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.

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.

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.

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.

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.

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.(AU)