Changes in gastric glandular cell kinetics after small bowel resection in the rat

The proliferation of gastric mucosal cells was studied in male Sprague-Dawley rats after a 50% resection involving the proximal small intestine. Two months after the operation, resected and control animals were given either a single or four successive intraperitoneal injections of 1 muCi of [3H]thymidine per g of body weight. Animals from each group were killed 1 hr after the single injection and 3, 7, 30, and 60 days after the first multiple dose injection [3H]thymidine. The number of labeled parietal, chief, and mucous cells of the gastric glands were measured. At the 3-, 30-, and 60-day periods, the percentages of labeled parietal and chief cells were significantly increased in animals with small bowel resection when compared with corresponding controls. The percentages of labeled mucous cells of the gastric glands were also increased in animals with small bowel resection at the 3- and 7-day periods. One hour after a single injection of [3H]thymidine, the percentage of labeled gastric mucosal progenitor cells in animals with small bowel resection, although increased, was not significantly different from that in controls. It is, therefore, believed that the increases in percentage labeling of gastric glandular epithelial cells at 3, 7, 30, and 60 days could not solely be explained on the basis of an increased turnover rate of gastric progenitor cells. Rather, it might be concluded that the increase in glandular epithelial cells is accomplished, in part, by increased differentiation of gastric progenitor cells.     

Ileal epithelial cell migration after 40% small-bowel resection

Adaptation in the small bowel after resection is associated with increases in crypt cell proliferation and villus height. This paper gives the results of an autoradiographic investigation with [³H]thymidine of epithelial cell migration 60 days after 40% small-bowel resection in the rat. The mean number of cell positions between the crypt-villus junction and the leading labeled cell 30 hr after injection was increased by 19.4% in the resected group (P<0.02). The mean total number of cells per villus column was increased by 27.8% (P<0.002). Migration rate estimated in cell positions per hour was accelerated by 18.9% (P<0.001) after resection. The 8.1% lengthened life span of villus cells was not statistically significant. The increased number of cells per villus column and unaltered life span of villus cells would facilitate functional adaptation. The causal relationship between the larger villus cell population and accelerated migration after resection and increased crypt cell proliferation is unknown.This investigation was supported by the National Health and Medical Research Council.     

Epithelial cell renewal and turnover and relationship to morphologic abnormalities in jejunal mucosa in tropical sprue

A morphometric study confirmed that increasing severity of the jejunal mucosal morphologic lesion is accompanied by increased crypt height and reduced villus length in patients with tropical sprue. Mitotic activity in the crypts was increased. Pulse labeling of jejunal mucosal biopsies cultured in vitro with [3H]thymidine confirmed that there was increased uptake of label in tropical sprue with more rapid migration of labeled cells to the villi. The label was also lost more rapidly. Earlier ultrastructural studies have shown enterocyte damage and extrusion in the crypt and villus epithelium. The present data suggest that in the jejunal mucosa of patients with tropical sprue, the loss of damaged enterocytes leads to villus shortening and increased cell production in the crypts, with hypertrophy of the crypts.     

Proliferating cell nuclear antigen expression in normal, preneoplastic, and neoplastic colonic epithelium of the rat.

Expression of the proliferating cell nuclear antigen (PCNA) was examined in normal rat intestinal tissues and in carcinogen-treated nonneoplastic and neoplastic colonic mucosa. In the normal intestine, PCNA expression was confined to the expected region of the proliferative compartment. A strong correlation was observed between PCNA labeling index and both [3H]thymidine labeling index (R = 0.993, P = 0.007) and percent of cells in S phase as determined by flow cytometry (R = 0.982, P = 0.018) and between the location of the maximal staining for PCNA and [3H]thymidine (R = 0.997, P less than 0.05). In animals treated with dimethylhydrazine (DMH), crypt hyperplasia, an increased PCNA labeling index, and shifts in both the region of maximal and the upper extent of PCNA expression were observed during DMH exposure; significant crypt hyperplasia and expansion of the PCNA-positive compartment persisted after completion of DMH injections. The patterns of PCNA expression and bromodeoxyuridine incorporation were similar in DMH-induced tumors. It is concluded that PCNA immunohistochemistry can be used as a reliable marker of the proliferative compartment in both normal and neoplastic colonic mucosa.     

