Trophic actions of E2 prostaglandins in the rat gastrointestinal mucosa. A quantitative morphologic study

Adult, male Sprague-Dawley rats in groups of 10 received one of the following treatments orally twice daily for 3 wk: prostaglandin E2 (PGE2) 7.5 mg X kg-1, 15(R)-15-methyl prostaglandin E2 (MePGE2) at 0.2 or 2.0 mg X kg-1, or vehicle. After 18 h of fasting and 10-12 h after the last dose, the rats were anesthetized, and the gastrointestinal tract was fixed and processed for macroscopic and microscopic investigations. Trophic changes were more pronounced in the gastric antrum than in the gastric corpus or small intestine. The thickness of the antral mucosa was significantly increased by PGE2 and in a dose-related way by MePGE2. The mucosa of the gastric corpus became significantly thicker only with the higher dose of MePGE2. In all the prostaglandin-treated groups, the proportion of endocrine cells was reduced. Small--but sometimes significant--changes were registered in the proportions of the various exocrine cells. The parietal cells became significantly larger (+88%) in the rats treated with high doses of MePGE2. The secretory surface of the parietal cells was markedly increased by PGE2 and MePGE2. The enlargement of the secretory surface in animals treated with prostaglandins corresponded to a marked elevation of the basal gastric acid secretion and an increase in plasma gastrin levels. Hypergastrinemia can explain some, but not all, of the trophic changes observed in this study. Light microscopic examination of the duodenal and jejunal mucosa showed dose-related increases in villus heights and crypt lengths after treatment with MePGE2. Only the duodenal villus heights were increased by PGE2.     

Cell kinetics of rat gastrointestinal mucosa. Autoradiographic study after treatment with 15(R)15-methyl-prostaglandin E2

Forty Sprague Dawley rats (120 g) were divided in groups of five rats each, and 2 mg , kg-1 15(R)15-methyl-prostaglandin E2 or vehicle was administered orally, twice daily for 5 days. On the 6th day, 1 mCi . kg-1 methyl-3H-thymidine was given intraperitoneally to all rats. Groups of rats were killed at 45 min and 24 h, 72, and 120 h after the labelling. Treatments were continued until death. Samples were taken from the corpus, antrum, and jejunum and processed for autoradiography. Microscopic evaluation of the proliferative and functional compartments included cell counts and determination of the labelling index (LI) and mitotic index (MI). Prostaglandin treatment increased the number of cells in the jejunal and gastric epithelia. The DNA synthesis, evaluated from the LI and 45 min after thymidine injection, was unaffected by treatment. The clearance of label from jejunal crypts and villi was significantly slower in the prostaglandin groups. Similar observations were made in the proliferative zone of the corporal and antral epithelia. The MI was unchanged or reduced by prostaglandin treatment, the reduction being significant after 8 to 10 days' treatment. It is suggested that the trophic action of prostaglandin E2 is produced by reduction of epithelial cell losses, thereby prolonging the cell survival time. The reduced MI may be secondary to negative feedback from the hyperplastic epithelium. Trophic actions are produced by short-term treatments.     

Initial kinetic changes of prostaglandin E2-induced hyperplasia of the rat small intestinal epithelium occur in the villous compartments

