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Methylene Blue and Proflavine As Intraarterial Marker for Functional

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Methylene Blue and Proflavine As Intraarterial Marker for Functional Journal of Personalized Medicine Article Methylene Blue and Proflavine as Intraarterial Marker for Functional Perforazome—Comparative Study Maria-Eliza Nedu 1, Mihaela Tertis 2 , Cecilia Cristea 2,* and Alexandru Valentin Georgescu 1 1 Department of Plastic Surgery, Faculty of Medicine, Iuliu Ha¸tieganuUniversity of Medicine and Pharmacy, 400347 Cluj-Napoca, Romania; [email protected] (M.-E.N.); [email protected] (A.V.G.) 2 Department of Analytical Chemistry, Faculty of Pharmacy, Iuliu Ha¸tieganuUniversity of Medicine and Pharmacy, 400349 Cluj-Napoca, Romania; [email protected] * Correspondence: [email protected]; Tel.: +40-721-375-789 Abstract: 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, Citation: Nedu, M.-E.; Tertis, M.; but PRO has the advantage of the absence of inflammatory reaction when using lower concentrations, Cristea, C.; Georgescu, A.V. while preserving unalerted its efficiency as tracer. Methylene Blue and Proflavine as Intraarterial Marker for Functional Keywords: perforasome tracer; methylene blue; proflavine; in vivo study; sentinel lymph nodes Perforazome—Comparative Study. J. Pers. Med. 2021, 11, 147. https:// doi.org/10.3390/jpm11020147 1. Introduction Academic Editor: Methylene blue (MB) also known as methylthioninium chloride is both a dye and a Kenneth P. H. Pritzker medicine known and used for a long time. The use of MB as a tracer has been known for almost 30 years when Morton et al. [1] introduced it in 1992 as intraoperative lymphatic Received: 3 January 2021 tracer in early-stage melanoma. A few years later, in 2001, Simmons et al. [2] proposed Accepted: 17 February 2021 Published: 19 February 2021 MB, as alternative to isosulfan blue, for identifying sentinel lymph nodes in T1 and T2 breast cancer patients [3] with 91%–93% efficiency [4,5]. The same researchers also used Publisher’s Note: MDPI stays neutral indocyanine green (ICG) [6,7] in combination with MB, the first being a well-known and with regard to jurisdictional claims in often used dye in perforator studies [8]. published maps and institutional affil- Tanaka et al. [9] used MB for coronary arteriography and cardiac perfusion assessment, iations. allowing direct visualization of the vessels. When intraarterially administrated, MB does not bind to proteins, and it has high tissue uptake and slow washout (40–60 min) [9]. Recently, ICG, a cyanine dye used in medical diagnostics, started to be used as perforator tracer. It was reported that ICG binds to plasmatic proteins and has a half- life of about 3–5 min in humans [10], and its concentration decreases rapidly in the first Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. five minutes after administration, after which the decrease is slower, and stabilizes after This article is an open access article 30 min [11]. One of the applications of the perfusion with dyes is the study and evaluation distributed under the terms and of perforators [12]. However, when the thickness of the flap is under 2 cm, the perfusion is conditions of the Creative Commons useful for perforators’ mapping [11]. Attribution (CC BY) license (https:// Proflavine (PRO) is a similar dye, whose concentration in the blood flow decreases creativecommons.org/licenses/by/ rapidly in the first 3–5 min after administration [13,14]; then, the decrease is slower and 4.0/). stabilizes for around 30 min after the initial dosing [14]. Confocal fluorescence microscopy J. Pers. Med. 2021, 11, 147. https://doi.org/10.3390/jpm11020147 https://www.mdpi.com/journal/jpm J. Pers. Med. 2021, 11, x FOR PEER REVIEW 2 of 11 J. Pers. Med. 2021, 11, 147 stabilizes for around 30 min after the initial dosing [14]. Confocal fluorescence2 of microscopy 11 tests were done in the presence of PRO as stain for the evaluation of the breast [15], for the in situ evaluation of the changes in adipocytes in invasive ductal carcinoma and ductal carcinomatests were [16] done and in thefor presencethe hyperspectral of PRO as stain imaging for the assessment evaluation of of the tumour breast [margin15], for in head andthe neck in situ cancer evaluation surgery of the[17,18]. changes The in same adipocytes dye was in invasive used for ductal in vivo carcinoma imaging and in micro- endoscopicductal carcinoma identification [16] and of for breast, the hyperspectral head and ne imagingck cancer assessment (18), dysplastic of tumour regions margin of colo- in head and