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Glycoprotein methods protocols - biotechnology
GI Adherent Mucous Gel Barrier 5757From: Methods in Molecular Biology, Vol. 125: Glycoprotein Methods and Protocols: The MucinsEdited by: A. Corfield © Humana Press Inc., Totowa, NJ5The Gastrointestinal Adherent Mucous Gel BarrierAdrian Allen and Jeffrey P. Pearson1. IntroductionThree phases of production of mucin can be identified in the gastrointestinal (GI)tract on the basis of their location in vivo: the stored, presecreted, intracellular mucin;the gel phase adherent to the epithelial surfaces; and the viscous, mobile mucin, whichis largely in soluble form and mixes with the luminal contents. The layer of adherentmucous gel that lines the epithelial surfaces throughout the gut from the stomach tothe colon marks the interface between the mucosal epithelium and the fluid luminalenvironment, which is teeming with nutrients, bacteria, destructive hydrolases, for-eign compounds and so on. The adherent mucous gel thus provides a protective barrierand a stable unstirred layer with its own microenvironment, between the mucosal sur-face and the lumen (1,2). In the mouth, where salivary mucins are secreted, and theesophagus, there is no discernible adherent mucous gel layer.A practical definition of the adherent mucous gel layer is that secretion of mucuswhich remains attached to the mucosal surface after washing away the luminal con-tents. This mucous gel layer is readily visible on unfixed, transverse sections of mu-cosa as a translucent layer of variable thickness (5–200 µm), between the densemucosal surface and the bathing solution (3). Mucous gel scraped from the mucosalsurface has the rheological characteristics of a true viscoelastic gel, although it has theability to slowly flow (30–120 min) and reform when sectioned (4,5). The concentra-tion of mucin in such gels scraped from the mucosal surface is high, e.g., ranging from50 mg/mL in gastric mucus to 20 mg/mL in colonic mucus (6). The extent of the mu-cus adherent to the mucosal surface in vivo is determined by a balance between therate of secretion by the underlying epithelium and the rate of erosion by mechanicalshear associated with the digestive processes and digestion by hydrolases, particularlyproteases (1,2). For full studies of mucous secretion in vivo, it is essential to quantitateboth the adherent mucous gel and mucin in the luminal solution since the latter canarise by degradation of the former as well as by secretion, and changes in these twophases do not necessarily parallel each other. Despite being the primary mucous bar- 58 Allen and Pearsonrier and the major secretion of mucin of the gut, the adherent mucous gel is frequentlyignored because of the practical difficulties in observing and quantitating it.Quantitative measurement of the amount of mucin in the adherent mucous gelsecretion in vivo can only be approximate. Although the mucous gel can be separatedfrom the mobile mucus by washing the mucosal surfaces, separation of the gel layerfrom the considerable intracellular mucin stores cannot be done satisfactorily. Themucous gel can be removed from the surface by gentle scraping, but inevitably eithernot all the adherent gel is removed or the scrapings contain substantial numbers ofepithelial cells with stored intracellular mucin (7). Researchers have attempted to esti-mate the amount of the adherent mucous gel by utilizing the binding by the dye alcianblue (AB) to the negatively charged mucin macromolecules (8). However, the amountof negative charge on mucins varies considerably according to the location in the gut,secretory status, and disease. Also other macromolecules present in the mucus, e.g.,negatively charged nucleic acids and cell surface components, will bind the dye, and ithas yet to be shown that the dye penetrates the mucous layer uniformly. Results fromthis method are therefore difficult to interpret and this method is not considered fur-ther here.The best approach to quantitating changes in the adherent mucous gel layer is bymeasuring its thickness. These measurements have the important advantage that con-tinuity and thickness of the layer can be directly related to functional efficacy (1,2).Routine histological methods for fixing and