Crypt-villus Architecture Research Articles

Supplementation of total parenteral nutrition with butyrate acutely increases structural aspects of intestinal adaptation after an 80% jejunoileal resection in neonatal piglets.

Supplementation of total parenteral nutrition (TPN) with a mixture of short-chain fatty acids (SCFA) enhances intestinal adaptation in the adult rodent model. However, the ability and timing of SCFA to augment adaptation in the neonatal intestine is unknown. Furthermore, the specific SCFA inducing the intestinotrophic effects and underlying regulatory mechanism(s) are unclear. Therefore, we examined the effect of SCFA supplemented TPN on structural aspects of intestinal adaptation and hypothesized that butyrate is the SCFA responsible for these effects. Piglets (n = 120) were randomized to (1) control TPN or TPN supplemented with (2) 60 mmol/L SCFA (36 mmol/L acetate, 15 mmol/L propionate and 9 mmol/L butyrate), (3) 9 mmol/L butyrate, or (4) 60 mmol/L butyrate. Within each group, piglets were further randomized to examine acute (4, 12, or 24 hours) and chronic (3 or 7 days) adaptations. Indices of intestinal adaptation, including crypt-villus architecture, proliferation and apoptosis, and concentration of the intestinotrophic peptide, glucagon-like pepide-2 (GLP-2), were measured. Villus height was increased (p < .029) within 4 hours by supplemented TPN treatments. Supplemented TPN treatments increased (p < .037) proliferating cell nuclear antigen expression along the entire intestine. Indicative of an antiapoptotic profile, jejunal Bax:Bcl-w abundance was decreased (p = .033) by both butyrate-supplemented TPN treatments, and ileal abundance was decreased (p = .0002) by all supplemented TPN treatments, regardless of time. Supplemented TPN treatments increased (p = .016) plasma GLP-2 concentration at all time points. Butyrate is the SCFA responsible for augmenting structural aspects of intestinal adaptations by increasing proliferation and decreasing apoptosis within 4 hours postresection. The intestinotrophic mechanism(s) underlying butyrate's effects may involve GLP-2. Ultimately, butyrate administration may enable an infant with short-bowel syndrome to successfully transition to enteral feedings by maximizing their absorptive area.

Weaning Is Associated with an Upregulation of Expression of Inflammatory Cytokines in the Intestine of Piglets

Cytokines play a central role in immune cell response, but they also participate in the maintenance of tissue integrity. Changes in the cytokine network of the pig gut may be expected at weaning, because abrupt changes in dietary and environmental factors lead to important morphological and functional adaptations in the gut. This study measured the gene expression of 6 inflammatory cytokines along the small intestine (SI) and the proximal colon in 28-d-old piglets (n = 45) at different time points (0, 1, 2, 5 and 8 d) postweaning, using RT-PCR. Villus-crypt architecture and enzymatic activities of lactase and sucrase in the SI were also examined. The results confirmed that weaning is associated with morphological and enzymatic changes in the SI. In addition, the data indicated that cytokine response in the gut could be divided into two periods: an early acute response (0 to 2 d postweaning) and a late long-lasting response (2 to 8 d postweaning). Between d 0 and d 2, the levels of IL-1beta, IL-6, and TNF-alpha messenger RNA (mRNA) increased. Marked upregulation of IL-1beta mRNA occurred in most parts of the intestine, whereas IL-6 and TNF-alpha mRNA markedly increased only at specific sites in the intestine. Between d 2 and d 8, the levels of IL-1beta, IL-6, and TNF-alpha mRNA rapidly returned to preweaning values, except that the level of TNF-alpha mRNA remained high in the distal SI. Levels of IL-12 subunit p40 (IL-12p40) and IL-18 mRNA also decreased, compared to those on d 0. Taken together, these results demonstrate that weaning in piglets is associated with an early and transient response in gene expression of inflammatory cytokines in the gut.

Jejunal brush border microvillous alterations in Giardia muris-infected mice: role of T lymphocytes and interleukin-6.

