Methylene blue reduces IL-1β levels by enhancing ERK1/2 and AKT phosphorylation to improve diabetic retinopathy in rats / 细胞与分子免疫学杂志

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1β (IL-1β) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1β and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1β level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.

An alternative approach for blocking the superior trunk of the brachial plexus evaluated by a single arm clinical trial

Abstract Background Interscalene brachial plexus block is associated with phrenic nerve paralysis. The objective of this study was to evaluate an alternative approach to interscalene brachial plexus blocks in terms of efficacy, grade of motor and sensory blockade, and phrenic nerve blockade. Methods The study was prospective and interventional. The ten living patients studied were 18 to 65 years old, ASA physical status I or II, and submitted to correction of rotator cuff injury. A superior trunk blockade was performed at the superior trunk below the omohyoid muscle, without blocking the phrenic nerve. The needle was advanced below the prevertebral layer until contacting the superior trunk. In order to guarantee the correct positioning of the needle tip, an intracluster pattern of the spread was visualized. The block was performed with 5 mL of 0.5% bupivacaine in ten patients. In the six cadavers, 5 mL of methylene blue was injected. Diaphragmatic excursion was assessed by ultrasonography of the ipsilateral hemidiaphragm. In three patients, pulmonary ventilation was evaluated with impedance tomography. Pain scores and analgesic consumption were assessed in the recovery room for 6 hours after the blockade. Results In the six cadavers, methylene blue didn't reach the phrenic nerve. Ten patients underwent arthroscopic surgery, and no clinically phrenic nerve paralysis was observed. No patient reported pain during the first 6 hours. Conclusions This study suggests that this new superior trunk approach to block the superior trunk may be an alternative technique to promote analgesia for shoulder surgery in patients with impaired respiratory function.

Distribution of various volumes of methylene blue injection in suprainguinal fascia iliaca block under ultrasound guidance: a cadaveric study / Distribución de varios volúmenes de inyección de azul de metileno en el bloque suprainguinal de la fascia ilíaca bajo guía por ultrasonido: un estudio cadavérico

SUMMARY: The local anesthetic volume for a single-shot suprainguinal fascia iliaca block (SFIB) is a key factor of a block success because the courses of the three target nerves from the lumbar plexus (LP), the lateral femoral cutaneous nerve (LFCN), femoral nerve (FN), and obturator nerve (ON), at the inguinal area are isolated and within striking distance. Thus, this cadaveric study aims to demonstrate the distribution of dye staining on the LFCN, FN, ON, and LP following the ultrasound-guided SFIB using 15-50 ml of methylene blue. A total of 40 USG-SFIBs were performed on 20 fresh adult cadavers using 15, 20, 25, 30, 35, 40, 45, and 50 ml of methylene blue. After the injections, the pelvic and inguinal regions were dissected to directly visualize the dye stained on the LFCN, FN, ON, and LP. All FN and LFCN were stained heavily when the 15-50 ml of dye was injected. Higher volumes of dye (40-50 ml) spread more medially and stained on the ON and LP in 60 % of cases. To increase the possibility of dye spreading to all three target nerves and LP of the SFIB, a high volume (≥40 ml) of anesthetic is recommended. If only a blockade of the FN and LFCN is required, a low volume (15-25 ml) of anesthetic is sufficient.

RESUMEN: El volumen de anestésico local para un bloqueo de la fascia ilíaca suprainguinal (FISI) de una sola inyección es un factor clave para el éxito del bloqueo, debido a que los cursos de los tres nervios objetivo del plexo lumbar (PL), el nervio cutáneo femoral lateral (NCFL), femoral (NF) y el nervio obturador (NO), en el área inguinal están aislados y dentro de la distancia de abordaje. Por lo tanto, este estudio cadavérico tiene como objetivo demostrar la distribución de la tinción de tinte en NCFL, NF, NO y PL siguiendo el FISI guiado por ultrasonido usando 15-50 ml de azul de metileno. Se realizaron un total de 40 USG-FISI en 20 cadáveres adultos frescos utilizando 15, 20, 25, 30, 35, 40, 45 y 50 ml de azul de metileno. Después de las inyecciones, se disecaron las regiones pélvica e inguinal para visualizar directamente el tinte teñido en NCFL, NF, NO y PL. Todos los NF y NCFL se tiñeron intensamente cuando se inyectaron los 15- 50 ml de colorante. Volúmenes mayores de colorante (40-50 ml) se esparcen más medialmente y tiñen el NO y la PL en el 60 % de los casos. Para aumentar la posibilidad de que el colorante se propague a los tres nervios objetivo y al PL del FISI, se recomienda un volumen elevado (≥40 ml) de anestésico. Si solo se requiere un bloqueo de NF y NCFL, un volumen bajo (15-25 ml) de anestésico es suficiente.