This study was performed to further identify the sequence of cell kinetics that occurs in the development of gastric and intestinal epithelial hyperplasia after orally administered prostaglandins of the E series. A high-dose, short-treatment schedule was used to examine the initial effects on kinetic parameters in the rat small intestinal epithelium. Groups of rats were killed after a single dose of oral prostaglandin E2 at 1 h after in vivo labeling with [methyl-3H]thymidine and during continued treatment at 6, 12, 24, 48, 72, and 96 h. As evidenced by autoradiography, the earliest change produced by prostaglandin E2 was an increased cellularity of the villous compartment (p less than 0.05 after 24 h). There was no change of labeling index of the villous compartment or of the leading edge of labeled cells within 24 h. At 48 h, the increased cellularity was accompanied by a significantly elevated labeling index of the villi. Throughout the study period no significant differences were observed between groups in the number of cells or labeling indices in the jejunal crypts, or in cellular input from the crypts to the villi. Epithelial turnover time in the placebo and treatment groups was 69 and 71 h, respectively. To exclude the possibility that prostaglandin E2 initially affects cell birth rate and mean cell cycle time, a metaphase blocker was given after 4 days of treatment in a second study. Animals were killed after 0, 0.5, 1.0, 1.5, and 2.5 h. The rate of entry into mitoses was 8.1% cells/h in controls compared with 8.2% cells/h in treated rats. The distribution of mitoses within crypts was identical in the two groups and the mean cell cycle time was 13.6 and 13.2 h, respectively. Also in this study there were trophic changes of the villi. It is concluded that the hyperplasia produced by oral prostaglandin E2 starts in the villi of the small intestine and is initiated by reduced cell exfoliation from the villous tips. Previously recorded retention of cellular elements in villi and crypts, increased cellularity of the proliferative compartments, and reduced mitotic index are secondary events.     

Quantitative alterations in the structural development of jejunal absorptive epithelial cells and their subcellular organelles in protein-energy-malnourished rats: a stereologic analysis

The effect of protein-energy malnutrition (PEM) on the structural development of rat jejunal absorptive epithelial cells was evaluated. Stereologic characteristics of jejunal histologic subcompartments, epithelial cell surfaces and volumes, and volumes of key subcellular organelles (nuclei, mitochondria, Golgi apparatus, lysosomes) in crypt and villus mid and tip cells were determined by morphometric analysis of light and electron micrographs in well-nourished rats and rats with PEM. In rats with PEM there were developmental alterations in cells migrating on villi that were not seen in crypt cells. The amplification of the apical microvillus surface between mid and tip levels in well-nourished rats (679-1085 micron2/cell) failed to occur in rats with PEM (807-729 micron2/cell). This resulted in an estimated 60% reduction of total jejunal absorptive surface, from 10,070 cm2 in well-nourished rats to 3975 cm2 in rats with PEM. In contrast, the development of the basolateral surface, which requires much less membrane accrual, was unaffected by PEM. Villus mid and tip cells of rats with PEM also had increased cell volume and mitochondrial volumes. Microvillus surface area per cell appears dependent on the number of microvilli per cell, which equals the cell flat surface times the microvillus numerical density (number of microvilli per square micrometer) in well-nourished rats. However, this relationship was not demonstrable in rats with PEM.     

Cell proliferation of the rat gastrointestinal mucosa after treatment with E2 prostaglandins and indomethacin

The frequency of arrested mitoses after vincristine injection was studied in the gastrointestinal mucosa of rats treated with either natural prostaglandin E2 (0.2-5.0 mg X kg-1, b.d.), 15-R-15 methyl prostaglandin E2 (2 mg X kg-1, b.d.) or indomethacin (1.0-3.0 mg X kg-1, b.d.). In addition to the mitotic index, morphometric measurements including the mucosal thickness and the thickness of the proliferative and functional zones of the gastric corpus, antrum and jejunum were performed. Natural prostaglandin E2, at the highest dose range, reduced significantly the mitotic index in the gastric antrum. Normal values were found in the gastric corpus and jejunum and in the antrum with the lower doses. The mitotic index was unaffected by treatment with 15-R-15 methyl prostaglandin E2. Natural prostaglandin E2 produced trophic changes (i.e. increased thickness and/or hyperplasia) in the antrum, functional epithelial zone of the gastric corpus and in the jejunum. More pronounced trophic changes were observed in the mucosa of rats treated with the analogue. Indomethacin reduced the mucosal thickness in all examined epithelia and lowered the mitotic index in the jejunum. It is concluded that the trophic effects of E2 prostaglandins on gastrointestinal epithelia are not caused by increased production of new cells. The reduced mitotic index observed in the antral mucosa of prostaglandin-treated rats could be secondary to a negative feedback from the hyperplastic epithelium. The antitrophic effects of the prostaglandin-synthesis blocker (indomethacin) indicates that endogenous prostaglandins may participate in the epithelial cell regulation of the gastrointestinal tract.     