staining of mucosal sections, althoughreadily demonstrating periodic acid-Schiff (PAS)/AB positive intracellular mucinstores, result in little or no adherent mucous gel being visible at the mucosal surfaces.This is because the prolonged use of fixatives and organic solvents together with con-ventional paraffin embedding causes denaturation, dehydration, and loss of the sur-face mucous gel layer (9–11). Similarly, standard preparation procedures for electronmicroscopy distort the mucous layer to give condensed strands or fenestrated patches,although a continuous mucous layer can be observed by scanning electron microscopyif it is first stabilized by antibodies (12). The paucity or absence of a mucous layer onbiological sections has led to controversy in the past as to whether an adherent mucouslayer existed over the gastric mucosal surface (13,14). However, a variety of differentmethods (discussed subsequently) have now demonstrated unequivocally that a thick(100–200 µm), continuous, adherent mucous layer exists over the mammalian gastricmucosal surface. A protective mucoid cap can be seen at the surface of damagedmucosa undergoing re-epithelialization, following preparation of sections by standardhistological procedures (1,13,15). This mucoid cap has been confused with the adher-ent mucus that covers the normal undamaged mucosa; however, it is a quite differentstructure consisting primarily of a fibrin gel and necrotic cells with some mucin stain-ing (15).Original methods for measuring adherent mucous thickness in situ were based on(1) differences in refractive index between the mucous layer and the mucosal surface,measured by a slit lamp and pachymeter (16); (2) the dimensions of the surface pHgradient measured using microelectrodes (17,18); and (3) observation of transversesections of unfixed mucosa (3). The first two of these methods also measure the GI Adherent Mucous Gel Barrier 59unstirred aqueous layer beyond the adherent mucous gel surface, the extent of whichdepends on the hydration of exposed mucosa and the degree of stirring of the solutionabove the mucosa, respectively. Consequently, the dimensions of mucous thicknessmeasured by the slit lamp and pachymeter or pH gradient methods can be substantiallygreater than the actual values for the gel layer itself (19). Since the mid-1980s, obser-vation of the translucent mucous layer on thick (1.6 mm) sections of unfixed mucosa(detailed subsequently) has been a simple method for measuring mucous thickness,although histological methodology has now reached the stage where preservation offull adherent mucous thickness can be maintained during processing (10).An elegant method for observing mucus and measuring its thickness has been devel-oped using in vivo microscopy in the anesthetized rat animal model (20–22). The GImucosa is exteriorized, with its blood and nerve supply intact, and the mucosa is placedover an illuminated lucite cone. A lucite chamber is fitted over the mucosa to exposeapprox 0.8 cm2of the surface and is filled with physiological saline. The mucous layercovering the mucosa can be observed microscopically from above, particularly if theluminal surface of the mucus is enhanced visually by the addition of carbon particlesto the bathing solution. The thickness of the mucous layer is best measured by record-ing the distance travelled by a glass microprobe moved by a micromanipulator at afixed angle through the mucous layer. A continuous mucous coat of mean thickness200–300 µm is seen in rat stomach and duodenum (21,22), and a mean thickness of800 µm is seen in the colon (23). Strong suction will remove about one-third of themucous layer in the stomach and nine-tenths of that in the colon to leave a firm adher-ent gel, whereas in the duodenum all the mucus can be sucked off. It is not yet clearwhether the mucus that can be sucked off in vivo is a continuum of the gel at a lowerconcentration or a separate secretion. This method, which for experimental practicali-ties can be applied only to the rat (and possibly other experimental animal models), isparticularly useful for validating other in vitro methods for measuring mucous thickness,for studying mucous secretion in vivo, and for studying the secretion of acid from thegastric glands through the covering