EARLY STAGES OF COLORECTAL CANCER CHARACTERIZATION BY AUTOFLUORESCENCE 3D MICROSCOPY: A PRELIMINARY STUDY.

BACKGROUND: Colorectal cancer is one of the most prevalent pathologies worldwide whose prognosis is linked to early detection. Colonoscopy is the gold standard for screening, and diagnosis is usually made histologically from biopsies. Aiming to reduce the inspection and diagnostic time as well as the biopsies and resources involved, other techniques are being promoted to conduct accurate in vivo colonoscopy assessments. Optical biopsy aims to detect normal and neoplastic tissues analysing the autofluorescence spectrum based on the changes in the distribution and concentration of autofluorescent molecules caused by colorectal cancer. Therefore, the autofluorescence contribution analysed by image processing techniques could be an approach to a faster characterization of the target tissue. OBJECTIVE: Quantify intensity parameters through digital processing of two data sets of three-dimensional widefield autofluorescence microscopy images, acquired by fresh colon tissue samples from a colorectal cancer murine model. Additionally, analyse the autofluorescence data to provide a characterization over a volume of approximately 50 µm of the colon mucosa for each image, at second (2nd), fourth (4th) and eighth (8th) weeks after colorectal cancer induction. METHODS: Development of a colorectal cancer murine model using azoxymethane/dextran sodium sulphate induction, and data sets acquisition of Z-stack images by widefield autofluorescence microscopy, from control and colorectal cancer induced animals. Pre-processing steps of intensity value adjustments followed by quantification and characterization procedures using image processing workflow automation by Fiji's macros, and statistical data analysis. RESULTS: The effectiveness of the colorectal cancer induction model was corroborated by a histological assessment to correlate and validate the link between histological and autofluorescence changes. The image digital processing methodology proposed was then performed on the three-dimensional images from control mice and from the 2nd, 4th, and 8th weeks after colorectal cancer chemical induction, for each data set. Statistical analyses found significant differences in the mean, standard deviation, and minimum parameters between control samples and those of the 2nd week after induction with respect to the 4th week of the first experimental study. This suggests that the characteristics of colorectal cancer can be detected after the 2nd week post-induction. CONCLUSION: The use of autofluorescence still exhibits levels of variability that prevent greater systematization of the data obtained during the progression of colorectal cancer. However, these preliminary outcomes could be considered an approach to the three-dimensional characterization of the autofluorescence of colorectal tissue, describing the autofluorescence features of samples coming from dysplasia to colorectal cancer. BACKGROUND: • A new digital image processing method was developed to measure intensity in 3D autofluorescence images of colorectal samples using a CRC mouse model. BACKGROUND: • This method showed that autofluorescence intensity in colon mucosa is similar in healthy tissue but changes significantly in tumor development. BACKGROUND: • Statistical analysis revealed CRC traits detectable from the second week post-induction, aiding in early CRC detection. BACKGROUND: • The study provides a basis for 3D autofluorescence characterization in colorectal tissue from dysplasia to cancer, although variability in autofluorescence limits data systematization during cancer progression.

Caracterização dos estágios iniciais do câncer colorretal utilizando microscopia de autofluorescência 3D: um estudo preliminar / Early stages of colorectal cancer characterization by autofluorescence 3d microscopy: a preliminary study