Intravenous infusion of methylene blue to visualize the ureter in laparoscopic colorectal surgery / 中华胃肠外科杂志

Objective: Intraoperative localization of the ureter can contribute to accurate dissection and minimize ureteral injury in colorectal surgery. We aim to summarize a single center's experience of fluorescence ureteral visualization using methylene blue (MB) and explore its visualization efficiency. Methods: This is a descriptive case-series-study. Clinical data of patients who had undergone laparoscopic colorectal surgery and fluorescence visualization of the ureter in the Gastrointestinal Surgery Department of Guangdong Provincial People's Hospital from March 2022 to May 2022 were retrospectively collected. Patients with incomplete surgery videos, renal insufficiency, or allergic reactions were excluded. MB was infused with 0.9% NaCl at 1.0 mg/kg in 100 mL of normal saline for 5 to 15 minutes during laparoscopic exploration. Imaging was performed using a device developed in-house by OptoMedic (Guangdong, China) that operates at 660nm to achieve excitation of MB. Clinical information, MB dosage, rate of successful fluorescence, time to fluorescence, operation time, blood loss, intraoperative blood oxygen levels, pathological staging, changes in renal function, and post-operative complications were retrospectively analyzed. Results: The study cohort comprised 27 patients (24 men and 3 women) with an average age of (60.25±16.95) years and an average body mass index of (21.72±3.42) kg/m2. The dosage of MB was 0.3-1.0 mg/kg and the infusion time was 5-15 minutes. Fluorescence signals were detected in all patients. The median time to signal detection was 20 (range, 10 to 40) minutes after MB infusion. The range of intraoperative blood oxygen fluctuation averaged 2.5% (range, 0 to 7.0%). The median change in creatine concentration was -1.3 (range, -17.2 to 29.2) µmol/L. No patients had complications associated with use of MB. Fluorescence visualization of the ureter was very valuable clinically in two patients (thick mesentery, stage T4). Conclusion: MB is a safe and effective means of visualizing the ureter by fluorescence during laparoscopic colorectal surgery, especially when the procedure is difficult. MB in a dosage of less than 1 mg/kg can slowly infused for more than 5 minutes during laparoscopic exploration. During the infusion, attention must be paid to blood oxygen fluctuations.

Novas estratégias terapêuticas para o controle de biofilmes orais: terapia fotodinâmica e sistema carreador de antimicrobianos / New therapeutic strategics for the control of oral biofilms: photodynamic therapy and drug delivery system

Os biofilmes orais possuem grande relevância clínica por estarem associados com o desenvolvimento de cárie dentária e candidose bucal, que são doenças infecciosas frequentemente encontradas na população. O presente trabalho foi dividido em dois estudos: Estudo 1 que teve como objetivo analisar os efeitos da terapia fotodinâmica antimicrobiana (TFDa), mediada por Fotoenticine (FTC) e Azul de Metileno (AM), sobre biofilmes microcosmos de cárie dentária; e Estudo 2 cujo objetivo foi avaliar o gellan gum como biomaterial para carreador do antifúngico Ester fenetil do ácido caféico (CAPE) contra Candida albicans. No estudo 1, amostras de dentina cariada foram coletadas de diferentes pacientes para formar biofilmes microcosmos in vitro. Os biofilmes foram tratados com FTC ou AM associado à irradiação LED a 660 nm (28,5 J/cm²). Os dados foram analisados pela contagem de Unidades Formadoras de Colônias (UFC/mL). Além disso, a biomassa, estrutura do biofilme e produção de ácidos pelos microrganismos foram determinadas por análises microscópicas ou espectrofotométricas. Os biofilmes de diferentes pacientes apresentaram variações na composição microbiana, sendo formados por estreptococos, lactobacilos e leveduras. No geral, a TFDa diminuiu 3,7 Log10 do total de microrganismos, 2,8 Log10 de estreptococos, 3,2 Log10 de lactobacilos e 3,2 Log10 de leveduras, e atingiu a erradicação de estreptococos do grupo mutans. A TFDa também foi capaz de reduzir a biomassa, desagregar os biofilmes e diminuir a concentração de ácidos em 1,1 a 1,9 mmol de lactato/L. Em relação ao estudo 2, inicialmente, foram preparadas formulações do CAPE em diferentes concentrações de gellan gum (0,6 a 1%). As formulações foram avaliadas em relação ao sistema de liberação e ação antifúngica contra C. albicans. Verificou-se que concentrações mais altas de gellan (0,9 e 1%) levaram a uma liberação mais prolongada do CAPE em relação as concentrações mais baixas. Os valores de concentração inibitória mínima do CAPE sobre C. albicans foram aumentados quando esse composto foi incorporado no gellan. As formulações de CAPE em gellan apresentaram atividade antifúngica tanto em culturas planctônicas como em biofilmes de C. albicans, sendo esses efeitos dependentes do tempo de tratamento. O CAPE e suas formulações em gellan também levaram a uma diminuição da atividade proteolítica de C. albicans. Concluiu-se que a TFDa mediada por Fotoenticine e o sistema carreador de gellan gum podem ser estratégias terapêuticas promissoras para o controle dos biofilmes na cavidade bucal, podendo ser usadas respectivamente no tratamento da cárie e candidose. (AU)