mucous layer (20). Full details of this method, whichrequires careful physiological experimentation to ensure full maintenance of in vivofunctions during the experimental period, can be found in refs. 20–22.Various groups have adapted histological procedures to observe adherent mucus onfixed mucosal sections. Modifications have included the use of cryostat sections (24)and fixation in Carnoy’s solution (11,25). However, the adherent mucous layer aftersuch procedures is very thin (typically about 25%) compared with that seen on unfixedmucosal sections and even less than that observed in vivo. A particularly interestingmethod developed by Ota and Katsuyama (11,25) shows alternating layers within bothhuman gastric and colonic adherent mucous layers following dual staining with galac-tose-oxidase-cold thionine Schiff and paradoxical concanavalin A. However, thismethod, which suggests some form of substructure within the mucous gel layer, usesCarnoy’s fixative, as well as clearing in xylene with paraffin embedding, and results inconsiderable shrinkage of the mucous gel to give a thickness less than one-fifth thatseen on unfixed sections. A recent method (detailed subsequently), using cryostat sec-tions, does give thickness values in the rat comparable with those measured for the 60 Allen and Pearsonfirm adherent mucous gel remaining after suction in vivo (10). In this method, cryostatsections receive no prefixation or extensive dehydration steps characteristically usedin conventional staining techniques. Instead, the tissue is given a brief ethanol pre-treatment followed by a prolonged staining with PAS/AB and a gentle postfixation inparaformaldehyde vapor, and finally is mounted in a water-soluble gelatin gel. Valuesfor mucous thickness over the rat gastric mucosa by this method give a mean of147 µm, which approximates that of the adherent gel remaining after suction in vivo.It would appear, therefore, that this method preserves intact the thickness of the stableadherent mucous gel layer. By contrast, mucous thickness by this method is twice thatseen using conventional PAS/AB staining techniques on cryostat sections of mucosaand 50% greater than that for unfixed rat gastric mucosal sections.2. Materials2.1. Mucous Thickness on Unfixed Sections1. NaCl solution: 150 mmol/L.2. Two parallel, sharp razor blades separated by spacers 1.6 mm apart such that one side ofeach of the two razor blades is left free for sectioning and held together by bolts tightenedby hand.3. Millipore filter paper to act as a backing for the mucosa during sectioning and subsequentmanipulation of the section.2.2. Histological Staining and Fixation of Cryostat Sections of Mucosa1. NaCl solution: 150 mmol/L.2. Acetic acid solution: 3% (v/v).3. Alcian Blue 8 GX (Sigma, Poole, Dorset, UK) 1% by weight in 3% acetic acid (pH 2.5).4. Periodic acid (aqueous): 1%.5. Schiff’s reagent (fuchsin-sulfite reagent) readily obtained, made up in solution (Sigma).6. Paraformaldehyde crystals placed in a desiccator for final postfixing of sections at 37°C.7. Gelatin for water-soluble mounting prepared by dissolving 10 g of gelatin in 60 mL ofdistilled water mixed with 250 mg of phenol in 70 mL of glycerol.8. Poly-L-lysine-coated slides: slides are immersed in 0.01% (w/v) poly-L-lysine for 5 minand dried at room temperature.3. Methods3.1. Observation and Measurementof Mucous Thickness on Unfixed Mucosal Sections (see Notes 1–4)This is a rapid and simple method for observing the adherent mucous layer andmeasuring its thickness on unfixed sections of mucosa (3). This method has been used tomeasure mucous thickness on resected human mucosa both in the stomach (26) and inthe colon (27) or in experimental animal models, particularly rat (3,9,28,29). Thismethod relies on the adherent gel being sufficiently firm that it is relatively undistortedwhen the mucosa is sectioned by parallel razor blades 1.6 mm apart.1. It is essential that the mucosa and sections are immersed in isotonic saline throughout theprocedure except during sectioning of the mucosa in order to maintain the gel in its fullyhydrated state. GI Adherent Mucous Gel Barrier 612. Gently wash the luminal mucosal surface with isotonic saline (150 mmol/L of NaCl) toremove food particles and so on as well as nonadhering, soluble