Methods for the determination of epithelial cell kinetic parameters of human colonic epithelium isolated from surgical and biopsy specimens

The purpose of this study is to introduce the application of new approaches to the determination of human colonic epithelial cell kinetic parameters. The isolation of pure and intact colonic epithelium from both surgical and biopsy specimens forms the basis of these approaches. The isolated epithelium is used in the determination of cell kinetic parameters by (a) flow cytometry, (b) Coulter counting, (c) dried cell preparations, and (d) crypt squashes. Using these methods, the following results were derived. The proportion of cells in the various phases of the cell cycle in the sigmoid epithelium was determined to be 81.6% +/- 2.15% (means +/- SE) in G1/G0 phase, 15.2% +/- 1.86% in S phase, and 3.2% +/- 0.62% in G2 + M phases. In the rectal epithelium, there were 79.6% +/- 3.35% in G1/G0 phase, 16.4% +/- 4.86% in S phase, and 4.08% +/- 1.90% in G2 + M phases. The total cell population in sigmoid epithelium was approximately 4.2 X 10(6) +/- 5.46 X 10(5) cells per cm2, and there were approximately 2.5 X 10(3) +/- 1.57 X 10(2) cells in each colonic crypt. Therefore, the number of crypts per square centimeter of human sigmoid mucosa could be estimated to be approximately 1.68 X 10(3). Lastly, in sigmoid epithelium, columnar cells of human sigmoid mucosa could be estimated to be approximately 1.68 X 10(3). Lastly, in sigmoid epithelium, columnar cells accounted for 76.3% +/- 6.18% of the epithelial cells, whereas the remaining epithelial cells, 23.7% +/- 4.08%, consisted of mucous cells.     

HLA-DR expression, natural killer cells and IgE containing cells in the jejunal mucosa of coeliac children.

The expression of HLA-DR by surface and crypt epithelium and the numbers of cells of natural killer (NK) phenotype and of IgE containing cells were studied with monoclonal antisera using the peroxidase technique. We examined 48 jejunal biopsy specimens taken from 35 coeliac children before treatment (11), during gluten free diet (20) and after gluten challenge (17), and 13 control specimens. The luminal surface of the epithelial cells stained with HLA-DR antiserum in all specimens, but the cytoplasm of the surface epithelial cells took up the stain more frequently in the specimens from the controls (5/13) than those from the coeliacs (2/48) (p less than 0.01). In 21/28 specimens taken from coeliacs when on a gluten containing diet the crypt epithelium showed strong HLA-DR expression, while only 4/20 (p less than 0.01) specimens of coeliacs on a gluten free diet and 1/13 specimens of controls had similar staining. Among the intraepithelial lymphocytes no cells of NK phenotype were found in specimens from patients or controls. As compared with control specimens biopsy specimens from untreated coeliac patients showed smaller numbers of NK cells in the lamina propria. No difference was found in the numbers of IgE containing cells between the patients and controls. The strong expression of HLA-DR by the crypt epithelial cells in coeliac children on a normal diet suggest that these cells are involved in the presentation of the antigen.     

HIV enteropathy: crypt stem and transit cell hyperproliferation induces villous atrophy in HIV/Microsporidia-infected jejunal mucosa

OBJECTIVES: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. DESIGN: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. METHODS: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. RESULTS: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. CONCLUSION: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.     

