Network Organization of Interstitial Connective Tissue Cells in the Human Endolymphatic Duct
Department of Medical Biochemistry and Microbiology (A-KHHE,KR), University of Uppsala, Sweden; Department of Otolaryngology (HRA), Head and Neck Surgery, University Hospital, Uppsala, Sweden; and Department of Physiology (VC), Hôpital Vichat, Paris, France
Correspondence to: Dr. Anna-Karin H. Ekwall, Dept. of Medical Biochemistry and Microbiology, University of Uppsala Biomedical Center, Box 582, SE-751 23 Uppsala, Sweden. E-mail: Anna-Karin.Ekwall{at}imbim.uu.se
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Summary |
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(J Histochem Cytochem 51:14911500, 2003)
Key Words: basal lamina intercellular adhesion cellECM contacts interstitial fluid pressure Ménière's disease
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Introduction |
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Several findings support the theory that the ED and ES are important for the regulation of endolymph pressure and volume (Kimura and Schuknecht 1965; RaskAndersen et al. 2000
; Salt and DeMott 2000
). It is believed that the pressure is balanced by high compliance of the "walls" of these structures and the volume to be controlled by both regulation of endolymph resorption and secretion. The ES has been suggested to be mainly phagocytotic, acting as a local organ involved in the immune defense of the inner ear, and to be involved in the degradation of waste products (RaskAndersen and Stahle 1980
; Tomiyama and Harris 1986
; Altermatt et al. 1990
). The ED, on the other hand, is believed to be responsible for the majority of resorption of water and solutes and equilibration of ions of the endolymph (RaskAndersen et al. 1981a
; BaggerSjöbäck and RaskAndersen 1986
; Wackym et al. 1986
).
In this investigation, human ED tissues were serially sectioned for analysis by light and transmission electron microscopy to establish a morphological background for the potential physiological functions of the interstitial connective tissue surrounding the ED. We focused on the structure of the periductal CT and on relationships between CT cells and the local extracellular matrix (ECM). In addition, immunohistochemical (IHC) stainings were carried out to characterize the periductal CT cells.
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Materials and Methods |
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Light and Transmission Electron Microscopy
The tissues were rinsed in saline, stained with 1% osmium tetroxide, and finally dehydrated in graded ethanols and embedded in Epon for semithin and thin sections. One ED was cut transversely from the vestibular orifice and every section was mounted on a coverglass and stained with toluidine blue. At every tenth µm a thicker section was taken and mounted on an Epon plastic column for further thin sectioning. Thin sections were cut with a diamond knife, attached to one-hole grids, and stained with uranyl acetate and lead citrate. The thin sections were viewed in a Jeol 100 SX electron microscope. Specimens were analyzed by both light and transmission electron microscopy at regular intervals throughout the entire length of the EDs.
Immunohistochemical Stainings
The decalcified ED tissues were frozen in liquid nitrogen and cut transversely in 6-µm sections with a cryostat. The IHC staining was carried out according to standard protocols (Sundberg et al. 2001). Briefly, nonspecific binding was blocked with 20% non-immune serum from the secondary antibody species in PBS supplemented with 0.2% bovine serum albumin (BSA) and 0.1% Tween-20 for 30 min. The sections were incubated with primary antibodies diluted in PBS with 0.2% BSA and 0.1% Tween-20 and supplemented with 4% of non-immune serum for 30 min. The anti-human vimentin (clone Vim3B4, dilution 1:400), the anti-human desmin (1:100), the anti-human macrophage CD68 (1:100), the anti-human fibroblast (clone 5B5, 1:200), and the anti-human CD31 endothelial cell (1:100) monoclonal antibodies were purchased from DAKO (Glostrup, Denmark). The anti-human
-smooth muscle actin (1:50) and the anti-pan-cytokeratin (1:400) monoclonal antibodies were purchased from SigmaAldrich (St Louis, MO). The anti-human fibroblast-specific antibody AS02 (1:600) was purchased from Dianova (Hamburg, Germany). After washing twice with PBS with 0.2% Tween-20 and once with PBS, the sections were incubated for 30 minutes with biotinylated F(ab')2 fragments of rabbit anti-mouse and swine anti-rabbit immunoglobulins, respectively, diluted 1:250 (DAKO) and then washed again. The staining was performed using the avidinbiotin complex method (Vectastain ABC-elite kit; Vector, Burlingame, CA) with diaminobenzidine (DAB substrate kit; Zymed, S. San Francisco, CA) as the peroxidase substrate. Sections were counterstained with Mayer's hematoxylin, dehydrated in graded ethanols, and mounted in Entellan.
