Experimental study of the removal of Candida albicans from mouse tongues using photoactivated disinfection / 口腔疾病防治

Objective @#To establish a mouse model of acute pseudomembranous stomatitis and to observe the effect of photoactivated disinfection (PAD) on the removal of Candida albicans in vivo, and initially explore the feasibility of this technology in the treatment of acute pseudomembranous stomatitis.@*Methods@#Six-week-old male ICR mice were selected and immunized with 1 × 107CFU/mL Candida albicans solution on the backs of the tongues of immunosuppressed mice. Thirty model mice with acute pseudomembranous stomatitis were successfully established and randomly divided into a control group and a photoactivated disinfection group, with 15 mice in each group. Mice in the photoactivated disinfection group were coated with 1 mg/mL toluidine blue solution on the back of the tongue, incubated for 1 min and irradiated with 750 mW LED red light for 1 min. Immediately after treatment, the tongue fungal load was measured in the photoactivated disinfection group and the control group. Tongue fungal load was measured again 48 h later, and tongue histopathological examination was performed in both groups. @*Results @# Forty-eight hours after PAD treatment, the white pseudomembrane on the back of the tongue in the photoactivated group was significantly less than the control group. The fungal load on the dorsum of the tongue in the treatment group was significantly lower than the control group immediately and 48 h after treatment for PAD, and the difference was statistically significant (P<0.05). Forty-eight hours after PAD treatment, HE staining showed that the epithelial structure of the PAD group was more regular than the control group, and no microabscesses were observed. PAS staining showed that the number of mycelia in the PAD group was significantly less than the control group. Mycelia occasionally invaded the keratinized layer but did not penetrate into the upper cortex.@*Conclusion@#PAD significantly removed Candida albicans from the tongues of mice with acute pseudomembranous Candida stomatitis.

Comparación de las tinciones con hematoxilina y eosina versus azul de toluidina en la evaluación de los carcinomas cutáneos durante la cirugía micrográfica de Mohs / Comparative study of toluidine blue and hematoxylin and eosin staining in evaluating skin-carcinoma during Mohs micrographic surgery / Comparação da coloração de hematoxilina e eosina versus azul de toluidina na avaliação de carcinomas cutâneos durante a cirurgia micrográfica de Mohs

Resumen: Introducción: la cirugía micrográfica de Mohs es una técnica quirúrgica especializada para el tratamiento del cáncer de piel no melanoma. La histopatología cumple un rol fundamental, y la elección de la tinción es un punto de controversia. Objetivos: comparar el rendimiento de las tinciones de hematoxilina y eosina (HyE) versus azul de toluidina (AT) durante la cirugía. Método: estudio observacional, descriptivo y transversal a partir de noviembre de 2017 hasta mayo de 2018. Se incluyeron las láminas empleadas durante la cirugía en el período mencionado. Estas fueron analizadas por el cirujano de Mohs, tres residentes y una dermopatóloga. Se valoró el rendimiento de ambas tinciones, teniendo en cuenta las características celulares y los elementos del estroma. Resultados: se estudiaron 23 tumores (16 carcinomas basocelulares y 7 carcinomas espinocelulares). Al observarse al microscopio óptico tanto con la tinción de AT como con HyE no se encontraron diferencias significativas entre ambos grupos en lo global, sólo en algunas características, especialmente con la HyE. Conclusiones: es el primer trabajo en Uruguay que compara la eficacia de las dos tinciones durante la cirugía micrográfica de Mohs. Como conclusión tanto la tinción de HyE como el AT son muy buenas técnicas para el diagnóstico de carcinomas cutáneos.

Abstract: Introduction: Mohs micrographic surgery is a specialized surgical technique used to treat nonmelanoma carcinoma. Histopathology plays a vital role in the diagnosis of this condition, and the choice staining method is controversial. Objective: to compare results in the use of hematoxylin and eosin (H&E) versus Toluidine blue (TB) staining during surgery. Method: observational, descriptive and transversal study conducted from November, 2017 until May, 2018 of the slides used during surgeries in the selected period. Slides were analysed by the Mohs surgeon, 3 residents and a dermopathologist to evaluate the results of both staining methods, in consideration of cell features and stromal elements. Results: 23 tumors were analysed (16 Basal Cell carcinomas and 7 Squamous Cell Carcinoma). Microscopic observation of slides prepared with Toluidine blue and hematoxylin and eosin stains did not show significant global differences between both groups, except in terms of a few characteristics, in particular with hematoxylin and eosin stains. Conclusions: this was the first study in Uruguay to evaluate the effectiveness of both staining methods during Mohs micrographic surgery, and it concluded that both Toluidine blue and hematoxylin and eosin stains are very good techniques in evaluating skin-cancer.

Resumo: Introdução: a cirurgia micrográfica de Mohs é uma técnica cirúrgica especializada para o tratamento do câncer de pele não melanoma. A histopatologia desempenha um papel fundamental, onde a escolha da coloração é um ponto de controvérsia. Objetivos: comparar o desempenho das colorações de hematoxilina e eosina versus azul de toluidina durante a cirurgia. Método: estudo observacional, descritivo e transversal de novembro de 2017 a maio de 2018. Foram incluídas as lâminas utilizadas durante as cirurgias no referido período. Estas foram analisadas pelo cirurgião especializado na técnica de Mohs, 3 residentes e um dermatopatologista onde foi avaliado o desempenho de ambas as colorações, levando em consideração as características celulares e os elementos do estroma. Resultados: foram estudados 23 tumores (16 carcinomas basocelulares e 7 carcinomas espinocelulares). Quando observados ao microscópio de luz para coloração AT e H&E, não foram encontradas diferenças significativas entre os dois grupos em geral, apenas em algumas características, especialmente com o H&E. Conclusões: é o primeiro estudo no Uruguai que compara a eficácia dos 2 corantes durante a cirurgia micrográfica de Mohs. Em conclusão, tanto a coloração com hematoxilina e eosina quanto com azul de toluidina são técnicas muito boas para o diagnóstico de carcinomas de pele.

