Caracterização morfológica da abertura de glândulas colônicas usando imagens de microscopia de autofluorescência / Morphological characterization of colorectal pits using autofluorescence microscopy images

ABSTRACT BACKGROUND: Colorectal cancer is one of the most prevalent pathologies. Its prognosis is linked to the early detection and treatment. Currently diagnosis is performed by histological analysis from polyp biopsies, followed by morphological classification. Kudo's pit pattern classification is frequently used for the differentiation of neoplastic colorectal lesions using hematoxylin-eosin stained samples. Few articles have reported this classification with image software processing, using exogenous markers over the samples. The processing of autofluorescence images is an alternative that could allow the characterization of the pits from the crypts of Lieberkühn, bypassing staining techniques. OBJECTIVE: Processing and analysis of widefield autofluorescence microscopy images obtained by fresh colon tissue samples from a murine model of colorectal cancer in order to quantify and characterize the pits morphology by measuring morphology parameters and shape descriptors. METHODS: Adult male BALB/cCmedc strain mice (n=27), ranging from 20 to 30 g, were randomly assigned to four and five groups of treated and control animals. Colon samples were collected at day zero and at fourth, eighth, sixteenth and twentieth weeks after treatmentwith azoxymethane. Two-dimensional (2D) segmentation, quantification and morphological characterization of pits by image processing applied using macro programming from FIJI. RESULTS: Type I is the pit morphology prevailing between 53 and 81% in control group weeks. III-L and III-S types were detected in reduced percentages. Between the 33 and 56% of type I was stated as the prevailing morphology for the 4th, 8th and 20th weeks of treated groups, followed by III-L type. For the 16th week, the 39% of the pits was characterized as III-L type, followed by type I. Further, pattern types as IV, III-S and II were also found mainly in that order for almost all of the treated weeks. CONCLUSION: These preliminaries outcomes could be considered an advance in two-dimensional pit characterization as the whole image processing, comparing to the conventional procedure, takes a few seconds to quantify and characterize non-pathological colon pits as well as to estimate early pathological stages of colorectal cancer.

RESUMO CONTEXTO: O câncer colorretal é uma das patologias mais prevalentes. Seu prognóstico é ligado à detenção e ao tratamento precoces. Atualmente o diagnóstico é realizado por análise histológica de biópsias de pólipo, seguida de classificação morfológica. A classificação de padrões de Kudo é frequentemente utilizada para a diferenciação de lesões colorretais neoplásicas usando amostras coradas por hematoxilina-eosina. Poucos artigos relatam esta classificação com utilização de processamento por software de imagem, utilizando marcadores exógenos sobre as amostras. O processamento de imagens de autofluorescência é uma alternativa que pode permitir a caracterização do padrão das criptas de Lieberkühn, contornando técnicas de coloração. OBJETIVO: Analisar, quantificar e caracterizar a morfologia do padrão das criptas medindo os parâmetros morfológicos e descritores de forma, através do processamento e análise de imagens de microscopia de autofluorescência de campo de Widefield obtidas em amostras de tecido de cólon fresco a partir de um modelo murino de câncer colorretal. MÉTODOS: Camundongos machos adultos BALB/cCmedc (n=27), variando de 20 a 30 g, foram distribuídos aleatoriamente em quatro e cinco grupos de animais tratados e de controle. As amostras de cólon foram coletadas no dia zero e na 4ª, 8ª, 16ª e 20ª semanas após o tratamento com azoxometano. Segmentação bidimensional (2D), quantificação e caracterização morfológica do padrão das criptas por processamento de imagem aplicados utilizando programação macro de FIJI. RESULTADOS: O tipo I é a morfologia da cripta prevalente entre 53% e 81% semanas do grupo controle. Os tipos III-L e III-S foram detectados em porcentagens reduzidas. A morfologia do tipo I entre os 33% e 56% foi constatada como a predominante para as 4ª, 8ª e 20ª semanas de grupos tratados, seguidos pelo tipo III-L. Para a 16ª semana, os 39% dos padrões das criptas foram caracterizados como tipo III-L, seguidos pelo tipo I. Além disso, os tipos de padrão como IV, III-S e II também foram encontrados principalmente nessa ordem para quase todas as semanas tratadas. CONCLUSÃO: Estes resultados preliminares podem ser considerados um avanço na caracterização bidimensional da cripta como um processamento integral da imagem, comparando-se ao procedimento convencional; demora-se alguns segundos a mais para quantificar e caracterizar pontos não-patológicos, bem como para estimar estágios patológicos precoces do câncer colorretal.

