The development of the Syrian hamster cheek pouch

The objective of this study was to provide a detailed account of the morphogenesis and early cytodifferentiation of the hamster cheek pouch. Although the newborn "cheek pouch" is used for in vitro studies of the effects of retinoids and carcinogens, its rudimentary structure has not been adequately described. Complete paraffin serial sections of the heads of 14- and 15-day fetuses were cut in three planes to determine the location and shape of the earliest pouch rudiments. Complete paraffin serial sections were prepared from pouch rudiments dissected from hamsters at birth and at daily intervals from 3 to 12 days postnatal. Semithin Epon sections were examined by light microscopy and ultrathin sections by transmission electron microscopy. The pouch can appear in the fetus as two solid epithelial ingrowths from the lining of the oral cavity. They are the margins of an ingrowing sheet of oral epithelium which becomes leaflike at about the time of birth, as it grows caudad into the tissue of the cheek. The central cells of the ingrowth accumulate large quantities of glycogen before differentiating as a stratum spinosum 5 days after birth. Within the stratum spinosum, groups of cells containing keratohyalin granules initiate the stratum granulosum. Keratinized cells appear within the stratum granulosum areas. Spaces appear between keratinized cells, and the spaces coalesce to form the pouch cavity between 7 and 12 days postnatal. Soon afterward, this cavity opens to the oral cavity to make a pouch, and the ultrastructure of the cheek pouch epithelium closely resembles that of the adult.     

Mucous metaplasia of the hamster cheek pouch epithelium under hypervitaminosis A.

Mucous metaplasia of the hamster cheek pouch epithelium induced by implantation of vitamin A pellet was studied electron-microscopically. After 5–10 days of vitamin A treatment, the epithelium was devoid of stratum corneum, and often infiltrated with inflammatory cells. The intercellular spaces were wide, and membrane-coating granules underwent modifications believed to be the earliest signs of mucous production. After 15 days of vitamin A treatment the inflammation had largely gone, and the epithelium was made up of several layers of cuboidal cells. The intercellular spaces and keratin fibrils had greatly decreased. Some mucous granules appeared for the first time in the cuboidal superficial cells. Various other changes in the cheek pouch epithelium were observed in animals treated with vitamin A for 20–25 days. In some regions hyperplasia or inflammatory reactions were observed, whereas in other regions complete mucous metaplasia was found. In completely metaplastic regions a surface layer of typical mucous cells was seen lying atop two or three layers of cuboidal cells.     

Electrical resistance of arterioles and venules in the hamster cheek pouch

The electrical resistance of the vascular endothelium was determined on single microvessels in the hamster cheek pouch in order to obtain information about this variable in a mammalian preparation. So far, the technique has only been applied to frog microvessels. The technique consists of injection of current into the vascular lumen via a microelectrode and recording of the ensuing intravascular potential distribution by a second microelectrode. Cable theory was used for the analysis. The average diameter of the vessels under study was 41 micron for arterioles and 28 micron for venules. The average resistance of the vessel wall at 37 degrees C was 19 omega cm2 and 3.3 omega cm2, respectively. For the venules this is somewhat lower than what has been recorded on muscle capillaries (Olesen & Crone 1983) in the frog at room temperature, whilst the values on arterioles are rather similar. The calculated sodium permeabilities, PNa+, were for arterioles 4 X 10(-5) cm X s-1 and for venules 23 X 10(-5) cm X s-1. The high permeability values for arterioles and venules indicate that the vascular exchange function may not be limited to capillaries only.     

Gender differences in microcirculation: Observation using the hamster cheek pouch

OBJECTIVES:

Estrogen has been shown to play an important protective role in non-reproductive systems, such as the cardiovascular system. Our aim was to observe gender differences in vivo with regard to the increase in macromolecular permeability and leukocyte-endothelium interaction induced by ischemia/reperfusion as well as in microvascular reactivity to vasoactive substances using the hamster cheek pouch preparation.

METHODS:

Thirty-six male and 36 female hamsters, 21 weeks old, were selected for this study, and their cheek pouches were prepared for intravital microscopy. An increase in the macromolecular permeability of post-capillary venules was quantified as a leakage of intravenously injected fluorescein-labeled dextran, and the leukocyte-endothelium interaction was measured as the number of fluorescent rolling leukocytes or leukocytes adherent to the venular wall, labeled with rhodamin G, during reperfusion after 30 min of local ischemia. For microvascular reactivity, the mean internal diameter of arterioles was evaluated after the topical application of different concentrations of two vasoconstrictors, phenylephrine (α₁-agonist) and endothelin-1, and two vasodilators, acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent).