ABSTRACT Background: Colorectal cancer is one of the most prevalent pathologies worldwide whose prognosis is linked to early detection. Colonoscopy is the gold standard for screening, and diagnosis is usually made histologically from biopsies. Aiming to reduce the inspection and diagnostic time as well as the biopsies and resources involved, other techniques are being promoted to conduct accurate in vivo colonoscopy assessments. Optical biopsy aims to detect normal and neoplastic tissues analysing the autofluorescence spectrum based on the changes in the distribution and concentration of autofluorescent molecules caused by colorectal cancer. Therefore, the autofluorescence contribution analysed by image processing techniques could be an approach to a faster characterization of the target tissue. Objective: Quantify intensity parameters through digital processing of two data sets of three-dimensional widefield autofluorescence microscopy images, acquired by fresh colon tissue samples from a colorectal cancer murine model. Additionally, analyse the autofluorescence data to provide a characterization over a volume of approximately 50 µm of the colon mucosa for each image, at second (2nd), fourth (4th) and eighth (8th) weeks after colorectal cancer induction. Methods: Development of a colorectal cancer murine model using azoxymethane/dextran sodium sulphate induction, and data sets acquisition of Z-stack images by widefield autofluorescence microscopy, from control and colorectal cancer induced animals. Pre-processing steps of intensity value adjustments followed by quantification and characterization procedures using image processing workflow automation by Fiji's macros, and statistical data analysis. Results: The effectiveness of the colorectal cancer induction model was corroborated by a histological assessment to correlate and validate the link between histological and autofluorescence changes. The image digital processing methodology proposed was then performed on the three-dimensional images from control mice and from the 2nd, 4th, and 8th weeks after colorectal cancer chemical induction, for each data set. Statistical analyses found significant differences in the mean, standard deviation, and minimum parameters between control samples and those of the 2nd week after induction with respect to the 4th week of the first experimental study. This suggests that the characteristics of colorectal cancer can be detected after the 2nd week post-induction. Conclusion: The use of autofluorescence still exhibits levels of variability that prevent greater systematization of the data obtained during the progression of colorectal cancer. However, these preliminary outcomes could be considered an approach to the three-dimensional characterization of the autofluorescence of colorectal tissue, describing the autofluorescence features of samples coming from dysplasia to colorectal cancer.

RESUMO Contexto: O câncer colorretal é uma das patologias mais prevalentes em todo o mundo, cujo prognóstico está ligado à detecção precoce. A colonoscopia é o padrão ouro para triagem, e o diagnóstico geralmente é feito histologicamente a partir de biópsias. Visando reduzir o tempo de inspeção e diagnóstico, bem como as biópsias e recursos envolvidos, outras técnicas estão sendo promovidas para realizar avaliações precisas de colonoscopia in vivo. A biópsia óptica visa detectar tecidos normais e neoplásicos analisando o espectro de autofluorescência com base nas mudanças na distribuição e concentração de moléculas autofluorescentes causadas pelo câncer colorretal. Portanto, a contribuição da autofluorescência analisada por técnicas de processamento de imagem poderia ser uma abordagem para uma caracterização mais rápida do tecido-alvo. Objetivo: Quantificar parâmetros de intensidade por meio do processamento digital de dois conjuntos de dados de imagens de microscopia de autofluorescência em campo amplo tridimensionais, adquiridas por amostras de tecido fresco de cólon de um modelo murino de câncer colorretal. Adicionalmente, analisar os dados de autofluorescência para fornecer uma caracterização em um volume de aproximadamente 50 µm da mucosa do cólon para cada imagem, na segunda (2ª), quarta (4ª) e oitava (8ª) semanas após a indução do câncer colorretal. Método: Desenvolvimento de um modelo murino de câncer colorretal usando indução de azoximetano/sulfato de sódio dextrano, e aquisição de conjuntos de dados de imagens Z-stack por microscopia de autofluorescência em campo amplo, de animais controle e induzidos ao câncer colorretal. Etapas de pré-processamento de ajustes de valores de intensidade seguidas por procedimentos de quantificação e caracterização usando automação de fluxo de trabalho de processamento de imagem por macros do Fiji, e análise estatística de dados. Resultados: A eficácia do modelo de indução de câncer colorretal foi corroborada por uma avaliação histológica para correlacionar e validar a ligação entre as mudanças histológicas e de autofluorescência. A metodologia de processamento digital de imagem proposta foi então realizada nas imagens tridimensionais de camundongos controle e das 2ª, 4ª e 8ª semanas após a indução química do câncer colorretal, para cada conjunto de dados. Análises estatísticas encontraram diferenças significativas nos parâmetros médios, desvio padrão e mínimos entre amostras de controle e aquelas da 2ª semana após a indução em relação à 4ª semana do primeiro estudo experimental. Isso sugere que as características do câncer colorretal podem ser detectadas após a 2ª semana pós-indução. Conclusão: O uso de autofluorescência ainda apresenta níveis de variabilidade que impedem uma maior sistematização dos dados obtidos durante a progressão do câncer colorretal. No entanto, esses resultados preliminares podem ser considerados uma abordagem para a caracterização tridimensional da autofluorescência do tecido colorretal, descrevendo as características de autofluorescência de amostras que vão da displasia ao câncer colorretal.