Dental caries and oral candidiasis are infectious diseases frequently found in the population. The present work is divided into two studies, study 1 time as objective: To analyze the effects of antimicrobial photodynamic therapy (aPDT), mediated by Fotoenticine (FTC) and Methylene Blue (MB), on dental caries microcosm biofilms. In study 2, the objective was to evaluate gellan gum as a biomaterial to carry the antifungal caffeic acid phenethyl ester (CAPE) on Candida albicans. To conduct study 1, carious dentin samples were collected from different patients to form in vitro microcosm biofilms. The biofilms were treated with FTC or MB associated with 660 nm red LED irradiation, with energy dose of 28.5 J/cm² and power dose of 40 mW/cm². The data were analyzed by the count of Colony Forming Units (CFU/mL). In addition, the biomass, biofilm structure and acid production of the microorganisms were determined by microscopic or spectrophotometric analysis. The biofilms from different patients showed variations in microbial composition, being formed by streptococci, lactobacilli, and yeasts. Overall, aPDT decreased 3.7 Log10 of total microorganisms, 2.8 Log10 of streptococci, 3.2 Log10 of lactobacilli and 3.2 Log10 of yeasts, and achieved eradication of mutans group streptococci. PDTa was also able to reduce biomass, disaggregate biofilms, and decrease acid concentration by 1.1 to 1.9 mmol lactate/L. For study 2 of this, first the standards of CAPE were determined, such as minimum inhibitory concentration, and absorption peak, then CAPE was incorporated into gellan gum, and then the standard curve test and analysis of CAPE release was performed, finally the formulations were tested on planktonic culture and biofilm of different strains of C. albicans, it was also analyzed the action of this drug on the production of Sap. The MIC found varied from 32 to 64 µg/mL, the release tests showed a gradual release in the higher formulations, finally in the CFU/mL count both in planktonic culture and biofilm the formulations were able to inhibit the fungus. With this it is concluded that both aPDT for oral microcosm and gellan gum as caregiver of CAPE for Candida albicans inhibition are promising. (AU)

Computer simulation of molecular docking between methylene blue and some proteins of Porphyromonas gingivalis / 北京大学学报(医学版)

OBJECTIVE@#To study the binding target of photosensitizer and bacteria in antimicrobial photodynamic therapy with computer-simulated target prediction and molecular docking research methods and to calculate the binding energy.@*METHODS@#The protein names of Porphyromonas gingivalis (Pg) were obtained and summarized in Uniprot database and RCSB PDB database; the structure diagrams of methy-lene blue were screened in SciFinder database, PubChem database, ChemSpider database, and Chemical Book, and ChemBioDraw software was used to draw and confirm the three-dimensional structure for target prediction and Cytoscape software was used to build a visual network diagram; a protein interaction network was searched and built between the methylene blue target and the common target of Pg in the String database; then we selected FimA, Mfa4, RgpB, and Kgp K1 proteins, used AutoDock software to calculate the docking energy of methylene blue and the above-mentioned proteins and performed molecular docking.@*RESULTS@#The target prediction results showed that there were 19 common targets between the 268 potential targets of methylene blue and 1 865 Pg proteins. The 19 targets were: groS, radA, rplA, dps, fabH, pyrG, thyA, panC, RHO, frdA, ileS, bioA, def, ddl, TPR, murA, lepB, cobT, and gyrB. The results of the molecular docking showed that methylene blue could bind to 9 sites of FimA protein, with a binding energy of -6.26 kcal/mol; with 4 sites of Mfa4 protein and hydrogen bond formation site GLU47, and the binding energy of -5.91 kcal/mol, the binding energy of LYS80, the hydrogen bond forming site of RgpB protein, was -5.14 kcal/mol, and the binding energy of 6 sites of Kgp K1 protein and the hydrogen bond forming site GLY1114 of -5.07 kcal/mol.@*CONCLUSION@#Computer simulation of target prediction and molecular docking technology can initially reveal the binding, degree of binding and binding sites of methylene blue and Pg proteins. This method provides a reference for future research on the screening of binding sites of photosensitizers to cells and bacteria.

Comparison of two volumes for sciatic perineural injection using 1% methylene blue in rabbit cadavers / [Comparação de dois volumes para injeção perineural no nervo isquiático utilizando azul de metileno 1% em cadáveres de coelhos]

The purpose of this study was to assess the efficacy of a blind technique for sciatic and femoral nerve block in rabbit cadavers by evaluating the spread of 1% methylene blue at two different volumes. Nine recently euthanized rabbits weighing 2.5(0.3kg were used. The sciatic (SN) and femoral (FN) nerves of each limb were randomly assigned for injection with 1% methylene blue at 0.2mL/kg (G0.2) or 0.3mL/kg (G0.3). Nerves were dissected and measured for depth and extension of staining (cm). Mean comparisons were performed using paired t test. The relation between volume and nerve staining ( 2cm was assessed using chi-square test. The mean depth of SN was 1.9±0.2 and 1.6±0.3cm and staining 1.9±1.4 and 2.0±1.2cm, respectively in G0.2 and G0.3. No relation was found between depth and dye spread and there was no association between nerve staining ( 2.0cm and volume of solution. The FN failed to be stained in all subjects. In conclusion, SN injection can be successfully performed without guidance in rabbits. The lower volume (0.2mL/kg) is recommended to avoid systemic toxicity.(AU)

O objetivo deste estudo foi avaliar a eficácia de uma técnica para bloqueio às cegas dos nervos isquiático e femoral em cadáveres de coelhos, por meio da avaliação da dispersão de azul de metileno 1% em dois volumes distintos. Nove coelhos recém-eutanasiados, com peso 2,5(0,3kg, foram utilizados. Os nervos isquiático (NI) e femoral (NF) de cada membro foram aleatoriamente designados para injeção com azul de metileno 1% a 0,2mL/kg (G0,2) ou 0,3mL/kg (G0,3). Em seguida, foram dissecados e mensurados em relação à sua profundidade e extensão corada (cm). As médias foram comparadas por meio de teste t pareado. A relação entre volume e extensão corada ( 2cm foi avaliada utilizando-se teste de qui-quadrado. A profundidade média do NI foi 1,9±0,2 e 1,6±0,3cm, e a extensão corada 1,9±1,4 e 2,0±1,2cm, respectivamente, no G0,2 e no G0,3. Não houve relação entre a profundidade e a extensão corada ou entre a extensão corada ( 2,0cm e o volume de solução. Não foi observada coloração do NF em nenhum cadáver. Concluiu-se que a injeção do NI pode ser realizada com sucesso sem auxílio de tecnologias em coelhos. O menor volume (0,2mL/kg) é recomendado para evitar toxicidade sistêmica.(AU)