RESULTS:

The increase in macromolecular permeability induced by ischemia/reperfusion was significantly lower in females compared with males [19 (17–22) leaks/cm² vs. 124 (123–128) leaks/cm², respectively, p<0.001), but the number of rolling or adherent leukocytes was not different between the groups. Phenylephrine-induced arteriolar constriction was significantly lower in females compared with males [77 (73–102)% vs. 64 (55–69)%, p<0.04], but there were no detectable differences in endothelin-1-dependent vasoreactivity. Additionally, arteriolar vasodilatation elicited by acetylcholine or sodium nitroprusside did not differ between the groups.

CONCLUSION:

The female gender could have a direct protective role in microvascular reactivity and the increase in macromolecular permeability induced by ischemia/reperfusion.     

Polycations induce microvascular leakage of macromolecules in hamster cheek pouch

The microvascular response to two polycationic proteins, poly-l-lysine (mol wt 104,000) and leukocyte elastase, was studied in the hamster cheek pouch microcirculation model. A 2-min topical application of polylysine (100g/ml) induced vigorous macromolecular leakage from venules only that declined within 30 min. A second application induced significantly less leakage. The leakage was inhibited by admixing polylysine with dextran sulfate prior to application or by giving hamsters an intravenous injection of dextran sulfate. The histamine antagonist pyrilamine did not interfere with the leakage, and only a few degranulated mast cells were found after polylysine application. No intravascular adhesion of leukocytes could be detected. Elastase (100g/ml) was deposited adjacent to venules with micropipets. The resulting leakage response was not inhibited by L658,758, an inhibitor of elastase enzymatic activity, but by dextran sulfate. These results may prove significant in light of the numerous polycationic proteins present within neutrophil granules.The material presented in this report is original and has not been submitted for publication elsewhere.This investigation complies with NIH guidelines for the care and use of laboratory animals.     

An intravital microscopic model for mast cell-dependent inflammation in the hamster cheek pouch

Topical antigen challenge in cheek pouches of immunized hamsters led to an acute inflammatory reaction which was characterized by intravital microscopy. The response consisted of short-lasting arteriolar spasm, followed by leakage of plasma, vasodilation, and accumulation of leucocytes. Several observations indicated that the reaction was due to mast cell activation. Thus, a very similar inflammatory response was seen after challenge with compound 48/80, and both antigen and compound 48/80 degranulated the numerous mast cells present in the cheek pouch. In addition, fluorescein-labelled antigen bound specifically to mast cells in cheek pouches of immunized animals, also suggesting the presence of mast cell-fixed antigen-specific antibodies, possibly immunoglobulin E. However, although antigen and compound 48/80 caused similar microvascular responses, cross-desensitization experiments indicated that the two stimuli activated mast cells via different mechanisms. The histamine antagonist mepyramine, which abolished plasma leakage induced by exogenous histamine, substantially inhibited the increase of microvascular permeability evoked by antigen or compound 48/80, but did not appear to affect the vasospasm and leucocyte accumulation. It is concluded that the hamster cheek pouch may be a most useful tool for investigation of dynamic microvascular events during allergic mast cell-dependent inflammation.     

Inhibition site of dexamethasone on extravasation of polymorphonuclear leukocytes in the hamster cheek pouch microcirculation.

A video system was used to investigate the inhibitory effect(s) of the glucocorticoid dexamethasone (DEX) on polymorphonuclear leukocyte (PMN) extravasation in the microcirculation of hamster cheek pouch. DEX was given intraperitoneally at 0.1 or 0.3 mg/kg body weight 2 h before induction of extravasation by topical application of leukotriene B4 (LTB4) or formyl-methionyl-leucyl-phenylalanine (fMLP) on the microvasculature. The number, time course, and behavior of PMNs were examined in the following five steps of extravasation: (1) rolling on the venular endothelium, (2) adhesion on the endothelium, (3) passage between the endothelial cells, (4) staying in the venular wall, and (5) migration from the venular wall into the interstitial space. In either the presence or absence of DEX, topical application of LTB4 (15 pmol/50 microliters) or fMLP (10 nmol/50 microliters) caused an increase in the number of PMNs that adhered to the venules. Thus, DEX did not inhibit the adhesion of PMNs on the endothelial cells. The adhered PMNs induced by these chemoattractants became gradually smaller, finally disappearing in the vascular lumen, as observed on the monitor screen. The whole process took about 10 min. This passage of PMNs between the endothelial cells was also not inhibited by DEX, as over 90% of the adhered PMNs passed through the endothelial cells and advanced to the next step of extravasation in the presence or absence of DEX. When these chemoattractants were applied to DEX-untreated animals, the PMNs that passed through the endothelial cells stayed for about 30 min in the venular wall. Thereafter, PMNs migrated into the interstitial space. The numbers of PMNs in the interstitial space were counted at 30, 60, and 90 min after the application of chemoattractants. In the DEX-untreated animals, PMNs in the interstitial space started to appear by 30 min, and thereafter the number further increased. However, in the DEX-treated animals, the number of PMNs at 60 and 90 min was significantly suppressed. Since DEX may inhibit the synthesis and/or release of a proteinase(s) in the PMN granules, which would normally degrade the basement membrane, this inhibition may be due to the inability of PMNs to penetrate the basement membrane.     