Irradiation Time of Photodynamic Therapy to the Number of Lactobacillus acidophilus

ABSTRACT@#Lactobacillus acidophilus (L. acidophilus) is one of the etiological agents for dental caries and dominant in the deep carious lesion. L. acidophilus has also been identified in persistent root canal infection and also related to the failure of endodontic treatment. Photodynamic therapy is a therapeutic process involving the combination of a nontoxic photosensitizer and a light source. The excited photosensitizer reacts with reactive oxygen species (ROS), which induce injury and death of the microorganism. This study aimed to prove the effect of irradiation time of photodynamic therapy to the number of L. acidophilus. Forty-two Eppendorf tubes were treated with 0.5 ml L. acidophilus distributed into seven groups. Group 1 as the control group received no treatment. Groups 2, 3, 4, 5, 6 and 7 were treated with a combination of 0.5 ml toluidine blue O (TBO) as a photosensitizer and 630 nm photoactivated (Fotosan®) exposure time for 10, 20, 30, 40, 50 and 60 sec. Then, all were stored in an incubator of 37ºC for 48 h. Later, the colony-forming unit (CFU) was counted for each group. There were significant differences in the number of L. acidophilus in CFU of the various irradiation times. The longer the photodynamic therapy irradiation was, the lesser the number of live L. acidophilus became. At 50 sec and 60 sec irradiation, none of the L. acidophilus was found alive.

Value of the toluidine blue test as an aid to determine the biopsy site in actinic prurigo cheilitis

Actinic prurigo (AP) is a type of photodermatosis that primarily affects the Latin American mestizo population. Histologically, AP cheilitis exhibits acanthosis with spongiosis and vacuolation of the basal cell layer overlying a dense lymphocytic inflammatory infiltrate that forms well-defined lymphoid follicles. Toluidine blue is a thiazide, acidophilic, and metachromatic dye used in vivo to selectively stain the acidic components of tissues such as sulfates, carboxylates, and phosphate radicals that are incorporated into DNA and RNA. It is necessary to develop a method that allows detecting, on clinical grounds the area of the lesion in which it is more feasible to find such structures. Thus to increase the sensitivity of the biopsy, in AP cheilitis to accurately identify where the lymphoid follicles reside, based on the higher concentration of DNA in such structures and thus confirm the diagnosis. In this study, staining was positive in 85% of patients with AP cheilitis, in 14 of whom 82% lymphoid follicles were observed by histopathology. One of the pathologist's problems in establishing the diagnosis of AP is that the main histopathological characteristics are not always identified in the submitted samples because it is not easy to clinically identify the most representative site of the lesion selected for performing a biopsy. Based on our results, we propose using toluidine blue as an auxiliary method to choose a tissue sample to facilitate the diagnosis and allow clinicians to make clinical correlations between the histopathological and therapeutic findings.

Simple Method For Optimization Of Heparin And Enoxaparin Detection Using Agarose Gel Electrophoresis

Objective: A simple gel electrophoresis method for low-molecular-weight heparins (LMWH) is required for use in a variety of laboratories to allow further identification and purification. This study aimed to optimize the detection of heparin and enoxaparin (low-molecular-weight heparin by gel electrophoresis. Methods: Several gel electrophoresis conditions were tested to optimize the detection of enoxaparin by using a simple method with a modified Volpi’s approach. Multiple gel thicknesses, voltage settings, and enoxaparin concentrations were tested in the optimization procedure. Enoxaparin was purchased from a local supplier as pre-filled pharmaceutical injections. Highly purified 0.5% and 1.0% agarose gels were prepared and a series of enoxaparin concentrations was added to both gels for comparison and optimization. The 0.2% toluidine blue stain was prepared by the addition of 1 ml in an ethanol-water-acetic acid mixture (50:49:1; v/v/w). The staining process comprised two steps: first, toluidine blue was added for 30 min and destained overnight in the solvent mixture. Subsequently, the following morning, the second step was conducted, in which the gel was restained for 30 min with the same concentration of toluidine blue. We continued to stain the gel until the bands were visible. Results: The gel electrophoresis results showed that clearest and sharpest bands were obtained using 65–75 mAh and 85 V settings. At 95 mAh, the bands were slightly washed out. Conclusion: This study successfully facilitated the detection of enoxaparin, a LMWH, and heparin in the laboratory by using simple tools and techniques available in most laboratories.

Comparative study of metachromatic staining methods in assessing the exfoliative cell types during oestrous cycle in Sprague-Dawley laboratory rats / Estudio comparativo de los métodos de tinción metacromática en la evaluación de los tipos de células exfoliativas durante el ciclo estral en ratas Sprague-Dawley de laboratorio

This study was aimed at comparing the commonly used metachromatic stains viz., Papanicolaou stain, WrightGiemsa, Toluidine blue and Methylene blue in the assessment of cell types of the oestrous cycle in rats. Eight female Sprague-Dawley rats aged 8-9 weeks were used for this assessment. Cotton Swabs were gently inserted in the animals vagina to obtain cells from which they were then transferred to glass slides for staining and evaluation under microscopy. The different cell types were compared for their morphological features and clarity of cellular detail under all four stains. The application, advantages and limitations of all stains were then discussed. It was concluded that the selection of the most effective stain in the assessment of vaginal cytology depends on their application to clinical or research which was based on the cellular detail of interest, time, cost and availability of each staining procedure.