neck cancer surgery [17,18]. The same dye was used for in vivo imaging in rectalmicro-endoscopic tissue [19], oral identification [20] and cervical of breast, epithelial head and neck cells cancer [21], (18),and dysplasticit was observed regions ofthat it had no colorectalinfluence tissue on the [19 ],evolution oral [20] and of the cervical tumour epithelial despite cells its [21 ability], and it to was intercalate observed thatinto it the DNA structurehad no influence[22–24]. on the evolution of the tumour despite its ability to intercalate into the DNADespite structure the [ability22–24]. of MB and PRO to intercalate in the structure of DNA, no negative influencesDespite on thetissues ability viability of MB and were PRO observed. to intercalate The in structural the structure formulas of DNA, of no the neg- two com- poundsative influencesare shown on in tissues Figure viability 1. were observed. The structural formulas of the two compounds are shown in Figure1. Figure 1. The chemical structure of methylene blue (left) and proflavine (right). Figure 1. The chemical structure of methylene blue (left) and proflavine (right). Leung et al. [25] introduced the concept of functional perforasome based on more anatomicLeung perforasomeet al. [25] introduced units. Its assessment the concept can of be functional done only inperfor vivo,asome and the based choke on more vessels which bind the anatomic perforasomes strongly influence the surface of functional anatomicperforasome perforasome and the number units. of Its irrigated assessment anatomical can be units, done respectively. only in vivo, and the choke ves- sels whichThe aimbind of the this studyanatomic was toperforasomes compare from strongly a clinical andinfluence histopathological the surface point of offunctional perforasomeview the use and of the the two number dyes: MB of and irriga PROted as anatomical functional perforasome units, respectively. tracers. In this study, levelsThe of aim the concentrationof this study of was infused to compare dye solutions from in a rats clinical were highand enoughhistopathological to allow the point of viewdirect the visualizationuse of the two with dyes: the nakedMB and eye, PRO namely: as fu 0.65nctional mg/mL perforasome for MB and 1tracers. mg/mL In for this study, PRO, respectively. levels of the concentration of infused dye solutions in rats were high enough to allow the direct2. Materials visualization and Methods with the naked eye, namely: 0.65 mg/mL for MB and 1 mg/mL for PRO, respectively.The study was conducted in the animal research laboratory, respecting all the regula- tions regarding animal studies, with the approval of the ethical committee certified with 2. Materialsthe authorization and Methods No. 199/07.02.2020. Twenty-eight male Wistar rats, weighting between 400–500 g, were used for this in vivo study. The study was conducted in the animal research laboratory, respecting all the regu- All the animals were housed under standard environmental conditions, in rat cages lationsand offered regarding standard animal rat chow studies, and tapwith water. the Theyapproval were housedof the ethical individually committee in the animal certified with thefacilities authorization of the institution No. 199/07.02.2020. under controlled Twenty-eight temperature male and Wistar humidity, rats, and weighting they were between 400–500photographed g, were at used different for this days in post vivo operatory. study. No prophylactic treatment with antibiotics wasAll used the for animals any of thewere animals housed in the under study standard group. The environmental rats were randomly conditions, assigned in in rat cages andtwo offered groups standard of two lots rat of 7 chow subjects, and based tap on water. the type They of dye were injected. housed Standard individually solutions ofin the ani- the two fluorescent dyes of 10 mg/mL were prepared in 0.9% serum solution for infusions mal(B. facilities Braun, Melsungen of the institution AG, Germany). under These controlled standard temperature solutions were and further humidity, diluted and with they were photographedserum to optimal at different concentrations days for postin vivooperatortests ony. rats,No prophylactic namely, 0.65 mg/mL treatment for MB with and antibiot- ics 1was mg/mL used for for PRO. any A of series the ofanimals solutions in with the concentrationsstudy group. betweenThe rats 0.16–1.6 were randomly mg/mL for assigned in twoMB, andgroups 0.1–1 of mg/mL two lots for of PRO, 7 subjects, respectively, based were on prepared the type and of injecteddye injected.
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