3.2 | Structural analysis of LF tissues
Growing evidence has demonstrated the structural disorder of LF tissues of LSCS patients (Sakai et al., 2017; Sun et al., 2020). In order to fully present the structural changes of LF tissues, we next conducted H&E, EVG, and MT staining, and transmission electron microscope observation experiments to detect the basic structure and ultrastructure of LF. As illustrated in Figure 2A, the normal LF from LDH group were rich in elastic fibers. The fibers were arrayed in a regular order (H&E and EVG staining, ×200). Nevertheless, consistent with those previous studies (Sakai et al., 2017; Sun et al., 2020), the hypertrophic LF showed marked loss of elastic fibers in the LSCS group. The morphology of the elastic fibers was uneven, fragmented, disorganized, and partially absent (H&E, EVG and MT staining, ×200). In the MT staining, the elastic fibers were stained pink, while the collagen fibers were stained blue. In the LDH group, a large area was stained pink. But in the LSCS group, most of the area was stained blue, showing fibrotic change (MT staining, ×200). As compared with LDH group, a large number of fibrosis-related proteins, including collagen I and Collagen III, were observed in LSCS group detected by immunohistochemistry.
Moreover, ultrastructural observation by the transmission electron microscopy (TEM) also showed that the LF tissues from LDH group abundantly contained elastic fibers with very few collagen fibers among them (Fig. 2B). However, there was a significant loss of elastic fibers and an increase in collagen fibers in the LSCS group. The morphology of elastic fibers on the coronal plane manily displayed long fusiform or irregular shapes in the LSCS group, while that of LDH group was round or short fusiform (Fig. 2B). The scale bar 5μm indicates 80000× magnification and the 1μm indicates 40000× magnification.