Colonic proliferation is increased in senescent rats

Our previous studies suggested that crypt size enlarged and that proliferation rate might be greater in the small intestine of rats during senescence. Crypt cell numbers and crypt cell proliferation rates, using the vincristine-induced metaphase arrest technique, now have been measured in the colon of aging and young Fischer 344 rats. The proximal colon of 26-28-mo-old unfasted rats had 10% more crypt cells and a higher proliferative rate than 3-4-mo-old young controls. In the distal colon, the crypt cell proliferation rate in aging rats was 56% greater than in the young. A 3-day fast reduced crypt cell proliferation about fourfold in young rats but only by 20% in aging rats. One-day refeeding abruptly increased the crypt cell population and proliferation rate in rats of both age groups. The crypt zone of proliferating cells from aging rats was broader than that seen in young rats. In addition, starvation lowered colonic crypt cell cycling rate much less in aging than in young animals. We conclude that the colons of aging rats demonstrate a hyperproliferative state and a failure to adapt appropriately to changes in food intake. These observations may be relevant to states of altered proliferation that occur in the premalignant colon.     

Adaptive changes of intestinal enzymes to nutritional intake in the aging rat

We previously have shown that aging alters the expression of several intestinal enzymes during cell migration from the crypt base to the villus tip. The activities of many mucosal enzymes are dramatically altered by starvation and refeeding. We compared the effects of starvation and refeeding on the activities of selected intestinal enzymes in young and aging Fischer 344 rats. Gut mass fell during starvation and rose during refeeding to a similar extent in both groups. Sucrase and maltase specific activities in control aging rats were lower than in young controls and, during starvation, enzyme activities declined at approximately similar rates in both groups. Total duodenal enzyme activities fell by about two-thirds in young animals and by greater than 80% in aged animals. Alkaline phosphatase and adenosine deaminase activities also were lower in aging than young animals. During refeeding, enzyme activities rose more in aging rats than in the young. In fact, the specific activities of sucrase and maltase in aging rats refed for 1 day exceeded the values found in fed aging controls. The adaptive responses of duodenal enzymes exceeded those in the jejunum. In conclusion, the aging intestine responds appropriately to starvation and refeeding. However, the fluctuations in brush-border enzyme activities are much greater in aging than in young rats. Such alterations may be an important influence of aging on gut differentiation and might have an adverse impact upon nutritional maintenance in aging animals.     

Aging and intestinal polyamine metabolism in the rat

Hyperproliferation and delayed expression of enzyme activity occur in small intestinal enterocytes of aging rats, and starvation and refeeding result in impaired control of these processes. Since altered polyamine metabolism may accompany changes in enterocyte proliferation, we studied the effects of nutrient manipulation upon cell numbers, ornithine decarboxylase (ODC) activity and polyamine content in jejunum and ileum of 4- to 5- and 26- to 27-month Fischer rats. In both groups, cell numbers fell during starvation and and increased during refeeding. Crypt cell hyperplasia was found in aging animals. Jejunal putrescine, spermine and spermidine content were greater in older rats, fell during starvation, and rose during refeeding. Ileal ODC activity was 66% greater in the aging rats, but jejunal ODC activity was modestly increased in young animals. Intestinal polyamine content correlates with proliferative changes and polyamine metabolism responds appropriately to nutrient manipulation during aging. Dissociation of ODC activity and polyamine content in aging jejunum probably occurred because enterocyte differentiation was delayed. Investigation of intestinal polyamine metabolism may be useful in elucidating deranged proliferative activities found in the intestine of aging rodents.     

