Methylene blue and proflavine are fluorescent dyes used to stain nucleic acid from the molecular level to the tissue level. Already clinically used for sentinel node mapping, detection of neuroendocrine tumors, methemoglobinemia, septic shock, ifosfamide-induced encephalopathy, and photodynamic inactivation of RNA viruses, the antimicrobial, anti-inflammatory, and antioxidant effect of methylene blue has been demonstrated in different in vitro and in vivo studies. Proflavine was used as a disinfectant and bacteriostatic agent against many gram-positive bacteria, as well as a urinary antiseptic involved in highlighting cell nuclei. At the tissue level, the anti-inflammatory effects of methylene blue protect against pulmonary, renal, cardiac, pancreatic, ischemic-reperfusion lesions, and fevers. First used for their antiseptic and antiviral activity, respectively, methylene blue and proflavine turned out to be excellent dyes for diagnostic and treatment purposes. In vitro and in vivo studies demonstrated that both dyes are efficient as perfusion and tissue tracers and permitted to evaluate the minimal efficient concentration in different species, as well as their pharmacokinetics and toxicity. This review aims to identify the optimal concentrations of methylene blue and proflavine that can be used for in vivo experiments to highlight the vascularization of the skin in the case of a perforasome (both as a tissue tracer and in vascular mapping), as well as their effects on tissues. This review is intended to be a comparative and critical presentation of the possible applications of methylene blue (MB) and proflavine (PRO) in the surgical field, and the relevant biomedical findings from specialized literature to date are discussed as well.

Background and aims. The aim of this study is to find the most suitable protocol based on an animal experimental model, through the use of a fluorescent dye, determining also its minimal concentration needed to stain the skin after arterial injection, in order to be evidence the functional perforasome by visual examination. Methods. Methylene blue solution was used in order to determine the territory vascularized by one perforator  on fresh cadavers in many studies which introduced, as a final result, the concept of perforasome. One of the most frequent complications of perforator flaps is partial flap necrosis which could be avoided by correctly assessing pre-operatively the functional perforasome surface. Two groups of seven rats were used in order to establish a proper surgical protocol to evaluate the functional perforasome in vivo by injecting the dye. Also, the minimal concentration for methylene blue was experimentally determined. Results. The direct injection into the femoral artery of the proper concentration of dye, 1mM for methylene blue and the clamping of all the branches except the medial branch of the superficial epigastric artery is a reliable model to study the functional perforasome. Conclusions. Our study demonstrates that the intraoperative assessment with fluorescent dye of the functional perforasome by intra-arterial injection of methylene blue is an easy, affordable and very efficient method to reduce the number of partial necrosis of the perforator flaps.

Methylene blue (MB) is both a dye and a medicine known and used for a long time including as lymphatic tracer in melanoma and breast cancer for revealing sentinel lymph nodes. Proflavine (PRO) is an acriflavine dye, used as bacteriostatic disinfectant against many gram-positive bacteria that was also successfully applied to evaluate morphopathological changes in tissues. This study was performed on a group of twenty-eight Wistar rats and had as its main objective the in vivo evaluation of the use of MB and PRO as perforator tracers. The two dyes proved to be effective functional perforasome tracers with medium inflammatory infiltrate in the skin of the island perforator flap which heals perfectly at 14 days with complete absence of the inflammatory reaction. At the same injected amount, PRO seems to determine a greater inflammatory reaction compared with MB, but in smaller concentration, the inflammatory response is absent in the case of PRO. In conclusion, both substances tested within this in vivo study are good functional perforasome tracers, but PRO has the advantage of the absence of inflammatory reaction when using lower concentrations, while preserving unalerted its efficiency as tracer.