Mucus Gel : a protective barrier
The primary, functional mucus secretion of the gastrointestinal tract is a layer of mucin gel adherent to the mucosal surface. This mucus layer acts as a protective barrier between the epithelium and lumen which is full of damaging hydrolytic enzymes, micro-organisms, toxins, free radicals and potential carcinogens. At the epithelial surfaces within the mucus gel, a stable, aqueous, unstirred layer exists, which is freely permeable to small sized ions and solutes but a barrier to larger sized molecules, eg, digestive enzymes and some toxins. In the stomach and proximal duodenum, the mucus bicarbonate barrier is the first line of defence against the gastric juice. The adherent mucus gel at the mucosal surface creates an unstirred layer which prevents the newly secreted mucosal bicarbonate secretion from immediately mixing with the vast excess of acid in the lumen. Surface neutralisation of acid occurs by epithelial bicarbonate diffusing into the gel and a pH gradient is established across the mucus layer from an acid lumen of pH 1-2 to a near neutral pH at the epithelial surface.
The gastric mucus layer also acts as a physical barrier preventing luminal pepsin from reaching and digesting the epithelial surface. Luminal pepsin does, however, digest the surface of the mucus gel to produce soluble degraded mucin in the gastric juice. The continuity and maintained thickness of the mucus gel layer seen in vivo is evidence that mucus secretion balances that lost by peptic digestion as well as that lost by mechanical erosion, etc.
The mucus layer throughout the gut acts as a barrier to pathogens. The oligosaccharide chains of the mucins mimic those of cell surface receptors and by binding microbes prevent them from reaching the surface. The mucus secretion is also impregnated with secretory IgA and lysosyme. The large, highly glycosylated mucin molecules readily quench free radicals although with this process extensive fragmentation to small degraded glycopeptides occurs. Specifically in the colon, the mucus layer provides a stable habitat and energy source for the prolific endogenous microflora which secrete a complete complement of mucin degrading glycosidases and proteases.
A key factor in assessing the functional effectiveness of the mucus barrier and the rate of its functional secretion is the thickness of the mucus gel layer. Clearly, the thicker the mucus barrier, the better will be its protective capability. Three phases of mucus can be identified (i) pre-secreted mucus, stored within intracellular vesicles in epithelial cells and clearly visible in histologically stained mucus secreting epithelia; (ii) the layer of mucus gel firmly adherent to the cell surface; and (iii) mobile luminal mucus mixed with the luminal contents.
The thickness of the adherent mucus gel layer is a balance between its rate of secretion from the intracellular mucus stores and its rate of erosion by luminal proteases, free radicals, mechanical erosion, etc. It is important to note that an increased level of luminal mucin is not a measure of mucus secretion since it frequently reflects increased erosion of the mucus barrier. It may well be indicative of a thinner, less effective mucus layer unless it is accompanied by a commensurate, increased rate of mucus secretion. Similarly, measurement of the rate of mucin biosynthesis by radioactive substrate incorporation or measurement of mucus secretion by observed depletion of intracellular, epithelial mucin stores cannot be directly equated to the thickness of the mucus barrier because neither considers the rates of mucus erosion.
Thickness
measurements of the gel barrier
For a direct measure of rate of mucus secretion it is essential, therefore, to measure changes in the thickness of the mucus gel barrier. This has until recently proved difficult and in the past led to controversy as to whether the gastroduodenal mucus layer is continuous and therefore an effective barrier. In fact some groups have even claimed that there is no gastric mucus barrier in vivo and that it is functionally redundant. This is because little or no adherent gel is visible at the gastroduodenal mucosal surface on classically fixed and stained histological sections. Whilst the intracellular stores of presecreted mucin characteristic of mucus secreting epithelia are clearly visible, the surface mucus gel layer is dehydrated, condensed and lost during the prolonged exposure to organic fixatives and mountants employed in these standard histological procedures.
The use of cryostat sections, prepared from snap freezing of mucosa in liquid nitrogen does retain the surface mucus gel but the subsequent histological processing used does not preserve the full thickness of the mucus layer. Similarly, standard preparation procedures for electron microscopy also distort the mucus layer to give condensed strands and fenestrated patches. However, a continuous extracellular mucus layer is seen by scanning electron microscopy if the mucus layer is first stabilised with antibodies and sections prepared from snap frozen samples.
There is now very good evidence from several different groups that
a continuous mucus gel layer exists adherent to the gastroduodenal
mucosal surface and the colon.
Various approaches have been used to overcome the lack of
histological methodology for observing the thickness of the mucus
layer. The observation of mucus on unfixed sections of mucosa has
proved effective in animal models and mucosal resections from human
subjects. This method, the success of which reflects the
firmness of the surface adherent gel, shows an uninterrupted mucus
layer over the stomach surface in man and rat with means of 180 mm
and 80 mm respectively. An early indirect method for mucus thickness
was to measure changes in refractive index using a slit lamp and
pachymeter on inverted mucosa. With this method, however, it is
not possible to distinguish between the mucus gel and the additional
aqueous surface layer attached to it.
An elegant approach for observing mucus thickness is that developed for in vivo using the anaesthetised rat gastroduodenal preparation. The mucus layer is observed in vivo by microscopy and the thickness can be measured by the distance moved by a micro-pipette from the luminal surface to the epithelial surface through the adherent gel layer. This method has not only been used to measure rat gastric mucus thickness in vivo but also to demonstrate acid secretion through channels in the mucus layer covering the gastric glands.
Recently, it was developed a histological method which enables the preservation and visualisation of the full thickness of the adherent mucus layer in cryostat sections of gastric mucosa. With this method, conditions that dehydrate and damage the mucus gel, namely, harsh fixation, the prolonged use of organic solvents and use of resinous mountants were avoided. These steps were replaced by a short 10 minute 100% ethanol pretreatment and a brief post-fixation (45 mins in paraformaldehyde vapour at 37 °C) and use of a water soluble mountant. By this method, a strongly stained PAS positive adherent mucus layer was observed over human antral biopsies with a mean (SD) thickness of 144 (52) mm. In validation experiments using rat gastric mucosa, a mean mucus thickness of 176 mm was measured. This was twice as thick as that previously measured on unfixed sections but approaching thickness values obtained in vivo (200-300 mm). The difference in mucus thickness observed by the in vivo method in rat compared with that seen on stained secretions is considered to reflect a viscous surface mucus that can be sucked off the luminal surface of the adherent gel in vivo. The particular advantage of this new histological method is that not only can it be readily applied to frozen resected mucosa but also to biopsy samples obtained at endoscopy.
With this new histological method, it was that the gastric mucus barrier in man is not only continuous but maintains a minimum thickness and therefore viable barrier across the surface of the mucosa. Gastric mucus thickness values varied from 30 to 320 mm over the mucosa, 96% of the readings were above 70 mm. Application of this method has shown that H. pylori infection per se does not decrease mucus thickness over the gastric mucosa although there is a significant 18% reduction with associated gastric atrophy.
Summary
In summary, if a comprehensive and dynamic picture of changes in the functional mucus barrier are to be obtained under different physiological and pharmacological conditions, then ideally the levels of all three phases of the mucus secretion should be monitored : biosynthesis, mucus gel thickness and mobile luminal mucus. Techniques for measuring mucin biosynthesis and luminal mucin have existed for some time. The methodology now exists for measuring the thickness of the mucus gel both in vivo and in vitro.