Intestinal morphology and cytokinetics in pancreatic insufficiency

Intraluminal pancreatic enzymes influence intestinal function, adaptation, and susceptibility to injury. These effects may be mediated partly through changes in the rate of epithelial cell turnover. We assessed intestinal morphology and cytokinetics in a rat model of exocrine pancreatic insufficiency that does not alter anatomic relationships or animal growth. Pancreatic duct occlusion was performed by applying metal clips on both sides along the common bile duct. Control animals underwent sham-operation with exposure and manipulation of the pancreas without duct occlusion. Twelve days later, pulse labeling with tritiated thymidine was performed, and mitotic arrest was induced with colcemid. Groups of animals were sacrificed at 0 and 2 hr after colcemid injection. Specimens for histopathology, morphometry, and autoradiography were obtained from duodenum, proximal jejunum, distal jejunum, and ileum. Labeling index, grain counts, mitoses per crypt, cells per crypt, cells per villus, crypt depth, villus height, and number of goblet cells per villus were used as end points. Pancreatic duct occlusion resulted in increased labeling index across intestinal segments relative to sham-operated controls (P<0.01) and increased labeling index and mitotic rate in distal compared to proximal intestine (P<0.05). Grain-count histograms were similar in the two experimental groups. There were no significant morphologic differences between pancreatic duct-occluded animals and controls. Exocrine pancreatic insufficiency increases crypt cell proliferation in distal small intestine but does not alter the duration of S phase. These changes are most likely due to an increase in the size of the proliferative compartment and may be partly responsible for changes in small bowel function and response to injury.Supported by grant 88.616 from The Norwegian Cancer Society.     

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.     

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.     

Age related increase of brush border enzyme activities along the small intestine.

Intestinal morphology and brush border hydrolase activities were determined along the small intestine of young adult (three months, n = 10), mature (12 months, n = 10), and senescent (29 months, n = 15) rats. The intestinal segments of the senescent rats contained higher mucosal mass and protein content (p less than 0.05) compared with the young and mature animals. A significant reduction of villus height and crypt depth (p less than 0.05) was found in the proximal intestine during aging. A 35% increase in villus height (p less than 0.05) without changes in crypt depth, was observed in the distal ileum in senescent rats. The activities of sucrase and isomaltase were significantly increased during aging in the duodenum and jejunum (p less than 0.05). Lactase and aminopeptidase activities which showed only minor changes between young and mature animals were significantly enhanced in senescent animals (p less than 0.05) with aminopeptidase exhibiting a three-fold increase in activity in the proximal ileum. The results when combined with those of previous studies suggest that in the aged animal, the increased level of intestinal hydrolase activities may be the consequence of prolonged cellular maturation along the villi in the proximal intestine, and of adaptation to increased concentrations of intraluminal substrates in the distal intestine.     

Small intestinal crypt cell proliferation rates are increased in senescent rats

Our previous studies implied that intestinal epithelial cell replication might be increased in senescent rats. Duodenal, jejunal, and ileal crypt cell production rates (CCPR) were measured in 3-4-mo and 26-28-mo female fed control, 3-day starved and 1-day refed, and in 4-5-mo and 26-28-mo male fed Fischer rats, using the vincristine-induced metaphase arrest technique. Fed aging rats had greater proximal intestinal crypt cell numbers which fell less during starvation than those of young controls. Metaphase accumulation also was higher in aging rat duodenum and jejunum, and CCPR were 30-100% more than in young rats. Starvation reduced CCPR by more than 40% in the duodenum of young, but only by 10% in older animals. Crypt proliferative patterns demonstrated a broadened proliferative zone in aging rats. These combined results directly demonstrate that small intestinal cell production is enhanced in senescent rats and that the nutritional controls of proliferation are blunted.     

Nimesulide inhibits crypt epithelial cell proliferation at 6 hours in the small intestine in CD-1 mice

To determine whether the gut-sparing selectivity of cyclooxygenase-2 inhibitors is related to early crypt kinetic mechanisms, this study compared the primary effects on small intestinal mucosal epithelial cell proliferation and morphometry of a nonselective dual cyclooxygenase inhibitor, indomethacin, with a cyclooxygenase-2 selective inhibitor, nimesulide. Indomethacin downregulated the crypt cell production rate in the proximal small intestine, and nimesulide reduced cell proliferation in the proximal and distal small intestine. Compared to controls, there were smaller proliferating compartments in the crypts in midintestinal segments in both indomethacin- and nimesulide-treated groups, but more dividing cells in the distal intestine in indomethacin-treated group. Crypt cellularity, numbers, and width were unchanged from control values in both treated groups, suggesting a reduction in crypt cell emigration. Despite its selectivity for inhibiting cyclooxygenase-2, nimesulide induces similar but widespread initial effects on intestinal cell kinetics when compared to indomethacin.     

