Applicability of the Ussing chamber technique to permeability determinations in functionally distinct regions of the gastrointestinal tract in the rat.

BACKGROUND: Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. METHODS: In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 mg kg(-1)), while ileal tissues were damaged in vitro by 0.4 M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). RESULTS: Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G (P < 0.001) and HRP (P < 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased (P < 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. CONCLUSION: We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.

IGFBP mRNA expression in small intestine of rat during postnatal development.

In contrast to the adult gut, the immature intestine is refractory to subcutaneously infused insulin-like growth factor I (IGF-I). IGF binding protein (IGFBP) mRNA expression was characterized in intestinal tissues from 6-, 19-, and 90-day-old rats to determine if changes in local expression could account for this age-related change in IGF-I potency. For all age groups, IGFBP-3 to -6, but not IGFBP-1 or -2, were detected by Northern blot analysis. IGFBP-3, -4, and -5 were more intensely expressed in the 6-day-old rat intestine compared with weanling or adult tissue. In contrast, IGFBP-6 expression peaked at the time of weaning. In situ hybridization showed IGFBP-3 to -6 expression was confined to cells of the lamina propria and submucosa and also in the muscularis layer for IGFBP-5. Furthermore, the pattern of IGFBP-5 localization in the intestine changed with development. The findings indicate that the expression of IGFBP-3 to -6 is higher in the immature intestine compared with the adult intestine, suggesting locally produced IGFBPs may inhibit systemically derived IGF-I action in the intestine. Therefore, changes to local IGFBP expression may contribute to the varying response of the rat intestine to IGF-I peptides during postnatal development.

Systemically but not orogastrically delivered insulin-like growth factor (IGF)-I and long [Arg3]IGF-I stimulates intestinal disaccharidase activity in two age groups of suckling rats.

The growth mitogenic properties of IGF-I on tissues of the gastrointestinal tract are well established; however, IGF effects on enzyme maturation are less clear. To test whether IGF-I peptide administration stimulates disaccharidase activity, we administered IGF-I or the more potent analog, long [Arg3]IGF-I, at doses ranging between 2 and 12.5 micrograms g-1 d-1 to suckling Wistar rat pups by either continuous s.c. infusion or by three times daily orogastric gavage. Peptides were administered for approximately 6 d starting on d 6 or 12 postpartum with six to nine rats per group. The results of the study demonstrated that systemically but not orally administered IGF-I stimulated duodenal wet tissue weight (up to 85%) and length (up to 36%). Enzyme maturation was assessed by measuring disaccharidase biochemically in tissue homogenates. Enzyme activity was also localized histocytochemically in cryostat-sectioned duodenum. After systemic infusion of IGF-I, intestinal lactase activity increased proportional to mucosal mass in both age groups. Systemic infusion of the more potent analog, long [Arg3]IGF-I, precociously induced the decline in lactase activity and accelerated the appearance of sucrase activity in the rat pups infused during the later suckling period. These findings indicate that enzyme maturation can be accelerated by systemically derived IGF-I peptides. Orogastrically IGF-I peptides, delivered at pharmacologic doses, did not affect intestinal growth or digestive enzyme maturation in suckling rat pups treated between 6 and 18 d postpartum, indicating the efficacy of IGF-I peptides may depend on the route of delivery and postnatal age of the recipient.

Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum.

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-beta1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-beta. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-beta1, and in situ hybridization for TGF-beta1 mRNA was also performed on the duodenum. TGF-beta levels in milk were assayed by ELISA. TGF-beta2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-beta1 was not detected. In contrast, the cell number and intensity of staining of TGF-beta1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-beta1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-beta2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-beta production in the small intestine increases during the weaning period.

Systemic infusion of IGF-I or LR(3)IGF-I stimulates visceral organ growth and proliferation of gut tissues in suckling rats.

