results Superficially, at minute 1, dark saturation covered 40% of nerve area in FD versus 19% in MB (p = 0.04) and 0% in TM (p < 0.0001). In bisected nerves, dark and medium-dark saturations occurred only in FD; medium-light saturation comprised anywhere from 4% to 22.5% over time in FD versus <1% at any time in MB (p = 1.000; p = 0.343; p = 0.383; p = 0.262). Deep staining was not found in TM at any point.Conclusion and clinical relevance Food dye rapidly stains superficial and deep nerve layers. Based on these characteristics, investigators can choose the appropriate dye for their study.