Food restriction retards age-related histological changes in rat small intestine

Previous studies have reported that small and large intestinal crypt hyperplasia and hyperproliferation occur in senescent rats about 27 mo of age. We have studied duodenal and ileal architecture in ad libitum chow-fed rats and have demonstrated that the increase in duodenal crypt depth and crypt hyperplasia do not develop throughout the life span, but become apparent at 21 and 27 mo of age. These crypt hyperplastic features occur without a change in duodenal villus cell number. Ileal villus cellularity increased throughout the life span, suggesting exposure to a gradually increasing luminal nutrient load. Diet restriction to 60% of the ad libitum feeding rate prolonged the life span of animals from 27 to greater than 33 mo and prevented both the duodenal hyperplasia and the increase in ileal villus cell numbers to the age of 27 mo. Thirty-three-month diet-restricted rats did show evidence of duodenal crypt hyperplasia. We conclude that proximal intestinal hyperplasia is a phenomenon that develops in advanced age, but that ileal villus cellularity increases throughout the ad libitum-fed rodent life span. Diet restriction dramatically retards these intestinal changes that are seen with ad libitum feeding and provides an experimental model for the study of age-related cellular changes of the rodent gastrointestinal tract.     

Analysis of the effects of food and of digestive secretions on the small intestine of the rat. 1. Mucosal morphology and epithelial replacement.

A modified Roux-en-Y repositioning of rat small intestine was performed so that the proximal segment of bowel (A) received only bile and pancreastic secretions, the second (B) received food direct from the stomach, and these two segments drained into a third (C). Four to five weeks after operation, cell production was assessed by injection of vincristine into operated, sham-operated and unoperated rats, and counts of blocked metaphases were made on isolated microdissected crypts. Villus height, crypt depth, and the number of crypts per villus (crypt/villus ratio) were also measured. Most of segment A showed no significant differences from sham-operated intestine, although the normal proximo-distal gradient of villus height was abolished. At the distal end (near the anastomosis with segments B and C), crypt depth and cell production were increased. The villus height gradient in segment B was also abolished, although crypt depth and cell production were significantly increased, especially at the proximal end. Crypt/villus ratio was also increased. Segment C showed all the characteristics of small bowel promoted to a more proximal position: increased villus height, crypt depth and cell production. Increased crypt/villus ratio was also observed. These results are discussed in terms of the role of food and of digestive secretions in the control of mucosal morphology and epithelial replacement.     

Indomethacin inhibits cell proliferation and increases cell losses in rat gastrointestinal epithelium

Gastrointestinal cell proliferation was estimated in histological sections of rats treated with low and high doses of parenteral indomethacin for 3 to 60 days. Mitoses were arrested with vincristine and cells in S phase were labeled with tritiated thymidine. Short-term, low-dose treatments reduced the mitotic activity in the oxyntic and small intestinal epithelium, whereas moderate doses restored the mitotic index and high doses increased the proliferative activity and produced epithelial hyperplasia. Long-term, low-dose treatments increased cell proliferation in the small intestine and reduced the number of villous cells. Indomethacin did not affect the proliferative response elicited by refeeding in the oxyntic mucosa, but the simultaneous administration of prostaglandin E₂ analog increased the number of arrested mitoses. The turnover of labeled cells was accelerated by indomethacin, particularly in the small intestine. These findings indicate that prostaglandins are regulators of the cell kinetics of the gastrointestinal epithelium but, at the same time, they disclose the presence of trophic mechanisms that are independent of the synthesis of endogenous prostaglandins.     

Does dietary fibre stimulate intestinal epithelial cell proliferation in germ free rats?

