Melanogenesis in melasma involves epithelial secretion of αMSH and activation of the Wnt pathway; nevertheless, it seems to be independent of the stimulation by ultraviolet radiation/p53, IL-1α, COX2/PgE , WIF-1 and ASIP. Damaged cells at upper dermis suggest the role of senescence/autophagy in sustained pigmentation in melasma.

Background: Melasma is a chronic acquired focal hypermelanosis which pathogenesis has not been fully elucidated. Classical pathophysiologic studies have analysed the affected and perilesional areas, but little is known about the status of sun-protected skin, which is subjected to the same endogenous and genetic factors. Objective: To assess the histological characteristics of melasma compared to adjacent and retroauricular skin. Methods: Skin samples were collected from 10 female from: melasma, perilesional area and retroauricular. The samples were stained (haematoxylin-eosin, periodic acid-Schiff, Fontana-Masson, picrosirius red, toluidine blue and Verhoeff), immunolabelled for CD34 and Wnt1. The data from the skin sites were analysed simultaneously by a multivariate model. Results: Melasma skin exhibited noteworthy stratum corneum compaction, greater collagen heterogeneity, solar elastosis, higher number of mast cells, basement membrane zone (BMZ) damage, Wnt1 expression, pendulum melanocytes, higher cellularity and vascular proliferation at the superficial dermis. Stratum cor-neum compaction, collagen heterogeneity and BMZ abnormalities were variables associated to melasma that not follow a continuum through retroauricular to adjacent skin. Mast cell count was the variable that disclosed correlation with the most other abnormalities as well as had the greater contribution in the multivariate model. Conclusion: In addition to melanocyte hyperactivity, melasma skin exhibits alterations in the epidermal barrier, upper dermis and BMZ, which differ from the adjacent sun-exposed skin and retroauricular skin, indicating a distinct phenotype, rather than a mere extension of photoageing or intrinsic ageing. Mast cells appear to play a central role in the physiopathology of melasma.

Apoptosis was prominent in hair follicles from the FPHL group, as well as in miniaturized ones. Moreover, it was also correlated with the inflammatory infiltrate, which suggests that inflammation can lead to apoptosis and play a role in the pathogenesis of follicle miniaturization.

Basal keratinocytes from facial melasma display changes in nuclear form and chromatin texture, suggesting that the phenotype differences between melasma and adjacent facial skin can result from complete epidermal melanin unit alterations, not just hypertrophic melanocytes.

Morphometric analysis of dermal collagen can provide quantitative support to dermatologic research. The authors of this article disclose a technique of digital image analysis which allows the identification of microscopic structures by color cluster segmentation regarding the estimate intensity and density of dermal collagen fibers. Keywords: Image cytometry; Cluster analysis; Collagen Resumo: Análise morfométrica do colágeno dérmico pode fornecer subsídio quantitativo para a pesquisa em dermatologia. Os autores demonstram uma técnica de análise de imagem digital que permite a identificação de estruturas microscópicas, a partir da segmentação por conglomerados (clusters), de cor aplicada à estimativa da intensidade e densidade das fibras colágenas da derme. Palavras-chave: Análise por conglomerados; Citometria por imagem, Colágeno Histological cuts of computational morphometry represent an important tool in biomedical research, integrating the objectiveness of the measurements, high level of reproducibility, low cost, independence of human subjectiveness and partiality, possibility of quantitative analysis of the variables and a great number of publications available. 2The estimate epidermic thickness, hyperkeratosis, parakeratosis, melanic pigmentation, depth of tumours, inflammatory infiltrate, volume of the glands, immunohistochemical marks, heterogeneity of chromatin, dermic elastosis and collagen alterations are some direct applications of morphometry to microscopic skin cuts. [3][4][5] In spite of the availability of specific commercial systems of morphometry, structures can be quantified using a simple microscope of light, attached to digital cameras and analyzed by free softwares such as the ImageJ, making it possible to promote the diffusion of quantitative research in dermatology. 6,7 In this study we present a strategy to estimate the density and intensity of collagenous fibers on the skin, which is an important variable in studies about ageing, genetic syndromes, fibromatosis and colagen diseases, besides therapeutic comparissons.There are various systems of color to operate morphometry, outstanding the HSB, the LAB, the XYZ and the RGB, the most commonly used. This means that the pixels of an image can be interpreted as shinning points with intensities of color that can be decomposed into channels such as: red (R), green (G) and blue (B). If each pixel projects its composition of color into a tridimensional orthogonal system RXGXB, it is possible to identify in this virtual space groups of points which are related to the shades of color of the image. Cluster analysis is a computational tool that can identify such groupings of points and substitute them by its median value (centroid), creat-

No abstract

Introdução: O ensaio do cometa ou técnica da eletroforese de células isoladas é largamente empregado para avaliação de danos e reparo do DNA em células individuais. O material pode ser corado por técnicas de fluorescência ou por sal de prata. Este último apresenta vantagens técnicas, como o tipo de microscópio utilizado e a possibilidade de armazenamento das lâminas. A análise dos cometas pode ser feita de modo visual, porém há a desvantagem da subjetividade dos resultados, que pode ser minimizada por análise digital automatizada. Objetivos: Desenvolvimento e validação de método de análise digital de cometas corados por sal de prata. Métodos: Cinquenta cometas foram fotografados de maneira padronizada e impressos em papel. Além de medidas manualmente, essas imagens foram classificadas em cinco categorias por três avaliadores, antes e depois de pré-processadas automaticamente pelo software ImageJ 1.38x. As estimativas geradas pelos avaliadores foram comparadas quanto sua correlação e reprodutibilidade. Em seguida, foram desenvolvidos algoritmos de análise digital das medidas, com base em filtros estatísticos de mediana e de mínimo. Os valores obtidos foram comparados com os estimados manual e visualmente após o pré-processamento. Resultados: As medidas manuais das imagens pré-processadas apresentaram maior correlação intraclasse do que as imagens preliminares. Os parâmetros automatizados apresentaram alta correlação com as medidas manuais pré-processadas, sugerindo que este sistema aumenta a objetividade da análise, podendo ser utilizado na estimativa dos parâmetros dos cometas. Conclusão: A presente análise digital proposta para o teste do cometa corado pela prata mostrou-se factível e de melhor reprodutibilidade que a análise visual. resumo unitermos

Type I collagen is the main dermal component, and its evaluation is relevant to quantitative studies in dermatopathology. However, visual gradation (0 to 4+) has low precision and high subjectivity levels. This study aimed to develop and validate a digital morphometric analysis technique to estimate type I collagen levels in the papillary dermis. Four evaluators visually quantified (0 to 4+) the density of type I collagen in 63 images of forearm skin biopsies marked by immunohistochemistry and two evaluators analyzed the same images using digital morphometric techniques (RGB split colors (I) and color deconvolution (II)). Automated type I collagen density estimation in the papillary dermis (two techniques) were correlated with visual evaluations (Spearman's rho coefficients of 0.48 and 0.62 (p<0.01)). With regard to the inter-observer repeatability, the four evaluators who used visual classification had an intraclass correlation coefficient (for absolute agreement) of 0.53, while the other two evaluators who used digital analysis (algorithm II) had an intraclass correlation coefficient of 0.97.