Ultrastructural and autoradiographic observations of hamster cheek pouch epithelium grown in vitro

Summary Epithelial outgrowths from hamster cheek pouch explants were cultured for varying peroids of time up to 22 days. Growth of the epithelial sheets was monitored, employing colcemid for demonstrating mitotic activity and tritiated thymidine for DNA synthesis. Mitoses and thymidine uptake were observed among epithelial outgrowths at a considerable distance from the original explant. The epithelial nature of the growing cell sheets was confirmed, employing electron microscopic techniques. The cells exhibited the presence of tonofilaments, desmosomes, ribosomes, Golgi, mitochondria, and rough endoplasmic reticulum. The cultured explants were treated with cyclic nucleotides in order to investigate their modulatory effects on epithelial cell differentiation. Dibutyryl cAMP induced marked mitotic inhibition (46.3%) in our assay, which was increased to 57% with the addition of theophylline. Dibutyryl cGMP showed only a mild (5%) stimulatory effect on mitotic activity. Dibutyryl cAMP enhanced keratinization in the epithelial cell out-growths with the biogenesis of keratohyalin granules, whereas dibutyryl cGMP did not produce any observable alterations.     

Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Abstract. Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine ([³H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.     

Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine [( 3H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.     

Histomorphology of the hamster cheek pouch

Histomorphology of the cheek pouch was studied in 14 hamsters by light and transmission electron microscopy. The cheek pouch wall was devoid of any lymphatic tissue and dense subepithelial tissue (i.e. the lamina propria) would render lymph drainage almost impossible and might constitute impermeable morphological barrier for non-recognition of transplants evoking a host immune response. Because in the literature it was reported that there is absence of any arteriovenous anastomoses on the pouch wall, and interruption of arterial supply failed to alter the growth rate of tissue grafts, we speculated that epidermal growth factors present in the saliva could play a role in maintaining the growth of tissue transplants.     

Circadian-stage dependence of methotrexate in a keratinized epithelium. An in-vivo study using flow cytometry on the hamster cheek pouch epithelium

The partially synchronized cell system of the hamster cheek pouch epithelium shows a characteristic diurnal rhythm of cell proliferation. Bolus injections of methotrexate (Mtx) in both lethal (10 g/m2) and non-lethal (2 g/m2) doses were found to inhibit cell-cycle progression primarily by impairing the G1/S transition. The results were obtained by flow cytometric DNA analysis. The inhibitory effect of Mtx manifested itself as a relative decrease of the S fraction (drug-effector phase), and was found to be dependent both on the dose and on the time of the day it was given. A bolus injection of Mtx was given either at 1200 hr (when a minimal number of cells are in S phase) or at 0200 hr (when a maximum number of cells are in S phase). The greatest cumulative decrease in S fraction was seen when the injection was given at 1200 hr. The time between injection and the effect (seen as a decrease in S fraction) was independent of the time of the Mtx injection, but seemed instead to be related to the natural diurnal period of increasing flux from G1 to S phase (at the onset of the dark period). The main effect (the relative decrease in S fraction) was repeated during the following 24-hr period, pointing to a protracted effect of Mtx on G1 cells. G1 cells affected by the initial high Mtx plasma concentration seem to be responsible for the reduced influx into S phase in both the first and second 24-hr period. In earlier toxicological studies, the survival rate of hamsters was dependent on the time of injection and was highest after injection at 1200 hr. Thus maximum cytokinetic effect on epithelial cells was found at the time of the day when there was a minimum lethal effect on the animal.     

Ultrastructural changes in contents of membrane-coating granules after extrusion from epithelial cells of hamster cheek pouch

Summary Membrane-coating granules of the epithelium of the hamster cheek pouch displayed a lamellated internal structure consisting of alternating thick and thin electron dense bands separated by translucent bands of equal width. The repeat distance of the contents was 8.5nm±sd 0.7nm and the thickness of the major electron dense band was 3 nm±sd 0.4 nm. After exocytosis from the cells the lamellated material became organised into extensive sheets and the dimensions changed so that the repeat distance was 10.9 nm±sd 0.9 nm but the major electron dense band was virtually unchanged at 3.3 nm±sd 1.1 nm. The narrower or intermediate dense band became progressively less easy to see and was virtually invisible at the surface of the epithelium.It is suggested that the changes observed may be due to an increase in hydration of a phospholipid/protein complex in the granules by permeation of water along the intermediate dense band.     

