Utility of Optical Density of Picrosirius Red Birefringence for Analysis of Cross-Linked Collagen in Remodeling of the Peripartum Cervix for Parturition

Abstract

We report on development of a rapid, quantitative analysis technique of collagen fibers in cross-linked structures to assess remodeling of the cervix during the transition from soft to ripening in preparation for birth. Optical density analysis of picrosirius red stain tissue using circular polarized birefringence light from fixed paraffin-embedded or frozen cervix from pregnant mice during phases of remodeling prior to birth. Data were analyzed using NIH Image J and extended recently to include studies of prepartum cervix in peripartum women. Our results, developed a rapid, consistent, technique to quantify cervical organization. This approach assesses the structure of collagen organization (the principle component of the cervix) and is essential for analysis of experimental outcomes that disrupt cervical morphology in rodent models of preterm birth. The technique, in this report has, for the first time permitted rapid, accurate assessment of the stages that define cervical ripening with large numbers of slides from individual animals. The approach integrates analysis of collagen organization, with distensability and inflammation, processes associated with cervical change before birth. This analysis further holds promise to evaluate other tissues, but also fibrolytic and fibrogenic changes in collagen associated with physiological or pathophysiological conditions.

Keywords: inflammation; pregnancy; remodeling; ripening.

Figure 1A.

Illustrative photomicrographs of Picrosirius Red stained, 10μm thick paraffin sections from the cervix of a non-pregnant (NP) and a day18 (d18) pregnant mouse cervix. Term gestation is 19–20 days. Note the ability to visualize individual collagen fibers and fascicles in both sections at this relatively low magnification (250X, using a 20 power objective). The NP section is substantially darker reflecting the denser collagen fiber organization and corresponding reduced light transmission of cross-polarized light through the tissue. In contrast, at d18, as term gestation approaches, the collagen fiber organization is less dense and less organized. Individual fascicles have increased spacing, allowing greater light transmission through the tissue. Within Individual fascicles there is less organization and numerous irregularities as denoted by the overall lighter appearance (increased transmitted light and increased disorganization of individual fibers. Scale bar=50μm. Figure 1 B and C. Photomicrographs of from nonpregnant (NP) and late term (d18) cervix from wild-type (WT) and progesterone receptor B knockout mice (PRBko). White areas illustrate birefringent illumination and more easily depicts differences in collagen organization. Fibers from non-pregnant animals have more linear structure, in contrast to late term D18 fibers which are dispersed and lack defined structure. Scale bar is 50μm for the two color images and 50μm for all grey scale images and is the same for all grey scale images.

Figure 2.

Histograms obtained from photomicrographs of Figure 1 A and B. Cervix optical density histograms (mean±SEM) from pregnant Prostaglandin Receptor 2 knockout (Ptgfr^−/−) and PRB ^−/−) mice following picrosirius red processing (see methods). Optical density values denote an inverse relationship to collagen structure and organization. The more structured a tissue, the lower optical density values for that tissue. All statistical comparisons were performed using analysis of variance (ANOVA) with Tukey’s post-hoc analysis. “a” equals comparison to non-pregnant, “b” denotes comparison to D15. [11, 14]