Intestinal colonization with the protozoan Giardia causes diffuse brush border microvillous alterations and disaccharidase deficiencies, which in turn are responsible for intestinal malabsorption and maldigestion. The role of T cells and/or cytokines in the pathogenesis of Giardia-induced microvillous injury remains unclear. The aim of this study was to assess the role of T cells and interleukin-6 (IL-6) in the brush border pathophysiology of acute murine giardiasis in vivo. Athymic nude (nu(-)/nu(-)) CD-1 mice and isogenic immunocompetent (nu(+)/nu(+)) CD-1 mice (4 weeks old) received an axenic Giardia muris trophozoite inoculum or vehicle (control) via orogastric gavage. Weight gain and food intake were assessed daily. On day 6, segments of jejunum were assessed for parasite load, brush border ultrastructure, IL-6 content, maltase and sucrase activities, villus-crypt architecture, and intraepithelial lymphocyte (IEL) infiltration. Despite similar parasitic loads on day 6, infected immunocompetent animals, but not infected nude mice, showed a diffuse loss of brush border microvillous surface area, which was correlated with a significant reduction in maltase and sucrase activities and a decrease in jejunal IL-6 concentration. In both athymic control and infected mice, jejunal brush border surface area and disaccharidases were high, but levels of tissue IL-6 were low and comparable to the concentration measured in immunocompetent infected animals. In both immunocompetent and nude mice, infection caused a small but significant increase in the numbers of IELs. These findings suggest that the enterocyte brush border injury and malfunction seen in giardiasis is, at least in part, mediated by thymus-derived T lymphocytes and that suppressed jejunal IL-6 does not necessarily accompany microvillous shortening.

A mouse model of intestinal stem cell function and regeneration.

We present here an in vivo mouse model for intestinal stem cell function and differentiation that uses postnatal intestinal epithelial cell aggregates to generate a differentiated murine small intestinal mucosa with full crypt-villus architecture. The process of neomucosal formation is highly similar to that of intestinal regeneration. Both in vivo grafting and primary culture of these cells reveal two different epithelial cell populations, which display properties consistent with intestinal epithelial transit amplifying and stem cell populations. Using this model system with a mixture of wild-type and transgene marked cells, we have shown that neomucosae originally develop from single aggregates, but that over time the mucosae fuse to form chimaeric mucosae. Despite fusion, the chimaeric mucosae maintain crypt clonality and villus polyclonality, demonstrating that clonal segregation persists during intestinal epithelial regeneration.

Changes in murine jejunal morphology evoked by the bacterial superantigen Staphylococcus aureus enterotoxin B are mediated by CD4+ T cells.

Bacterial superantigens (SAgs) are potent T-cell stimuli that have been implicated in the pathophysiology of autoimmune and inflammatory disease. We used Staphylococcus aureus enterotoxin B (SEB) as a model SAg to assess the effects of SAg exposure on gut form and cellularity. BALB/c, SCID (lacking T cells) and T-cell-reconstituted SCID mice were treated with SEB (5 or 100 microg intraperitoneally), and segments of the mid-jejunum were removed 4, 12, or 48 h later and processed for histochemical or immunocytochemical analysis of gut morphology and major histocompatibility complex class II (MHC II) expression and the enumeration of CD3+ T cells and goblet cells. Control mice received saline only. SEB treatment of BALB/c mice caused a time- and dose-dependent enteropathy that was characterized by reduced villus height, increased crypt depth, and a significant increase in MHC II expression. An increase in the number of CD3+ T cells was observed 48 h after exposure to 100 microg of SEB. Enteric structural alterations were not apparent in SEB-treated SCID mice compared to saline-treated SCID mice. In contrast, SEB challenge of SCID mice reconstituted with a mixed lymphocyte population or purified murine CD4+ T cells resulted in enteric histopathological changes reminiscent of those observed in SEB-treated BALB/c mice. These findings implicate CD4+ T cells in this SEB-induced enteropathy. Our results show that SAg immune activation causes significant changes in jejunal villus-crypt architecture and cellularity that are likely to impact on normal physiological processes. We speculate that the elevated MHC II expression and increased number of T cells could allow for enhanced immune responsiveness to other SAgs or environmental antigens.

Effects of methotrexate on the intestinal epithelium are influenced by environmental changes