The aim of the present experiment was to investigate the role of hind gut fermentation in the proliferative response of the intestinal epithelium to dietary fibre. We have previously shown that refeeding starved rats with an elemental diet supplemented with fermentable dietary fibre (but not inert bulk) is capable of stimulating intestinal epithelial cell proliferation throughout the gastrointestinal tract. Three groups of 10 germ free (GF) rats and three groups of 10 conventional (CV) rats, were used. All groups were starved for three days and then refed for two days with either an elemental diet (Flexical); Flexical plus 30% kaolin; or Flexical plus 30% of a fibre mixture. Cell production was determined by the accumulation of vincristine arrested metaphases in microdissected crypts. There was no significant difference between refeeding the rats with an elemental diet alone or with kaolin supplementation, however, the addition of fibre in CV rats was associated with a significant increase in intestinal crypt cell production rate in both the small intestine (p less than 0.01) and the colon (p less than 0.001). This marked proliferative effects of fibre was abolished in the GF rats. It can be concluded that it is the products of hind gut fermentation, not fibre per se that stimulate intestinal epithelial cell proliferation in the colon and small intestine.     

Experimental studies of immunologically mediated enteropathy. Development of cell mediated immunity and intestinal pathology during a graft-versus-host reaction in irradiated mice.

The intestinal component of a graft-versus-host reaction (GvHR) provides a useful experimental model to elucidate the pathogenesis of clinical enteropathies which cause villus atrophy and crypt hyperplasia and which are associated with a local immune response. One to three days after induction of GvHR in heavily irradiated (CBAxBALB/c)F1 mice, a proliferative form of enteropathy developed. Compared with controls, these mice had increased counts of jejunal intraepithelial lymphocytes and had a four-fold increase in crypt cell production rate as well as an increase in crypt length. These changes were accompanied by a marked enhancement of splenic natural killer cell activity. After day three, the crypt cell production rate fell to zero and cytotoxic T lymphocytes (CTL) which could lyse targets of host origin appeared. In parallel, mice with GvHR developed significant villus shortening and their clinical condition deteriorated. Further experiments showed that increased counts of intraepithelial lymphocytes, villus atrophy and crypt hyperplasia also occurred in grafts of fetal CBA intestine implanted under the kidney capsule of (CBAxBALB/c)F1 mice with GvHR. As these grafts are syngeneic to the injected CBA spleen cells, they should not be attacked by anti-host cytotoxic T lymphocytes. We suggest that the proliferative and destructive components of enteropathy in GvHR are caused by lymphokines released by an anti-host delayed type hypersensitivity reaction.     

Changes in Cell Proliferation and Differentiation of Adult Rat Small Intestine Epithelium After Adrenalectomy

The effects of 10-day bilateral adrenalectomy on morphometry, proliferation, and apoptosis were determined in the small intestine of 3-month-old Sprague–Dawley rats. The activities of sucrase, lactase, and its respective mRNA, aminopeptidase N, and intestinal alkaline phosphatase were also evaluated. Adrenalectomy lead to partial atrophy and disorganization of the epithelium, with an increased number of goblet and Paneth cells and a reduction of crypt cell proliferation paralleled by a marked increase in villus apoptosis. Biochemical assays revealed that aminopeptidase N and intestinal alkaline phosphatase activities were significantly decreased, whereas disaccharidases were increased by adrenalectomy. The corresponding induction of lactase mRNA suggests an active response of the epithelium. In conclusion, adrenalectomy modified maturation and the differentiation processes of the small intestinal mucosa, especially in the proximal part of the small intestine. This result points to an important role of adrenals and glucocorticoids in the trophic status of the adult small intestinal mucosa.     

HLA-DR expression in human fetal intestinal epithelium.

Villus epithelial cells in fetal human small intestine are HLA-DR- until 17 weeks gestation. At 18 weeks HLA-DR begins to be expressed in the epithelial cells, usually at the villus tips. Of 13 specimens examined between 18 and 22 weeks gestation, two were HLD-DR-, seven had HLA-DR expressed only at the villus tips and in four most of the villus epithelial cells were HLA-DR+. The epithelium overlying the Peyer's patches in fetal intestine was also HLA-DR+. T cells in explant cultures of fetal intestine were activated in situ using pokeweed mitogen. The local cell mediated immune reaction increased expression of HLA-DR on the villus and crypt epithelial cells. Organ culture supernatants from explants treated with pokeweed mitogen induced HLA-DR expression on the HT-29 epithelial cell line; an effect inhibited by antibody against human interferon gamma.     

