A METHOD FOR QUANTITATION OF PROLIFERATIVE INTESTINAL MUCOSAL CELLS ON A WEIGHT BASIS: SOME VALUES FOR C57BL/6

A method for determining the number of intestinal mucosal crypts, S cells, and total proliferative cells, on a weight basis has been presented. The number of crypts was obtained (following injection of tritiated thymidine) by dividing the disintegrations per minute (dpm) per mg intestine by the dpm per crypt. Multiplication of the number of crypts per mg by the number of labeled cells per crypt (determined radioautographically) resulted in the number of S cells per mg intestine. Division of the number of S cells per mg by the fraction of proliferative cells in S (obtained by cell cycle analysis) resulted in the number of proliferative cells per mg intestine. Values for duodenum, jejunum, and ileum of male C57BL/6 mice are given.     

POPULATION DYNAMICS OF INTESTINAL EPITHELIA IN THE RAT TWO MONTHS AFTER PARTIAL RESECTION OF THE ILEUM

Sprague-Dawley rats that had been subjected 2 months previously to partial resection (10 per cent) of the small intestine and an equal number of control rats were injected with tritiated thymidine and sacrificed at intervals during the subsequent 16 hours. Segments of duodenum, jejunum and ileum were prestained by the Feulgen technique and radioautographed. The proportion of crypt cells bearing labeled nuclei, the percentage of labeled crypt cells in mitosis and the appearance of labeled crypt cells on the villi were determined. Comparison of control and resected rats showed that (a) the proportion of intestinal crypt cells incorporating thymidine was considerably greater and uniformly high throughout the shortened intestine, (b) the life cycle of crypt cells was slightly reduced, and was uniform throughout the shortened intestine, and (c) the time during which cells were retained in crypts was markedly reduced. On the basis of persistent, generalized increase in the production of crypt cells, and on prior evidence that the epithelial cells of shortened intestine continue to have a brief life span and evidence of metabolic immaturity, the existence of a humoral factor, tentatively called "intestinal epithelial growth hormone," is postulated.     

Histological characteristics of the intestinal mucosa of the rat during the first year of life

In spite of the widespread use of rats in gastrointestinal research, there is a lack of information on the qualitative and quantitative histological characteristics. Therefore, a study was performed in 69 male Wistar rats with ages ranging from one day to one year old. The features studied included: height and number of villi in the duodenum, jejunum and ileum, and depth and number of crypts in the duodenum, jejunum, ileum, colon and rectum. Morphometric observations were expressed in a mathematical logarithmic curve that showed a normal, pattern of intestinal growth for each intestinal level. The number of villi in the small intestine decreased from 1 to 35 days of age, whereas the other intestinal parameters all increased during the same period. After 35 days the rates of increase or decrease were lower. The quantification of these intestinal changes provides a new complementary pattern as a reference for research as indicators of normality or malfunction in the rat intestine.     

The distribution of endocrine cells along the mouse intestine: a quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30-60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the bottom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3-4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

THE EFFECT OF INTESTINAL RESECTION ON THIRY-VELLA FISTULAE OF JEJUNAL AND ILEAL ORIGIN IN THE RAT: EVIDENCE FOR A SYSTEMIC CONTROL MECHANISM OF CELL RENEWAL

Local and systemic control mechanisms have been postulated to explain the maintenance of steady state cell renewal in intestinal epithelium. Permanent alterations of cell renewal resulting in a new steady state imply alterations in control. Intestinal resection appears to cause such alterations resulting in hyper-plasia of the residual intestine. To test the hypothesis of a systemic control, the effect of 60% mid-intestinal resection on Thiry-Vella fistulae of both jejunal and ileal origin was observed in rats. Results showed that hypoplasia occurred in fistulae without resection of the remaining intestine in continuity. Cell counts of crypt and villus columns and tritiated thymidine uptake in isolated whole crypts were reduced. Scanning electron microscopy showed marked hypoplastic alterations in villi. However, when 60% of the intestine in continuity was resected, hyperplasia occurred not only in the residual intestine but in the fistulae of both jejunal and ileal origin. Cell counts of villus and crypt columns were increased along with increased tritiated thymidine uptake per crypt. Neutral cc-glucosidase and non-specific esterase activities did not change as a result of resection but the activities of both enzymes were greater in ileal fistulae than in ileum in situ. Observations on the different resection response of the jejunal versus ileal fistulae lead to a distinction between inherent and induced differences within the small intestine. This study suggests a systemic control of cell renewal. A possible mechanism involving intestinal vascular physiology is discussed.     