The functions of the intestinal epithelium of patients receiving cytostatic drugs is severely impaired, leading to malabsorption and malnutrition. More insight in mechanisms that mediate mucosal damage and repair is needed for the development of prophylactic regimes. Aim: To further investigate the effects of the cytostatic drug methotrexate (MTX) on functional capacities of intestinal epithelial cells during damage and regeneration by studying epithelial morphology and gene expression. Methods: Rats were subjected to a single or double dose of intraveneously injected MTX ranging from 10 to 60 mg/kg. Rats were kept in cages either alone or grouped (n=4) and sacrificed at 2, 4, 6 or 8 days after the final injection. Jejunal segments were isolated and processed for histochemical analysis. Next to morphology, changes in epithelium-specific differentiation markers were studied at the level of mRNA (in situ hybridization) and protein (immunohistochemistry). Sucraseisomaltase (SI), intestinal (i-) and liver (1-) fatty acid binding protein (FABP) and sodium-glucosetransporter 1 (SGLT1) were used as markers for enterocytes. Mucin (Muc2) and the protease matrilysin were used for goblet cells and Paneth cells, respectively. Results: After a week of adaptation to solitary caging, a single dose of 30, 45 or 60 mg/kg MTX did not lead to any epithelial alterations. However, when two injections of 30 mg/kg MTX were given with a 24 hour interval, 100% mortality occurred at day 6 after the last injection. In rats adapted to group-caging, two-day treatment with 20 mg/kg and 10 mg/kg per day led to villus atrophy that was maximal at day 4 after the last dose and associated with severe cryptloss. In contrast to iand 1-FABP mRNA's, no expression of SI mRNA and SGLT1 mRNA were detected at day 4. At the level of protein, changes in gene expression parallelled the mRNA patterns. Between day 6 and 8, expression 'patterns of all markers returned to normal as the crypt villus architecture restored. Expression patterns of MUC2 and matrilysin mRNA and protein did not show major changes during the period analysed. When rats were caged alone when treatment with MTX started, a single dose of 30 mg/kg affected the epithelium comparable to treatment with 20 mg/kg + 10 mg/kg in rats adapted to group-caging. Conclusions: The observed differences in intestinal epithelial damage between rats adapted to solitary or group-caging versus rats suffering social loss at the moment of MTX treatment, indicate that environmental changes induce a condition of stress leading to increased epithelial sensitivity towards MTX. Still, epithelial damage and mortality also occur in rats kept in a socially known environment using longer period of treatment (2 days). During MTX-induced mucosal damage, enterocyte function is affected with respect to SI and SGLTI expression whereas iand 1-FABP expression is unaltered, suggesting changes in enterocyte metabolism from sugar to fatty acid absorption. Expression of luminal cytoprotective MUC2 and basolateral metalloprotease matrilysin are maintained during MTX-indnced epithelial damage, since these markers remained detectable throughout the study.

DISTINCT CHANGES IN INTESTINAL EPITHELIAL GENE EXPRESSION UPON METHOTREXATE TREATMENT

Introduction. Structure, function and differentiation of the intestinal epithelium are severely affected in patients receiving cytostatic drugs. Especially in pediatric patients, major side effects like malabsorption and malnutrition are of great concern. Little is known about effects of cytostatics on gene expression in the intestinal epithelium and about the mechanisms that mediate the mucosal damage and regeneration. Aim. In order to get insight in the functional capacities of intestinal epithelium during tissue damage and subsequent regeneration, we focussed on rats treated with methotrexate (MTX) as a model to investigate quantitavely the changes in epithelium specific gene expression. Methods. Rats were injected i.v. with a single dose of MTX (30 mg/kg). Up to 10 days after treatment rats were sacrificed and jejunal segments were removed and processed for histological and biochemical analysis. Gene expression of cell type specific differentiation markers was quantified at the level of mRNA, protein and enzyme activity using Northern blots, Western blots and enzyme assays, respectively. Results. Histologically, mucosal damage resulted in villus shortening at day 1 that increased to almost complete villus atrophy at day 4. During day 1 to day 4, enterocyte markers carbamoyl phosphate synthese (CPS) and sucrase-isomaltase (SI) decreased one after another to undetectable levels, whereas the enterocyte marker intestinal fatty acid binding protein (iFABP) increased to 120% of control values. In these tissue samples, expression of the mucin MUC2, a goblet cell marker, increased 1.5-fold. Furthermore, expression of chromogranin A (chromA), a marker for differrentiated entero-endocrine cells, decreased 2.5-fold during villus shortening. Expression of lysozyme, a marker for Paneth cells, increased 3-fold at day 2. The crypt villus architecture was regenerated at day 6. During regeneration, expression of all differentiation markers returned to normal values. Expression patterns of mRNA, protein and enzyme activity of each marker gene were parallel during the course of the experiment. Conclusions. Our results indicate a distinct effects of MTX on gene expression in different intestinal epithelial cell types. Decreased expression of CPS and SI together with an increased expression of iFABP after MTX treatment, suggests selective changes in enterocyte metabolism during damage and regeneration. Furthermore, goblet cells and Paneth cells contribute to increased synthesis of the cytoprotective molecules, MUC2 and lysozyme, during epithelial damage. Since effects of MTX parallelled at the different levels of expression, changes in epithelial gene expression seem to be mainly regulated at pretranslational level.