Localización intraoperatoria de las recurrencias en cáncer de tiroides mediante la inyección de azul de metileno guiada por ecografía / Intraoperative localization of recurrences in thyroid cancer by ultrasound-guided injection of methylene blue

Introducción. La cirugía para extirpación de metástasis en un cuello previamente intervenido afronta un reto para lograr una resección exitosa. El presente estudio pretende demostrar la utilidad de la técnica de inyección de azul de metileno, guiada por ecografía, para la localización intraoperatoria de lesiones recurrentes en cáncer de tiroides, para facilitar su resección. Métodos. Se realizó un estudio observacional, descriptivo y retrospectivo, en pacientes reintervenidos por recurrencia de carcinoma diferenciado de tiroides, durante un periodo de dos años y medio. Se utilizó la inyección intratumoral de azul de metileno guiada por ecografía para su identificación intraoperatoria de recurrencia. Se hizo análisis de variables demográficas y clínicas. Resultados. Este estudio incluyó 10 procedimientos en 9 pacientes, 77,8 % mujeres, con una media de edad de 54 años. Todos tenían un nivel de tiroglobulina detectable y elevado antes de la intervención; posteriormente, el 89 %presentó un descenso y el 33 % una adecuada respuesta bioquímica. La técnica agregó 10 minutos al tiempo quirúrgico. En el 100 % se identificaron de manera intraoperatoria los ganglios marcados; el promedio de ganglios resecados fue de 12, de los cuales, 6 fueron positivos, todos con carcinoma papilar de tiroides. Esta técnica se consideró de gran utilidad y de bajo costo en todos los casos. Discusión. Esta técnica se muestra como una estrategia efectiva para la identificación intraoperatoria de las recurrencias corregionales en carcinoma de tiroides, permitiendo una disección ganglionar exitosa, disminuyendo complicaciones, tiempo quirúrgico y, especialmente, costos frente a otras intervenciones

Introduction. The approach of a previously operated neck for metastasis resection faces a challenge to achieve a successful resection. The present study aims to demonstrate the usefulness of the ultrasound-guided injection of methylene blue technique for the intraoperative localization of recurrent lesions in thyroid cancer to facilitate their resection. Methods. An observational, descriptive and retrospective study was conducted in patients reoperated for recurrences of differentiated thyroid carcinoma over a period of two and a half years, using ultrasound-guided intratumoral injection of methylene blue for its intraoperative identification. An analysis of demographic and clinical variables was carried out and its advantages over other methods were identified. Results. This study included 10 procedures in nine patients, 77.8% women and 22.2% men, with a mean age of 54 years. All had a detectable and elevated thyroglobulin level before the intervention, 89% had a decrease in its level and 33% had an adequate biochemical response. The technique added 10 minutes to the surgical time. All marked lymph nodes were identified intraoperatively. The average number of lymph nodes resected was 12, of which six were positive, all with papillary thyroid carcinoma. It was considered of great utility and low cost in all cases. Discussion. This technique shows to be an effective strategy for the intraoperative identification of locoregional recurrences in thyroid carcinoma, allowing a successful lymph node dissection, reducing complications, surgical time and especially costs compared to other interventions

After Thirty Years, We Still Cannot Understand Why Methylene Blue is not a Reference to Treat Vasoplegic Syndrome in Cardiac Surgery

Abstract Vasoplegic syndrome (VS) comprises a constellation of concurrent signs and symptoms: hypotension, high cardiac index, low systemic vascular resistance, low filling pressures, the tendency to occur diffuse bleeding, and sustained hypotension. All of these parameters may persist even despite the use of high doses of vasoconstrictor amines. VS arises from vasoplegic endothelial dysfunction with excessive release of nitric oxide by polymorphonuclear leukocytes mediated by the nitric oxide synthase's inducible form and is associated with systemic inflammatory reaction and high morbimortality. The achievements regarding the treatment of VS with methylene blue (MB) are a valuable Brazilian contribution to cardiac surgery. The present text review was designed to deliver the accumulated knowledge in the past ten years of employing MB to treat VS after cardiac surgery. Considering that we have already published two papers describing acquired experiences and concepts after 15 and 20 years, now, as we achieve the 30-year mark, we compose a trilogy.

Methylene blue for treatment of hospitalized Covid-19 patients: a randomized, controlled, open-label clinical trial, phase 2