Effects of fasting and refeeding on jejunal morphology and cellular activity in rats in relation to depletion of body stores

BACKGROUND: Intestinal mucosa atrophy following a period of starvation characterized by the mobilization of fat stores for energy expenditure (phase II) worsen after a long fast marked by an increase in protein catabolism (phase III). However, the morphology of the jejunum is completely restored after 3 days of refeeding. The aim of this study was to determine the mechanisms involved in the rapid jejunal restoration following the critical phase III. METHODS: Jejunal structure was observed through conventional and environmental scanning electron microscopy, whilst cellular dynamics were studied using classical optic microscopy tools and immunohistochemistry. RESULTS: Mucosal structural atrophy during fasting proved to worsen over the two phases. During phase II, apoptosis is still present at the tip of the villi, the number of mitosis in crypts showed a 30% decrease and a transient drop in cell migration is observed. During phase III, however, an 85% rise in mitosis was noticed along with an increase in cell migration and the disappearance of apoptotic cells at the villus tips. This increased cell renewal continues after food ingestion. CONCLUSIONS: Starved rats appeared to be in a phase of energy sparing in phase II, with depressed cellular events in the intestinal mucosa. In phase III, however, the preservation of functional cells and the early increase in crypt cell proliferation should prepare the mucosa to refeeding and could explain why jejunal repairs are complete after 3 days of refeeding following either phase II or phase III.     

Effects of amidated gastrin and glycine-extended gastrin on cell proliferation and crypt fission in parenterally and orally fed rats

BACKGROUND/AIMS: It has been suggested that processing variants of gastrin, such as glycine-extended gastrin (G17-Gly), are enterotrophic to the colon. METHODS: Cell proliferation and crypt branching were studied in total parenteral nutrition (TPN) and orally fed rats after infusion of G17-Gly or gastrin-17. RESULTS: Gastrin produced an increase in the weight of the stomach and small intestine and a marked proliferative action on the proximal small intestine, which diminished distally. No proliferative effects of gastrin were seen in the colon. G17-Gly was associated with a small, but significant, increase in colonic weight but had little effect on cell proliferation, except in the gastric fundus. In the distal colon, G17-Gly was associated with a significant decrease in proliferation. Neither agent affected crypt branching in the small intestine or colon, but both proliferation and branching were significantly decreased by TPN. CONCLUSION: Gastrin was trophic to the stomach and the proximal small intestine but not the colon. G17-Gly had only modest proliferative actions on the intestinal epithelium in this study.     

Gene expression profiling of intestinal epithelial cell maturation along the crypt-villus axis

BACKGROUND & AIMS: To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. METHODS: Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. RESULTS: A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. CONCLUSIONS: This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.     

Cholesterol synthesis and high density lipoprotein uptake are regulated independently in rat small intestinal epithelium.

The rates of high density lipoprotein HDL uptake and cholesterol synthesis were compared in the normocholesterolaemic (SW) and genetically hypercholesterolaemic (RICO) rat intestine. The RICO rat has a hyperintestinal cholesterol synthesis. 14C sucrose, a marker which becomes irreversibly entrapped within the cells, was used to measure total rat HDL uptake over 24 hours in the various cells of the small intestinal mucosa. The rates of sterol synthesis were estimated in vivo with 1-14C acetate, as previously validated. The rates of HDL uptake in the upper villus cells were similar along the length of the small intestine in both types of rat, but the rates of sterol synthesis varied up to eightfold. When the mucosal epithelium was divided along the villus/crypt axis, HDL uptake increased two to threefold and cholesterol synthesis two to fivefold in the upper villus compared with the crypt cells in both SW and RICO rats. The high cholesterogenesis in the mucosal cells of the RICO rat is not related to a modified HDL cholesterol uptake. Thus, cholesterol synthesis and HDL uptake seem to be regulated independently in the rat small intestinal mucosa.