Ionizing radiation reduces neurally evoked electrolyte transport in rat ileum through a mast cell-dependent mechanism

Background/Aims: Mechanisms of neuroimmune regulation of intestinal electrolyte transport under pathophysiological conditions are unclear. This study investigated the effect of ionizing radiation on ileal electrolyte transport. Methods: Rats were exposed to 10 Gy γ-radiation and were killed 2, 24, and 48 hours later. Ileal segments were either mounted in Ussing chambers and exposed to electrical field stimulation, prostaglandin E2, leukotriene D4, or theophylline, or they were assayed for biochemical indices of inflammation. Other segments were processed for routine histological screening, mast cell counts, or immunohistochemical analysis of the distribution of vasoactive intestinal polypeptide or substance P immunoreactivity. Results: Basal short-circuit current was unchanged 2, 24, or 48 hours postirradiation. However, there was a reduction of tissue responsiveness to electrical field stimulation, prostaglandin E2, and theophylline but not to leukotriene D4. Decreased responsiveness at 2-hours postirradiation was blocked by pretreatment with the H1 antagonist pyrilamine. Tissue myeloperoxidase activity and 5-hydroxytryptamine content were not altered post-irradiation, but tissue histamine and mucosal mast cells were significantly reduced at 24 and 48 hours. There were no significant changes in villus-crypt architecture until 48 hours postirradiation. There was no significant alteration in the distribution of immunoreactive vasoactive intestinal polypeptide or substance P. Conclusions: Ionizing radiation reduced the transport response to neural stimulation. The effect correlated temporally with decreased mast cells and histamine, suggesting a functional role for previously reported mast cell-nerve interactions.

Adaptation of the small intestine in desert-dwelling animals: Morphology, ultrastructure and electrolyte transport in the jejunum of rabbits, rats, gerbils and sand rats

1. 1. The aim of this study was to characterize adaptive alterations of the small intestine in desert-dwelling animals by comparing jejunal morphology, ultrastructure and function in mammalian species with or without desert habitat origins. 2. 2. Crypt-villus architecture, brush border surface area, and electrolyte transport were measured and compared in the jejunum of rabbits (Oryctolagus cuniculus), rats (Rattus rattus), gerbils (Meriones unguiculatus and sand rats Psammomys obesus). 3. 3. In addition, transport of 3-0-methyl-D-glucose was compared in rats, rabbits and gerbils. 4. 4. Gerbils and sand rats had significantly longer microvilli and villi ( P < 0.01), increased brush border surface area ( P < 0.01), and greater absorption of Na + and Cl - ( P < 0.05) than rabbits and rats. 5. 5. Absorption of3-0-methyl-D-glucose was also significantly ( P < 0.05) greater in gerbils than in rabbits or rats. 6. 6. The findings demonstrate enhanced small intestinal absorption of electrolytes and nutrients in desert-dwelling animals. 7. 7. This increase was associated with an enlarged mucosal absorptive surface area. 8. 8. The findings suggest that intestinal mucosal adaptation allows desert-dwelling animals to compensate for the limited availability of water and nutrients in an arid environment.

Upper intestinal mucosal proliferation in the newborn guinea pig: effect of composition of milk feeds.

To examine the effects of first enteral feeds on the development of the gastrointestinal tract, the changes in upper intestinal mucosal morphology and kinetics were studied during the 1st wk of postnatal life in neonatal guinea pigs. Animals were reared either on mother's milk or on a cow's milk formula isocaloric with guinea pig milk. Mucosal crypt-villus architecture was measured by microdissection, and mucosal kinetics were measured using a metaphase arrest technique. Comparable growth was achieved between the two feeding groups. There were no significant differences in the villus heights, crypt depths, or crypt:villus ratios between the naturally fed and formula-fed guinea pigs. The formula fed had a crypt cell production rate twice as high as the naturally fed animals throughout the study period (p less than 0.001). The higher mucosal proliferation rate of the formula-fed animals may be due to the absence of growth-modulating factors in this milk, or a regenerative response to "damage" of the upper intestinal mucosa by cow's milk proteins.

Ontogenic Timing Mechanism Initiates the Expression of Rat Intestinal Sucrase Activity

Morphologic and enzymic differentiation occurs in rat small intestinal epithelium during 16-20 days of postnatal life. This change is considered to be initiated by an ontogenic timing mechanism and is modulated by extrinsic systemic and luminal factors. The importance of the ontogenic timing was tested directly using a transplantation technique in which jejunal isografts from newborn (day 0) and 5-day-old (day 5) rats were implanted under the skin of newborn (day 0) hosts. Isografts showing cryptvillus architecture were obtained in 44% and 21% of transplants, respectively. Day 0 isografts and host intestine expressed sucrase activity at about 16-18 days of age and showed similar crypt cell labeling and epithelial migration after [3H]thymidine injection. Day 5 isografts expressed sucrase activity when the hosts were 13 days of age, whereas host intestine showed no detectable sucrase activity. Isograft lactase activities in both experimental transplant models were significantly higher than host intestinal lactase up to 28 days of age, suggesting that luminal factors are important in modulating lactase activity during the first 4 wk of postnatal life. It is concluded that (a) no systemic factors at day 13 inhibit the expression of sucrase activity and (b) an ontogenic timing mechanism in the jejunum initiates the expression of sucrase activity.