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Results |
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The periductal connective tissue was of the loose interstitial type, containing collagen fiber bundles (Figures 1 and 2D) . In the subepithelial area, particularly in the mid and distal parts of the ED where the stretched epithelium formed protuberances into the lumen of the duct, the CT was very loose and CT cells were sparse (Figure 1C). The collagen fibers appeared in bundles, which ran in several directions but mostly parallel to the duct (Figures 2B and 2C). The CT cells formed a network via cytoplasmic branches, which ramified and connected to other cell ramifications via small membrane densifications (Figures 2A2D and 2F). Each individual CT cell therefore made contact with three or four other CT cells in the 2D pictures (Figures 2A and 2B). The tissue cells also formed electron-dense contacts with adjacent collagen fiber bundles or other ECM components, forming a network between cells and ECM fibers (Figures 2D and 2E). In addition, the cytoplasmic processes of the CT cells often formed direct physical connections to the basal lamina underlying the epithelial cells (Figure 3) and to the bone matrix of the surrounding vestibular aqueduct (Figure 4F) .
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General Morphology of the ED Epithelium
All four EDs were funnel-shaped and wide in the proximal portion near the vestibular orifice (Figure 1A). The ED narrowed continuously until it was transformed into the ES (Figure 1C), in agreement with earlier observations. In the proximal portion, the ED consisted mostly of a flat, thin epithelial layer (Figures 1A and 4B). However some cuboidal or low columnar epithelial cell types could be found (Figures 1A and 4A). In the mid and distal parts of the duct, most of the epithelial cells consisted of cuboidal or low columnar epithelial cells (Figures 1B and 1C). From the mid to the distal part of the duct, the epithelium also showed some large polyp-like formations. At certain sites these formations took up most of the luminal surface and the epithelial cells appeared stretched or were flattened (Figure 1C). The epithelial cells were interconnected by tight junctions and adherens junctions (Figure 4C). The tight junctions were of the shallow type, with only one to four parallel junctional strands (Figure 4D), in agreement with results from studies on the ES (BaggerSjöbäck and RaskAndersen 1986). In the zonula adherens, typical desmosomes were present (Figure 4D). The columnar cells were electron-translucent or electron-dense, as described earlier (Wackym et al. 1986
). Both cell types had extensive basal infoldings covered by a basal lamina (Figures 3A and 3B). The epithelial cell membrane facing the basal lamina contained many micro-pinocytotic vesicles (Figure 3B). Long, slender basal cytoplasmic epithelial cell protrusions formed deep projections into the surrounding loose CT. These protrusions were always covered by a basal lamina.
Immunohistochemical Stainings of the Periductal CT Cells and the ECM
The CT cells, as well as the endothelium of large vessels in the bone channels, were vimentin-positive (Figure 5A
; Table 1). The endothelial cells showed stronger homogeneous vimentin staining, whereas the CT cells displayed a granular staining pattern. In addition, some but not all epithelial cells were vimentin-positive, with a staining pattern varying from homogeneous to granular. The endothelial cells of the periductal capillaries and larger vessels in the neighboring bone channels were CD31-positive (Figure 5B; Table 1). The CT cells and the ED epithelium were negative for -smooth muscle actin and desmin antibodies (Table 1). Both the CT cells and the ED epithelial cells were stained positive by the anti-human fibroblast antibody (Figure 5C; Table 1). Again, the CT cells presented a more granular staining pattern. The anti-human fibroblast-specific antibody showed a strong homogeneous staining of a majority of the ductal epithelial cells and a weaker staining of some but not all CT cells (Figure 5D; Table 1). The epithelial cells exclusively were stained positive with the anti-pan-cytokeratin antibody (Figure 5E; Table 1). A few cells in the periductal tissue and in the CT surrounding the vessels of the bone channels were stained specifically with the macrophage CD68 antibody (Figure 5F; Table 1). Positive cells were found in immediate contact with the epithelium as well as in the larger veins. Control experiments with secondary antibodies alone and normal mouse serum were negative (data not shown). Positive controls of the anti-fibroblast antibodies on human skin showed no staining of the epidermis but specific staining of fibroblasts in dermis (data not shown).