Small intestinal villous pattern and mucosal dynamics in healthy Jamaicans

Twenty intestinal biopsy specimens from Jamaican subjects who did not have evidence of gastrointestinal disease were examined under the dissecting microscope and histologically. The appearances were normal in all 20. The most common finding was a mixture of finger- and leaf-shaped villi. Histologically, the biopsy specimens were also normal, consisting of long slender villi with short crypts and very few inflammatory cells. The crypt length; adult crypt cell ratio, mitotic index and turnover time were normal. These findings indicate that the set of circumstances causing and perpetuating the abnormalities seen in the jejunal biopsies of subjects living in the tropics, do not exist in Jamaica, and that the small intestinal villous appearance and mucosal dynamics in the healthy Jamaican Negro are similar to those of Europeans and North Americans.     

Differentiation status of rat enterocytes after intestinal adaptation to jejunoileal bypass.

The differentiation status of epithelial cells in intestinal adaptation remains unclear. To determine whether enterocytes reach optimum maturity following adaptation after 85% shortening of the rat gut by jejunoileal bypass surgery, activities of two brush border enzymatic markers of differentiation, alkaline phosphatase and sucrase, were examined in subpopulations of epithelial cells isolated sequentially from the villus/crypt axis of normal (sham operated) and hyperplastic mucosa. In jejunal villi, adaptational hyperplasia was associated with an increase in total epithelial alkaline phosphatase, but not total sucrase, activity; alkaline phosphatase activity increased most obviously in cells at the 11-50% position (from the tip) on villi. In hyperplastic ileal villi, total alkaline phosphatase activity fell, although sucrase activity did not change significantly. Specific activity (per mg protein) of sucrase on jejunal villus epithelium was reduced by the adaptational changes to bypass; alkaline phosphatase specific activity remained unchanged. In the ileum, despite adaptational changes to bypass, there was no increase in the normally low specific activities of sucrase and alkaline phosphatase. Bypass surgery did not change the major site of expression of either enzyme on jejunal or ileal villi. In conclusion, enzymatic markers of functional differentiation are not all equally affected by adaptational hyperplasia. Hypertrophy of villi and increased cell proliferation seen in jejunum remaining exposed to luminal contents resulted in an increase in the alkaline phosphatase but not the sucrase content. This is not, therefore, the result of a simple immaturity of villus cells. Morphological adaptation in the ileum, however, is not accompanied by adaptation of brush border enzyme markers of differentiation, confirming a functional immaturity of these cells. Strategies for increasing the expression of these markers may have clinical value.     

Response of the rat small-intestine epithelium to methotrexate.

We studied jejunal epithelial structure and function in rats 24, 48, 96, and 192 hours after a single intravenous injection of methotrexate (MTX) 30 mg/kg. The acute effect of the drug on the gut at 24 and 48 hours was characterised, as expected, by reduced mitoses in crypts, shortened villi, and depressed activity of thymidine kinase (an enzyme normally confined to intestinal crypt cells). At 96 hours, when MTX was no longer detectable in serum, the intestine had entered a proliferative phase characterised by increased crypt mitoses, accelerated migration of enterocytes along villi, and the presence on villi of epithelial cells with the enzyme profile of crypt cells, decreased disaccharidase, alkaline phosphatase, and Na+-K+ATPase activities and increased thymidine kinase activity. Although the enzyme data suggested that enterocyte maturation was defective during this proliferative phase, glucose-stimulated Na+ transport, normally a function of fully differentiated villus cells, was normal at 96 hours. Measured both in Ussing chambers and in suspensions of enterocytes isolated from villi, Na+ transport responded normally to glucose at 96 hours, although the response had been significantly depressed at 24 hours. These findings cannot be attributed to MTX-induced malnutrition, as all comparisons included pair-fed controls. We conclude that, in the MTX-induced malnutrition, as all comparisons included pair-fed controls. We conclude that, in the small intestine under conditions of altered epithelial renewal, some components of enterocyte function may be affected more than others. Comparing the present experimental model with another intestinal disorder, acute viral enteritis, in which proliferative activity is excessive, it is clear that the nature of the original intestinal injury is a significant determinant of the pattern of enterocyte response.     