ABSTRACT Background: There is no pharmacological intervention on the treatment of hypoxemia and respiratory distress in COVID-19 patients. Objective: The objective of the study was to study the effect of the reduced form of methylene blue (MB) on the improvement of oxygen saturation (SpO2) and respiratory rate (RR). Methods: In an academic medical center, 80 hospitalized patients with severe COVID-19 were randomly assigned to receive either oral MB along with standard of care (SOC) (MB group, n = 40) or SOC only (SOC group, n=40). The primary outcomes were SpO2 and RR on the 3rd and 5th days. The secondary outcomes were hospital stay and mortality within 28 days. Results: In the MB group, a significant improvement in SpO2 and RR was observed on the 3rd day (for both, p < 0.0001) and also the 5th day (for both, p < 0.0001). In the SOC group, there was no significant improvement in SpO2 (p = 0.24) and RR (p = 0.20) on the 3rd day, although there was a significant improvement of SpO2 (p = 0.002) and RR (p = 0.01) on the 5th day. In the MB group in comparison to the SOC group, the rate ratio of increased SpO2 was 13.5 and 2.1 times on the 3rd and 5th days, respectively. In the MB group compared with the SOC group, the rate ratio of RR improvement was 10.1 and 3.7 times on the 3rd and 5th days, respectively. The hospital stay was significantly shortened in the MB group (p = 0.004), and the mortality was 12.5% and 22.5% in the MB and SOC groups, respectively. Conclusions: The addition of MB to the treatment protocols significantly improved SpO2 and respiratory distress in COVID-19 patients, which resulted in decreased hospital stay and mortality. ClinicalTrials.gov: NCT04370288

The routine use of the methylene blue test in sleeve gastrectomy: why not? / Uso rotineiro do teste do azul de metileno na gastrectomia vertical: por que não?

ABSTRACT Background: Although considered a safe procedure, sleeve gastrectomy (SG) has a non-negligible risk of major postoperative complications related to it, with special attention to gastric leaks. Aim: Evaluate the clinical value of the methylene blue test (MBT) in predicting the occurrence of post-SG leaks. Methods: Retrospective study that included 1136 patients who underwent SG with intraoperative MBT between 2012 and 2016. Sensitivity, specificity, positive predictive value (PPV) and negative predicted value (NPV) were calculated to determine the clinical correlation between the MBT and the occurrence of postoperative leaks. Staple line oversewing was performed in all patients who presented positive MBT. Results: Laparoscopic SG was performed in 97.0% of cases; open in 2.3%, and robotic in 0.7%. MBT was positive in 19 cases (1.67%). One positive MBT occurred during an open SG and the other 18 at laparoscopy. Moreover, there were nine cases (0.8%) of postoperative leaks, among which, only two presented positive MBT. MBT diagnostic value was evaluated through the calculation of sensitivity (22.0%), specificity (98.0%), PPV (11.0%) and NPV (99.0%). There were no cases of allergic reaction or any other side effect with the use of the methylene blue solution. Conclusion: MBT showed high specificity and negative predictive value, thus presenting an important value to rule out the occurrence of postoperative leaks.

RESUMO Racional: Embora considerada procedimento seguro, a gastrectomia vertical (GV) apresenta risco não desprezível de complicações pós-operatórias importantes relacionadas a ela, com atenção especial para fístulas gástricas Objetivo: Avaliar a aplicabilidade clínica do teste do azul de metileno (TAM) na predição da ocorrência de fístulas após a GV. Método: Estudo retrospectivo que incluiu 1136 pacientes operados entre 2012 e 2016 com aplicação do TAM intraoperatório. Sensibilidade, especificidade, valor preditivo positivo (VPP) e valor preditivo negativo (VPN) foram calculados na tentativa de determinar a correlação clínica entre os resultados do TAM e a ocorrência de fístulas pós-operatórias. Sobressutura da linha de grampos foi realizada em todos os pacientes que apresentaram TAM positivo. Resultados: GV laparoscópica foi realizada em 97,0% dos casos; por laparotomia em 2,3% e roboticamente em 0,7%. TAM foi positivo em 19 casos (1,67%). Um dos casos positivos ocorreu na laparotomia e os outros 18 na laparoscopia. Ainda, houve nove casos (0,8%) de fístulas pós-operatórias, dentre os quais, apenas dois apresentaram TAM positivo. O valor diagnóstico do TAM foi avaliado através dos cálculos de sensibilidade (22,0%), especificidade (98,0%), VPP (11,0%) e VPN (99,0%). Não houve casos de reação alérgica ou qualquer outro efeito colateral advindo do uso da solução de azul de metileno. Conclusão: TAM mostrou alta especificidade e VPN, apresentando importância em descartar a ocorrência de fístulas pós-operatórias.

Antimicrobial photodynamic inactivation mediated by methylene blue: Analysis of inactivation kinetics and biochemical mechanisms / Inativação fotodinâmica antimicrobiana mediada por azul de metileno: Análises de cinética de inativação e mecanismos bioquímicos

The widespread use of antimicrobial chemotherapy in medicine and livestock production imposed an evolutive selection of drug-resistant strains worldwide. As a result, the effectiveness of our current antimicrobial armamentarium is constantly being reduced to alarming levels. Therefore, novel antimicrobial therapeutic strategies are urgently needed. Antimicrobial photodynamic therapy (APDT) comes to this scenario as a powerful tool to counteract the emergence of microbial drug-resistance. Its mechanisms of action are based on simultaneous oxidative damage of multiple targets and, therefore, it is much less likely to allow any type of microbial resistance. Therefore, the objectives of this study were focused into establishing 1) a mathematical tool to allow precise analysis of microbial photoinactivation; 2) a broad analysis of APDT effectiveness against global priority drug-resistant pathogens; 3) inhibition of ßlactamase enzymes; and 4) how the biochemical mechanisms of APDT avoid emergence of resistance. The main results obtained through the investigation led by this thesis were divided into 4 scientific articles regarding each of the above-mentioned objectives. In summary, we discovered that 1) a power-law function can precisely fit all microbial inactivation kinetics data and provide insightful information of tolerance factors and lethal doses; 2) there is no correlation between drug-resistance and APDT sensitivity, i.e., extensively drug resistant microorganisms are killed in the same kinetics as drug-sensitive controls; 3) ß-lactamases are very sensitive to photodynamic inhibition; 4) biochemical mechanisms of APDT promote oxidative damages to external cell membranes, DNA and proteins whereas the main cause of microbial death seems to be directly associated with protein degradation. Thus, we conclude that APDT is effective against a broad-spectrum of pathogens and has minimum chances of promoting resistance mechanisms