Delayed enzyme expression: a defect of aging rat gut

To evaluate the effect of aging upon the small intestine, the distribution, content, and concentration of epithelial cell enzymes at different levels along the crypt-villus column were measured in aging and young adult, male, Fisher 344 rats. Specific activities of sucrase, maltase, lactase, and adenosine deaminase in mucosal homogenates were lower in the upper intestines of aging than in young animals, whereas the specific activity and content of thymidine kinase was higher. Enzyme activities were measured in cells obtained by cryostat sectioning from villus tip to crypt base. Sucrase and maltase activities were fully expressed nearest the crypt, alkaline phosphatase in cells higher on the villus, and adenosine deaminase higher still, whereas thymidine kinase activity was limited to the crypts. The ordered pattern of enzyme expression was maintained in aging rats but the initiation and duration were delayed. Because peak specific enzyme activities were similar in young and aging animals, the reduced specific activities in mucosal homogenates from aging animals were due to an increase in the proportion of relatively undifferentiated villus epithelial cells. These findings are of importance in explaining altered intestinal function during aging without a concomitant change in intestinal structure.     

Effects of parenteral hydrocortisone sodium succinate on epithelial renewal in hamster gastric mucosa

The aim of this study was to determine whether parenteral administration of steroids affects epithelial renewal in hamster stomach. Male golden hamsters received either hydrocortisone sodium succinate or saline intraperitoneally for three days. In the first experiment, hamsters were sacrificed 1 hr after injection of tritiated thymidine [( 3H]TdR) to label proliferating cells. In the second experiment, hamsters were sacrificed hourly after a single [3H]TdR injection up to 48 hr in order to determine cell cycle time by the method of fraction of labeled mitoses. In the third experiment, hamsters were sacrificed 1, 24, and 72 hr after [3H]TdR injection for the study of epithelial migration and cell turnover time. Sections of fundic and antral mucosae were prepared for light autoradiography. Steroid treatment caused no gross or microscopic injury to gastric mucosa, but the number of [3H]TdR-labeled cells as well as the thickness of the proliferative zone were reduced significantly in fundic mucosa, but not in antral mucosa. The study of the fraction labeled mitoses indicated that steroid treatment lengthened the cell cycle time in fundic mucosa, which was due primarily to prolonged G1 and DNA synthesis phases. Furthermore, epithelial migration was significantly slower in fundic mucosa after steroid treatment, which was associated with a prolonged cell turnover time. Thus, parenteral steroids depress the entire process of epithelial renewal in hamster fundic mucosa.     

Migration of epithelial cells in the small intestine of mice perorally infected with coxsackievirus B5.

The rate of cell migration in the small intestine during enteric viral infections has not been assessed previously. CD-1 mice (33 days old) were infected perorally with 1.0 X 10(8) plague-forming units of coxsackievirus B5 and 12 hr later were injected intraperitoneally with 2 micron Ci of [3H]thymidine/g of body weight. After 2, 12, 24, 48, 60, and 72 hr, mice were killed, and the small intestine was removed. Specimens obtained at each interval were examined by radioautography; similar specimens were titrated for virus by plaque assay in HeLa cells. In mice perorally infected with coxsackievirus B5, epithelial cells migrated from crypt to villus tip in 60 hr, as compared with 48 hr in uninfected control mice and 24 hr previously reported for mice perorally infected with enteric bacteria (e.g., Salmonella typhimurium). Virus was recovered from intestinal tissue, but no inflammatory response in the limina propria was apparent. These observations are consistent with previous report that substrate absorption rates may be altered during viral and bacterial enteric infection.     