Three-dimensional morphological characterization of colorectal pits from label-free microscopy images.

The prognosis of colorectal cancer (CRC), one of the most prevalent pathologies worldwide, is linked to early detection. Kudo's pit pattern classification states morphological pit patterns of the Lieberkühn crypts by analyzing the superficial mucosa, predicting the histology of colorectal lesions. Its use as a highly accurate two-dimensional diagnostic criterion has increased, mostly involving expert endoscopists' judgment. The processing of autofluorescence images could allow the diagnostic, bypassing staining techniques and decreasing the biopsies, resources and times involved in the inspection. That criterion could be extended by data of the pit three-dimensional (3D) morphology. Thus, this work was aimed at obtaining 3D morphological information by quantifying geometrical and shape descriptors through software processing and analysis of widefield autofluorescence microscopy image stacks acquired by fresh colon tissue samples from a murine model of CRC. Statistical analyses included pits from control mice and from the second (2nd), fourth (4th), and eighth (8th) weeks of treatment. Statistically significant differences were found for almost all parameters between the pits from control and from the 4th treated week, stating that the major morphological changes begin after the 2nd week. In particular, pits from control or initial treatment time points were more tubular, straighter and less rough than the ones from later treatment points. Therefore, they may be more associated to normal or non-neoplastic crypt lumens than linked to adenomas or even cancer crypts. These preliminary outcomes could be considered an advance in 3D pit morphology characterization.

MORPHOLOGICAL CHARACTERIZATION OF COLORECTAL PITS USING AUTOFLUORESCENCE MICROSCOPY IMAGES.

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.

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.

The exocrine pancreas is an extracardiac source of atrial natriuretic peptide.

Previous studies have shown that atrial natriuretic peptide (ANP) regulates exocrine pancreatic function in health and disease. As extracardiac sources of ANP have been identified and ANP-like immunoreactivity has been reported in the exocrine pancreas, in the present work we sought to establish whether ANP was produced in the rat exocrine pancreas and if conditions like fasting/feeding or acute pancreatitis were reflected on ANP expression. By using RT-PCR, immunoblotting, and immunofluorescence microscopy assays, it was found that both mRNA and protein ANP were present in the acinar cells of the exocrine pancreas. The amount of ANP in the pancreas was lower in than the atrium but similar to other tissues like the kidney and liver. Immunogold labeling electron microscopy studies revealed that ANP was localized in zymogen granules and the endoplasmic reticulum suggesting local synthesis and package into granules. ANP protein expression was significantly increased not only in fasting but also in acute pancreatitis, the latter probably related to impaired secretion. Natriuretic peptide receptor type C which mediates ANP biological effects in the exocrine pancreas was also present in acinar cells and its expression did not change with either fasting or acute pancreatitis. Present findings show that the exocrine pancreas is a relatively important extracardiac source of ANP and further support previous studies strongly suggesting the active role of the peptide in pancreatic physiology and pathophysiology.

Epithelial Ovarian Cancer Diagnosis of Second-Harmonic Generation Images: A Semiautomatic Collagen Fibers Quantification Protocol.