El presente estudio tuvo como objetivo comparar las tinciones metacromáticas comúnmente utilizadas, Wright's-Giemsa, azul de toluidina, azul de metileno y tinción de Papanicolaou, en la evaluación de los tipos de células del ciclo estral en ratas. El estudio se realizó en ocho ratas hembras SpragueDawley, con edades entre 8 y 9 semanas, y se usaron hisopos vaginales de algodón para preparar portaobjetos. Los diferentes tipos de células se compararon por sus características morfológicas y claridad en las cuatro tinciones. La aplicación, ventajas y limitaciones de todas las tinciones fueron discutidas. Se concluye que la selección de la tinción más efectiva en la evaluación de la citología vaginal depende de su uso, es decir, clínico o de investigación, el detalle celular de interés, tiempo, costo y disponibilidad.

Caracterização histoquímica no diagnóstico da babesiose bovina por Babesia bovis / Histochemical characterization in the diagnosis of bovine babesiosis by Babesia bovis

A babesiose é uma doença hemolítica transmitida por carrapatos e causada por protozoários intraeritrocitários do gênero Babesia. Esta é uma doença de incidência elevada na Região Sul do Brasil e responsável por perdas econômicas consideráveis. O diagnóstico clínico-patológico pode ser feito através da demonstração do parasito pelo exame de esfregaços sanguíneos ou de tecidos frescos. Entretanto, com frequência, somente órgãos fixados em formol são remetidos para laboratórios de patologia, o que impossibilita a realização do esfregaço. O principal objetivo deste estudo foi encontrar técnicas histoquímicas alternativas e capazes de aprimorar a evidenciação de Babesia bovis intraeritrocitária em tecidos fixados em formol. Para este estudo, foram analisadas retrospectivamente amostras de tecidos de 50 casos de necropsias de bovinos, as quais haviam sido fixadas em formol e processadas rotineiramente para histopatologia. Os casos foram divididos em um grupo controle, constituído por 12 casos de babesiose cerebral com substância cinzenta encefálica róseo-cereja característica (grupo A), e 38 casos sugestivos de tristeza parasitária bovina (grupo B), conforme os protocolos de necropsias. Foram testadas as técnicas histoquímicas de Azul Alciano, Azul de Metileno, Azul de Toluidina, Giemsa, Gram (método de McCallum-Goodpasture), Grocott, Ácido Periódico de Schiff e Ziehl-Neelsen. Dentre estas, observou-se que as técnicas de Azul de Metileno e Azul de Toluidina permitiram observar características morfológicas e tintoriais de maneira mais nítida, auxiliando na identificação de B. bovis. Adicionalmente, foram estabelecidos vários parâmetros clínico-epidemiológicos e anatomopatológicos da babesiose por B. bovis.(AU)

Babesiosis is a hemolytic tick-borne disease caused by intraerythrocytic protozoal parasites of the genus Babesia. This is a disease of high incidence in the southern Brazil and responsible for considerable economic losses. Clinical-pathological diagnosis can be made by demonstrating the parasite by examining blood smears or fresh tissues. However, frequently, only formalin-fixed organs are sent to pathology laboratories, which makes it impossible to perform the smears. The main objective of this study was to find alternative histochemical techniques capable to improve the identification of intraerythrocytic Babesia bovis in histological sections. For this study, tissue samples from 50 bovine necropsy cases were retrospectively analyzed, which had been fixed in formalin and routinely processed for histopathology. The cases were divided into a control group, consisting of 12 cases of cerebral babesiosis with characteristic pink-cherry gray matter (group A), and 38 cases suggestive of cattle tick fever (group B), according to necropsy protocols. Histochemical techniques of Alcian Blue, Methylene Blue, Toluidine Blue, Giemsa, Gram (McCallum-Goodpasture method), Grocott, Periodic Acid of Schiff and Ziehl-Neelsen were tested. Among these, it was observed that the techniques of Methylene Blue and Toluidine Blue allowed to observe morphological and dye characteristics in a clearer way, aiding in the identification of B. bovis. In addition, several clinical-epidemiological and anatomopathological parameters of babesiosis caused by B. bovis were established.(AU)

Correlation between clinical findings, mast cell count and interleukin 31 immunostaining in the skin of dogs with atopic dermatitis / Correlação entre achados clínicos, contagem de mastócitos e imunomarcação de interleucina 31 na pele de cães com dermatite atópica

ABSTRACT: In this study the correlation between the clinical score, mast cell count and interleukin 31 (IL-31) immunostaining in the skin of dogs with atopic dermatitis was determined. A total of 31 dogs of different breeds, from one to eight years of age, were chosen for the study. The 20 females and 11 males were categorized based on the CADESI-4 system, as having discrete, moderate or marked atopic dermatitis. Skin samples were collected from the axillary and interdigital regions and stained with hematoxylin and eosin for cytohistomorphological analyses and toluidine blue to evaluate the mast cell counts, and immunohistochemistry for the IL-31 immunostaining. Animals revealing higher atopic dermatitis scores had greater numbers of mast cells and IL-31 immunolabeled cells. More numbers of cells immunolabeled for IL-31 were evident in the axillary skin compared with the interdigital skin in dogs having this condition. A correlation was identified between the clinical scores and mast cell numbers in the interdigital region, as well as between the clinical scores and number of cells immunolabeled for IL-31 in the axillary area. A correlation was also reported between the mast cell numbers and IL-31 immunolabeled cells only in the axillary skin, and none in the interdigital regions. It was thus concluded that the mast cells and IL-31 are involved in the pathogenesis of the canine atopic dermatitis (CAD), as well as lymphocytes and plasma cells. It was also observed that the higher the degree of clinical severity of the disease, the more the numbers of mast cells and IL-31 in the skin of those animals suffering from CAD, which implies the influence of these immunological constituents on the genesis of pruritus and disease progression.