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.     

Indomethacin accelerates clearance of labeled cells and increases DNA synthesis in gastrointestinal mucosa of the rat

Sprague-Dawley rats treated with placebo, parenteral indomethacin, or oral prostaglandin E₂ for six days were given an intraperitoneal injection of [³H]methyl-thymidine and killed at 45 min and 96 hr after labeling. Treatments were continued, until death. The dpm/DNA index was determined in mucosal scrapings of the stomach, duodenum, and jejunum and used to estimate DNA synthesis (45 min) and the clearance of labeled cells (96 hr). Indomethacin increased the DNA synthesis in both the duodenal and jejunal mucosa (P<0.05). In comparison to the controls, the clearance of labeled cells from the antral, duodenal, and jejunal mucosa was accelerated by indomethacin treatment, whereas the elimination of labeled cells from the antral and jejunal mucosa was slowed by PGE₂ treatment (P<0.05). DNA synthesis of the antral mucosa was significantly reduced by PGE₂ (P<0.05). The cyclooxygenase blocker did not affect the cell kinetic parameters of the oxyntic mucosa. The plasma levels of somatostatin were significantly higher both in PGE₂- and indomethacin-treated rats than in controls (P<0.05). It is concluded that indomethacin treatment increases the cell losses from the epithelial surface, which in turn trigger a compensatory trophic reaction. It is suggested that an important physiological action of endogenous prostaglandins is to regulate the outflow of cells from the superficial zones of the epithelium. Finally, this study disclosed the presence of hitherto unknown regulatory mechanism that promote cell proliferation in the gastrointestinal, mucosa despite inhibition of the synthesis of endogenous prostaglandins.     

Effects of indomethacin on cellular resistance and synthesis of prostaglandins in epithelial cells isolated from rat gastric mucosa

This study was done to test effects of indomethacin (IND) on cell damage and prostaglandin (PG) synthesis in mucosal epithelial cells isolated from rat stomach in vitro. IND caused the cell damage in a dose-related way at concentrations over 5 X 10(-4) M. This damage was inhibited by 16,16-dimethyl PGE2 (10(-6) M). IND abolished the synthesis of PGE2, PGI2, and TXA2 at the concentration of 10(-4) M at which IND alone did not cause cell damage. The cells treated with 10(-4) M IND were significantly susceptible to damage caused by 15% ethanol compared to the cells not treated with IND. 16,16-Dimethyl PGE2 also inhibited the damage caused by IND + ethanol. These results suggest that the IND-induced susceptibility of the cells to damage is related to PG deficiency.     

Prostaglandin E2 induces proliferation of glandular epithelial cells of the human endometrium via extracellular regulated kinase 1/2-mediated pathway

In this study, we investigated the effect of prostaglandin E(2) (PGE(2)) on MAPK ERK1/2 protein phosphorylation and on proliferation of epithelial cells of the human endometrium. Treatment of proliferative phase endometrium with PGE(2) induced rapid phosphorylation of ERK1/2 proteins in glandular epithelial and endothelial cells. Treatment of human endometrial tissue with PGE(2) for 24 h resulted in increased incorporation of 5-bromo-2'-deoxyuridine (a marker of cellular proliferation) in glandular epithelial cells. To investigate further the effect of PGE(2) on proliferation of epithelial cells, we used an endometrial epithelial cell line (HES). HES cells express functional EP4 (with absence of expression of EP1, EP2, and EP3) receptors and stimulate cAMP release and rapid phosphorylation of ERK1/2 proteins in response to PGE(2) or forskolin. Treatment of HES cells with PGE(2) or forskolin alone resulted in a significant increase in HES cell proliferation compared with control untreated cells (P < 0.05). Cotreatment of the cells with PGE(2) or forskolin and PD98059 abolished the increase in cellular proliferation. These data demonstrate ERK1/2 phosphorylation in response to PGE(2) in the human endometrium and suggest that PGE(2) via EP4 receptor may induce glandular epithelial cell proliferation in ERK1/2- dependent manner during the proliferative phase of the menstrual cycle.     

