Rat Oral Mucosa Research Articles

Immunocytochemical analysis of early focal cellular infiltrates in experimental oral contact hypersensitivity.

The cellular infiltrates of early contact hypersensitivity reactions to 2,4-dinitrofluorobenzene (DNFB) in rat oral mucosa were phenotypically characterized using serial frozen tissue sections and monoclonal antibodies. CD2+CD45RB- lymphocytes and ED1+RT1B/D+ monocytes/macrophages appeared in focal collections at the epithelium-connective tissue interface 2-6 h after challenge with DNFB. These foci also contained CD4+, CD5+, CD8+, TCR alpha beta+ cells. CD45RB+ "naive" T cells were difficult to detect at 2-6 h but appeared in significant numbers at 24 h post-challenge. At this stage, the number of all the other phenotypes also was increased. CD2+ cells were approximately twice as many as CD5+ or TCR alpha beta+ respectively, indicating that TCR gamma delta+ lymphocytes might be involved. An additional observation was the presence of increased numbers of CD2+ lymphocytes in the oral mucosa of sensitized but not challenged animals. Our findings indicate that the oral mucosa of skin DNFB pre-sensitized animals may be "contact hypersensitivity conditioned" by migration-prone memory T cells, and that these cells may rapidly interact with locally resident epithelial Langerhans' cells following antigen restimulation, creating the very initial antigen-specific part of experimental oral contact hypersensitivity.

Irritant and sensitizing potential of copper, mercury and tin salts in experimental contact stomatitis of rat oral mucosa

HgCl2, CuSO4, SnCl2, SnCl4 or sodium lauryl sulphate (SLS) were openly applied to rat oral mucosa for 1 min, followed 6 h later by histologic examination of the tissue response. Granulocytes were the predominant inflammatory cells and no lymphocytic infiltration could be seen with any of the substances tested. Irritant threshold levels were defined histologically for each of the substances. CuSO4 was found to be non-irritant at all concentrations. The addition of non-irritant concentrations of SLS lowered the threshold levels for HgCl2 and SnCl2, but CuSO4/SLS was non-irritant at all concentrations tested. Preapplication to the mucosa of SLS at non-irritant concentrations gave results with HgCl2, SnCl2 and CuSO4 similar to those with SLS added to the metal salt solutions. Lesions of allergic contact type could not be induced in the oral mucosa to any of the metal salt preparations.

Experimentally‐induced neurogenic inflammation in the rat oral mucosa

Blood flow and vascular permeability in the gingiva and alveolar mucosa of the lower jaw were studied after the antidromic (electric) or orthodromic (topical administration of capsaicin onto the oral mucosa) stimulation of the inferior alveolar nerve (IAN) in rats with or without capsaicin pretreatment. Blood flow was determined using 86Rb isotope dilution technique, while vascular permeability was assessed by the Evans blue extravasation. Both antidromic or orthodromic stimulation of the IAN increased blood flow and Evans blue extravasation. Capsaicin pretreatment abolished the increase in gingivo-mucosal blood flow and vascular permeability induced by nerve stimulation. The results of the present study appear to confirm the possibility of the development of neurogenic inflammation in the oral cavity. The mechanism observed may play a role in the development of certain inflammatory reactions in the oral mucosa frequently encountered in human clinical practice.

Oral Cancer

In the U.S. oral cancer accounts for 2.1% of all cancers and 1% of cancer deaths. Two to three times as many males as females are affected. Blacks have more intra-oral cancer than whites, and their incidence and mortality rates have increased in recent years. The etiologic process very likely involves several factors. The major etiologic agents are tobacco (all types) and alcoholic beverages. Herpes simplex virus, human papilloma virus, and Candida have been implicated. Host factors include poor state of dentition, nutritional aberrations, cirrhosis of liver, lichen planus, and immunologic impairmant. Cellular changes include amplification of some oncogenes, alterations in antigen expression, production of gamma-glutamyl transpeptidase, and disturbance of keratin and involucrin production. Experimentally, cancer is readily produced on the hamster cheek pouch and rat oral mucosa. Unlike oral cancer in humans, most experimental lesions are exophytic, and they rarely metastasize.