mucus.3. Remove outer muscle layers from serosal side of mucosa by blunt dissection.4. Mount the mucosa, luminal surface up, on a Millipore filter on a corkboard and cut sec-tions with a pair of parallel, sharp razor blades separated by a distance of 1.6 mm. Therazor blades are held together with spacers (1.6 mm) between them by bolts tightened byhand. It is often useful to loosen partially the bolts in order to remove the newly cutmucosal section from between the razor blades.5. Position sections transversely on a microscope slide. Check the positioning of the sec-tions with a low-powered (e.g., × 3.5 ) stereoscopic microscope. Care should be taken notto distort or stretch the sections during manipulation and positioning.6. The surface mucous layer can be observed under an inverse microscope (e.g., × 200) usingeither light- or dark-field illumination or phase contrast. The adherent mucous layerappears as a translucent layer between the dense mucosa and the clear bathing solution.The thickness of the mucous layer is measured using an eyepiece graticule.7. Individual mucous thickness values should be measured on at least three to six sectionsper mucosal specimen. Measurements of gel thickness should be taken at regular inter-vals along each section; A good distance is 500 µm between each reading (about 6–10readings per section, depending on its length).8. Mucous thickness values on unfixed mucosal sections usually show a nonparametric dis-tribution with more readings at the lower end of the range. Therefore, results are besttreated for statistical significance by methods that compare nonparametric distributionssuch as the Mann-Whitney U-test. Values for thickness should therefore be expressed interms of medians and upper and lower quartiles.9. When this technique is used for the first time on a new tissue, it is useful to validate themethod by checking that the thickness of the adherent mucous layer on the sections doesnot change significantly over a period of 45 min and that the same mean values for mucousthickness are obtained for individual sections when read from either side (sections turnedover 180° and remounted).3.2. Histological Fixation and Staining of Adherent Mucous Gelon Cryostat Sections of Mucosa (see Notes 5–9)Jordan et al.’s (10) method preserves the mucous layer on cryostat sections ofresected mucosa or biopsies without apparently causing shrinkage and retaining itsoriginal thickness in situ. The cryostat sections undergo no prefixation or long ethanoldehydration steps characteristic of previous methods. Instead, the tissue is given abrief ethanol pretreatment followed by a prolonged aqueous staining, and a gentlepostfixation in paraformaldehyde vapor (37°C for 45 min), and is finally mounted in awater-soluble gelatin gel.3.2.1. Preparation of SectionsMucosal segments are snap-frozen in liquid nitrogen and stored at –20°C. Forsmaller gut segments, e.g., rat, a good method is to leave the stomach and intestineintact, ligate one end, fill with 0.9% NaCl, and ligate the other end before snap-freez-ing. This ensures minimum disturbance of the mucous layers and prevents contact ofthe adjacent luminal walls of the tissue. Biopsies, e.g., from human endoscopy(e.g., 2 mm2) need support, which can be provided by wrapping them in a 1–cm2sand- 62 Allen and Pearsonwich of a suitable piece of tissue. Pig’s liver is satisfactory for this procedure. Thesandwiched biopsies are immediately snap-frozen and stored at –20°C. Serial cryostatsections, 6–20 µm thick, are cut from representative portions of the tissue and mountedon poly-L-lysine–coated slides and stained. Sections on slides can be convenientlystored at –20°C before staining.3.2.2. Staining ProcedureSections are stained by the following sequence of steps:1. Defrost: 20 min2. Pretreatment in 100% ethanol: 10 min.3. Rinse in running tap water: 10 min.4. Rinse in 3% (v/v) acetic acid: 2 min.5. Stain in 1% Alcian Blue 8GX in 3% acetic acid (pH 2.5): 2.5 h.6. Rinse in 3% acetic acid and then rinse in running tap water: 2 min.7. Oxidize in 1% periodic acid (aqueous) at room temperature: 10 min.8. Wash in running tap water: 5 min.9. Immerse in Schiff’s reagent: 15 min.10. Rinse in running tap water: 5 min.11. Rinse in 0.5% sodium metabisulfite, repeat three