Olena Prykhodko

Induced growth and maturation of the gastrointestinal tract after Phaseolus vulgaris lectin exposure in suckling rats.

Objectives: In mammals, the postnatal development of the gastrointestinal tract is characterized by vast structural and functional changes. Using a suckling rat model, we investigated whether red kidney bean lectin, phytohemagglutinin (PHA), a potent gut mitogen in adult rats, can accelerate the growth and maturation of the gastrointestinal tract. Methods: At either 10 or 14 days of age, suckling rats were daily gavage fed with PHA (0.05 mg/g body weight) or saline for 3 days. At 1 or 3 days after this treatment, gastrointestinal organ growth, intestinal morphology, disaccharidase pattern, macromolecular absorption capacity, and pancreatic enzyme contents were studied. Results: After PHA exposure, increased small intestinal growth and number of crypt cells were observed, whereas the proportion of enterocytes with supranuclear vacuoles in the distal intestine was decreased. The macromolecular absorption of the markers bovine immunoglobulin (Ig)G and bovine serum albumin and plasma levels of maternal IgG decreased, and intestinal disaccharidases switched toward an adult-like pattern. The pancreas weight and pancreatic protein and trypsin contents increased. These changes were partly reversible when the PHA treatment began at 10 days of age, but they persisted when the treatment began at 14 days of age. Conclusions: PHA induced enhanced growth and precocious functional maturation of the gastrointestinal tract in suckling rats. The effects persisted if the PHA treatment started at 14 days of age, but not before, suggesting an age dependent mechanism. These findings may lead to a better understanding of gastrointestinal maturation and constitute a basis for the treatment of mammals having an immature gut.

Binding and the effect of the red kidney bean lectin, phytohaemagglutinin, in the gastrointestinal tract of suckling rats

Enteral exposure of suckling rats to phytohaemagglutinin (PHA) has been shown to induce growth and precocious functional maturation of the gastrointestinal tract. The aim of the present study was to explore the mechanism of this action. Suckling rats, 14 d old, were fed a single dose of PHA (0.05 mg/g body weight) or saline. The binding of PHA to the gut epithelium and its effect on the morphology and functional properties of the gut and pancreas were studied up to 3 d after treatment. Initially, at 1-24 h, the PHA bound along the gut mucosal lining, resulting in disturbed gut morphology with villi shortening and rapid decreases in disaccharidase activities and macromolecular absorption capacity. During a later phase, between 1 and 3 d, the PHA binding had declined, and an uptake by enterocytes was observed. An increase in crypt cell proliferation and gut growth became evident during this period, together with a functional maturation, as indicated by increases in disaccharidase (maltase and sucrase) activities and the low macromolecular absorption capacity. Pancreas growth also increased, as did its content of digestive enzymes. We conclude that enteral exposure to PHA in suckling rats temporarily causes mucosal disarrangement and functional impediment of the gut, which may be explained by binding to and disruption of the gut mucosa and a two-fold increase in the plasma corticosterone concentration. These findings may lead to a better understanding of the role of diet in gastrointestinal maturation and may constitute a basis for the treatment of mammals having an immature gut.

Enterally but Not Parenterally Administered Phaseolus vulgaris Lectin Induces Growth and Precocious Maturation of the Gut in Suckling Rats