Immunohistochemical changes in the jejunum in first degree relatives of patients with coeliac disease and the coeliac disease marker DQ genes. HLA class II antigen expression,interleukin-2 receptor positive cells and dividing crypt cells.

The staining of HLA class II antigens, the presence of cells positive for interleukin-2 receptors, the proportion of crypt cells in mitosis in the jejunal biopsy specimens, and the dose of coeliac disease marker HLA-DQ genes were studied in 75 healthy family members of coeliac disease patients. Eleven had silent coeliac disease; in the rest the morphology of the jejunum was normal. In the specimens from family members, staining of epithelial cells with HLA-DP and -DR antibodies was more widely distributed and stronger than in those from 19 controls. Interleukin-2 receptor+ cells were seen in the epithelium of all eight specimens from subjects with silent coeliac disease, and also in 24 morphologically normal specimens from family members, but not in the 19 control specimens. The proportion of crypt cells in mitosis was increased only in the specimens from the subjects with silent coeliac disease. The staining intensity of the epithelial cells with HLA-DP and -DR antibodies, the presence of interleukin-2 receptor+ cells and the percentage of crypt cells in mitosis were significantly associated with the number of coeliac disease marker DQB genes. Many family members of patients with coeliac disease have signs of inflammation even in morphologically normal jejunum; these inflammatory changes together with coeliac disease marker DQ genes may point to latent disease in these subjects.     

On the frequency of metaphase chromosome aberrations in the small intestine of aged rats

Abnormalities in DNA synthesis and cell proliferation are characteristic of aged populations of proliferating cells. Cytogenetic analysis of jejunal crypt cells from young and senescent rats indicates that the imbalance in cell production in vivo is associated with an age-dependent increase in metaphase chromosome aberrations. Furthermore, the frequency of these chromosome aberrations is correlated with histologic evidence of cell death. These results demonstrate that the maintenance of genomic integrity is disturbed in the aged small intestine and may explain the age-related increase in the proportion of relatively undifferentiated villus epithelial cells in the small intestine of senescent rats.     

Effects of glucagon, vasoactive intestinal peptide, and vasopressin on villous microcirculation and superior mesenteric artery blood flow of the rat

The effects of the peptide hormones glucagon, vasoactive intestinal peptide, and vasopressin on the microcirculation of single jejunal villi were studied in anesthetized rats. By means of a recently developed in vivo video-microscopy technique, the red blood cell velocity (pretreatment value: 2.1 +/- 0.1 mm X s-1) and the diameter of the red blood cell column (5.5 +/- 0.2 micron) were measured in the villous arcade vessels. From these parameters, an index of blood flow was calculated in order to determine changes in response to intravenous infusions of the peptides. During the infusions of glucagon and vasopressin, simultaneous measurements were made of superior mesenteric artery blood flow and villous arcade flow. Glucagon (1 microgram X kg-1 X min-1) increased villous arcade flow markedly to 150.1 +/- 13.7% of control, while superior mesenteric artery flow remained unchanged. Vasoactive intestinal peptide (1 microgram X kg-1 X min-1) produced a dilation of the arcade vessel with a commensurate reduction of red cell velocity, leaving the flow index unaltered. Vasopressin (14.3 mU X kg-1 X min-1) was found to be a potent vasoconstrictor at the mucosal level, and since red cell velocity also decreased, villous flow was reduced substantially, paralleling a reduction of superior mesenteric artery flow. After the vasopressin infusion, a reactive hyperemia occurred in the villous arcades. No such increase in blood flow was observed in the superior mesenteric artery. From these findings, we conclude that the villous microvasculature is influenced by various hormones and, therefore, must occupy a prominent position in control of the circulation of the small intestine.