Cell migration pathway in the intestinal epithelium: an in situ marker system using mouse aggregation chimeras

The cell migration pathway in the intestinal epithelium of DDK in equilibrium C57BL/6JLac mouse chimeras is demonstrated using Dolichos biflorus agglutinin-peroxidase as strain-specific marker. Cell sheets of one genotype extend in relatively straight lines from crypt to villus apex. Narrow sheets are mostly interrupted in the distal two-thirds of duodenal but not ileal villi, suggesting that in the duodenum cell loss occurs below the apical extrusion zone. These differences between duodenum and ileum correspond to differences in villus shape. The pattern of cell migration in Peyer's patch epithelium is consistent with that of the duodenum. In chimeric colon, sharply demarcated territories of crypts with a narrow cuff of surface epithelium represent the counterpart of the villus/crypt unit of the small intestine.     

The distribution of endocrine cells along the mouse intestine: A quantitative immunocytochemical study

The topographical distribution of endocrine cells in the crypt and villus epithelium along the length of the mouse intestine was studied. Argyrophil reactivity using the Grimelius stain was used to estimate the total endocrine population of the intestine. Comparisons were then made with the fraction of endocrine cells containing glucagon like material, stained immunocytochemically using rabbit anti-glucagon antisera. A highly significant reduction in the incidence of endocrine cells (argyrophil reactive) from the proximal to distal end of the intestine was noted. However, only 10-30% of these cells contained glucagon like material in the crypts of the duodenum, jejunum and ileum, compared to 30–60% in the crypts of the colon and rectum. The distribution of endocrine cells (argyrophil reactive) was maximal in the lower regions of the proliferative zone of the crypts but showed no significant variation along the length of the villi. Cells containing glucagon like material were also most frequent in the lower regions of the proliferative zone of the crypts, but were not generally found above the botom third of the villi. Each crypt in the small intestine contains between 3 and 5 endocrine cells one of which contained glucagon like immunoreactive material. In the colon and rectum each crypt contains about 6-8 endocrine cells, of which 3–4 contained glucagon like immunoreactive material. These results indicate that a sub-set of cells containing glucagon like material, differentiate early in the lineage of endocrine cells within the proliferative zone of the intestinal crypts.     

Cell proliferation in the small intestine and colon of intravenously fed rats: effects of urogastrone-epidermal growth factor

Abstract. There is marked intestinal hypoplasia in the intestine of intravenously fed (TPN) rats. Recombinant urogastrone-epidermal growth factor (URO-EGF) reversed these changes by significantly increasing the length of the intestinal crypts. Crypt diameter, however, was not affected to the same extent. Few differences in labelling indices were seen between the orally fed and TPN groups, however, this was the consequence of the concomitant changes in crypt population.
The number of mitoses and labelled cells per crypt, and thus the crypt cell production rates, were significantly decreased in the TPN group when compared to the orally fed. URO-EGF significantly increased both proliferative indices and the number of dividing cells per crypt. Crypt cell production in the small intestine was restored to those levels seen in the orally fed rats, moreover, labelling per crypt in the colon was increased to more than twice that of orally fed rats. The location of the mean labelling position and the half maximum labelling position followed the changes in crypt length in the small intestine, but to a lesser extent; thus the growth fraction was significantly increased in the TPN rats in comparison with the orally fed and the URO-EGF treated groups. Similar changes in these positions were seen in the colon, but the growth fraction was much reduced in the URO-EGF treated rats, as a consequence of the large increase in crypt length without a concomitant alteration in label distribution.     

