Three-dimensional analysis of fibroblast-like cells in the lamina propria of the rat ileum using serial block-face scanning electron microscopy

Abstract

Serial block-face scanning electron microscopy (SBF-SEM) is useful for three-dimensional observation of tissues or cells at high-resolution. In this study, SBF-SEM was used to three-dimensionally analyze the characteristics of fibroblast-like cells (FBLCs) in the rat ileal lamina propria (LP). The results revealed that FBLCs in LP could be divided into four types, tentatively named FBLC type I-IV, based on the external cellular appearance, abundance or shape of each organelle, detailed distribution in the LP and relationship with surrounding cells. FBLC-I and -II localized around the intestinal crypt (InC), FBLC-III localized from the lateral portion of InC to the apical portion of the intestinal villus (InV), and FBLC-IV localized in the InV. FBLC-I, -II and -III, but not FBLC-IV, localized beneath the epithelium. FBLC-II possessed thin lamellar-shaped endoplasmic reticula, whereas FBLC-I possessed expanded endoplasmic reticula that occasionally showed a spherical shape. FBLC-III and -IV possessed a cytoplasmic region with high-electron density and no organelles immediately beneath the cellular membrane; this region was found at the epithelial sides in FBLC-III and scattered in FBLC-IV. FBLC-IV were in constant close proximity to villous myocytes throughout the InV and also in contact with FBLC-III especially in the apical portion of the InV. FBLC-I, -II and -IV, but not -III, were constantly found to be in contact with various immunocompetent cells in the LP. Three-dimensional analysis using SBF-SEM indicates that four types of FBLC localized in the rat ileal LP.

Three-dimensional analysis of neural connectivity with cells in rat ileal mucosa by serial block-face scanning electron microscopy

“…In this study, we reused the data stacks obtained by SBF-SEM in our earlier study [ 25 ]; therefore, animals, tissue blocks and data stacks are the same as our earlier study [ 25 ].…”

“…For this investigation, we reused the data stacks obtained in our earlier investigation [ 25 ] for the 3D reconstruction of nerve fibers and nerve bundles. Briefly, ileal blocks from six rats were fixed with 2.5% glutaraldehyde and 2.0% paraformaldehyde fixative in 0.1 M cacodylate buffer (CB; pH 7.4) and postfixed with 2.0% OsO 4 /1.5% potassium ferrocyanide in CB.…”

“…Cells other than pericytes, vascular endothelial cells, and lymphatic endothelial cells in the lamina propria could be divided into two types by their external appearance: cells with highly irregular shapes and cells with simple shapes (sphere or oval). Irregularly shaped cells that had one or more long cellular processes were classified as FBLCs and MLCs: FBLCs, which had been already defined and identified in our earlier study [ 25 ], possessed abundant and expanded endoplasmic reticula and consisted of four types, whereas MLCs were characterized by both the existence of lysosomes and/or phagosomes and a lesser expansion of endoplasmic reticula compared to FBLCs ( unpublished data ).…”

“…In a previous study, we had already classified FBLCs in the data stacks used in the present study into four types based on the external cellular appearance, the abundance or shape of each organelle, the detailed distribution, and relationship with surrounding cells ( Fig. 1B ) [ 25 ]. Briefly, the type I and type II FBLCs were localized in the subepithelial region around the intestinal crypts, whereas the type III FBLCs were located in the subepithelial region, closer than the type I and II FBLCs, from the ICL to the villous apex.…”

“…Our earlier study revealed that the rat ileal lamina propria contains four morphologically distinct types of FBLCs, macrophage-like cells (MLCs), lymphocyte-like cells, eosinophils, plasma cells, and other cells [ 25 ], and these findings provided useful information for evaluations of the connective targets of the nerve fibers in the intestinal mucosa. Here, we focused on the innervation by direct contact and comprehensively evaluated the connectivity of nerve fibers running in the lamina propria for each type of cells in the rat ileum.…”