Cell kinetics of slow renewing cell populations in mice stomach

BACKGROUND: The renewal rates of parietal and chief cells in the gastric mucosa and smooth muscle cells of muscularis propria have not been examined as precisely as superficial epithelial cells. To examine cell renewal of these cells, continuous labeling with tritiated ([3H])-thymidine was performed. METHODS: Mice received 112 repeated injections of [3H]-thymidine at 6-hour intervals for 28 days after birth and were killed immediately thereafter, or 60, 120, 200 or 300 days after the last injection. RESULTS: After continuous labeling, most cells in the stomach were labeled. At 60 days, unlabeled parietal cells in the neck area of the gland and unlabeled chief cells in the middle part of the gland appeared. Thereafter, the area of unlabeled cells expanded downwards to the bottom of the gland. Times required for labeling of total cell populations of parietal and chief cells to half were less than 60 days and more than 200 days, respectively. At 300 days, most parietal cells and about half of the chief cells remained labeled in the bottom of the gland. The labeling index of smooth muscle cells was about 100% for 300 days. CONCLUSIONS: The time required for the newly formed parietal and chief cells to reach the lower end of the gland was more than 300 days. As a total cell population, the renewal rate of parietal cells was more rapid than that of chief cells. However, in terms of the downward migrating cell population, the renewal rate of parietal cells was a little slower than that of chief cells. Smooth muscle cells showed almost no renewal.     

Changes in the structure and regeneration mode of the rat small intestinal mucosa following benzalkonium chloride treatment

Tritiated thymidine was administered IP to rats that had been exposed to benzalkonium chloride in the duodenum, jejunum, and ileum, resulting in neuronal ablation. Epithelial cell proliferation and migration were studied 21 and 7 days after treatment. Significant hyperplasia and hypertrophy of the villi and crypts was seen from day 7 on. This was half as pronounced as that of the muscle layer, whose maximal percent increase was not seen until day 21. In the crypt, the proliferation had increased significantly (65% 3H index corrected) and its zone had expanded proportionally to the total crypt depth. After an average of 36 hours in the ileum (48 hours in normal rats), labeled cells reached the tip of the lengthened villi, reflecting significantly accelerated migration. Concerning the distributional pattern of the labeled cells in the crypt, a nonsignificant shift to the lower two thirds of the crypt could be distinguished. From this the author concludes that treatment with benzalkonium chloride influences the proliferation and migration of the epithelial cells in the treated area. These alterations may result from loss of the myenteric plexus, but other factors cannot be excluded.     

Dominance of the Senescent Phenotype in Heterokaryons Between Replicative and Post-Replicative Human Fibroblast-Like Cells

In heterokaryons between senescent and young diploid fibroblast-like cells, dominance of the former with respect to nuclear DNA synthesis (incorporation of [³H]thymidine) was demonstrated. For identification of the respective partners, double-layer autoradiography was used after the old cells were labeled with [³H]methionine and the young cells were labeled with [¹⁴C]thymidine. Synchrony of nuclear labeling (i.e., all nuclei in a cell labeled with [³H]thymidine) was observed in the majority of di- and polykaryons during the second and third of three 24-hr periods of labeling with [³H]thymidine. The results are compatible with either terminal differentiation or error theories of clonal senescence.     

Prostaglandins and the colonic epithelium. Effects of misoprostol on crypt size, cell production, and cell migration in the dog

Colonic epithelial cell division, cell migration, and cell transit were investigated in dogs given 300 micrograms.kg-1.day-1 of the prostaglandin E1 analogue, misoprostol, for 11 weeks. The animals were then injected with [3H]thymidine and killed at timed intervals. The distribution of labeled and mitotic cells within the crypts was determined by scoring autoradiographs. There were no significant differences in mitotic index or labeling index between the two groups. The data were pooled and converted to give crypt cell production rates of 51.1 +/- 11.1 (control) and 58.24 +/- 8.6 cells per crypt per hour (test). However, the crypt length and cell population were slightly, but significantly, greater in the misoprostol-treated group (P less than 0.01). The movement of the wave of labeled cells with time after injection was used to calculate the median cell migration rates, which were 23.50 +/- 3.03 cell positions per day (control) and 18.30 +/- 2.56 (test). Thus, misoprostol had no significant effect on either the cell migration rate or the transit rates.     