times: 1 min/rinse.12. Rinse in running tap water: 5 min.13. Postfix in paraformaldehyde vapor, at 37°C: 45 min.14. Mount in gelatin.4. Notes1. Removal of the muscle layers first makes sectioning of unfixed mucosa easier. However,the mucosa can be sectioned with the muscle layer intact, and previous studies have shownthat doing so does not affect the thickness of the adherent mucous layer.2. Various stains, e.g., toluidine blue and PAS have been used to improve visualization ofthe mucous layer on unfixed sections. However, it is essential that the solutions used areisotonic and do not contain any organic solvent (e.g., alcohol), which will cause dehydra-tion and distortion of the mucous gel layer.3. The unfixed section method is most satisfactory for gastric mucosa where there is a thicklayer of firm adherent gel. Although this method has been used successfully to look atsmall intestinal and colonic mucous gel, it is less satisfactory for these tissues since themucous gel is not so firm and the mucosa is more elastic than in the stomach.4. The unfixed section method has been applied to human biopsy specimens from the gastricmucosa. However, it is very difficult to obtain satisfactory sections from biopsies, andhence, consistent results for mucous thickness. It is necessary to obtain resected mucosato use this method for human studies.5. The distribution of mucous thickness readings on histologically stained and fixed sec-tions is usually nonparametric. Therefore, thickness values obtained by this method shouldbe expressed as medians with upper and lower quartiles and comparisons among groupsmade by methods that compare nonparametric distributions, such as the Mann-WhitneyU-test.6. The thinner the cryostat sections are cut (down to 6 µm) for histological processing thebetter, because this provides clearer details of cell structure. It may be necessary, in somecases, to use thicker sections (up to 20 µm) in order to obtain a good mucous gel layer. GI Adherent Mucous Gel Barrier 63Where detailed mucosal structure is required, parallel histological methodology can beapplied to adjacent mucosal sections from that on which mucous thickness is measured.Note, however, that on such sections, the adherent mucous gel layer will be greatlyreduced and discontinuous.7. Careful handling of the cryostat sections on the slide during fixing and staining is impor-tant because excessive washing and so on can remove mucus from the section.8. Full validation of the staining and fixing methodology for unfixed sections and compari-son of results by this method with those of previous methods used for staining the mucousgel can be found in Jordan et al. (10). An example of the use of this method for studies inhumans is given in Newton et al. (30).9. This histological and fixation method can be adapted to staining the adherent mucous gelwith biotinylated antibodies and lectins coupled to avidin-horseradish peroxidase usingconventional methodology in place of steps 4–11.References1. Allen, A. (1989) Gastrointestinal mucus, in Handbook of Physiology, The Gastrointesti-nal Physiology. Salivary, Gastric and Hepatobiliary Secretions, vol. III (Forte, J. G., ed.),American Physiology Society, Bethesda, MD, pp. 359–382.2. Allen, A., Flemstrom, G., Garner, A., and Kivilaakso, E. (1993) Gastroduodenal mucosalprotection. Physiol. Rev. 73(4), 823–857.3. Kerss, S., Allen, A., and Garner, A. (1982) A simple method for measuring thickness ofthe mucosal gel layer adherent to rat, frog and human gastric mucosa: influence of feedingprostaglandin, N-acetyl-cysteine and other agents. Clin. Sci. 63, 187–195.4. Bell, A. E., Allen, A., Morris, E. R., and Ross-Murphy, S. B. (1984) Functional interac-tions of gastric mucus glycoprotein. Int. J. Biol. 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(1998) Helicobacter pylori in vivo structural changes in the adherent gastricmucus gel layer but barrier thickness is not compromised. Gut 43, 470–475. . Ross-Murphy S. B. (1988) Mucus glycoproteingels: role of glycoprotein polymeric structure and carbohydrate side-chains in gel-forma-tion. Carbohydrate Res. 178,. water-soluble mounting prepared by dissolving 10 g of gelatin in 60 mL ofdistilled water mixed with 250 mg of phenol in 70 mL of glycerol.8. Poly-L-lysine-coated