Experimental paracoccidioidomycosis of hamster inoculated in the cheek pouch

We compared the granuloma morphology and immune response of hamsters inoculated withParacoccidioides brasiliensis (Pb) into the cheek pouch, which lacks lymphatic drainage, and into the footpad, which is rich in lymphatics. Our objective was to better understand the modulation ofPb granuloma in an immunocompetent animal inoculated in an immunologically privileged site. The humoral immune response (ELISA) and cell mediated immunity (footpad test) became positive on days 7 and 14, respectively in animals inoculated into footpad and on days 35 and 60 in animals inoculated into the pouch. Typical epithelioid granulomas were observed at both sites on day 14. The number of fungi gradually decreased from the beginning of the experiment in footpad lesions, but only after day 35 in pouch granulomas, when cell mediated immunity was detectable. The results indicate that typical epithelioid paracoccidioidomycotic granulomas may develop in the absence of a detectable immune response; however, they are incapable of controlling fungal reproduction. Lack of lymphatic drainage delays the appearance of a detectable immune response, but with time fungi escape from the pouch, elicit an immune response and reach other organs. Our results further indicate the importance of the lymphatics in the pathogenesis of paracoccidioidomycosis.Abbreviations HCP hamster cheek pouch - Pb Paracoccidioides brasiliensis - Pbmycosis Paracoccidioidomycosis     

Free-radical inhibition of ATPase in hamster cheek pouch homogenates

The effects of free-radicals generated by either the oxidation of hypoxanthine by xanthine oxidase (HX/XO) or the lipoxidation of arachidonic acid (AA) on the ATPase of the hamster cheek pouch has been studied. Cheek pouches were removed from female golden syrian hamsters and homogenized. ATPase activity was measured by the production of Pi at 37 degrees. HX/XO and AA were added at a final concentration of 9.6 X 10(-5) M HX with 5 X 10(-2) units HX and 5 X 10(-5) M AA with and without 1 X 10(-4) M ouabain. HX/XO produced a 24.7% inhibition alone and 35.0% when combined with ouabain. Ouabain alone produced a 7.1% inhibition. AA produced a 23.6% inhibition alone and 24.3% inhibition when combined with ouabain. Ouabain alone produced a 5.4% inhibition in this series. When AA was added in doses ranging from 1 X 10(-5) to 2 X 10(-3) M, a plot of percent inhibition versus log dose followed a typical sigmoid type curve. The IC50 was 1.5 X 10(-4) M. These results suggest that free-radicals are capable of inhibiting the ATPase found in the hamster cheek pouch tissues. The possible modes of action of the free-radicals in producing this inhibition are discussed.     

Low-level X-radiation effects on functional vascular changes in Syrian hamster cheek pouch epithelium during hydrocarbon carcinogenesis