Background: The lectin, phytohemagglutinin (PHA) has been shown to induce growth and functional maturation of the gastrointestinal (GI) tract in suckling rats. Objectives: To investigate the effect of the administration route, and whether enteral exposure to PHA was necessary to induce functional maturation. Methods: Fourteen-day-old rats were daily administered PHA via orogastric feeding (0.05 mg PHA/g BW) or via subcutaneous injection (0.05 or 0.005 mg PHA/g BW) for 3 days, while the controls received saline orogastrically. At 17 days of age, organ weight, intestinal and pancreatic function, and plasma corticosterone levels were analyzed. Moreover, 14-days old pups receiving a single dose of PHA, enterally or parenterally, were sacrificed after 12 h and examined for organ PHA binding using immunohistochemistry. Results: Enteral PHA exposure resulted in PHA binding in the epithelial lining of the small intestine, increased gastrointestinal growth, reduced intestinal macromolecular absorption, altered the disaccharidase expression towards an adult-like pattern, and increased the pancreatic protein and trypsin contents. In contrast, parenteral PHA exposure (high dose) resulted in PHA-binding in extra-intestinal organs, increased liver and spleen weight, and decreased thymus weight. Moreover, the intestinal maltase activity increased moderately, and the transfer of BSA to blood plasma was partially reduced. Both PHA treatments led to elevated plasma corticosterone levels. Conclusion: These results demonstrated that enteral exposure to PHA was necessary to induce the precocious maturation of the GI tract and the pancreas, while parenteral administration affects the extra-intestinal organs. Furthermore, the enteral effects were probably not mediated via a corticosteroid dependent pathway.

Intake of Blueberry Fermented by Lactobacillus plantarum Affects the Gut Microbiota of L-NAME Treated Rats

Prebiotics, probiotics, or synbiotics can be used as means to regulate the microbiota to exert preventative or beneficial effects to the host. However, not much is known about the effect of the gut microbiota on hypertension which is a major risk factor of cardiovascular disease and also a symptom of the metabolic syndrome. The NG-nitro-L-arginine methyl ester (L-NAME) induced hypertensive rats were used in order to test the effect of a synbiotic dietary supplement of Lactobacillus plantarum HEAL19 either together with fermented blueberry or with three phenolic compounds synthesized during fermentation. The experimental diets did not lower the blood pressure after 4 weeks. However, the fermented blueberries together with live L. plantarum showed protective effect on liver cells indicated by suppressed increase of serum alanine aminotransferase (ALAT) levels. The diversity of the caecal microbiota was neither affected by L-NAME nor the experimental diets. However, inhibition of the nitric oxide synthesis by L-NAME exerted a selection pressure that led to a shift in the bacterial composition. The mixture of fermented blueberries with the bacterial strain altered the caecal microbiota in different direction compared to L-NAME, while the three phenolic compounds together with the bacteria eliminated the selection pressure from the L-NAME.

Pancreatic and pancreatic-like microbial proteases accelerate gut maturation in neonatal rats.

Objectives Postnatal gut maturation in neonatal mammals, either at natural weaning or after precocious inducement, is coinciding with enhanced enzymes production by exocrine pancreas. Since the involvement of enzymes in gut functional maturation was overlooked, the present study aimed to investigate the role of enzymes in gut functional maturation using neonatal rats. Methods Suckling rats (Rattus norvegicus) were instagastrically gavaged with porcine pancreatic enzymes (Creon), microbial-derived amylase, protease, lipase and mixture thereof, while controls received α-lactalbumin or water once per day during 14–16 d of age. At 17 d of age the animals were euthanized and visceral organs were dissected, weighed and analyzed for structural and functional properties. For some of the rats, gavage with the macromolecular markers such as bovine serum albumin and bovine IgG was performed 3 hours prior to blood collection to assess the intestinal permeability. Results Gavage with the pancreatic or pancreatic-like enzymes resulted in stimulated gut growth, increased gastric acid secretion and switched intestinal disaccharidases, with decreased lactase and increased maltase and sucrase activities. The fetal-type vacuolated enterocytes were replaced by the adult-type in the distal intestine, and macromolecular transfer to the blood was declined. Enzyme exposure also promoted pancreas growth with increased amylase and trypsin production. These effects were confined to the proteases in a dose-dependent manner. Conclusion Feeding exogenous enzymes, containing proteases, induced precocious gut maturation in suckling rats. This suggests that luminal exposure to proteases by oral loading or, possibly, via enhanced pancreatic secretion involves in the gut maturation of young mammals.

Precocious gut maturation and immune cell expansion by single dose feeding the lectin phytohaemagglutinin to suckling rats.

