Collagen organization and structure in FBLN5-/- mice using label-free microscopy: implications for pelvic organ prolapse

Abstract

Pelvic organ prolapse (POP) is a gynecological disorder described by the descent of superior pelvic organs into or out of the vagina as a consequence of disrupted muscles and tissue. A thorough understanding of the etiology of POP is limited by the availability of clinically relevant samples, restricting longitudinal POP studies on soft-tissue biomechanics and structure to POP-induced models such as fibulin-5 knockout (FBLN5^-/- ) mice. Despite being a principal constituent in the extracellular matrix, little is known about structural perturbations to collagen networks in the FBLN5^-/- mouse cervix. We identify significantly different collagen network populations in normal and prolapsed cervical cross-sections using two label-free, nonlinear microscopy techniques. Collagen in the prolapsed mouse cervix tends to be more isotropic, and displays reduced alignment persistence via 2-D Fourier transform analysis of images acquired using second harmonic generation microscopy. Furthermore, coherent Raman hyperspectral imaging revealed elevated disorder in the secondary structure of collagen in prolapsed tissues. Our results underscore the need for in situ multimodal monitoring of collagen organization to improve POP predictive capabilities.

Fig. 1.

In this study, we compared endocervical collagen organization and structure in heterozygous fibulin-5 (HET) mice and homozygous fibulin-5 knockout (KO) mice with pelvic organ prolapse (A). From excised murine reproductive tracts, axial cross-sections 5 $\mu$ m in thickness were made at the external os and internal os of the cervix (B). Each cross-section was imaged using two-photon excited fluorescence (TPEF) of endogenous fluorophores and second harmonic generation (SHG) of collagen. A TPEF and SHG stitched image of a HET internal os cross-section is shown in C, highlighting collagen alignment perpendicular to the cervical canal. The image was formed using high-resolution tiles to resolve tissue structures and thin collagen fibers (D and E).

Fig. 2.

The collagen orientation index (COI) indicates the degree of collagen anisotropy within a selected window. The binarized power spectrums of anisotropic (A) and isotropic (B) collagen windows reveal distinct spatial frequency distributions (C and D). By fitting an ellipse to the power spectral densities, we extract the collagen orientation index as one minus the ratio of the minor axis length to the major axis length. More aligned collagen (C) has a greater COI than isotropic collagen (D). Furthermore, the orientation angle of the major axis (-90° and 90°) is extracted from ellipse fitting for LMAAD quantification. The COI was computed on distinct 64 x 64-pixel windows across every SHG image. The spatial COI values for the HET internal os are displayed in E, highlighting greater COIs for the relatively more anisotropic collagen near the cervical canal. (F) The HET internal (N=4, n=13,716) and external os (N=4, n=17,430) display significantly greater mean COIs compared to the KO internal (N=4, n=31,272) and external os (N=4, n=51,727). * Indicates a P < 0.001 via two-tailed Mann-Whitney-Wilcoxon test.

Fig. 3.

The local mean absolute angular difference (LMAAD) quantifies the persistence of collagen orientation. Using each window’s (64 x 64 pixels) major axis orientation angle, we calculate the mean absolute angular difference between the center window (0) and the nearest neighbor windows (NNW) (1 $\le$ d < 2), the second NNW (2 $\le$ d < 3), and third NNW (3 $\le$ d < 4) (A and B). The result is assigned to the center window. (D and C) Applying this processing (1 $\le$ d < 2) to one of the HET and KO internal os samples indicates low LMAAD near the cervical canal. Violin plots of compiled LMAAD distributions for the nearest neighboring windows (1 $\le$ d < 2) are highlighted in E (HET: int. os, n=12,913; ext. os, n=15,815; KO: int. os, n=29,989; ext. os, n=48,970). (F) The HET internal (N=4) and external os (N=4) display significantly different LMAAD compared to the KO internal (N=4) and external os (N=4) as a function of NNW displacement. * Indicates a P < 0.001 via two-tailed Mann-Whitney-Wilcoxon test.

Fig. 4.

The structural order of collagen in internal os (HET, N=4; KO, N=4) and external os (HET, N=3; KO, N=4) cross-sections probed using BCARS microscopy. (A) TPEF and SHG images are used to locate subepithelial collagen for BCARS imaging. (B) Molecular pseudocolor of the ROI of the whole image in A. Red is protein signal arising in the amide I (C=O stretch at 1659 cm^-1), green is assigned to the amide III of collagen (1246 cm-1), and blue is assigned to phenylalanine (1002.2 cm^-1). (C) Using the intensity of amide III of collagen (1246 cm-1) as a mask, the ratio of disordered to ordered collagen is quantified as the sum of 1246 $\pm$ 5 cm^-1 over the sum of 1271 $\pm$ 5 cm^-1. Representative normalized, averaged (5 x 5 pixels) collagen spectra in the internal os (D) and external os (E). The combined amide III ratio distributions (20 bins, bin size is 0.1) indicate significantly different collagen structural disorder in HET (int. os, n=154,833; ext. os, n=75,365) and KO (int. os, n=128,599; ext. os, n=135,142) tissues (F). * Indicates a P < 0.001 via two-tailed Mann-Whitney-Wilcoxon test.