A vast number of human pathologic conditions are directly or indirectly related to tissular collagen structure remodeling. The nonlinear optical microscopy second-harmonic generation has become a powerful tool for imaging biological tissues with anisotropic hyperpolarized structures, such as collagen. During the past years, several quantification methods to analyze and evaluate these images have been developed. However, automated or semiautomated solutions are necessary to ensure objectivity and reproducibility of such analysis. This work describes automation and improvement methods for calculating the anisotropy (using fast Fourier transform analysis and the gray-level co-occurrence matrix). These were applied to analyze biopsy samples of human ovarian epithelial cancer at different stages of malignancy (mucinous, serous, mixed, and endometrial subtypes). The semiautomation procedure enabled us to design a diagnostic protocol that recognizes between healthy and pathologic tissues, as well as between different tumor types.

E-cadherin roles in animal biology: A perspective on thyroid hormone-influence.

The establishment, remodeling and maintenance of tissular architecture during animal development, and even across juvenile to adult life, are deeply regulated by a delicate interplay of extracellular signals, cell membrane receptors and intracellular signal messengers. It is well known that cell adhesion molecules (cell-cell and cell-extracellular matrix) play a critical role in these processes. Particularly, adherens junctions (AJs) mediated by E-cadherin and catenins determine cell-cell contact survival and epithelia function. Consequently, this review seeks to encompass the complex and prolific knowledge about E-cadherin roles during physiological and pathological states, particularly focusing on the influence exerted by the thyroid hormone (TH).

Mouse colorectal cancer an early detection approach using nonlinear microscopy.

Murine induced colon cancer has been used to demonstrate that Second Harmonic Generation (SHG) microscopy images, combined with Two-Photon Excitation Fluorescence (TPEF) and specific quantization scoring methods allow distinguishing early alterations in colon mucosa. TPEF was used only to identified crypts and submucosa regions, whereas the image analysis was used to get quantitative data (Integrated Intensity and Aspect Ratio scoring) of different cancer stages. The submucosa amount of collagen fibers was significant and their orientation suffering proportional changes with the development of the pathological processes. Both after the fourth and eighth weeks after colon cancer induction, integrated intensity and aspect ratio values have shown significant statistical differences compared with control samples. Thus, SHG microscopy has proved to be a useful quantitative tool to highlight early changes of submucosa and the progression of these through the cancer development.

Nonlinear optical microscopy signal processing strategies in cancer.

This work reviews the most relevant present-day processing methods used to improve the accuracy of multimodal nonlinear images in the detection of epithelial cancer and the supporting stroma. Special emphasis has been placed on methods of non linear optical (NLO) microscopy image processing such as: second harmonic to autofluorescence ageing index of dermis (SAAID), tumor-associated collagen signatures (TACS), fast Fourier transform (FFT) analysis, and gray level co-occurrence matrix (GLCM)-based methods. These strategies are presented as a set of potential valuable diagnostic tools for early cancer detection. It may be proposed that the combination of NLO microscopy and informatics based image analysis approaches described in this review (all carried out on free software) may represent a powerful tool to investigate collagen organization and remodeling of extracellular matrix in carcinogenesis processes.

Endothelin system in intestinal villi: A possible role of endothelin-2/vasoactive intestinal contractor in the maintenance of intestinal architecture.

The endothelin system consists of three ligands (ET-1, ET-2 and ET-3) and at least two receptors (ETA and ETB). In mice ET-2 counterpart is a peptide originally called "vasoactive intestinal contractor" (VIC) for this reason, this peptide is frequently named ET-2/VIC. In intestinal villi, fibroblasts-like cells express endothelin's receptors and response to ET-1 and ET-3 peptides, changing their cellular shape. Several functions have been attributed to these peptides in the "architecture" maintenance of intestinal villi acting over sub-epithelial fibroblasts. Despite this, ET-2/VIC has not been analyzed in depth. In this work we show the intestine gene expression and immunolocalization of ET-1, ET-2 and the ETA and ETB receptors from duodenum to rectus and in the villus-crypt axis in mice, allowing a complete analysis of their functions. While ET-1 is expressed uniformly, ET-2 had a particular distribution, being higher at the bottom of the villi of duodenum, ileum and jejunum and reverting this pattern in the crypts of colon and rectus, where the higher expression was at the top. We postulated that ET-2 would act in a cooperative manner with ET-1, giving to the villus the straight enough to withstand mechanical stress.