Cellular Proliferation of Intestinal Epithelia in the Rat Two Months after Partial Resection of the Ileum

Sprague-Dawley rats subjected 2 months previously to partial resection (10 per cent) of the small intestine and their controls were injected with tritiated thymidine and sacrificed at 2 and 23 hours. Segments of the duodenum, jejunum, and ileum were autoradiographed, and the migration of the labelled cells during the period between 2 and 23 hours was measured with an eyepiece micrometer. The cells had migrated 35, 42, and 34 per cent of the total distance from the crypts to the tips of the villi in the control segments of duodenum, ileum, and jejunum respectively, and 43, 90, and 82 per cent, respectively, in similar segments from resected animals. The rate of migration in the portion of the intestine remaining after resection was approximately three times the normal rate in the ileum, twice the normal rate in the jejunum, and showed an increase of one-third in the duodenum. These results demonstrate that the rate of cell renewal is considerably greater in the remaining portion of the intestine of resected animals than in normal intestine. The increased rate of migration after resection, together with the increase in the height of the villi, resulted in an increase in the rate of cell renewal amounting to 141 per cent in the ileum, 114 per cent in the jejunum, and 23 per cent in the duodenum when compared with control segments.     

A cytokinetic study of small-intestinal and colonic mucosa after resection of 70% of the small intestine

Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied. In the distal ileum there is a significant reduction in cell cycle time (Tc) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in Tc at these sites, but there does appear to be a significant reduction in Tc on the part of the cells in the stem-cell zone at the crypt base. In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

Effects of whole wheat feeding on the development of the digestive tract of broiler chickens

The objective of the current study was to investigate the impact of including whole wheat in broiler diets on the development of the digestive tract. Chickens were fed a standard feed containing 400 g ground wheat/kg or the same diet with a part of the wheat given separately as whole grains that increased progressively from 200 g/kg at 8 d to 400 g/kg at 22 d. Every week, from 16 to 44 d, growth performance, modifications of the size of the digestive tract organs and intestinal enzyme activities were investigated. Morphology of villi and crypts in the small intestinal segments (duodenum, jejunum, ileum) were analyzed at 23 and 44 d. Microbacterial counts were performed in jejunal, ileal and caecal contents weekly from 16 to 44 d.During the adaptation period from 8 to 15 d, the birds fed the whole wheat diet had lower feed intake and lower weight gain. Thereafter, they showed improved growth performance so that by the end of the experiment, they had higher body weight compared to the standard-fed birds, 2430 ± 29 versus 2331 ± 36 g.Higher relative weights of gizzard (+26%) and pancreas (+12%) were observed from 16 to 44 d for whole wheat-fed birds compared to standard-fed birds. No differences in relative size of the different intestinal segments were observed, except that the jejunum was shorter. Increased villus to crypt length and surface ratios were observed at 23 d in the duodenum of whole wheat-fed birds, with no differences in morphometry between groups thereafter. Alkaline phosphatase activity was higher from 16 to 44 d in the duodenum and jejunum of whole wheat-fed birds. However, the activities of the digestive enzymes, leucine aminopeptidase and maltase, were similar between the two diets in the measured intestinal segments.A lower number of facultative anaerobic bacteria was found in the ileum of the whole wheat-fed birds, with no differences between treatments for Escherichia coli and for Lactobacillus counts. In the jejunum and the caeca, no differences in microflora counts were observed.The present results showed that feeding whole grains to broilers led mainly to modifications in the upper part of the digestive tract (gizzard, pancreas) and had little influence on the small and large intestine.     

Effects of reduced food intake on morphometry and cell production in the small intestine of the rat

The present study examined the effects of a 60% reduction in food intake on kinetic and morphometric parameters in the small intestine of adult male Lewis rats. We observed that, after 20 days, the wet weight of jejunum and ileum, thickness of muscularis externa of duodenum, crypt depth throughout the intestine, and DPM/mg and DPM/crypt in ileum were decreased in animals on reduced food intake when compared to their paired, normally fed controls. These results demonstrated that reduced food intake caused distinctive effects focused primarily in the ileum, thus opening to question the use of the technique of pair feeding as a control for studies of intestinal cell proliferation in which manipulations of the animals result in altered food intake or body weight.     