Distribution and properties of Ca2+-ATPase, phytase, and alkaline phosphatase in isolated enterocytes from normal and vitamin D-deficient rats.

The effects of vitamin D-deficiency and repletion on the distribution and activities of Ca2+-ATPase, phytase, and alkaline phosphatase in intact epithelial cells isolated from different regions of the villi and the crypts of the rat jejunum were studied. Similar distribution patterns of activities were found for the three enzymes. In all cases, the enzyme levels were the highest at the villus tip and gradually declined to low activities in the crypt. The Kms were very different between cells in the crypt base and those at the villus tip, the highest Kms being found in the crypt. The activities of these enzymes were reduced in the entire length of the villus in vitamin D-deficient rats. Recovery of the enzymatic levels was observed on vitamin D repletion, but at different rates. Total recovery of activity of Ca2+-ATPase, phytase, and alkaline phosphatase was observed after 18, 24, and 36 hours, respectively, after a single dose of 6.5 nmol (2.5 micrograms) vitamin D3. Enzymatic activities in the crypt cells were not affected by vitamin D3 treatment. These data suggest that Ca2+-ATPase, phytase, and alkaline phosphatase may be distinct entities, and that their activities in the crypt cells may not be vitamin D-dependent.     

Spatial differentiation of the intestinal epithelium: analysis of enteroendocrine cells containing immunoreactive serotonin, secretin, and substance P in normal and transgenic mice.

The mammalian intestinal epithelium undergoes continuous and rapid renewal of its four principal terminally differentiated cell types. These cells arise from multipotent stem cells located at or near the base of the crypts of Lieberkühn. The differentiation process is precisely organized along two spatial dimensions (axes)--from the crypt to the villus tip and from the duodenum to the colon. The enteroendocrine cell population provides a sensitive marker of the intestine's topologic differentiation. At least 15 different regionally distributed subsets have been described based on their principal neuroendocrine products. We have used immunocytochemical methods to characterize the spatial relationships of the serotonin-, secretin-, and substance P-containing enteroendocrine cell subsets in normal adult C57BL/6J x LT/Sv mice as well as in transgenic littermates that contain rat liver fatty acid-binding protein-human growth hormone fusion genes. Our results reveal precise spatial interrelationships between these populations and suggest a differentiation pathway that may involve the sequential expression of substance P, serotonin, and secretin.     

The Profile of Antioxidant Systems and Lipid Peroxidation Across the Crypt-Villus Axis in Rat Intestine

The distribution of lipid peroxiation and profile of antioxidant-pro-oxidant enzyme systems have been studied in rat intestinal enterocyte across the length of villi. The MDA levels estimated as a measure of lipid peroxidation, under induced or uninduced in vitro conditions, indicated markedly high levels at villus tip cells compared to that in the crypt base. The activities of glutathione-S-transferase and glutathione reductase were three- to sixfold higher in villus tip cells compared to that in the crypt base. However the levels of catalase and superoxide dismutase showed a reverse pattern, being high in the crypt base and lowest in the villus tip region. Feeding coconut oil, sunflower oil, or groundnut oil did not modify the distribution pattern of these systems across crypt-villus unit in rat intestine. These findings suggest that the large amount of free radicals generated in villus tip cells may be responsible for the release of enterocytes from the villus tip as a consequence of apoptosis.     