Effects of systemic administration of nicotine on capillaries in rat oral mucosa : Johnson GK, Fung YK, Squire CA. J Oral Pathol Med 18:230, 1989

Effects of systemic administration of nicotine on capillaries in rat oral mucosa : Johnson GK, Fung YK, Squire CA. J Oral Pathol Med 18:230, 1989

A relationship found between intra‐oral sites of 4NQO reductase activity and chemical carcinogenesis

Topical application on rat oral mucosa of the chemical 4-nitroquinoline 1-oxide (4NQO) has been shown to produce squamous cell carcinomas on the posterior tongue and/or the posterior hard palate. 4NQO is broken down in vivo by a diaphorase, 4NQO reductase (E.C.1.6.99.2), to produce an active molecule believed to be responsible for carcinogenesis. It has been shown that there are higher concentrations of 4NQO reductase in oesophageal mucosa compared with elsewhere in the gastrointestinal tract. The purpose of these experiments was to compare the distribution of certain diaphorases in the oral mucosa. Samples of rat tongue and cheek epithelia were homogenized, then ultracentrifuged to provide mixed cytosol and microsome fractions from the epithelial cells. A spectrophotometer was used to measure the variation in absorbance at 340 nm of NADH consumed by reduction of 4NQO by enzymes present in the tissue extracts. A histochemical technique was used to compare the activity of NADH diaphorase, NADP diaphorase and glucose-6-phosphate dehydrogenase at different sites of the oral mucosa. Statistical analysis showed that there were significant (P less than 0.01) differences between the activities of all three enzymes at different sites of the oral mucosa. In each case, a higher activity was found at the sites of high incidence of squamous cell carcinoma. A lower activity was found at sites where carcinomas did not occur.

Effects of systemic administration of nicotine on capillaries in rat oral mucosa.

This study examined the reaction of the local vasculature of the oral mucosa in 16 Sprague Dawley rats receiving systemic nicotine delivered (1.5 mg/kg/day) via subcutaneous minipumps for 24 h or 2wk. Control animals received saline. After treatment animals were killed and biopsies taken from palate, maxillary gingiva and buccal mucosa, frozen and cryostat sections incubated to demonstrate alkaline phosphatase, which is a capillary marker. The total length of the capillary fragments in the nicotine treated groups was significantly less than of the control group. There was also a decrease in capillary height in both of the nicotine groups when compared to the control animals. This study indicates that morphologic alterations occur in the microvasculature of the oral mucosa following systemic nicotine administration. This may have implications for the role of chronic tobacco use in the etiology of oral mucosal disease, including periodontal disease.

Chemically induced inflammation in rat oral mucosa.

The toxic, irritative, and sensitizing effects of topically applied sodium lauryl sulfate (SLS), dithranol triacetate (DTA), nonanoic acid in methyl- or propyl ester (NAM, NAP) in the buccal mucosa were investigated in a Sprague-Dawley rat model. Semi-quantitative evaluations of cellular infiltrates were performed in routine histologic preparations. The toxic potential was tested with 2% and 0.2% solutions. All substances, except 0.2% SLS, caused an increased cellularity, mainly of a mononuclear cell type. The low dose of NAM induced stronger inflammatory reactions than the high dose. Repeated applications of 2% solutions decreased the response compared to one application, except for NAM, where a clear irritative potential was observed. Pre-exposure of dorsal skin prior to buccal painting resulted in an enhanced reaction to NAM and NAP, whereas no sensitizing capacity was noted in SLS or DTA in this model.