Epithelial cell proliferation and uptake of radiolabeled urogastrone in the intestinal tissues following abdominal irradiation in the mouse

We have evaluated the rate of crypt cell production and uptake of radiolabeled recombinant human urogastrone (125I-rhUG) in the intestinal tissues of the mouse at 3, 5, 7, 9, and 12 days following irradiation of the abdomen with 9 Gy. At autopsy, the animals were injected intraperitoneally with 1 microgram/g body weight of the metaphase arrest agent, vincristine sulfate, and 25 muCi of 125I-rhUG (specific activity 1.7 muCi/micrograms) to quantify the rate of crypt cell production and uptake of radiolabeled urogastrone, respectively. The results indicated that the rate of crypt cell production was increased significantly in the irradiated animals compared to the unirradiated animals and showed a peak on the 3rd and 5th postirradiation days in small intestine and colon, respectively. The uptake of 125I-rhUG was increased significantly on the 3rd postirradiation day in the intestinal tissues but showed a bimodal pattern with peaks on the 3rd and 9th postirradiation days. These results suggest that there may be a close association between epithelial cell proliferation and uptake of 125I-rhUG, particularly in the early part of recovery of intestinal mucosa following irradiation. However, these data do not discriminate whether the increased uptake of 125I-rhUG is the cause or the effect of proliferation induced by an irradiation stimulus. Further analysis also revealed that there was no relationship between crypt depth and 125I-rhUG uptake. However, crypt depth was inversely correlated with villus height in the proximal small intestine but not in the ileum. Villus height was correlated inversely with 125I-rhUG uptake in the ileum and jejunum but not the duodenum. The rate of crypt cell production was strongly correlated with crypt depth throughout the intestine and inversely correlated with villus height. This suggests that villus-to-crypt inhibitory feedback may be a primary regulator of cellular proliferation in the crypts and the association of 125I-rhUG uptake with proliferation indirectly reflects this interaction.     

Morphological effects of a large single dose of cycloheximide on the intestinal epithelium of the rat.

Adult male rats received 15 mg/kg cycloheximide and the subsequent morphological effects at three and six hours after injection were evaluated using histometry, light and electron microscopy, histological demonstration of terminal web and acid phosphatase, and radioautography with tritiated thymidine. Rapid atrophy of the villi took place, progressing from the villus tip by premature exfoliation of epithelial cells. The crypts also diminished by random exfoliation of many crypt cells and by partial or complete disintegration. Mitosis and epithelial cell migration were absent. By six hours, the area occupied by the villi and the crypts per unit length of histological section was decreased by about 70-90% in most of the small intestine but only by about 40-60% in the duodenum and the terminal ileum. In the upper half of the villi, the epithelium was strongly positive for acid phosphatase and contained large numbers of round bodies resembling primary lysosomes. In the lower half, the microvillous border and terminal web were found to be disrupted. Animals receiving only 5 mg/kg cycloheximide also showed the atrophy of villi and crypts, and the round bodies resembling lysosomes. Evidence from several sources has indicated that protein synthesis in normal villus epithelial cells subsides toward the villus tip and becomes minimal at exfoliation. At exfoliation, proteins responsible for epithelial cohesion probably fail because they are no longer replenished. Cycloheximide appears to accelerate this process.     

A Cytokinetic Study of Small-Intestinal and Colonic Mucosa After Resection of 70% of the Small Intestine

Abstract. Pulse-labelling with tritiated thymidine and a fraction of labelled mitoses experiments have been performed in order to investigate the proliferative changes induced at various sites in the hyperplastic small-intestinal mucosa of rats previously subjected to resection of 70% of the small intestine. Proliferative activity in the colon was also studied.
In the distal ileum there is a significant reduction in cell cycle time (T_c) of cells at all levels within the crypt and the growth fraction falls. In the jejunum and proximal ileum the crypts contain an increased number of proliferating cells, but as the size of the maturation zone is also increased, there is no significant alteration in the relative number of proliferating cells per crypt. Nor does the distribution of proliferating cells in these crypts seem to alter. There is no general reduction in T_c at these sites, but there does appear to be a significant reduction in T_c on the part of the cells in the stem-cell zone at the crypt base.
In neither proximal nor distal colon was there any significant proliferative change apparent after small-intestinal resection.     