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Discussion |
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In this study we show that the interstitial CT cells are non-endothelial, non-smooth muscle mesenchymal cells, based on the fact that they stained negative for the vascular endothelial cell marker CD31, -smooth muscle actin, and desmin, but were vimentin-positive. Vimentin is an intermediate filament protein present in cells of mesenchymal origin. Furthermore, based on the fact that CT cells were stained positive by the fibroblast-specific (AS01) antibody and anti-fibroblast antibody (clone 5B5), it is reasonable to conclude that these cells are fibroblastoid cells. The anti-human fibroblast antibody reacts specifically with prolyl 4-hydroxylase, an enzyme of collagen synthesis, in CT cells and myoepithelial cells. The anti-human fibroblast-specific antibody recognizes fibroblasts of different origin but also weakly stains kidney tubule epithelial cells (Saalbach et al. 1996
).
The ED epithelium appears to consist of different cell types because the anti-vimentin and fibroblast-specific antibodies stained some but not all epithelial cells. This is consistent with findings with transmission electron microscopy, in which the columnar epithelial cells have an electron-translucent or electron-dense appearance. The vimentin-positive epithelial cell type appears to be of mesenchymal origin because it contains vimentin intermediate filaments. Kidney tubule epithelium develops from mesenchyme (Ekblom 1989). It may be that one or more cell types of the ED epithelia are derived from a similar mesenchyme-to-epithelium transition. Both kidney tubule epithelia and ED epithelia are important for water and ion regulation. Bauwens et al. (1991)
found co-expression of vimentin and cytokeratin in the epithelium of the human ED and ES, probably reflecting a dual origin of the cells constituiting the epithelium of the endolymphatic duct and sac. These data further support our findings.
Available data suggest that there is an electrolyte shift along the length of the ED, because the ES endolymph, unlike that of the inner ear, contains a high concentration of sodium but is low in potassium (Miyamoto and Morgenstern 1979). The transepithelial flow of water is probably driven by the osmotic potential formed by such an active equilibration of ions. The epithelium of the human ED, with its conspicuous polarity, probably helps to maintain a proper ion balance between the endolymph and the interstitium. Na+/K+-ATPase has earlier been localized in the epithelium of the ES in guinea pig but only at low concentration in the guinea pig ED (Ichiyama et al. 1994
). However, the structure of the epithelial cells, with the extensive basal infoldings, suggests the presence of an active metabolic mechanism for equilibration of electrolytes at this site. The gradual change in epithelial structure along the length of the ED may indicate that more passive mechanisms are initially involved in the transepithelial flow, whereas distally active mechanisms may be more important.
New concepts in physiology suggest that loose interstitial connective tissue, with its fibroblasts and pericytes, actively and dynamically controls interstitial fluid pressure and thereby tissue fluid volume (Rubin et al. 1996; Reed et al. 2001
; Wiig et al. 2003
). It is tempting to speculate that the ED connective tissue network, via intercellular and cellECM contacts, participates in the control of interstitial fluid pressure in the periductal tissue (Figure 6)
. A pressure gradient may be necessary for the transport of water and solutes from the ED lumen, through the CT, and into the draining veins of the neighboring bone channels. A slow flow from the membranous labyrinth to the intracranial veins through the CT network may form an important pathway for both regulation of inner ear pressure and endolymph outflow. In addition, there are still controversies concerning the degree of longitudinal flow under normal and pathological conditions (Salt 2001
). Under normal conditions, the flow appears to be nonexistent or extremely low. On the other hand, under pathological conditions leading to increased pressure and volume of endolymph, this flow may be substantial.
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Acknowledgments |
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Footnotes |
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