RESUMO: Este estudo avaliou a correlação entre o escore clínico, a contagem de mastócitos e a imunomarcação de interleucina 31 (IL-31) na pele de cães com dermatite atópica. Foram selecionados 31 cães de diferentes raças, com idade entre um e oito anos, sendo 20 fêmeas e 11 machos, divididos em discretamente, moderadamente e acentuadamente acometidos por dermatite atópica segundo o sistema CADESI-4. Amostras da pele das regiões axilar e interdigital foram colhidas e submetidas às colorações de hematoxilina e eosina para a avaliação cito-histomorfológica e azul de toluidina para a contagem de mastócitos, bem como a técnica de imunoistoquímica para a imunomarcação de IL-31. Os animais com maior escore de dermatite atópica apresentaram maior número de mastócitos e de células imunomarcadas para IL-31. Houve maior número de células imunomarcadas para IL-31 na pele da axila em relação à interdigital nos cães com a doença. Foi constatada correlação entre o escore clínico e a quantidade de mastócitos no interdígito, bem como entre o escore clínico e a quantidade de células imunomarcadas para IL-31 na axila. Também foi verificada correlação entre a quantidade de mastócitos e células imunomarcadas para IL-31 na pele da região axilar, mas não da interdigital. Conclui-se que mastócitos e a IL-31 estão envolvidos na patogenia da DAC, assim como linfócitos e plasmócitos. Também, quanto maior o grau de severidade clínica da doença, maior a quantidade de mastócitos e IL-31 na pele dos animais com DAC, o que remete à influência desses componentes imunológicos na gênese do prurido e progressão da doença.

The Modified Toluidine Blue Staining Method for Mast Cell / 现代生物医学进展

Objective:This study aimed to identify the morphology of mast cells by using a modified toluidine blue staining scheme,so as to provide a powerful reference for the experimental basis research of mast cells.Methods:Bone marrow-derived mast cells were induced in vitro.After 4 weeks,the cells were collected,fixed,and stained.Mast cells were fixed at different temperature during different time.The optimum condition was determined by comparing the effects of toluidine blue staining.Results:Bone marrow cells were induced to differentiate into mast cells by SCF and IL-3 in vitro.When mast cells were stained with modified toluidine blue staining,the staining effect was better.Mast cells were round or oval and the cell membrane was complete and the cytoplasm was filled with a large number of purple particles.Conclusion:In this study,we successfully applied a modified toluidine blue staining method to mast cells cultured in vitro.The results showed that the condition at 37 ℃ full fixation with staining could reduce the degeneration of mast cells.This method was easy to operate with good stability.It was suitable for the morphological observation of mast cells cultured in vitro.

Analysis of the presence and location of mast cells in periapical cysts and periapical granulomas / Análise da presença e localização de mastócitos nos cistos periapicais e granulomas periapicais

ABSTRACT Objective: The aim of the present study was to locate mast cells in chronic periapical lesions (granulomas and cysts) by using histochemical techniques and toluidine blue staining. Methods: A quantitative, descriptive, cross-sectional and retrospective research was performed. The sample was obtained from histopathological reports in the archives of the laboratory of surgical pathology of the University of Pernambuco between November 2014 and May 2015. Results: Sixteen cases of granuloma and 21 cases of periapical cysts were selected. The stained slides were analyzed by two examiners at different times, in a double-blind study. Mast cells were found in 13 (61.9%) of the periapical cyst cases, located in the capsule of the lesion. In the periapical granuloma cases, mast cells were found in eight cases (50%), located in the granulation tissue. Conclusion: Mast cells were detected in both cysts and periapical granuloma, located in the capsule and granulation tissue, respectively. Mast cells were more prevalent in periapical cysts than in periapical granuloma.

RESUMO Objetivo: Localizar mastócitos em lesões periapicais (granulomas e cistos) através de técnicas histoquímicas e corante azul de toluidina. Métodos: Uma pesquisa quantitativa, descritiva, transversal e retrospectiva foi realizada. As amostras foram obtidas de documentos histopatológicos nos arquivos do laboratório de patologia cirúrgica da Universidade de Pernambuco entre Novembro de 2014 e Março de 2015. Resultados: Dezesseis casos de granuloma e 21 casos de cistos periapicales foram selecionados. As lâminas coradas foram analisadas por dois examinadores em tempos diferentes, em um estudo duplo-cego. Mastócitos foram encontrados em 13 (61,9%) dos casos de cistos periapicales, localizados na cápsula da lesão. Nos granulomas periapicais, mastócitos foram encontrados em oito casos (50%), localizados no tecido de granulação. Conclusão: Mastócitos foram detectados tanto em cistos quanto em granulomas periapicais, localizados na cápsula e no tecido de granulação respectivamente. Os mastócitos estavam mais presentes nos cistos do que nos granulomas periapicais.

Avaliação dos efeitos da terapia fotodinâmica utilizando compostos fenotiazínicos sobre melanoma in vitro / Assessment of the effects of photodynamic therapy using phenothiazine compounds on in vitro melanoma

