This study obtained visual evidence of novel cellular and extracellular matrix-level structural alterations in term and preterm human fetal amniochorionic membranes. Amniochorions were collected from term cesarean (not in labor) or vaginal (labor) deliveries, preterm premature rupture of membranes, and spontaneous preterm birth. To determine the effect of oxidative stress on membranes at term or preterm labor, term not in labor samples in an organ explant culture in vitro were exposed to cigarette smoke extract. Tissues were imaged using multiphoton autofluorescence and second harmonic generation microscopy. Images were analyzed using ImageJ and IMARIS software. Three-dimensional microscopic analysis of membranes revealed microfractures that were characterized by amnion cell puckering, basement membrane degradation, and tunnels that extended into the collagen matrix with migrating cells. Numbers of microfractures were similar at term regardless of labor status; however, morphometric measures (width and depth) were higher in term labor membranes. Oxidative stress induced higher numbers of microfractures in term not in labor membranes, with morphometry resembling that seen in term labor membranes. Preterm premature rupture of the membranes had the highest number of microfractures compared to membranes from term and other preterm births. Microfractures are structural alterations indicative of areas of tissue remodeling during gestation. Their increase at preterm and in response to oxidative stress may indicate failure to reseal, predisposing membranes to rupture.
Early neoplastic features in oral epithelial dysplasia are first evident at the basal epithelium positioned at the epithelial-connective tissue interface (ECTI), separating the basal epithelium from the underlying lamina propria. The ECTI undergoes significant deformation in early neoplasia due to focal epithelial expansion and proteolytic remodeling of the lamina propria but few studies have examined these changes. In the present study, we quantitated alterations in ECTI topography in dysplasia using in vivo volumetric multiphoton autofluorescence microscopy and second harmonic generation microscopy. The label-free method allows direct noninvasive visualization of the ECTI surface without perturbing the epithelium. An image-based parameter, ‘ECTI contour’, is described that indicates deformation of the ECTI surface. ECTI contour was higher in dysplasia than control or inflammed specimens, indicating transition from flat to a deformed surface. Cellular parameters of nuclear area, nuclear density, coefficient of variation in nuclear area in the basal epithelium and collagen density in areas adjacent to ECTI were measured. ECTI contour differentiated dysplasia from control/benign mucosa with higher sensitivity and specificity than basal nuclear density or basal nuclear area, comparable to coefficient of variation in nuclear area and collagen density. The presented method offers a unique opportunity to study ECTI in intact mucosa with simultaneous assessment of cellular and extracellular matrix features, expanding opportunities for studies of early neoplastic events near this critical interface and potentially leading to development of new approaches for detecting neoplasia in vivo.
Injury occurring on the surface of the rectal mucosal lining that causes defects in barrier function may result in increased risk for transmission of infection by HIV and other pathogens. Such injury could occur from microbicidal or other topical agents, mechanical trauma during consensual or nonconsensual intercourse, or inflammatory conditions. Tools for evaluation of rectal mucosal barrier function for assessing the mucosa under these conditions are lacking, particularly those that can provide in vivo structural and functional barrier integrity assessment and are adaptable to longitudinal imaging. We investigated confocal endomicroscopy (CE) as a means for in vivo imaging of the rectal epithelial barrier in the ovine model following spatially confined injury to the surface at a controlled site using a topical application of the microbicide test agent benzalkonium chloride. Topical and intravenous (i.v.) fluorescent probes were used with CE to provide subcellular resolution imaging of the mucosal surface and assessment of barrier function loss. A 3-point CE grading system based on cellular structure integrity and leakage of dye through the mucosa showed significant differences in score between untreated (1.19 ؎ 0.53) and treated (2.55 ؎ 0.75) tissue (P < 0.0001). Histological grading confirmed findings of barrier compromise. The results indicate that CE is an effective means for detecting epithelial injury and barrier loss following localized trauma in a large-animal model. CE is promising for real-time rectal mucosal evaluation after injury or trauma or topical application of emerging biomedical prevention strategies designed to combat HIV.
<p>a) Spatial variation of collagen density is shown by reduced SHG signal in the compromised LP beneath neoplastic foci relative to neighboring LP outside the foci. b) Average collagen density in control, inflammation and OED. "*" and "**" represent p< 0.05 and p<0.01 respectively.</p>
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