O amplo uso da quimioterapia antimicrobiana impôs uma seleção evolutiva de cepas resistentes a medicamentos. Como resultado, a eficácia dos fármacos antimicrobianos tem sido reduzida a níveis alarmantes. Portanto, novas estratégias terapêuticas antimicrobianas são urgentemente necessárias. A terapia fotodinâmica antimicrobiana (TFDA) entra neste cenário como uma ferramenta poderosa para combater a resistência microbiana. Seus mecanismos de ação são baseados no dano oxidativo sobre múltiplos alvos e, portanto, é muito menos provável que permita o surgimento de qualquer tipo de resistência. Os objetivos deste estudo foram focados ao estabelecimento de 1) modelo matemático para análise precisa da fotoinativação microbiana; 2) ampla análise da eficácia da TFDA contra patógenos resistentes a fármacos antimicrobianos de prioridade global; 3) inibição de ß-lactamases por TFDA; e 4) como os mecanismos bioquímicos da TFDA evitam o surgimento de resistência. Os principais resultados obtidos através da investigação conduzida por esta tese foram divididos em 4 artigos científicos. Em resumo, descobrimos que 1) uma função de lei de potência pode ajustar com precisão todos os dados de cinética de inativação microbiana e fornecer informações detalhadas sobre fatores de tolerância e doses letais; 2) não há correlação entre resistência à quimioterapia antimicrobiana e sensibilidade à TFDA, isto é, cepas extensivamente resistentes aos antimicrobianos são inativadas sob a mesma cinética que controles sensíveis aos antimicrobianos; 3) ß-lactamases são altamente sensíveis à inibição fotodinâmica; 4) os mecanismos bioquímicos da TFDA promovem danos oxidativos às membranas celulares e DNA, porém, a principal causa de morte microbiana é diretamente associada à degradação das proteínas. Assim, concluímos que a TFDA é eficaz contra um amplo espectro de patógenos e tem chances mínimas de promover mecanismos de resistência

Associação da quitosana à terapia fotodinâmica para controle de Candida albicans: estudo em culturas planctônicas, biofilmes e células persistentes / Association of chitosan and photodynamic therapy to control Candida albicans: study in planktonic cultures, biofilms and persister cells

A terapia fotodinâmica (TFD) vem se mostrando como um método eficaz no controle de micro-organismos patogênicos, sendo investigada para o tratamento de diversas doenças infecciosas, como a candidose bucal. Recentemente, alguns agentes potencializadores dessa terapia têm sido estudados, incluindo a Quitosana (QT), um polímero natural extraído do exoesqueleto de crustáceos. O objetivo desse estudo foi avaliar o efeito potencializador da QT na TFD mediada pelo fotossensibilizador Azul de Metileno (AM) sobre cepas de Candida albicans, investigando sua ação em culturas planctônicas, biofilmes e células persistentes ao fluconazol. Além disso, foi avaliado a capacidade da QT em interferir na absorção do AM pelas células de Candida. Foram utilizadas duas cepas de C. albicans, sendo uma padrão (ATCC 18804) e uma clínica isolada de candidose orofaríngea (70). Para os ensaios em culturas planctônicas, as cepas de C. albicans foram cultivadas em caldo Yeast Nitrogen Base (YNB) por 24 horas. Os biofilmes foram formados por 48 horas no fundo das placas de 96 poços. Para indução de células persistentes, C. albicans foi cultivada com altas concentrações de fluconazol por 48 horas. A seguir, foram realizados os tratamentos com QT a 5 mg/mL (pH de 6,5), AM nas concentrações de 300 ou 600 µM, e irradiação com LED (660 nm) na densidade de energia de 30 J/cm2. Foram incluídos oito grupos experimentais: TFD com AM e QT na presença de Luz (AM+QT+L+), AM e QT sem Luz (AM+QT+L-), QT e Luz (QT+L+), QT sem Luz (QT+L-), TFD com AM e Luz (AM+L+), AM sem Luz (AM+L-), Solução Fisiológica com Luz (F-L+) e apenas Solução Fisiológica (F-L-). Após os tratamentos, foi realizada contagem de unidades formadoras de colônias (UFC/mL). Para determinar a absorção do fotossensibilizador pelas células de C. albicans, as células foram lisadas e centrifugadas para leitura da densidade óptica. Os dados foram analisados por ANOVA e teste de Tukey (p<0,05%). Na TFD em culturas planctônicas, o AM (300 µm) reduziu as células de Candida em 1,6 log (UFC/mL), enquanto que a associação AM+QT levou à redução de 4,7 log. Na TFD em biofilmes, ocorreu redução microbiana de 2,9 log para o tratamento com AM (600 µm) e de 5,3 log para AM+QT. Em relação às células persistentes, a redução encontrada foi de 0,8 log para AM e 1,5 log para AM+QT. No teste de absorção, a penetração do AM nas células de Candida (DO 0,02) foi aumentada na presença da QT (DO 0,39). Concluiu-se que a QT potencializou o efeito antimicrobiano da TFD em culturas planctônicas, biofilmes e células persistentes de C. albicans, provavelmente por facilitar a penetração do AM nas células fúngicas.