High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis.

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.

[Structural and functional polarity of porcine hepatocyte cultured spheroids]. / Análisis de la polaridad estructural y funcional de estructuras tridimensionales (esferoides) de células hepáticas porcinas cultivadas in vitro.

Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functionalpolarities of pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Well-maintained cellular organelles, as mitochondria, nucleus, nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculi network was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.

Análisis de la polaridad estructural y funcional de estructuras tridimensionales (esferoides) de células hepáticas porcinas cultivadas in vitro / Structural and functional polarity of porcine hepatocyte cultured spheroids

Los hepatocitos son células epiteliales polarizadas que, al ser aisladas y cultivadas, pierden la polaridad y las propiedades de célula diferenciada. El cultivo de células hepáticas como esferoides permite obtener estructuras con organización de tipo tisular. En este trabajo se analizó estructural y funcionalmente la polaridad de esferoides porcinos. Para ello, las células hepáticas porcinas fueron aisladas y cultivadas en agitación constante. La actividad metabólica de los esferoides fue probada mediante el metabolismo de diazepam y de amonio, así como con síntesis de albúmina. Sus características estructurales mostraron la polaridad de las células. Fueron observados paquetes de fibras de colágeno distribuidas irregularmente y fibras reticulares en formahomogénea en todo el volumen del esferoide. Se hallaron células Ck19+ formando estructuras tipo ducto biliar, así como también _ y _-cateninas y pan-cadherinas en diferentes zonas, especialmente en las laminas externas, con características de epitelio cuboidal. Por microscopía electrónica de barrido se observaron estructuras muy compactas con superficie lisa, y por microscopía electrónica de transmisión, canalículos biliares con microvellosidades, uniones tight, zonula adherens y desmosomas. Las organelas celulares como mitocondrias, núcleos, nucleolos, peroxisomas, retículo endoplásmico estaban bien conservadas. Una compleja red de canalículos biliares fue observada mediante la incorporación y excreción de un análogo de sal biliar fluorescente. El análisis de los ácidos biliares excretados mostró un patrón normal. La morfología y funcionalidad de los esferoides puede aportar un modelo apropiado para aplicaciones en las que es primordial mantener las funciones específicas del hígado, como un dispositivo de hígado bioartificial.

Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functional polaritiesof pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Wellmaintained cellular organelles, as mitochondria, nucleus,nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculinetwork was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.

Análisis de la polaridad estructural y funcional de estructuras tridimensionales (esferoides) de células hepáticas porcinas cultivadas in vitro / Structural and functional polarity of porcine hepatocyte cultured spheroids

Los hepatocitos son células epiteliales polarizadas que, al ser aisladas y cultivadas, pierden la polaridad y las propiedades de célula diferenciada. El cultivo de células hepáticas como esferoides permite obtener estructuras con organización de tipo tisular. En este trabajo se analizó estructural y funcionalmente la polaridad de esferoides porcinos. Para ello, las células hepáticas porcinas fueron aisladas y cultivadas en agitación constante. La actividad metabólica de los esferoides fue probada mediante el metabolismo de diazepam y de amonio, así como con síntesis de albúmina. Sus características estructurales mostraron la polaridad de las células. Fueron observados paquetes de fibras de colágeno distribuidas irregularmente y fibras reticulares en formahomogénea en todo el volumen del esferoide. Se hallaron células Ck19+ formando estructuras tipo ducto biliar, así como también _ y _-cateninas y pan-cadherinas en diferentes zonas, especialmente en las laminas externas, con características de epitelio cuboidal. Por microscopía electrónica de barrido se observaron estructuras muy compactas con superficie lisa, y por microscopía electrónica de transmisión, canalículos biliares con microvellosidades, uniones tight, zonula adherens y desmosomas. Las organelas celulares como mitocondrias, núcleos, nucleolos, peroxisomas, retículo endoplásmico estaban bien conservadas. Una compleja red de canalículos biliares fue observada mediante la incorporación y excreción de un análogo de sal biliar fluorescente. El análisis de los ácidos biliares excretados mostró un patrón normal. La morfología y funcionalidad de los esferoides puede aportar un modelo apropiado para aplicaciones en las que es primordial mantener las funciones específicas del hígado, como un dispositivo de hígado bioartificial. (AU)

Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functional polaritiesof pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Wellmaintained cellular organelles, as mitochondria, nucleus,nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculinetwork was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.(AU)

Análisis de la polaridad estructural y funcional de estructuras tridimensionales (esferoides) de células hepáticas porcinas cultivadas in vitro / Structural and functional polarity of porcine hepatocyte cultured spheroids

Los hepatocitos son células epiteliales polarizadas que, al ser aisladas y cultivadas, pierden la polaridad y las propiedades de célula diferenciada. El cultivo de células hepáticas como esferoides permite obtener estructuras con organización de tipo tisular. En este trabajo se analizó estructural y funcionalmente la polaridad de esferoides porcinos. Para ello, las células hepáticas porcinas fueron aisladas y cultivadas en agitación constante. La actividad metabólica de los esferoides fue probada mediante el metabolismo de diazepam y de amonio, así como con síntesis de albúmina. Sus características estructurales mostraron la polaridad de las células. Fueron observados paquetes de fibras de colágeno distribuidas irregularmente y fibras reticulares en formahomogénea en todo el volumen del esferoide. Se hallaron células Ck19+ formando estructuras tipo ducto biliar, así como también _ y _-cateninas y pan-cadherinas en diferentes zonas, especialmente en las laminas externas, con características de epitelio cuboidal. Por microscopía electrónica de barrido se observaron estructuras muy compactas con superficie lisa, y por microscopía electrónica de transmisión, canalículos biliares con microvellosidades, uniones tight, zonula adherens y desmosomas. Las organelas celulares como mitocondrias, núcleos, nucleolos, peroxisomas, retículo endoplásmico estaban bien conservadas. Una compleja red de canalículos biliares fue observada mediante la incorporación y excreción de un análogo de sal biliar fluorescente. El análisis de los ácidos biliares excretados mostró un patrón normal. La morfología y funcionalidad de los esferoides puede aportar un modelo apropiado para aplicaciones en las que es primordial mantener las funciones específicas del hígado, como un dispositivo de hígado bioartificial. (AU)

Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functional polaritiesof pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Wellmaintained cellular organelles, as mitochondria, nucleus,nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculinetwork was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.(AU)

Natriuretic peptide system gene expression in human coronary arteries.

The natriuretic peptides (NPs) ANF, BNP, and CNP have potent anti-proliferative and anti-migratory effects on vascular smooth muscle cells (SMCs). These properties make NPs relevant to the study of human coronary atherosclerosis because vascular cell proliferation and migration are central to the pathophysiology of atherosclerosis. However, the existence and cytological distribution of NPs and their receptors in human coronary arteries remain undetermined. This has hampered the development of hypotheses regarding the possible role of NPs in human coronary disease. We determined the pattern of expression of NPs and their receptors (NPRs) in human coronary arteries with atherosclerotic lesions classified by standard histopathological criteria as fatty streak/early atherosclerotic lesions, intermediate plaques, or advanced lesions. The investigation was carried out using a combination of immunocytochemistry (ICC), in situ hybridization (ISH), and semi-quantitative polymerase chain reaction (PCR). Both by ICC and ISH, ANF was found in the intimal and medial layers of all lesions. BNP was highly expressed in advanced lesions where it was particularly evident by a strong ISH signal but weak ICC staining. CNP was demonstrable in all types of lesions, giving a strong signal by ISH and ICC. This peptide was particularly demonstrable in the endothelium, as well as in the SMCs of the intima, media, and vasa vasorum of the adventitia and in macrophages. By ISH, NPR-A was not detectable in any of the lesions but both NPR-B and NPR-C were found in the intimal and the inner medial layers. By RT-PCR, mRNA levels of all NPs tended to be increased in macroscopically diseased arteries, but only the values for BNP were significantly so. No significant changes in NPR mRNA levels were detected by PCR. In general, the signal intensity given by the NPs and their receptors by ICC or ISH appeared dependent on the type of lesion, being strongest in intermediate plaques and decreasing with increasing severity of the lesion. This study constitutes the first demonstration of NPs and NPR mRNAs in human coronary arteries and supports the existence of an autocrine/paracrine NP system that is actively modulated during the progression of atherosclerotic coronary disease. This suggests that the coronary NP system is involved in the pathobiology of intimal plaque formation in humans and may be involved in vascular remodeling.