This study describes developmental changes in gastrointestinal response to insulin-like growth factor I (IGF-I) peptide administration. Neonatal rats were infused with IGF-I or long [Arg3]IGF-I (LR(3)IGF-I) for 6.5 days starting on day 6 or 12 postpartum. Peptides were delivered by mini osmotic pumps at 0, 2, 5, or 12.5 microg x g(-1) x day(-1). IGF-I infusion increased plasma IGF-I levels in both age groups but stimulated body weight gain only in the older rats. Infusion of LR(3)IGF-I did not change plasma IGF-I levels. Both peptides enhanced expression of IGF-binding proteins (IGFBP) 1 and 2 and induced IGFBP-3 in the older rats. For both age groups, weights of the kidney and spleen increased by up to 85 and 76%, respectively. IGF-I treatment also stimulated gut weight and length by up to 60 and 32%, respectively, but dose dependency was observed only in the older rats. LR(3)IGF-I was more potent for all growth parameters in both age groups. Histological observations included thickening of the mucosa and muscularis externa after infusion of IGF-I peptides. Thymidine labeling in the younger rats indicated that proliferative activity increased proportionately with crypt cell growth. These results show that IGF-I peptides selectively stimulate growth of gastrointestinal tissues in suckling rats and that the proximal gut was the most peptide-responsive region.

Administration of insulin-like growth factor-I (IGF-I) peptides for three days stimulates proliferation of the small intestinal epithelium in rats.

It has previously been shown that longterm administration of insulin-like growth factor-I (IGF-I) or the analogue Long R3 IGF-I (LR3IGF-I) selectively stimulate growth of the gastrointestinal tract in gut resected, dexamethasone treated, and normal rats. In this study, the short-term effects of IGF-I administration on intestinal proliferation have been investigated. Female rats (110 g, five-six/group) were infused for three days with 2.5 mg/kg/day of either IGF-I or LR3IGF-I and compared with vehicle treated or untreated control rats. LR3IGF-I but not IGF-I increased body weight and wet tissue weight of the small and large intestine (+20%), compared with controls. Tissue weight responses were independent of food intake and were reflected in the histology of the tissue. In LR3IGF-I treated animals, duodenal and ileal crypts length were increased by 13 and 22%, respectively, associated with an increase in crypt cell number. No such histological changes were seen in IGF-I treated rats. Tritiated thymidine labelling indices were significantly increased after administration of either IGF-I or LR3IGF-I (up to 14%) in both the duodenum and ileum. In IGF-I treated rats, increased nuclear labelling was not associated with an increase in the crypt compartment. In contrast, LR3IGF-I induced proportional increments in thymidine labelling and crypt size, suggesting that LR3IGF-I is not only more potent than the native peptide but also induced proliferative events more rapidly. In the colon, the thymidine labelling index was low, however, a non-significant increase in the number of cells labelled with thymidine was seen. These results suggest that within a three day treatment period intestinal mitogenesis is more advanced in animals treated with LR3IGF-I. The differences in proliferative response between the two peptides may be accounted for by variations in pharmacokinetics, clearance rates, and interactions with circulating and tissue specific binding proteins.

Prolonged administration of IGF peptides enhances growth of gastrointestinal tissues in normal rats.

To investigate the effect of insulin-like growth factor (IGF) peptide infusion on the gastrointestinal tract, female rats (115 g, 6/group) were treated for 14 days with IGF-I or long R (LR3IGF-I; 0, 44, 111, or 278 micrograms/day) delivered by osmotic minipumps. Both peptides induced a dose-dependent increase in gastrointestinal tissue weight. Total gut weight, small intestinal weight, and small intestinal length increased by 43, 47, and 13%, respectively, after treatment with 278 micrograms/day of LR3IGF-I. Crypt depth and villus height increased after peptide treatment with an associated increased crypt cell population (+33%), cells per villus column (+34%), and villus cell density (+20%). Proportional increments in proliferating cell nuclear antigen labeling and an unaltered crypt growth fraction indicated that the balance between the proliferative and maturation compartment of the crypt was maintained. Fecal nitrogen excretion was significantly reduced in rats treated with LR3IGF-I, suggesting an increased absorptive capacity of the duodenum. The enhanced potency of LR3IGF-I supports previous findings that the gut is especially responsive to analogues with reduced binding affinity to IGF-binding proteins.