Photodynamic therapy (PDT) has been shown to be an effective method to control pathogenic microorganisms, being investigated for the treatment of several infectious diseases, such as oral candidiasis. Recently, some agents that enhance this therapy have been studied, including Chitosan (CS), a natural polymer extracted from the exoskeleton of crustaceans. The aim of this study was to evaluate the potentiating effect of CS on Methylene Blue (MB) photosensitizer-mediated PDT on Candida albicans strains, investigating its action on planktonic cultures, biofilms and persister cells to fluconazole. In addition, the ability of CS to interfere with MB absorption by Candida cells was evaluated. Two strains of C. albicans were used, one standard (ATCC 18804) and one isolated clinical of oropharyngeal candidosis (70). For assays in planktonic cultures, C. albicans strains were cultivated in Yeast Nitrogen Base broth (YNB) for 24 hours. Biofilms were formed for 48 hours at the bottom of 96-well plates. For the induction of persister cells, C. albicans was cultivated with high concentrations of fluconazole for 48 hours. Next, treatments were performed with CS at 5 mg/mL (pH 6.5), MB at concentrations of 300 or 600 µM, and irradiation with LED (660 nm) at an energy density of 30 J/cm2. eight experimental groups were included: PDT with MB and CS in the presence of Light (MB+CS+L+), MB and CS without Light (MB+CS+L-), CS and Light (CS+L+), CS without Light (CS +L-), PDT with MB and Light (MB+L+), MB without Light (MB+L-), Physiological Solution with Light (F-L+) and Physiological Solution only (FL-). After the treatments, colony forming units (CFU/mL) were counted. To determine the absorption of the photosensitizer by C. albicans cells, the cells were lysed and centrifuged for optical density reading. Data were analyzed by ANOVA and Tukey test (p<0.05%). In PDT in planktonic cultures, MB (300 µm) reduced Candida cells by 1.6 log (CFU/mL), while the MB+CS association led to a 4.7 log reduction. In PDT in biofilms, there was a microbial reduction of 2.9 log for the treatment with MB (600 µm) and of 5.3 log for MB+CS. Regarding persister cells, the reduction found was 0.8 log for MB and 1.5 log for MB+CS. In the absorption test, the penetration of MB into Candida cells (OD 0.02) was increased in the presence of CS (OD 0.39). It was concluded that CS potentiated the antimicrobial effect of PDT in planktonic cultures, biofilms and persister cells of C. albicans, probably by facilitating the penetration of MB into fungal cells .

Electrochemical DNA Biosensor for Mycobacterium leprae Identification

Abstract Nowadays, a prompt and reliable diagnosis is one of the most critical measures for leprosy control. The current diagnostic is based on clinical exams by a health care professional, and it may not recognize early signs of the disease. Therefore, other leprosy diagnosis methods are needed that are sensitive, disease-specific, and easy to deliver to the end-user. This study describes the construction of an electrochemical DNA biosensor to detect PCR products of Mycobacterium leprae using methylene blue as an indicator of the hybridization. The capture probe was immobilized on the graphite electrode modified with poly(4-aminophenol). The electrode surface was morphologically characterized by atomic force microscopy. Linear voltammetry was used to monitor the concentration of methylene blue on the DNA biosensor, which indicated a limit detection of 1 x 10-10 mol/L. The biosensor showed selective when placed to hybridize with a non-complementary sequence. This study suggests that the electrochemical DNA biosensor developed is promising for detecting DNA of Mycobacterium leprae.

Methylene Blue for the Treatment of Health Conditions: a Scoping Review

HIGHLIGHTS This scoping review summarizes the findings of clinical trials using methylene blue (MB) for the treatment of various health conditions. This research method allowed mapping main findings, clarifying research topics, and identifying gaps in the literature.

Abstract studies evaluating effective drugs for health conditions are of crucial importance for public health. Methylene blue (MB) is an accessible synthetic drug that presents low toxicity and has been used in several health areas due to its effectiveness. Objective: this scoping review aims to provide a comprehensive overview of relevant research regarding the use of MB for the treatment of health conditions. Methods: a five-stage framework Arksey and O'maley scoping review was conducted. The literature was searched in Cochrane Library database using Mesh term "methylene blue". Data were collected by two independent reviewers and submitted to descriptive synthesis. Results: The search resulted in 429 records, from which 16 were included after exclusion criteria were applied. The therapeutic use of MB was identified for acute conditions (malaria and septic shock), chronic conditions (discogenic back pain, bipolar disorder, refractory neuropathic pain, and post-traumatic stress disorder), and postoperative care (vasoplegic syndrome, and pain after haemorrhoidectomy, lumbar discectomy, and traumatic thoracolumbar fixation). Conclusion: there is much evidence emerging from clinical trials about the therapeutic use of MB for acute, chronic, and postoperative conditions; however, many gaps were identified, which open further avenues for future research.