The dietary lectin phytohaemagglutinin (PHA) induces gut growth and precocious maturation in suckling rats after mucosal binding. The present study investigated the dose range in which PHA provokes gut maturation and if it coincided with immune activation. Suckling rats, aged 14 d, were orogastrically fed a single increasing dose of PHA: 0 (control), 2, 10, 50 or 250 microg/g body weight (BW) in saline. The effect on gut, lymphoid organs and appearance of CD3+ (T-lymphocyte) and CD19+ (B-lymphocyte) cells in the small-intestinal mucosa was studied at 12 h (acute) and 3 d (late phase) after treatment. The low PHA doses (2 and 10 microg/g BW) induced intestinal hyperplasia without mucosal disarrangement but did not provoke gut maturation. Only the high PHA doses (50 and 250 microg/g BW) temporarily disturbed the intestinal mucosa with villi shortening and decrease in disaccharidase activities, and later after 3 d provoked precocious maturation, resulting in an increase in maltase and sucrase activities and decrease in lactase activity and disappearance of the fetal vacuolated enterocytes in the distal small intestine. Exposure to the high, but not to the low, PHA doses increased the number of mucosal CD19+ and CD3+ cells in the small intestine after 12 h, a finding also observed in untreated weaned rats aged 21-28 d. In conclusion, there was a dose-related effect of PHA on gastrointestinal growth and precocious maturation that coincided with a rapid expansion of mucosal B- and T-lymphocytes, indicating a possible involvement of the immune system in this process.

A piglet with surgically induced exocrine pancreatic insufficiency as an animal model of newborns to study fat digestion.

The maldigestion and malabsorption of fat in infants fed milk formula results due to the minimal production of pancreatic lipase. Thus, to investigate lipid digestion and absorption and mimic the situation in newborns, a young porcine exocrine pancreatic insufficient (EPI) model was adapted and validated in the present study. A total of thirteen EPI pigs, aged 8 weeks old, were randomised into three groups and fed either a milk-based formula or a milk-based formula supplemented with either bacterial or fungal lipase. Digestion and absorption of fat was directly correlated with the addition of lipases as demonstrated by a 30% increase in the coefficient of fat absorption. In comparison to the control group, a 40 and 25% reduction in total fat content and 26 and 45% reduction in n-3 and n-6 fatty acid (FA) content in the stool was observed for lipases 1 and 2, respectively. Improved fat absorption was reflected in the blood levels of lipid parameters. During the experiment, only a very slight gain in body weight was observed in EPI piglets, which can be explained by the absence of pancreatic protease and amylase in the gastrointestinal tract. This is similar to newborn babies that have reduced physiological function of exocrine pancreas. In conclusion, we postulate that the EPI pig model fed with infant formula mimics the growth and lipid digestion and absorption in human neonates and can be used to elucidate further importance of fat and FA in the development and growth of newborns, as well as for testing novel formula compositions.

Effects on gut properties in exocrine pancreatic insufficient (EPI) pigs, being growth retarded due to pancreatic duct ligation at 7 weeks but not at 16 weeks of age.

PURPOSE Exocrine pancreatic insufficiency (EPI) induced in young pigs by pancreatic duct ligation (PDL) early after weaning result in total growth deprivation while it has little effect in somewhat older pigs. The main objective was to study effects of EPI on gut structure and function in littermate pigs underwent to PDL at different age. MATERIAL/METHODS Pigs, duct-ligated at either 7 (2 weeks post-weaning, PDL-7) or 16 weeks of age (PDL-16), and euthanized at an age of 21-23 weeks together with un-operated littermates were studied. The intestinal in vitro permeability was studied in separate PDL-pigs and compared to un-operated. RESULTS Morphometric analysis showed gut mucosal atrophy in the PDL-7 as compared to PDL-16 pigs, while no differences in mucosal disaccharidase activities. The intestinal permeability for different-sized markers was significantly increased in the PDL-pigs compared to the un-operated controls. Analyses of the intestinal digesta showed a total lack of pancreatic enzymes in all PDL-pigs, while instead new, as yet unidentified, enzyme-activities appeared. CONCLUSIONS All EPI-pigs, independent of age at PDL-operation, displayed adaptive gut changes, however the EPI-pigs operated early after weaning appeared more sensitive, probably related to their gut maturity and possibly explaining the growth arrest seen in these pigs.