Effects of repeated low-level X radiation on functional microvascular changes in hamster cheek pouch epithelium during and following carcinogenesis by 7,12-dimethylbenz[a]anthracene (DMBA) were studied. Prior studies showed enhancement of such carcinogenesis by repeated 20 rad head and neck X-radiation exposures, and it was proposed that one possible mechanism was radiogenic alteration of the functional microvasculature in a manner which favored subsequent tumor development. Hamsters were treated with either radiation, DMBA, radiation + DMBA, or no treatment. Animals were sacrificed at 3-week intervals from 0 to 39 weeks after treatments began. Pouch vascular volume and permeability changes were studied by fractional distributions of radiotracers and were analyzed by a variety of statistical methods which explored the vascular parameters, treatment types, elapsed time, presence of the carcinogen, and histopathologic changes. All treatments resulted in significant changes in vascular volume with time, while only DMBA treatments alone resulted in significant changes in vascular permeability with time. Prior to the appearances of frank neoplasms, volumetric changes in DMBA only and radiation only groups were similar, while volume changes in DMBA + radiation groups increased slowly to a peak later than in other groups and then declined steadily to levels similar to the radiation only group. As in prior studies, there were significant vascular volume differences between DMBA and DMBA + radiation groups of tumor-bearing cheek pouches. DMBA maxima were significantly higher than those of DMBA + radiation. Radiation significantly affected DMBA-associated vascular volume and permeability changes during carcinogenesis. Several possible explanations for the relationship of these changes to the enhancement of DMBA carcinogenesis include: radiation blocking normal capillary proliferative and/or dilatory responses to inflammation secondary to neoplastic changes; radiation-induced focal increases in the pericapillary connective tissue histohematic barrier, stimulating angiogenesis but reducing nutrient diffusion; radiation exposures sensitizing vascular endothelium to subsequent angiogenic stimulation from premalignant tissues; DMBA vascular and epithelial effects partially or completely blocking radiation effects on epithelial and/or endothelial cells; and radiation damage to vessel walls partially or fully inhibiting normal physiologic mechanisms of repairing DMBA damage to the vessels.     

Tyrosine kinase inhibitors modulate agonist-induced vasodilation in the hamster cheek pouch.

The purpose of this study was to determine whether inhibitors of tyrosine kinase attenuate vasodilation elicited by endogenously elaborated and exogenously applied nitric oxide in the in situ peripheral microcirculation. Using intravital microscopy, we found that pretreatment with genistein (1.0 microM) and tyrphostin 25 (10.0 microM), two structurally unrelated tyrosine kinase inhibitors, significantly attenuated acetylcholine-, bradykinin- and nitroglycerin-induced dilation of second-order arterioles (51 +/- 1 microm) in the in situ hamster cheek pouch (P < 0.05). Both inhibitors nearly abrogated acetylcholine-induced responses but only partially blocked bradykinin- and nitroglycerin-induced vasodilation. Genistein and tyrphostin 25 alone had no significant effects on resting arteriolar diameter and on adenosine-induced vasodilation in the cheek pouch. On balance, these data indicate that tyrosine kinase inhibitors attenuate endogenously elaborated and exogenously applied nitric oxide-induced vasodilation in the in situ hamster cheek pouch. However, the extent of tyrosine kinase inhibitor-sensitive pathway involvement in this response appears to be agonist dependent.     

Effects of tetraiodothyronine and triiodothyronine on hamster cheek pouch microcirculation

The aim of the present study was to assess the effects of topically applied triiodothyronine (T(3)) and thyroxine (T(4)) on the arterioles of hamster cheek pouch microcirculation in vivo. Microvessels were visualized using a fluorescent microscopy technique. Topical application of T(3) (3.08, 30.8, 61.5, 307, 615, and 6,150 nM/l) consistently induced dose-dependent dilation of arterioles within 2.0 +/- 0.5 min of administration. The application of T(4) (150, 257, 514, and 5,140 nM/l) caused different dose-dependent effects: dilation at the three lower doses within 16 +/- 2 min and rhythmic diameter changes at the highest dose. Aging of hamsters did not alter the arteriolar responses to T(3) and T(4). T(3)-induced dilation was countered by the inhibition of nitric oxide synthase with N(G)-nitro-L-arginine-methyl ester or N(G)-nitro-L-arginine. Iopanoic acid (IPA), which inhibits types I and II 5'-deiodinase, abolished the dilation elicited by 514 nM T(4) but did not affect T(3)-dependent dilation. 6-Propyl-2-thiouracil (PTU), which inhibits type I 5'-deiodinase only, did not affect the dilation induced by T(4). IPA and PTU did not impair arteriolar dilation induced by acetylcholine or sodium nitroprusside. These results indicate that T(3) induces arteriolar dilation, likely through nitric oxide release. The local conversion of T(4) to T(3) appears to be crucial for the dilation induced by T(4).