O câncer é considerado a segunda maior causa de morte em países ocidentais. Nos Estados Unidos da América (EUA) as mortes por câncer anualmente superam a soma das mortes provocadas em guerras como Vietnã, Coreia e as Grandes Guerras Mundiais. Apresentando-se como a mais agressiva das neoplasias dermatológicas, o melanoma cutâneo está associado à cerca de 75% das causas de morte por câncer de pele. Um dos tratamentos estudados para aplicação em pacientes com esta e outras patologias é a Terapia Fotodinâmica (TFD), que é baseada no uso de corantes de baixa toxicidade, que tem seletividade por alguns tecidos ou células e quando ativados por baixas doses de luz visível induzam alterações celulares como a produção ERO. Os fenotiazínicos são moléculas catiônicas com, absorção de luz na região entre 620-660nm, espectro que permite maior penetração nos tecidos, promovendo maior ação do composto em tecidos mais profundos. A irradiação foi feita com luz lazer com λ= 660nm, com potência de 100 mW, densidade de energia de 150 J/cm2 por 1 min. As quantificações de viabilidade celular foram feitas por método colorimétrico, e realizada a leitura em leitor de microplaca a 655 nm. A determinação das IC50 do Azul de Metileno e Azul de Toluidina O foi 2,5 μM e 8,4 μM, respectivamente. Utilizando a TFD observamos que os efeitos citotóxicos dos compostos fenotiazínicos apresentam significância estatística (p<0,05) tanto em AM quanto em ATO. Em microscopia óptica com coloração por May-Grunwald-Giemsa observamos que existe uma aparente diminuição do conteúdo citoplasmático e preservação nuclear nas células tratadas com TFD, além da diminuição da quantidade de células por campo. A avaliação ultraestrutural por Microscopia de varredura mostra células com visível extração citoplasmática após a TFD, apontando uma lesão membranar. Novas avaliações precisam ser feitas como estabelecer um melhor protocolo para aumentar os efeitos da TFD.

Cancer is considered the second leading cause of death in Western countries. In the United States of America (USA) cancer deaths annually exceed the total of deaths in wars like Vietnam, Korea and the Great World Wars. Presenting as the most aggressive of the skin neoplasms, cutaneous melanoma is associated with circa 75% of the deaths from skin cancer. One of the therapies used in patients with melanoma and other pathologies is Photodynamic Therapy (PDT), which is based upon the use of dyes of low toxicity, which has selectivity for certain tissues or cells and low doses when activated by visible light induce cellular changes such as ROS production. The phenothiazic dyes are cationic molecules, absorption of light in the region between 620-660 nm, allowing greater spectrum tissue penetration, promoting higher activity of the compound in deeper tissues. The irradiation was performed with laser light with λ = 660 nm with 100 mW, energy density of 150 J/cm2 for 1 min. Quantification of cell viability was performed by the colorimetric method, and performed a reading in a microplate reader at 655nm. Determination of IC50 of Methylene Blue and Toluidine Blue O was 2.5 μM and 8.4 μM respectively. Using the protocol of PDT observed that there is one of the cytotoxic effects of phenothiazine compounds with statistical significance (p <0.05) in both MB and in TBO. Through optical microscopy by staining with fast Panotic was observed that there is an apparent decrease in the cytoplasm in cells treated with MB and TBO as well as reduced number of cells per field. The Scanning Electron Microscopy, shows cells with cytoplasm extraction, after PDT, indicated possibility, membrane damage. New assessments need to be made to establish a better protocol to potentiate the effects of PDT.

Laser terapêutico e células inflamatórias - parte 1: mastócitos* / Therapeutic laser and inflammatory cells - part 1: mast cells

O presente estudo avaliou a quantidade de mastócitos após irradiação do laser não cirúrgico λ685 nm. Dezoito ratos machos, linhagem Wistar (250 a 300 gramas), nos quais foram realizadas feridas cirúrgicas padronizadas, foram distribuídos em seis grupos, três experimentais (com quatro ratos) e três controles (com dois ratos). Os grupos experimentais foram irradiados em quatro pontos perilesionais equidistantes (0,5 J/cm², 35 mW, 35 Hz e 17 segundos). Os grupos-controles não foram irradiados. Os animais foram mortos 6, 12 e 24 horas após a irradiação, sendo obtidas peças que seguiram para processamento laboratorial de rotina. O estudo das lâminas foi realizado por meio da contagem absoluta dos mastócitos pelo método esteriológico. Os resultados obtidos demonstraram que, nas primeiras 6 e 12 horas, após a irradiação laser, existe uma diminuição no número total de mastócitos, estatisticamente significante, quando comparados aos grupos controles, porém, após 24 horas, não existem diferenças no número de mastócitos entre os grupos experimental e controle. Baseados nos resultados obtidos com o protocolo de aplicação da luz laser utilizado, para esse modelo animal, pôde-se concluir que a luz laser é capaz de diminuir o número total de mastócitos após uma única irradiação.

This study measured the variation in the total number of mast cells after low level laser therapy (λ= 685 nm) using a stereological method. Eighteen male Wistar rats (250 to 300 grams) with standardized surgical wounds were randomly distributed into 6 groups - 3 study groups with 4 rats each, and 3 control groups with 2 rats each. In the study groups, four equidistant spots around the standardized wound were irradiated (0.5 J/cm², 35 mW, 35 Hz, 17 seconds). The control groups were not irradiated. The animals were killed at 6, 12 and 24 hours after laser therapy. Biological specimens were harvested and routinely processed; slides were prepared and stained with toluidine blue. The total number of mast cells was counted using the stereological method. A statistically significant decrease in the total number of mast cells was observed at 6 and 12 hours after irradiation. However, there was nodifference in the number of mast cells between study and control groups at 24 hours. Our results showed that one single irradiation with laser light reduced the total number of mast cells in the animal model used in this study.