Mucosal cell proliferation of the rectal stump in ulcerative colitis patients after ileorectal anastomosis

The proliferative activity and polyamine levels of the rectal epithelium in unoperated ulcerative colitis patients and in ulcerative colitis patients after total colectomy and ileorectal anastomosis were determined and compared with control subjects. Cell proliferation was evaluated in rectal biopsies by in vitro 3H thymidine incorporation by measuring the labeling index and the position of labeled cells along the crypt; polyamines were determined with a chromatographic method. In ulcerative colitis patients the labeling index was significantly increased, and labeled cells were shifted toward the upper part of the crypt when compared with controls. Ileorectal anastomosis patients showed a normalization of the labeling index and a distribution of labeled cells similar to controls. Polyamine levels were also increased in ulcerative colitis patients; in ileorectal anastomosis patients, the level of polyamines was decreased in respect to unoperated patients and return to normal values except for spermine. Because the increased proliferation and higher polyamine levels are related to increased colon cancer risk, our results confirm that ulcerative colitis is a risk factor for the development of carcinoma. Ileorectal anastomosis may reduce this risk through a normalization of mucosal cell proliferative activity and of some polyamine levels.     

Glutathione and gamma-glutamyl cycle enzymes in crypt and villus tip cells of rat jejunal mucosa.

Villus tip cells and crypt cells of rat jejunal mucosa were separated by the planning procedure of Imondi et al. and were studied with respect to their activities of the enzymes of the gamma-glutamyl cycle and glutathione content. The villus tip cells exhibit much higher gamma-glutamyl transpeptidase activities than do the crypt cells: thus, gamma-glutamyl trnaspeptidase appears to be a villus-specific enzyme. gamma-Glutamyl cyclotransferase and the enzymes required for glutathione synthesis are not specifically localized to either the crypt or villus tip cells but are present in both. The crypt cells have a high concentration of glutathione (4-5 mM) comparable to the levels found in liver and kidney; in contrast, the villus tip cells have much lower concentrations. On fasting, the glutathione concentration decreased markedly in both villus tip and crypt cells; feeding of protein, but not of sucrose, led to increased glutathione concentrations. The migration of cells from the undifferentiated crypt cell region to the villus tip is associated with structural and biochemical changes that equip the cell for its mature functional activities, which include transport. The present findings indicate that such cellular differentiation and migration is associated with a marked increase in gamma-glutamyl transpeptidase activity and in the utilization of glutathione.     

Effect of Fat Feeding on Pro-oxidant and Anti-oxidant Enzyme Systems in Rat Intestine: Possible Role in the Turnover of Enterocytes

Immature epithelial cells generated in the crypt base undergo differentiation while progressing to the villus tip, where the cells upon apoptosis are detached from the underlying muscular tissue. We previously reported that lipid peroxidation might be involved in the turnover of enterocytes across the crypt–villus axis in rat intestine (Dig Dis Sci 52:1840–1844, 2007). To examine whether long-term feeding of fat with different fatty-acid composition influences this process, in the present study we investigated the effect of feeding fish oil (n − 3) and corn oil (n − 6) polyunsaturated fatty acids on lipid per-oxidation and anti-oxidant systems in different epithelial cell fractions isolated in rat intestine. Feeding fish oil or corn oil markedly enhanced lipid per-oxidation levels of enterocytes throughout villus height compared with control, but there was no difference in the distribution profile of pro- and anti-oxidant enzyme systems and lipid per-oxidation across the crypt–villus axis under these conditions. Analysis of lipid peroxidation levels in different cell fractions revealed that the thiobarbituric acid reactive substance were 9- to 11-fold higher at the villus tip compared with at the crypt base. The activities of glutathione reductase and glutathione-S-transferase were 2- to 5-fold higher in villus tip compared to the crypt region. However, the activities of superoxide dismutase and catalase were 6- to 8-fold high at the crypt base compared with at villus tip cells. Immunocytolocalization of superoxide dismutase showed high staining in crypt base compared with that in villus, tip cells. These findings further suggest that generation of reactive oxygen species in enterocytes across the crypt–villus axis may be involved in turnover of enterocytes across the crypt–villus unit in rat intestine.     

Birth of projection neurons in the higher vocal center of the canary forebrain before, during, and after song learning.