Retrospective analysis of sentinel lymph node biopsy using methylene blue dye for early breast cancer / 中华医学杂志(英文版)

BACKGROUND@#Methylene blue is the most commonly used tracer for sentinel lymph node (SLN) biopsy (SLNB) in China. This study aimed to investigate the feasibility of clinical application of SLNB using methylene blue dye (MBD) for early breast cancer and the prognosis of patients with different SLN and non-SLN statuses.@*METHODS@#We retrospectively analyzed the clinicopathological data of patients with early breast cancer treated at the Peking University First Hospital between 2013 and 2018. We calculated the SLN identification rate (IR) in SLNB with MBD and the false-negative rate (FNR), and analyzed the prognosis of patients with different SLN and non-SLN statuses using Kaplan-Meier curves.@*RESULTS@#Between January 2013 and December 2018, 1603 patients with early breast cancer underwent SLNB with MBD. The SLN IR was 95.8% (1536/1603). Two SLNs (median) were detected per patient. There were significant differences in FNR between patients with SLN micrometastasis and macrometastasis (19.0% vs. 4.5%, χ2 = 12.771, P < 0.001). Chi-square test showed that there were significant differences in SLN successful detection rates among patients with different vascular tumor embolism status (96.3% vs. 90.8%, χ2 = 9.013, P = 0.003) and tumor (T) stages (96.6% vs. 94.1%, χ2 = 5.189, P = 0.023). Multivariate analysis showed that vascular tumor embolism was the only independent factor for SLN successful detection (odds ratio: 0.440, 95% confidence interval: 0.224-0.862, P = 0.017). Survival analysis showed a significant difference in disease-free survival (DFS) between patients with non-SLN metastasis and patients without non-SLN metastasis (P = 0.006).@*CONCLUSION@#Our single-center data show that, as a commonly used tracer in SLNB in China, MBD has an acceptable SLN IR and a low FNR in frozen sections. This finding is consistent with reports of dual tracer-guided SLNB. Positive SLNs with non-SLN metastasis are associated with DFS.

Evaluación por gastroscopia simple y cromoendoscopia convencional de la superficie gastroesofágica y duodenal proximal del equino. Estudio piloto / Evaluation by simple gastroscopy and conventional chromoendoscopy of the gastro-esophageal and proximal duodenal surface of equine. Preliminary study

RESUMEN Las enfermedades gastrointestinales equinas tienen una alta incidencia con un pronóstico variable en la práctica clínica. La mayoría de los estudios se limitan a describir lesiones ulcerativas y lesiones inflamatorias. El objetivo de este estudio fue evaluar el potencial diagnóstico complementario de la cromoendoscopia convencional en la mucosa gas-troesofágica y duodenal proximal del equino. El estudio incluyó 20 caballos criollos colombianos de ambos sexos (12 hembras y 8 machos), con edades entre 5 y 20 años, peso entre 250 y 350 kilogramos, condición corporal 4-5/9 y con historial de alteraciones digestivas en los últimos 3 meses; quienes previo a la evaluación por gastroscopia y cromoendoscopia se sometieron a ayuno (sólidos 12h y líquidos 4h) y sedación (xilacina 0,5 mg/kg/iv). Se utilizaron tinciones como rojo fenol, lugol, índigo carmín, azul de metileno y ácido acético y se tomaron biopsias de los segmentos que mostraron reacción. El azul de metileno reveló 52% de las lesiones, el lugol 19%; por su parte, el rojo fenol, el índigo carmín y el ácido acético revelaron el 9,5% restante. El epitelio escamoso fue el más afectado (66,6%), el glandular (19%), antro pilórico (9,5%) y duodeno proximal (4,7%). Los hallazgos histopatológicos fueron hiperplasia, hipertrofia, hiperqueratosis, congestión, degeneración vacuolar, infiltrados celulares, fibrosis, necrosis y atrofia en diferentes grados de severidad. La cromoendoscopia reveló lesiones prematuras, que pasaron desapercibidas con las técnicas convencionales de endoscopia del tracto digestivo. Este es el primer estudio que emplea la cromoendoscopia en equinos; a pesar de que la técnica mejoró la visualización y facilitó la ubicación y descripción de lesiones ulcerativas prematuras a través de la histopatología, se recomiendan mayores estudios controlados y con un número más amplio de muestras.

ABSTRACT Equine gastrointestinal diseases have a high occurrence with a variable prognostic in clinic practice. Most of the studies limits to describe ulcerative and inflammatory lesions. The objective of this study was to evaluate the potential complementary diagnostic of conventional chromoendoscopy on the gastroesophageal and proximal duodenal mucosa of the equine. 20 Colombian creole horses, of both sexes (12 females and 8 males), with ages between 5 and 20 years old, weight between 250 and 350 kilograms, body condition 4-5/9, that had presented digestive alterations in the last 3 months, were subjected to fasting (solids 12h and liquids 4h) and sedated (xylazine 0,5 mg/kg/iv) to be evaluated by gastroscopy and chromoendoscopy, using for stains phenol red, lugol, indigo carmine, methylene blue and acetic acid, taking biopsy samples of the segments that showed reaction. The methylene blue revealed 52%, lugol 19%, and phenol red, indigo carmine and acetic acid revealed only 9,5% of the lesions, being the squamous epithelium the most affected (66,6%), glandular epithelium (19%), pyloric antrum (9,5%) and proximal duodenum (4,7%), where histopathological findings were hyperplasia, hypertrophy, hyperkeratosis, congestion, vacuolar degeneration, cellular infiltrates, fibrosis, necrosis and atrophy in different degrees of severity. Chromoendoscopy revealed lesions premature, which go unnoticed with conventional light endoscopy techniques. This is the first study using chromoscopy in horses to show that the reagents used allow a better visualization of injuries than the conventional technique, helping histopathological studies and molecular biology to understand ulcerative premature injuries and possible pathophysiological pathways. However, larger controlled studies and a larger number of samples are needed.