Age-related changes in capillaries of rat oral mucosa. A quantitative electron microscopic study

Ultrastructural age changes in capillaries of the buccal mucosa were examined in montages of cross sections made from electron micrographs at 37 500 x. Six rats aged 6 months and 6 aged 30 months were perfused with glutaraldehyde and conventional thin sections obtained. Two capillaries located within a connective tissue papilla were studied from each rat. Capillaries of the old group differed from those of the young group by statistically significant increases in several parameters. The endothelial cell was increased in thickness, especially in the vicinity of junctions. The frequency of pinocytotic vesicles/unit length of the cell circumference was nearly doubled. Junctions were of nearly double length and took a more oblique course. All parts of the basement membranes were thickened, though perhaps less than seems true in skin. In striking contrast to epidermis, the epithelium of oral mucosa undergoes no appreciable thinning with age. We suggest that the observed age increases in frequency of pinocytotic vesicles and in the length of junctions may facilitate blood/tissue exchange, thus compensating for impaired exchange due to the thickened basement membranes. These compensatory changes in conjunction with the unchanged size of the mucosal capillary bed in the aged rat (demonstrated previously) could explain the unchanged thickness of the oral epithelium.

Immunogenic potential of some mercury compounds in experimental contact allergy of the rat oral mucosa.

The contact allergenic potential of substances included in a "dental screening test" has been investigated in a previously established animal model for producing contact allergy. We were unable to produce contact hypersensitivity with any of the original formulas. HgCl2 was selected for further studies and, in order to improve its penetration, butanol was used as a solvent. Sensitization was, however, not obtained with any of the HgCl2/butanol formulas. Our results are discussed in relation to the requirements of reaching immunogenic threshold levels of mercury in the tissue.

Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase.

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.

Local variations in the staining patterns of keratin antigens and blood group antigens in normal rodent oral epithelia

All rodent oral epithelia are orthokeratinized. However, morphological, immunohistochemical and biochemical studies have shown that regional differences exist. In the present study, intraregional variations in differentiation patterns of rat oral epithelia are demonstrated using monoclonal anti-keratin antibodies AE1 and AE2 and antibodies to blood group antigens B and H. Well-defined areas of rat buccal and hard palate epithelium differed from the general staining patterns of these epithelia. These areas were associated with a papillary surface contour. These local variations were not found in the strain of mice examined. The results suggest that physiologically different vertical compartments of keratinocytes exist within one and the same region of rat oral mucosa, a phenomenon previously recognized in detail only in the epithelium of dorsal tongue. The papillary structures may have some functional significance related to the processing of food similar to that suggested for lingual filiform papillae.

Tissue pressure in rat oral mucosa measured by micropuncture technique

Tissue pressure or interstitial fluid pressure (IFP) was measured in 68 rats in different areas of the oral mucosa (free gingiva, attached gingiva in front and molars, alveolar mucosa, hard palate, cheek) by glass pipettes (diameter 2‐4 μm) connected to a servocontrolled counterpressure system. All the measurements of IFP were considerably above ambient pressure. The highest IFP was measured in the hard palate (14.8 mm Hg) and in attached gingiva (8.6, 9.2 mm Hg), while free gingiva, alveolar mucosa and cheek showed lower values (2.3, 3.6, 1.5 mm Hg respectively). Venous stasis resulted in an immediate rise in IFP in the attached gingiva. Repeated measurements of IFP at the same location gave the same results 3 h after induction of anesthesia, and there was no change in IFP in continuous recordings for up to 30 min. Administration of indomethacin did not change IFP. The results show that IFP in oral mucosa is high and that there is a marked regional variation in IFP, which may reflect regional differences in aspects of anatomy and function. The great difference between IFP in attached and free gingiva shows that there is a pressure gradient between these tissues, possibly maintained by the anatomical structure and the drainage of gingival fluid into the gingival sulcus.

Progesterone metabolism by rat oral mucosa. III. Participation of granulation tissue and fibroblasts.