The higher vocal center (HVc) of the canary brain projects to two forebrain nuclei: robustus archistriatalis (RA) and area X of lobus parolfactorius. The time of birth of HVc neurons projecting to these two regions was determined by combining [3H]thymidine autoradiography and retrograde fluorogold uptake. Birds were sacrificed at 13 months of age, 4 days after fluorogold injections into area X or RA. A single injection of [3H]thymidine in ovo (embryonic day 9) labeled 76% of area X-projecting cells and 0.8% of cells projecting to RA. The great majority of RA-projecting cells were produced during posthatching development (posthatching day 10-240; P10-P240), with a peak at P60 and a hiatus at P120. HVc reaches full adult size by P240, yet at that age the production of new RA-projecting cells continued at a rate comparable to that recorded during posthatching development. Late production of neurons interconnecting two distant regions of the brain may regulate source to target cell population size. Male canaries start to sing at P40. During subsequent months, they imitate external models and their song becomes more structured and stereotyped. At sexual maturity (P240), song is stable. Three interpretations are offered: (i) neurogenesis of RA-projecting cells is related to learning, and learning continues even after achievement of pattern stability; (ii) neurogenesis of RA-projecting cells is not related to learning; (iii) the production of RA-projecting cells serves different purposes during development and after sexual maturity.     

Development of a new Leydig cell population after the destruction of existing Leydig cells by ethane dimethane sulphonate in rats: an autoradiographic study

The formation of new Leydig cells in adult male rats was studied after the complete destruction of the original population by ethane dimethane sulphonate (EDS). Following administration of EDS, proliferating interstitial cells were labelled in a pulse-chase experiment by way of three [3H]thymidine injections on days 2, 3 and 4 after EDS administration. Some of the newly formed Leydig cells found 14 days after EDS administration were labelled with [3H]thymidine, indicating that these Leydig cells were derived from precursor cells, most likely mesenchymal cells, that had incorporated [3H]thymidine at days 2, 3 or 4 after EDS administration. At 21 days after EDS administration, the total number of Leydig cells (labelled plus unlabelled) had increased 7- to 16-fold compared with the number of cells that were present 14 days after EDS had been administered. In a second series of experiments, [3H]thymidine was given 2 h before the rats were killed (short-term labelling experiment). In this experiment it was shown that the proliferative activity of the mesenchymal cells, which are presumed to be the precursors of the Leydig cells, after a considerable increase at day 2 after EDS administration, had returned to the control level at day 7. However, the total number of mesenchymal cells (labelled plus unlabelled) remained increased from 2 to 49 days after EDS administration. This indicated that the majority of the new Leydig cells which were formed from day 14 onwards probably did not derive from differentiating mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of isolated duodenal epithelial cells along a cryptvillus axis in rats fed diets with different iron content

The intestinal mucosa is characterized by cell proliferation, commitment, differentiation, digestion and absorption. These processes occur at specified locations along the crypt to villus axis. A technique is reported for the isolation of cells along this axis which allows the study of any one of these processes in an enriched population of cells. As an example, the uptake of transferrin-bound iron by enterocytes was studied. Rats were fed diets normal, high (3% carbonyl iron) or low in iron for 12 days. Cells from either the duodenum or ileum were isolated by incubating in a Ca²⁺-, Mg²⁺-free, cation chelating solution for varying periods. The incorporation of thymidine into DNA was measured in these cells as a marker of the crypt region, while alkaline phosphatase and sucrase activities marked mature enterocytes. The in vivo uptake of transferrin-bound ⁵⁹Fe was measured in cells isolated either 2 or 4 h after intravenous injection. This procedure resulted in the isolation of 10 fractions of viable cells. Earlier fractions were enriched at least 10-fold in villus cells and the last fractions in crypt cells. Cells in intermediate fractions were at various stages of development. Uptake of transferrin-bound iron into enterocytes was highest with feeding an iron-loaded diet compared with control or iron-deficient diets. However, with all diets uptake was highest in crypt cells and this fell at the crypt-villus junction to be only 25%, as high at the villus tip as the crypt. A technique for the reproducible isolation of viable enterocytes along a crypt-villus axis is described. Transferrin receptor activity changes with maturation of the enterocyte.