Ultrastructural study on thyroid glands of Bufo arenarum larvae kept in potassium perchlorate solution

The ultrastructure of thyroid glands in Bufo arenarum tadpoles was studied with inhibited thyroid function. After hatching, larvae were kept in potassium perchlorate solution, a goitrogenic substance known to prevent absorption of iodine from water or food, resulting in impaired thyroid hormone synthesis. Increased thyroid gland volume and follicular cell height were observed in treated larvae. The changes in size and volume of follicular cells in experimental tadpoles appeared to be mainly a function of the overstimulation of proteinpoietic apparatus. The cell shows a conspicuous development of endoplasmic reticulum and Golgi complex, as well as an increase in number of mitochondria and colloid droplets. These observations in follicular thyroid cells from Bufo arenarum, obtained under such experimental conditions, were interpreted in terms of interruption of negative feedback mechanisms involving the decrease in the thyroxine circulatory level, and therefore the increase in hypothalamo-hypophyseal endocrine secretory stimulation.

Ultrastructural study on thyroid glands of Bufo arenarum larvae kept in potassium perchlorate solution

The ultrastructure of thyroid glands in Bufo arenarum tadpoles was studied with inhibited thyroid function. After hatching, larvae were kept in potassium perchlorate solution, a goitrogenic substance known to prevent absorption of iodine from water or food, resulting in impaired thyroid hormone synthesis. Increased thyroid gland volume and follicular cell height were observed in treated larvae. The changes in size and volume of follicular cells in experimental tadpoles appeared to be mainly a function of the overstimulation of proteinpoietic apparatus. The cell shows a conspicuous development of endoplasmic reticulum and Golgi complex, as well as an increase in number of mitochondria and colloid droplets. These observations in follicular thyroid cells from Bufo arenarum, obtained under such experimental conditions, were interpreted in terms of interruption of negative feedback mechanisms involving the decrease in the thyroxine circulatory level, and therefore the increase in hypothalamo-hypophyseal endocrine secretory stimulation.(AU)

Presence and release of immunoreactive atrial natriuretic peptide (ANP) in Bufo arenarum spermatozoa after HCG treatment

Atrial natriuretic peptide (ANP)-like immunoreactivity has been demonstrated in mature spermatozoa of Bufo arenarum. However, after spermiation induced by Gonadotropin Releasing Hormone (GnRH), no ANP immunoreactivity was detected in testicular spermatozoa. Recently, the presence of GnRH and GnRH receptors in amphibian testes has been demonstrated. To clarify if the loss of ANP-like immunoreactivity in spermatozoa is a direct effect of GnRH or pituitary gonadotropins, a study on Bufo arenarum adult males, has been performed. The in vivo treatment with Human Chorionic Gonadotropin (HCG) induced spermiation and loss of ANP-like immunoreactivity. The in vitro treatment with HCG showed the same results. However, in vitro GnRH treatment failed to cause spermiation and loss of ANP-like immunoreactivity. The present results indicate that ANP from mature spermatozoa is regulated via gonadotropic hormones and may be involved in the spermiation process.