ABCG2 expression and uric acid metabolism of the intestine in hyperuricemia model rat

Abstract

To elucidate roles of the intestine in uric acid (UA) metabolism, we examined ABCG2 expression, tissue UA content and xanthine oxidoreductase (XOR) activity in different intestinal segments. Male SD rats were assigned to control group or oxonic acid-induced hyperuricemia (HUA) group. In control rats, ABCG2 was present both in villi and crypts in each segment. Tissue UA content and XOR activity were relatively high in duodenum and jejunum. However, in HUA rats, tissue UA content was significantly elevated in the ileum, whereas it remained unaltered in other segments. Moreover, ABCG2 expression in the HUA group was upregulated both in the villi and crypts of the ileum. These data indicate that the ileum may play an important role in the extra-renal UA excretion.     

Diet, mucosal architecture and epithelial cell production in the small intestine of specified-pathogen-free and conventional rats.

Upper jejunum and terminal ileum were examined in specified-pathogen-free (SPF), conventional and conventional after SPF rearing (ex-SPF) rats. The effect of 2 differential diets on the last 2 groups was examined. Ex-SPF rats had taller villi and deeper crypts than SPF rats, but similar crypt to villus ratios and cell production rates. Ex-SPF rats had similar crypt depth and jejunal villus height to conventional rats on the same diet, but taller ileal villi and a lower cell production rate. Even after 6-8 weeks, in a conventional environment, ex-SPF rat intestine was still not identical with conventional rat intestine. Diet had a significant effect on mucosal architecture, and a smaller effect on cell production rate. It is concluded that diet, microbiological status of colony of origin, and environment after weaning, can all affect mucosal architecture and epithelial cell production, and should be properly controlled in experimental studies.     

Radioprotection to small intestine of the mice against ionizing radiation by semiquinone glucoside derivative (SQGD) isolated from Bacillus sp. INM-1

Ionizing irradiation induces severe damage to the intestinal crypt cells which are responsible for renovation and maintenance of the intestinal cellular architecture. Therefore, protection of intestinal cells and tissue against lethal irradiation using a semiquinone glucoside derivative (SQGD) isolated from radioresistant bacterium Bacillus sp. INM-1 is the prime focus of the present investigation. BALB/c mice were administered by SQGD (50?mg/kg.b.wt. i.p.) 2?h before whole body irradiation (10?Gy), and histological analysis of the jejunum section was carried out and compared to the irradiated mice. Significant (p?<?0.0001) increase in villus length, number of cells per villus, crypts numbers per villus section, total cells counts and mitotic cell counts per crypt and low goblet cells per villus section, and low apoptotic index per crypt section were observed in the irradiated mice pre-treated by SQGD at 48–168?h. Significant induction in NF-kβ at 24?h and Bcl-2/Bax ratio was observed in irradiated mice pre-treated by SQGD compared to only irradiated animals. SQGD pre-treatment before irradiation was found instrumental to reverse the radiation-induced degenerative changes by replenishment of the damaged cells by enhancing mitotic, proliferating, pro-survival, and apoptosis inhibitory activities probably through modulation of cell cycle arrest in G₁/S phase in the intestinal cellular milieu.     

Distribution of 3-hydroxy-3-methylglutaryl-CoA reductase in isolated villus and crypt cells of chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), the major rate-limiting enzyme of cholesterogenesis, was studied in epithelial cells isolated in a villus to crypt gradient from chick duodenum, jejunum and ileum, in order to resolve the apparent controversy that exists on the anatomical localization of sterol synthesis in the intestine. Consistent separation was demonstrated by using the marker enzymes alkaline phosphatase, specific to the villus cells, and thymidine kinase, specific to the crypt cells. No relative difference in stability was observed, as shown by the equal distribution of acid phosphatase. Cells were 90-95 per cent viable. The highest specific activity of reductase was located in the microsomal fraction (41 per cent of the total). The mitochondria had lower specific activity (8 per cent of the total). The distribution of reductase activity in epithelial cells of the villus-crypt axis was also studied. The specific activity in each cell fraction from chick duodenum was clearly lower than that in jejunum and ileum. The jejunal and ileal crypt regions showed lower specific activity than the villus cells. About 70 per cent of total reductase activity was found in cells from the upper and the mid villus fraction in each intestinal segment.