The utility of sperm DNA damage assay using toluidine blue and aniline blue staining in routine semen analysis / 대한생식의학회지

OBJECTIVE: The aim of the present study was to examine the relationship among male age, strict morphology, and sperm chromatin structure and condensation. METHODS: Sperm samples from a total of 100 men underwent semen analysis, and sperm chromatin structure and condensation were assessed with toluidine blue (TB) and aniline blue (AB) tests. RESULTS: Prevalence of strict morphology of less than 4%, and abnormal sperm chromatin structure and condensation did not show any statistically significant differences according to male age (p=0.605, p=0.235, and p=0.080). No significant correlation was demonstrated among age of male partners, strict morphology, and abnormal sperm chromatin structure using TB and AB tests. However, abnormal sperm chromatin condensation was positively associated with sperm chromatin structure (r=0.594, p=0.000) and showed negative correlation with strict morphology (r=-0.219, p=0.029). CONCLUSION: The tests for sperm chromatin condensation showed a significant association with strict morphology. Further study is needed to elucidate the relationship between clinical outcome and sperm chromatin tests.

Evaluation of Infiltration of Immunological cells (Tumour Associated Tissue Eosinophils and Mast cells) in Oral Squamous Cell Carcinoma by Using Special Stains.

Aim: Our study aimed to evaluate the infiltration of tissue eosinophils and mast cells in oral squamous cell carcinoma (OSCC) by using special stains. Study Design: Comparative study. Place of Study: Sharad Pawar Dental College, Sawangi (Meghe), Wardha, Maharashtra. Methodology: The study was carried out with the sample size of 30 histopathologically diagnosed cases of oral squamous cell carcinoma and comparison of infiltration of these (tissue eosinophil and mast cells) inflammatory cells with control (normal) group of patients, was done by using special stains. Special stains are wonderful because they allow us to see which we cannot see with routine Haematoxylin and eosin stain. Special stains were used to demonstrate tissue eosinophils and mast cells. Carbol Chromotrope and congo red were used for tissue eosinophil and for mast cells staining toluidine blue and thionin were used. Results: The comparison of infiltration of tissue eosinophil and mast cell in OSCC with control group (normal) of patients shows significantly increased infiltration of these immunological cells in OSCC group of patients (P<0.05). We also found that among special stains carbol chromotrope is better than congo red for demonstration of tissue eosinophils and toluidine blue shows better staining intensity for mast cells than thionin. Conclusion: We conclude that both inflammatory cells i.e. number of tissue eosinophils and mast cell infiltration is increased in OSCC. Special stains (carbol chromotrope and toluidine blue) are inexpensive and time saving rapid process for microscopic evaluation of infiltration of immunological cells (tissue eosinophil and mast cell) in tumour stroma.

Relationship among damaged chromatin, motility and viability in cryopreserved spermatozoa from Brahman bulls / Relación entre la alteración de la cromatina, movilidad y viabilidad en espermatozoides criopreservados de toros Brahman / Relação entre a alteração da cromatina, motilidade e viabilidade dos espermaotozoides criopreservados de touros Brahman

The aim of this study was to determine the percentage of sperm with damaged chromatic measure with toluidine blue stain and it´s relationship with motility and viability in criopreserverd semen from Brahman bulls. Three ejaculates from six Brahman bulls were used. Immediately after thawing, sperms were stained with toluidine blue to establish chromatin integrity (sperms with normal chromatin were light blue or green while sperms with damaged chromatin were dark blue or violet). Sperms were also stained with eosin-nigrosin to determine viability (live sperms were unstained while dead sperms were pink). Motility was measured under light microscope. Effects of bull, ejaculate, and the interaction between variables were assessed. The percentage of live sperms was 50.02 ( ± 14.13%). The mean motility was 33.88 (± 12.43%), while the percentage of sperms with damaged chromatin was 4.17 ( ± 2.96%). Viability was positively correlated with motility (r=0.77217, p=0.0002), and negatively correlated with damaged chromatin sperms (r= -0.43104, p=0.0087). Motility percentage was negatively correlated with the percentage of sperms with damaged chromatin (r=-0.48337, p=0.0421). In conclusion, cryopreserved semen of Brahman bulls presented a low level of chromatin damage, and this trait was negatively correlated with sperm motility and viability.

El objetivo del presente trabajo fue determinar el porcentaje de espermatozoides con cromatina dañada medida con la tinción de azul de toluidina, y su relación con la motilidad y la vitalidad del semen criopreservado de toros Brahma. Para ello, se utilizó semen de tres eyaculados de seis toros Brahman, el cual una vez descongelado se procedió a teñir con azul de toluidina para determinar la integridad de la cromatina (espermatozoides con cromatina normal teñidos de azul o verde claro; espermatozoides con cromatina anormal teñidos de azul oscuro o violeta), también se tiñeron con eosinanigrosina para determinar la viabilidad (espermatozoides vivos permanecen blancos; espermatozoides muertos se tiñen de rosado) y se estimó la motilidad espermática mediante microscopía óptica. Se evidenciaron las diferencias en todos los parámetros evaluados debidas al efecto toro y al eyaculado, así como a la interacción entre estas dos variables. El porcentaje de espermatozoides vivos fue de 50.02 ± 14.13% y la motilidad espermática promedió un 33.88 ± 12.43%, mientras que el porcentaje de espermatozoides con cromatina dañada fue de 4.17 ± 2.96%. El porcentaje de espermatozoides vivos se correlacionó positivamente con la motilidad (r=0.77217, p=0.0002), y negativamente con el porcentaje de espermatozoides con cromatina dañada (r= -0.43104, p=0.0087), mientras que el porcentaje de motilidad se correlacionó negativamente con el porcentaje de espermatozoides con cromatina anormal (r= -0.48337, p=0.0421). En conclusión, el semen criopreservado de toros Brahman presenta un bajo nivel de espermatozoides con daño en la cromatina, lo cual se correlaciona negativamente con la motilidad y la vitalidad espermática.