Effects of biliary diversion to the ileum on the in situ disaccharidase activities of duodenal and ileal mucosa in the rat: a study on enzyme kinetics in tissue sections.

In order to study the influence of biliary secretions on the in situ kinetic data of brush border disaccharidases in relation to small intestinal villus-crypt architecture, an anastomosis was constructed between the common bile duct, which had been divided before its entrance into the pancreatic head, and the ileum in adult female Wistar rats. Simple attachment of the terminal ileum to the liver hilum was performed in the controls. Six weeks after the operation, tissue sections were prepared from duodenum and ileum of biliary-diverted and control animals (n = 5 in each group) and examined by section biochemistry and morphometry. In both groups the apparent Vmax values of neutral alpha-glucosidase and lactase/beta-glucosidase, measured at the villus base and at its apex, and the villus height were decreased from the duodenum towards the distal ileum. After biliary diversion to the ileum, an increase in villus height ensued in this segment, and the increasing gradient of glucosidase activity towards the villus apex, as seen in the ileum of the controls, was no longer detectable. In the duodenum, however, villus height remained unaltered and the in situ activity of the disaccharidases was increased at both villus sites when compared with the controls. The results indicate different effects of bile on mucosal morphology and function in the proximal and distal small intestine.

Adaptative response of alpha- and beta-glucosidase kinetics along the villi of rat self-filling jejunal blind loops.

Four and 12 days after the construction of self-filling jejunal blind loops in the rat, the apparent Km- and Vmax-values of lactase/beta-glucosidase and neutral alpha-glucosidase were determined by in-situ quantitative enzyme histochemistry, and changes in the villus-crypt architecture of the mucosa were examined by microdissection. The results were compared with corresponding data from sham-operated controls. The kinetics data were obtained from the base and the transition zone between medium and apical villus third by the use of a microdensitometric technique. The apparent Vmax of lactase/beta-glucosidase is significantly smaller than in the control rats at both measuring sites of the villi and even decreases from day 4 to day 12. The apparent Vmax of neural alpha-glucosidase is not affected, and thus the same increase in enzyme activity along the villi as in the controls is observed. The apparent Km of this enzyme, however, is already significantly increased on day 4 at both villus positions in the blind loops. A pronounced increase in villus surface area is detected in the blind loops as a result of an increase in crypt cell proliferation. The results indicate that enzymatic adaptation in the self-filling blind loops of rat jejunum exhibits different patterns for brush border alpha- and beta-glucosidases and is at least in part accomplished independently of the pronounced mucosal transformation occurring in this experimental condition.

Giardiasis: Clinical Spectrum and Functional-Structural Abnormalities of the Small Intestinal Mucosa

We studied 17 adult and 11 pediatric patients with giardiasis in order to examine the clinical spectrum and the functional-structural changes of the small intestinal mucosa. The most common clinical manifestations were diarrhea, weight loss, abdominal cramps, and, in children, failure to thrive. Giardiasis was frequently associated with hypogammaglobulinemia (HGG) and intestinal nodular lymphoid hyperplasia (47% and 41% of adults, respectively). Malabsorption of D-xylose and vitamin B12 occurred in 55% and 60% of patients, respectively, steatorrhea in 64%, and hypocarotinemia and low serum folate in 36%. No or only slight mucosal abnormalities were present in pretreatment jejunal biopsies of 75% of those adults on whom biopsies were performed, whereas 25% (all with HGG) had marked inflammation and changes of the villus-crypt architecture. Marked structural changes were seen in 43% of subjects with HGG. Estimates of brush border enzymes showed a significant reduction in lactase, sucrose, and leucyl-naphthylamidase specific activity in about 80% of patients assayed, whereas maltase, isomaltase, and alkaline phosphatase were not affected. Five patients were studied prospectively and absorption tests and jejunal mucosal biopsies were repeated after treatment with metronidazole or quinacrine. Resolution of symptoms with restoration of the morphologic and functional abnormalities of the small intestinal mucosa occurred after treatment in all but 1 patient. These observations indicate that Giardia cause brush border damage, which, is usually reversible with eradication of the parasite. Other factors, alone or in combination with Giardia, may be responsible for the cell damage beyond brush border injury found in some patients.