A quantitative study of lamina densa alterations in hamster cheek pouch carcinogenesis

Lesions produced by topical application of 0.5 per cent. DMBA to the hamster cheek pouch epithelium were classified as hyperplasia, dysplasia and carcinoma groups using strict histological criteria. Untreated epithelium served as a control. Tissue samples from five animals in each group were processed for electron microscopy and electron micrographs from the epithelialconnective tissue junction were obtained from 5 blocks per animal. The micrographs were subjected to stereological intersection counting to determine the relative surface (S_SLD,BM) of lamina densa which was in normal relationship to the basal cell plasma membranes. Quantitative results indicated a progressive loss of lamina densa during carcinogenesis and this was accompanied by the extrusion of pseudopodia from the basal cells through the gaps. The pseudopodia were frequently related to peripheral cytoplasmic microfilaments. Quantitative data confirmed the progressive nature of this loss, with values for S_SLD,BM being of the order of 98 per cent., 88 per cent., 76 per cernt. and 42 per cent. for normal epithelium and for the hyperplastic, dysplastic and carcinomatous lesions respectively. The loss of lamina densa is discussed in relation to the specificity of the response and to the development of features indicative of motility in transforming cells.     

Stereological analysis of histological parameters in experimental premalignant hamster cheek pouch epithelium

Hamster cheek pouch mucosa was treated with the carcinogen DMBA in order to provide samples of epithelium in the various pathological stages of carcinogenesis. Stereological techniques were applied to histological sections of normal and carcinogen-treated epithelium in order to quantify cytological and tissue parameters that may be related to the features of epithelial atypia. The results were compared with values previously obtained for benign epithelial hyperplasia induced by a solution of oil of turpentine. Of the parameters investigated, the nuclear-cytoplasmic ratio, nuclear numerical density and volume-to-interface area ratios were best able to distinguish between benign and premalignant epithelium. The ability to ascribe actual numerical values to histological features which are usually only subjectively evaluated may be a useful diagnostic aid.     

Periarteriolar localization of mast cells promotes oriented interstitial migration of leukocytes in the hamster cheek pouch.

As studied by intravital microscopy, mast cell-dependent inflammatory reactions evoked by antigen or compound 48/80 in the hamster cheek pouch involved leakage of plasma and emigration of leukocytes exclusively from the venules. The leukocyte diapedesis and subsequent tissue migration induced by antigen or compound 48/80 were oriented from the venules towards adjacent arterioles. In contrast, leukocyte emigration induced by a mast cell-independent stimulus, leukotriene B4, did not show preferential orientation towards arterioles. Moreover, mast cells were abundant in the hamster cheek pouch, and they were localized predominantly along arterioles, rather than along venules. Because mast cells are considered to be the source of the chemotactic mediators causing the leukocyte emigration, the periarteriolar mast cell localization may be of functional significance by creating chemotactic gradients between arterioles and venules, thereby promoting oriented and effective interstitial migration of leukocytes. Whether or not a similar mechanism is operative in other species and tissues remains to be established, however, arteriolar predominance of mast cells was observed also in rat calvarial periosteum and in mouse skin.     

A quantitative cytochemical study of isolated epithelial cells of the hamster cheek pouch

1. Dissociation of the hamster cheek pouch epithelium by a method combining trypsin attack and EDTA exposure, yielded a mixture f highly viable isolated cells (basal, spinous, and granular cells). 2. Such heterogeneous cell population was reproducibly separated into 4 fractions by pycnic sedimentation: in order of increasing density, Fraction I and Fraction II predominantly contained cells of the basal layer (93% and 76%, respectively), Fraction III basal, spinous and granular cells in equivalent concentration, Fraction IV mainly granular cells (69%). Horny squamae sedimented at the bottom of the tube. 3. Individual cells of the whole population and those of the population fractions separated by density gradient, were analyzed and charcterized as regards morphology, viability, volume, dry mass, density and DNA synthesizing ability. 4. A positive correlation was found between morphology, dry mass and density of the cells, showing that differentiation occurs by increments in mass and density. The weights of the basal, spinous and granular cells were distributed within ranges well defined and scantily overlapping, and were positively correlated with cell differentiation. Also cell density increased with differentiation degree. On the contrary, volume distribution of the various types of cells showed differences of minor importance, indicating for an increase of solids concentration in the more differentiated cells. 5. DNA synthesis took place only in basal cells, the density of which was generally very low.