O objectivo deste estudo foi determinar a percentagem de espermatozóides com cromatina danificada, determinada pela coloração com azul de toluidina e sua relação com a viabilidade e a mobilidade do esperma cripreservado de touros Brahman. Para isso, foram utilizados três ejaculados de sêmen de seis touros Brahman, que uma vez descongelado foram coradas com azul de toluidina para determinar a integridade da cromatina (espermatozóides com cromatina normal coloream de azul ou verde; cromatina de espermatozóides con cromatina danificada, coloream de azul escuro ou violeta). Também foram corados com eosina nigrosina para determinar a viabilidade (espermatozóides vivos permanecem brancos e os mortos de cor rosa) e a motilidade espermática foi estimada por microscopia de luz. Foram encontradas diferenças significativas em todos os parâmetros, devido ao efeito de touro e o ejaculado, bem como a interacção entre essas duas variáveis. A percentagem de espermatozóides vivos foi de 50.02 ± 14.13% e motilidade espermática média de 33.88 ± 12.43%, enquanto a percentagem de espermatozóides com cromatina danificada foi de 4.17 ± 2.96%. A percentagem de espermatozóides vivos foi positivamente correlacionada com a motilidade (r=0.77217, p=0.0002) e negativamente com a porcentagem de espermatozóides com cromatina danificada (r = -0.43104, p= 0.0087), enquanto que a percentagem de motilidade correlacionou negativamente com a percentagem de espermatozóides com cromatina danificada (r = -0.48337, p=0.0421). Em conclusão, o sêmen de touros Brahman criopreservados tem um baixo nível de dano da cromatina, que está correlacionada negativamente com a motilidade e a vitalidade do esperma.

Avaliação computacional da compactação da cromatina e de características morfométricas da cabeça de espermatozoides de coelho (Oryctolagus cuniculus) / Computational evaluation of chromatin condensation and morphometric characteristics of rabbit (Oryctolagus cuniculus) sperm head

Investigou-se a correlação entre a morfometria da cabeça e a intensidade da condensação e heterogeneidade da cromatina em espermatozoides de coelho (Oryctolagus cuniculus). Para tal, utilizaram-se 35 esfregaços de sêmen de coelhos da raça Nova Zelândia, corados com azul de toluidina e avaliados por análise de imagem computacional. As imagens foram obtidas digitalmente em tons de cinza e avaliadas por algoritmos desenvolvidos em ambiente de programação Scilab. As mensurações obtidas da cabeça dos espermatozoides foram área, perímetro, comprimento, largura, relação comprimento largura, elipsidade, fator de forma, descritores Fourier e simetria lateral e anteroposterior. Também foram avaliadas a intensidade da compactação e a heterogeneidade da cromatina espermática. Os espermatozoides de coelho apresentaram compactação e heterogeneidade cromatínica mais intensas do que os de touro e observou-se correlação significativa entre características morfométricas da cabeça e compactação e heterogeneidade cromatínica. Conclui-se que a cromatina é importante para a constituição morfológica da cabeça de espermatozoides de coelho e que a cromatina espermática de coelho é naturalmente mais heterogênea e menos compactada que a de touro.

The correlation between the head morphometry and the intensity of condensation and heterogeneity of sperm chromatin were investigated in rabbit (Oryctolagus cuniculus). To this, 35 semen smears from New Zealand rabbits were stained with toluidine blue and evaluated by computer image analysis. The images were obtained in digital grayscale and analyzed by algorithms developed in the Scilab programming environment. The measurements obtained from the sperm heads were area, perimeter, length, width, length:width ratio, ellipticity, shape factor, lateral and anterior-posterior symmetries, and Fourier descriptors. The intensity and heterogeneity of the compaction of sperm chromatin was also evaluated. The rabbit spermatozoa showed chromatin heterogeneity and condensation more intense than the bull spermatozoa, and it was observed correlation between morphometric characteristics of the head and chromatin compaction and heterogeneity. The results suggest that chromatin is important for the morphological constitution of the head morphology spermatozoa head, as well as, rabbit sperm chromatin is inherently more heterogeneous and less condensed than the bull sperm chromatin.

Diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignant and malignant lesions.

Background and Objectives: In vivo stains are prompt resources, which have emerged, in the recent years, to aid as clinical diagnostic tools in detecting early premalignant and malignant lesions. The aim of the study was to determine the diagnostic efficiency of toluidine blue with Lugol's iodine in oral premalignancies and malignancies and to evaluate the reliability of in vivo staining with toluidine blue and Lugol's iodine in the lesions at risk of malignancy. Materials and Methods: The study group comprised 30 subjects with clinically suspicious premalignant lesions and 30 subjects with clinically suspicious malignant lesions. All the lesions were stained consecutively with toluidine blue and Lugol's iodine and the dye retention were recorded with photographs. Depending on the retention of the dyes, the biopsy site was determined. The biopsy specimens were sent for histological confirmation and results were statistically analyzed. Results: The overall diagnostic accuracy of Lugol's iodine when used consecutively with toluidine blue stain in distinguishing premalignant lesions and malignant lesions was 90%. As the degree of differentiation of malignant lesions progressed toward more severity, they failed to show the retention of Lugol's iodine and the result was highly significant statistically, with a P value < 0.001. Interpretation and Conclusion: Lugol's iodine when used with toluidine blue helped in delineating the inflammatory lesions and was the mean source in determining clinically the degrees of differentiation of malignant lesions as the poorly differentiated malignant lesions without glycogen content failed to show Lugol's iodine retention. Toluidine blue with Lugol's iodine can be used as a pretherapeutic assessment of the biologic aggressiveness of the disease.