The metabolism of progesterone by rat granulation tissue was studied. Experimental granulation tissue was produced by implanting viscose‐cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules and fibroblasts in the sponges were homogenized, and mitochondrial, microsomal and soluble fractions were prepared with differential centrifugation. The subcellular Preparations were incubated with 4‐14C‐progesterone and NADPH for 30 min at PH 7.4 and 37°C. The metabolites were identified with column and multiple thinlayer chromatography and radioautography and quantitated with liquid scintillation counting. The granulation tissue and fibroblasts showed much less activity in metabolizing progesterone than the gingival tissue of either sex. As reported earlier, gingival tissues contain Δ4‐5α‐ and,Δ4‐5β‐steroid hydrogenases and 3α‐, 3β‐, 20α‐ and 20β‐hydroxysteroid dehydrogenases. The granulation tissue and fibroblasts lack 3 β‐hydroxysteroid dehydrogenase activity. In addition, the fibroblasts show no 20 β‐hydroxysteroid dehydrogenase activity. The enhanced metabolism of progesterone during gingival inflammation is thus probably not due to the formation of granulation tissue.

Pigmentary changes in rat oral mucosa following antimalarial therapy

Pharmacogenic pigmentation of the oral mucosa has been reported following the use of a number of anti-malarial drugs. The nature and distribution of the pigment is inconclusive in the literature. The aim of the present study was to document pigment deposition within the oral mucosa of DA rats following prolonged chloroquine and pyrimethamine administration. The drugs were given as a combined dosage and separately to different groups via stomach gavage tube. After 12 weekly administrations the palatal mucosa was examined histochemically and ultrastructurally for changes in numbers and size of active melanocytes using the dopa-oxidase technique. The serum was analysed for changes in ACTH and testosterone levels. Morphometric analysis of cells incubated for dopa-oxidase showed a significant increase in the size of dopa positive cells with both drugs but an increase in the number of active melanocytes with chloroquine only. Serum levels of ACTH remained unchanged with both drugs but pyrimethamine caused an elevation in testosterone.

Site-specific distribution of epithelial cell-surface carbohydrates in rat oral mucosa

The binding of two fluorescein-labelled lectins to epithelial cell surfaces was examined microspectrofluorimetrically in rat oral mucosa. Griffonia simplicifolia (GS-I-B4), which is specific for α-D-galactosyl end groups, labelled only basal cells, while Ulex europaeus (UEA-I), which is specific for α-L-fucosyl groups, labelled only spinous cells. The degree of binding of the lectins was dependent on the lectin concentration and the lectin pH. Different sites were examined. The labelling of basal cells by GS-I-B4 was maximal on the lateral borders of the tongue, and the fluorescence diminished medially; in contrast, the UEA-I labelling of the corresponding spinous cells was of undiminished intensity in the mediolateral direction across the entire lingual epithelium. There was a gradual increase in the binding of GS-I-B4 and UEA-I towards the posterior aspect of the tongue. In the mid-palate, there was stronger staining both of basal cells by GS-I-B4 and of spinous cells by UEA-I in the gingivae as compared to the centre of the palate. In anteroposterior sections of the fore- and mid-palate, the fluorescence intensity of basal cells was inversely related to that of spinous cells, with maximal labelling of basal cells by GS-I-B4 and corresponding minimal binding of spinous cells by UEA-I being evident at the crests of the transverse rugae, and the opposite pattern of staining by both lectins being noted at the bases of the rugae. In sections of the soft palate and buccal mucosa, there was uniform labelling of basal cells by GS-I-B4 and of spinous cells by UEA-I. There were no significant differences in cell numbers at sites where there was low and high lectin binding. The findings indicate that, in the rat oral mucosa, there is a specific topographical distribution in the expression of α-D-galactosyl groups on epithelial basal-cell surfaces (as demonstrated by GS-I-B4) and α-L-fucose residues on spinous cells (as shown by UEA-I).

Changes in cAMP phosphodiesterase activity during oral mucosal regeneration.

