Light microscopical observations on luminally administered dyes, dextrans, nanospheres and microspheres in the pre-epithelial mucus gel layer of the rat distal colon

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Abstract

The permeability in vivo of the pre-epithelial mucus gel layer (PML) of the rat distal colon to several intraluminally administered dyes, dextrans, nano- and microspheres was studied by transmission and fluorescence light microscopy. Methylene Blue (MW 356) passed through the colonic PML in less than 1 min and stained mucosal as well as propria cells. Light Green SF yellowish (MW 792.9) was found in all sublayers of the PML and had stained the superficial cells of the colon mucosa 5 min after luminal administration. Alcian Blue (MW 1300), Evans Blue (MW 960.8) and Trypan Blue (MW 960.8) seemed to be trapped within the PML when intraluminally administered as 1% solution. Dextran 10 000 was transferred through all of the PML-sublayers within 1 min of its application into the gut lumen and it was found in the nuclei of the superficial cells of the distal colonic mucosa. Dextran 40 000 and Dextran 70 000 also entered the PML, but they were not taken up into the epithelial cells. Small polymeric nanosphere particles reached the apical membranes of the surface epithelium cells less than 2 min after luminal administration and were stuck to them. Midsize nanospheres (415 nm diameter) were also found to be adhered to the apical membranes of the surface epithelium cells 30 min after dosage. Instead of `mucoadhesive' properties, these latex nanospheres were found rather to possess `mucosa-adhesive' or `cell-adhesive' properties. In contrast, there was no accumulation of large microspheres (1.09 μm diameter) on the surface cell membranes within the observation period of 30 min following administration. They were found within the PML, but away from the mucosa. The PML of the rat distal colon represents a diffusion barrier for solutions of the cationic dyes Alcian Blue, Evans Blue and Trypan Blue and for large molecules. On the other hand, surprisingly the PML does not represent a barrier for Dextran 10 000. Furthermore, Dextran molecules of MW 40 kDa and higher pass the PML sublayers slowly in the direction of the superficial cells of the gut epithelium. The PML is not a diffusion barrier for virus size latex particles (nanospheres), nor even an absolute barrier for near bacterium-size latex particles (microspheres), although these do seem to be slowed down in their diffusion through the PML due to their large size.

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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