Alterações de cromatina em espermatozóides de ovinos e caprinos avaliadas por azul de toluidina e alaranjado de acridina / Chromatin alterations in ram and goat spermatozoa evaluated by toluidine blue and acridine orange

Reprodutores com espermograma normal podem se comportar como subférteis ou passarem por períodos de subfertilidade. As alterações na cromatina dos espermatozóides são possíveis explicações encontradas para tais comportamentos. Conduziu-se este trabalho, com o objetivo de testar a eficiência de azul de toluidina (AT) e do alaranjado de acridina (AA) na identificação de alterações na compactação de cromatina em espermatozóides de ovinos e caprinos, além de avaliar a correlação entre essas alterações e as de morfologia espermática. Para tal, foram avaliadas amostras de sêmen de 15 ovinos e de 15 caprinos, com dez repetições para cada método por animal. Calcularam-se a média, o desvio padrão (DP) e o coeficiente de variação (CV) para cada técnica e animal. Utilizou-se o teste t-Student para avaliar diferença entre as médias obtidas nos dois métodos. Também foram calculados a correlação de Pearson e os coeficientes kappa ponderado e não ponderado para avaliar a concordância entre os métodos com AT e AA. Foi verificado que nem sempre as anomalias morfológicas de cabeça são acompanhadas por alterações na cromatina identificáveis pelos métodos utilizados neste trabalho. O método AT é mais estável e possui maior sensibilidade do que AA para ambas as espécies, sendo o mais indicado para caprinos. Contudo, em razão de apresentar repetibilidade muito baixa, ambos os métodos não são indicados para avaliação espermática em ovinos.

Males with normal spermogram can behave as subfertile or pass for periods of subfertility. Chromatin alterations of spermatozoa can account for such behavior. The objective of the present work was to test the efficacy of toluidine blue (TB) and acridine orange (AO) in the identification of alterations in chromatin compaction in spermatozoa from rams and goats, in addition to evaluate the correlation between those alterations and the ones of spermatic morphology. In order to do that, samples of semen from 15 rams and 15 goats were evaluated with 10 replications for each method. Mean, standard deviation and coefficient of variation were calculated for each animal and technique. Student's t-test was used to evaluate differences between the averages of two methods. Pearson correlation coefficient, weighted and non weighted kappa coefficient were also calculated to evaluate the agreement between methods with TB and AO. It was verified that morphological alterations of head not always are accompanied by alterations in chromatin identified by the methods used in this work. The TB method is more stable and sensitive than AO method for both species and this is most appropriate for goats. However, due to the very low repeatability, both methods are not indicated for evaluating ram spermatozoa.

Análise computacional da compactação da cromatina de espermatozoides de galo / Computational analysis of chromatin condensation of rooster spermatozoa

Testaram-se variantes metodológicas utilizando azul de toluidina (AT), até se estabelecer um protocolo confiável para a avaliação computacional da compactação da cromatina em espermatozoides de galo. Para tal, foram utilizados sêmen de 10 galos com 35 semanas de idade e sêmen de 10 galos com 60 semanas de idade. O melhor método foi o de hidrólise com ácido clorídrico 1N por 10 minutos, coloração em cubeta com AT 0,025 por cento, pH 4,0, por 20 minutos, desidratação em álcool, diafanização em xilol e montagem com bálsamo do Canadá. Todas as amostras de sêmen foram submetidas a este protocolo e posteriormente avaliadas por análise de imagem computacional, em que foram feitas mensurações da área, comprimento, largura, perímetro, homogeneidade da compactação da cromatina dentro de cada cabeça e intensidade de compactação da cromatina. Os espermatozoides de galos velhos apresentaram mais alterações na cromatina que os de galos jovens. Os galos jovens apresentaram cabeça dos espermatozoides maior que os galos mais velhos. A análise computacional da compactação da cromatina mostrou-se um método menos subjetivo e mais preciso que a avaliação visual das cabeças dos espermatozoides.

The methodological variants using toluidina blue (AT) to establish a trustworthy protocol for the computational analysis of chromatin condensation of rooster spermatozoa were studied. Twenty semen samples were used: ten from 35-week-old roosters and ten from 60-week-old roosters. Different methods of denaturation and staining were tested. The best method was hydrolysis with 1N HCl for 10 minutes, staining in bucket with 0.025 percent AT, pH 4.0, for 20 minutes, dehydration in alcohol, clearing in xylol, and mounted with Canada balsam. All the semen samples were submitted to this protocol and later evaluated by computational image analysis. Area, length, width, perimeter, and chromatin compaction homogeneity of head spermatozoa were measured. The sperm of older roosters presented more chromatin changes than the ones of younger ones. The spermatozoa of younger roosters presented bigger heads than the ones of older roosters. The computational analysis of chromatin compaction showed to be less subjective and more precise than the visual evaluation for identification of chromatin alterations of rooster spermatozoa.

Studies of the bactericidal effects of a He-Ne laser combined with toluidine blue O / 中华物理医学与康复杂志

Objective To investigate the effects of photodynamic therapy (PDT) on Staphylococcus aureus and Escherichia coli strains and their laboratory-developed biofilms in vetro and the basic mechanisms of its action.Metods The bacteria were irradiated with a He-Ne laser in the presence of toluidine blue O (TBO).The colony forming units (CFU) of each strain were countde before and after treatment,and the uptakd of TBO by the bacteria was determined using fluorospectrophotometry. Results With a constant light dose,the bactericidal effects of PDT against the two bacteria cultures increasde woth the concentration of TBO,with the strongest bactericidal effect after PDT treatment more than 90%.S.aureus seemed to be more sensitive to PDT than E.coli,though the amount of TBO absorbed by the E.coli cells was significantly larger.The effects of PDT on mature and immature biofilms formed by the two strains showed no significant difference.The bactericidal rate was 20% to 30%.Conclusion Ge-Ne laser irradiation associated with TBO demonstrated significcant photodynamic bactericidal effects on S.arueus and E.coli strains cultivatde in vitro,but the iffects on their biofilms wer limitde.The efficacy of PDT was independent of the absorption of TBO into the bacterial cells.