Evaluación de formulaciones de ketoconazol para uso tópico en leishmaniasis cutánea por Leishmania(Viannia) / Evaluation of ketoconazole formulations for topical use in cutaneous leishmaniasis caused by Leishmania (Viannia)

RESUMEN Se evaluó la efectividad de diversas formulaciones farmacéuticas de ketoconazol en modelos experimentales de leishmaniasis cutánea (LC) en ratones BALB C. Fueron preparadas formulaciones tópicas tipo gel, lipogel y crema conteniendo potenciadores de la permeación y diferentes concentraciones de ketoconazol. Se determinó la estabilidad, la toxicidad y la actividad anti-Leishmania in vitro. Además, se evaluó in vivo la efectividad de las formulaciones aplicadas tópicamente en ratones con LC infectados con Leishmania (Viannia) braziliensis. Las formulaciones tipo crema fueron evaluadas adicionalmente en ratones infectados con L. (V.) panamensis. Los sistemas evaluados mantuvieron in vitro la actividad del ketoconazol contra los parásitos; sin embargo, ninguna de las formulaciones fue efectiva en curar las lesiones de LC en los ratones. El tratamiento tópico con miltefosina (utilizado como control) curó las lesiones. Se concluye que las formulaciones que contienen ketoconazol diseñados en este estudio, no fueron efectivos contra la LC en los ratones infectados.

ABSTRACT The effectiveness of various pharmaceutical formulations of ketoconazole was evaluated in experimental models of cutaneous leishmaniasis (LC) in BALB C mice. Topical gel, lipogel, and cream formulations containing permeation enhancers and different concentrations of ketoconazole were prepared. Stability, toxicity and anti-Leishmania activity were determined in vitro. In addition, the effectiveness of topically applied formulations in LC-infected mice infected with Leishmania (Viannia) braziliensis was evaluated in vivo. Cream formulations were additionally evaluated in mice infected with L. (V.) panamensis. The systems evaluated maintained in vitro the activity of ketoconazole against parasites; however, none of the formulations were effective in curing LC lesions in mice. Topical treatment with miltefosine (used as a control) cured the lesions. It is concluded that the ketoconazole-containing formulations designed in this study were not effective against LC in infected mice.

The role of apoptosis in the cellular response of liver and spleen of BALB/c mice in cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis is a common parasitic disease in Iran being mainly caused by Leishmania (L.) major. The aim of this study was to investigate the occurrence of apoptosis in the spleen and liver of female mice infected with L. major. METHODS: BALB/c mice were randomly assigned into the control and experimental groups (ten mice per group). The experimental groups were subcutaneously inoculated with promastigotes of L. major at stationary phase. The animals were sacrificed after 20, 40, 60, 90, and 120 days of injection. The liver and spleen were analyzed for various parameters of apoptosis. RESULTS: Activities of superoxide dismutase and caspase-3, levels of superoxide anion production and malondialdehyde, and the percent of DNA fragmentation were increased in the liver and spleen of the infected mice. Catalase activity in the liver was increased, while glutathione level in both tissues was decreased after 90 and 120 days of infection. The numbers of apoptotic nuclei in the spleen were higher than the liver at 90 and 120 days post-infection using the TUNEL method. CONCLUSION: L. major infection induces a time-dependent increase in apoptosis in the liver and spleen as evidenced by the production of ROS, increasing activation of caspase-3, elevated DNA fragmentation, and increasing lipid peroxidation. Induction of oxidative stress was observed in the liver and spleen after 90 and 120 days of initiation of the infection. However, the spleen tissue appears to be more sensitive to the infection to L. major on oxidative stress and apoptosis induction compared with the liver tissue.