Exogenous pancreatic-like enzymes are recovered in the gut and improve growth of exocrine pancreatic insufficient pigs

The exocrine pancreatic insufficient (EPI) pigs grow less due to different disturbances in feed digestion, absorption, and retention. Use of pancreatic-like enzymes of microbial origin in pigs may improve feed use and performance in slow-growing pigs. The aim was to study gut recovery and effectiveness of pancreatic-like enzymes of microbial origin supplementation on pig performance. Six male pigs 10 to 12 kg BW underwent pancreatic duct ligation surgery to induce total exocrine pancreatic insufficiency (EPI). Three cannulas to access the gastrointestinal tract content were installed in stomach, duodenum, and ileum in EPI pigs and in 3 control (healthy) pigs. One month after surgery, enzymes were given before feeding and digesta samples were collected for analyses. The BW of EPI pigs did not increase during 1 mo following surgery (11.7 vs. 11.6 kg BW); however, BW increased after 1 wk of enzyme supplementation (12.1 kg BW). Coefficient of fat and N absorption increased (P < 0.05) in EPI pigs after enzyme supplementation. Activity of amylase, lipase, and protease in chyme samples of EPI pigs was very low compared to controls. In EPI pigs after enzyme supplementation, amylase activity increased from 5.32 to 72.9 units/mL but remained lower than that of healthy pigs (162.7 units/mL). Lipase activity increased from 79.1 to 421.6 units/mL, which was similar to that of controls (507.3 units/mL). Proteolytic activity increased from 7.8 to 69.7 units/mL but still did not reach control pigs (164.3 units/mL). In conclusion, exogenous microbial enzymes mimic endogenous pancreatic enzymes being recovered along the lumen of the gastrointestinal tract. These enzymes might be a useful tool to stimulate growth of slower-growing pigs after the weaning period.

The effect of long-term lactobacilli (lactic acid bacteria) enteral treatment on the central nervous system of growing rats.

The aim of this study was to explore the relationship between consumption of large doses of lactic acid bacteria (LAB) and the behaviour and brain morphobiochemistry of normal growing rats. Four groups of rats were treated with LAB cultures twice daily for 6 months. The control group received 1 ml of saline per treatment, while two experimental groups received 1 ml of living bacteria (Lactobacillus plantarum and Lactobacillus fermentum, respectively) and the remaining group received a heat-treated (inactivated) L. fermentum culture. After 2 and 6 months of treatment, respectively, eight animals from each group were sacrificed, and specimens were taken for further analyses. The behaviour of the rats was evaluated five times in an open-field test at monthly intervals throughout the study. Lactobacilli treatment for 2 months induced changes in the motoric behaviour of the rats. The concentration of the astrocytesoluble and filament glial fibrillary acidic protein (GFAP) decreased in the posterior part of the hemispheres, including the thalamus, hippocampus and cortex of the rats treated with L. fermentum. A greater decrease in filament GFAP (up to 50%) was shown in the group receiving the live form of L. fermentum. In contrast, the GFAP in the live L. plantarum-treated group increased, showing elevated levels of the soluble and filament forms of GFAP in the posterior part of the hemispheres. A 60-66% decrease in the amount of the astrocyte-specific Ca-binding protein S-100b was shown in the posterior parts of the hemispheres and in the hindbrain of rats given LAB for 2 months. Prolonged feeding with LAB for 4 months up to full adulthood led to a further decrease in astrocyte reaction, reflected as an additional decrease in the amount of soluble GFAP and locomotor activity in all experimental groups. The changes in filament GFAP and S-100b appeared to disappear after prolonged feeding (total of 6 months) with LAB. In summary, LAB dietary treatment affected the ontogenetic development of the astrocytes, with the highest intensity observed in the early stages of rat development. It can be postulated that LAB treatment may play a preventive role in neurological diseases by decreasing astrocyte reaction and, consequently, lowering locomotor activity.