Light microscopical observations on luminally administered dyes, dextrans, nanospheres and microspheres in the pre-epithelial mucus gel layer of the rat distal colon
Introduction
The mucus layer adherent to the epithelial surface of the intestine (PML) represents an extracellular diffusion barrier for some hydrophilic nutrient molecules and ions dissolved in the ingesta, in addition to its functions as a lubricant and as a protective blanket against pathogen penetration. The mucus layer covering the colonic mucosa is a stable viscoelastic gel, built of a mucin glycoprotein framework. Some 90–98% is composed of an `unstirred water layer', while other contents include desquamated epithelial cells, secretory IgA, lysozyme, lactoferrin, Na+-, K+- and Ca++-ions [1]. The gel is mainly impermeable to slowly diffusing large molecular weight solutes, such as proteins 1, 2. The mucus layer seems to have some importance as an absorption barrier, at least for lipophilic substances like testosterone [3], in addition to the epithelial cell layer (the major barrier to nutrient absorption in the intestine). Glycolipids in the mucus gel layer play a prominent role as a barrier to luminal acid, similar to the function of lipidic elements as surface barriers in the skin [4]. The significance of the mucus gel layer as a diffusion barrier for hydrophilic solutes is not yet clear. Evans Blue was demonstrated to be trapped by the rat colonic PML when administered as a 1% solution, but was only partly trapped when administered as a 3% solution [5]. For hydrophilic solutes, intestinal mucus seems to serve as a molecular sieve, cutting off the permeation of large molecules with a molecular mass more than 600–700 Da. Absorption of substances with higher molecular weights remains at a low level [6], but mucus does not constitute an absolute barrier even to high molecular weight solutes 7, 8, 9.
In vitro studies have clearly demonstrated a slow diffusion of macromolecules through mucus gel models [8]. Latex (polystyrene) nanospheres and microspheres have been shown to be translocated through the PML and to be present in Peyer's patches of the rat colon 12 h following oral administration; smaller particles gained more rapid access to the lymphatic gut cells than larger particles of the colloid marker. The uptake was size-dependent in the range 50 nm to 3 μm [10].
The present study was carried out to investigate the binding capabilities and barrier function of the PML of the rat distal colon in vivo. Several dyes, dextrans, nano- and microspheres were administered directly into the colonic lumen and their passage through the PML was observed and qualitatively compared using light microscopy.
In the rat distal colon, a distinct and entirely continuous mucus layer of fairly homogenous thickness (30–50 μm) can be observed at the mucosal surface separated from the digesta, showing a kind of stratification, containing up to eight sublayers [11]. In the light microscope, the mucus layer can be observed in freshly cut thin razorblade sections of pellet-containing rat distal colon, as well as in cryostat sections. In air-dried rat distal colon sections the mucus gel can also be stabilized by celloidin for further histochemical staining [12].
In this present work, cryostat sections were studied over the time period 1–30 min after luminal administration of the various marker substances.
Section snippets
Materials and methods
Experiments were carried out on 20 adult Wistar AS/Ztm and Lewis rats of both sexes with a mean body weight of 302±52 g, kept under standardized conditions. The rats were anaesthetized with sodium pentobarbital (35 mg/kg), and laparotomised and 200 μl of a tracer containing solution was applied intraluminally through an anal catheter into subsequent segments of the distal colon (Fig. 1). The tracer containing gut segments were ligated, and excised at 1, 2, 4, 5, 7, 10, 15, or 30 min after
Dyes in the mucus layer
In gut segments excised 1 min after luminal application of the dye Methylene Blue (MW 355.89), the pre-epithelial mucus layer (PML) and the surface epithelial cells were already stained (Fig. 2B; Table 1). This indicates that the 30- to 50-μm-thick PML of the rat distal colon was permeated by Methylene Blue molecules in less than 1 min. In gut segments taken 5, 7, and 30 min after application, Methylene Blue was present in superficial and in deep cells of the epithelium. No stain was observed
Conclusions
The PML in the rat distal colon is a significant diffusion barrier for some solute molecules and luminal particles, but not for others.
Methylene Blue (MW 356) and also FiTC-Dextran 10 000 penetrated the 30- to 50-μm-thick PML to reach the epithelium cells in 1, and 2 min, respectively. For Light Green SF yellowish (MW 792.9) it took 5 min to reach the surface cells of the mucosa. Evans Blue (MW 960.8), Alcian Blue (MW 1300) and Trypan Blue (MW 960) entered the PML from the luminal solution in
Acknowledgements
The author thanks Mrs S. Staacke and Mrs K. Risse for technical assistance and Mrs B. Leppich for photographic and graphical help.
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