Preliminary studies (Lineweaver-Burk, Ca2+ calmodulin sensitivity) suggest that oral mucosa contains at least two cyclic AMP phosphodiesterases, one a high affinity (low Km) enzyme and the other a low affinity (high Km) enzyme. Analysis of the distribution of both enzymatic forms during oral mucosal regeneration revealed that the low Km, and high Km cAMP phosphodiesterase activities were significantly elevated prior to the first wave of cAMP accumulation and during the second cAMP wave. Although the cAMP peaks declined between 20-24 h, both cAMP phosphodiesterases remained significantly elevated at the wound site. The apparent Km of the low Km form increased from 5.3 to 7.5 microM, while that of the high Km form remained essentially unaltered 20 h after wounding. The low Km, and high Km cAMP phosphodiesterase activities in normal rat oral mucosa were not affected by epinephrine or insulin; were slightly inhibited by glucagon, and significantly inhibited by methylprednisolone. Imidazole and histamine activated both forms and theophylline was inhibitory to the enzyme. The high Km cAMP phosphodiesterase was sensitive, and the low Km form insensitive to Ca2+ calmodulin stimulation.

Morphology of the oral mucosa in rats exposed to high concentrations of phosphorus

Morphology of the oral mucosa in rats exposed to high concentrations of phosphorus

Changes in the rate of epithelial proliferation of rat oral mucosa in response to acute inflammation induced by turpentine

Inflammatory lesions were produced in the buccal mucosa by a subepithelial injection of turpentine; animals were killed 24 h later, 1 h after an intravenous injection of tritiated thymidine ([ 3H]-Tdr). Control rats were given [ 3H]-Tdr but no turpentine. Lesions comprised a turpentine pool surrounded by a dense layer of inflammatory cells, beyond which the tissues were more diffusely inflamed. The labelling index (L.I.) for mitotic activity in overlying epithelium was determined in a region (C) close to the layer of dense infiltration and in a region (D) more distant. The L.I. in region D was over four times greater than in region C, and nearly four times greater than that of the contralateral, uninjected cheek. The L.I. in the uninjected cheek was significantly lower than that in controls, which may indicate a systemic depression of proliferative activity in the experimental animals, probably due to stress. Thus mild inflammatory injury stimulates epithelial proliferation, whereas more severe inflammation depresses it, perhaps due to more extensive progenitor-cell damage.

Preferential Lectin Binding to Specific Layers of Rat Oral Epithelium and Modification by Enzyme Pretreatment

Three fluorescein-labeled lectins were shown to bind differentially to cell surfaces in different epithelial layers of rat oral mucosa regardless of the age or the site of origin of the tissue. Griffonia simplicifolia (GS-1-B4), specific for alpha-D-galactosyl end groups, labeled basal cells only; Ulex europeus (Ulex 1) specific for alpha-L-fucosyl groups labeled spinous cells; and Bandeiraea simplicifolia (BSII), specific for N-acetyl-D-glucosamine, labeled cornified cells. Pretreatment of sections with alpha-galactosidase completely abolished the staining of basal cells by GS-1-B4, but had no effect on the staining of spinous cells by Ulex 1. In contrast, alpha-fucosidase abolished the staining of spinous cells by Ulex 1 and caused staining of both basal and spinous cells by GS-1-B4. Neuraminidase and chondroitinase ABC produced results similar to one another, with staining of basal cells by GS-1-B4 and labeling of both basal and spinous cells with Ulex 1. beta-galactosidase, beta-glucuronidase, and testicular hyaluronidase did not affect the staining pattern of GS-1-B4 or Ulex 1, whereas chymotrypsin completely abolished any staining with either lectin. The results demonstrate a complex arrangement of cell surface carbohydrates in the epithelium of rat oral mucosa. The findings indicate a possible simplification in the spatial arrangements of cell surface carbohydrates during the differentiation of basal to spinous cells.