Multispectral Imaging Method for Rapid Identification and Analysis of Paraffin-Embedded Pathological Tissues.

The study of the interaction between light and biological tissue is of great help in the identification of diseases as well as structural alterations in tissues. In the present study, we have developed a tissue diagnostic technique by using multispectral imaging in the visible spectrum combined with principal component analysis (PCA). We used information from the propagation of light through paraffin-embedded tissues to assess differences in the eye tissues of control mouse embryos compared to mouse embryos whose mothers were deprived of folic acid (FA), a crucial vitamin necessary for the growth and development of the fetus. After acquiring the endmembers from the multispectral images, spectral unmixing was used to identify the abundances of those endmembers in each pixel. For each acquired image, the final analysis was performed by performing a pixel-by-pixel and wavelength-by-wavelength absorbance calculation. Non-negative least squares (NNLS) were used in this research. The abundance maps obtained for the first endmember revealed vascular alterations (vitreous and choroid) in the embryos with maternal FA deficiency. However, the abundance maps obtained for the third endmember showed alterations in the texture of some tissues such as the lens and retina. Results indicated that multispectral imaging applied to paraffin-embedded tissues enhanced tissue visualization. Using this method, first, it can be seen tissue damage location and then decide what kind of biological techniques to apply.

Gestational folic acid deficiency alters embryonic eye development: Possible role of basement membrane proteins in eye malformations.

OBJECTIVES: Folic acid (FA) is crucial before and during early pregnancy. FA deficiency can occur because dietary FA intake is low in mothers at the time of conception. Likewise, various ocular pathologies are related to the alteration of extracellular matrices. The present study aimed to investigate the association between maternal FA deficiency and congenital eye defects. We also investigated whether maternal diet deficient in FA alters the expression of collagen IV and laminin-1 as a possible mechanism responsible for the appearance of ocular malformations. Both proteins are the main components of the basal lamina, and form an interlaced network that creates a relevant scaffold basement membrane. Basal laminae are involved in tissues maintenance and implicated in regulating many cellular processes. METHODS: A total of 57 mouse embryos were classified into the following groups: Control group, (mothers were fed a standard rodent diet), and D2 and D8 groups (mothers were fed FA-deficient [FAD] diet for 2 or 8 wk, respectively). Female mice from group D2 were fed a FAD diet (0 mg/kg diet + 1% succinyl sulfathiazole used to block the synthesis of FA) for 2 wk from the day after mating until day 14.5 of gestation (E14.5). On the other hand, female mice from group D8 were fed a FAD diet for 8 wk (6 wk before conception and during the first 2 wk of pregnancy). For the data analysis, we first estimated the incidence of malformations in each group. Then, the statistical analysis was performed using IBM SPSS Statistics, version 25.0. Expression patterns of collagen IV and laminin-1 were examined with the immunohistochemical technique. RESULTS: Our results showed that mice born to FA-deficient mothers had several congenital eye abnormalities. Embryos from dams fed a short-term FAD diet were found to have many significant abnormalities in both anterior and posterior segments, as well as choroidal vessel abnormalities. However, embryos from dams fed a long-term FAD diet had a significantly higher incidence of eye defects. Finally, maternal FA deficiency increased the expression of both collagen IV and laminin-1. Likewise, changes in the spatial localization and organization of collagen IV were observed. CONCLUSIONS: A maternal FAD diet for a short-term period causes eye developmental defects and induces overexpression of both collagen IV and laminin-1. The malformations observed are probably related to alterations in the expression of basement membrane proteins.

Alteration of lens and retina textures from mice embryos with folic acid deficiency: image processing analysis.

PURPOSE: Folic acid (FA) is an essential vitamin for embryonic development. It plays particularly a critical role in RNA, DNA and protein synthesis. On the other hand, the collagen IV and laminin-1 are important proteins during embryonic development. This study was done to find if FA deficiency at a short and a long term in mothers could alter the tissue texture of retina and lens of the progeny. METHODS: Collagen IV and laminin-1 were localized by immunohistochemistry in the lens and retina of the FA-deficient embryos. To carry out the image processing, texture segmentation was performed through canny edge detection and Fourier transform (FT). We defined a parameter, the grain size, to describe the texture of the lens and retina. A bootstrap method to estimate the distribution and confidence intervals of the mean, standard deviation, skewness and kurtosis of the grain size has been developed. RESULTS: Analysis through image processing using Matlab showed changes in the grain size between control- and FA-deficient groups in both studied molecules. Measures of texture based on FT exhibited changes in the directionality and arrangements of type IV collagen and laminin-1. CONCLUSIONS: Changes introduced by FA deficiency were visible in the short term (2 weeks) and evident in the long term (8 weeks) in both grain size and orientation of fibre structures in the tissues analysed (lens and retina). This is the first work devoted to study the effect of FA deficit in the texture of eye tissues using image processing techniques.

Biometric Alterations of Mouse Embryonic Eye Structures Due to Short-Term Folic Acid Deficiency.

PURPOSE: Folic acid (FA) is an essential nutrient for normal embryonic development. FA deficiency (FAD) in maternal diet increases the risk of several defects among the progeny, especially, neural tube defects. The eye begins its development from the neural tube; however, the relationship between FAD and ocular development in the offspring has been little explored and it isn't known how the FAD affects the formation of the eye. Our objective was to analyze the effect of maternal FAD on mouse embryos ocular biometry. METHODS: Female mice C57/BL/6J were distributed into three different groups, according to the assigned diet: control group fed a standard FA diet (2 mg FA/kg), FAD group for short term fed (0 mg FA/kg + 1% succinylsulfathiazole) from the day after mating until day 14.5 of gestation, and FAD group for long term fed the same FA-deficient diet for 6 weeks prior mating and continued with this diet during gestation. A total of 57 embryos (19 embryos of each dietary group) at 14.5 gestational days were evaluated. As indicators of changes in ocular biometry, we analyze two parameters: area and circularity of the lens and whole eye, and the area of the retina. The program used in the treatment and selection of the areas of interest was ImageJ. The statistical analysis was performed by IBM SPSS Statistics 19. RESULTS: Regarding the measures of the area, FA-deficient lenses and eyes were smaller than that of controls. We have also observed increase in the size of the neural retina, spatially, in embryos from females fed FAD diet during long term. On the other hand, as regard to circularity measures, we have seen that eyes and lenses were more circular than control. CONCLUSION: Maternal FAD diet for a very short term generates morphological changes in ocular structures to the offspring.