Electron microscopic studies on the small intestine of rats after mechanical intestinal obstruction.

The morphologic changes of the small intestine after the mechanical obstruction were studied by light and electron microscopy. After the ligation of the upper small intestine, segments of jejunum, both proximal and distal to the site of obstruction, were removed at intervals varying from 45 min to 24 h. The essential changes were found in the epithelial cells of the tips of villi, and few morphologic differences were recognized between samples proximal and distal to the site of obstruction. The most remarkable changes in the mucosa were the pseudopodlike extension of the cytoplasm, vacuolar alteration of the villus epithelial cells, desquamation of these degenerated cells, and the dilatation of the epithelial intercellular spaces. A few epithelial cells showed hypertrophy of the smooth endoplasmic reticulum. In the submucosa, vascular stasis and edema were observed throughout the course. The mechanism of fluid loss into the intestinal lumen was discussed briefly.     

Selective expression of brush border hydrolases by mouse Peyer's patch and jejunal villus enterocytes

Developmental profiles describing the expression of lactase, alpha-glucosidase, and alkaline phosphatase activities have been determined quantitatively in mouse jejunal enterocytes during migration over villi and Peyer's patch lymphoid tissue. The predicted maximal lactase and alpha-glucosidase activities expressed by enterocytes migrating over Peyer's patch follicles were about one-quarter and one-half of values found in control villi. Alkaline phosphatase activity was, on the other hand, one third greater in Peyer's patch compared with villus enterocytes. Expression of lactase and alpha-glucosidase activities was initially less in enterocytes migrating along interfollicular compared with control villi. Subsequent increase in hydrolase activities occurred during the later stages of enterocyte migration over interfollicular villi. Lactase activity in athymic mice Peyer's patch enterocytes was identical to that recorded for control mice. The corresponding value for villus lactase was, however, only half that found in control tissue. Factors produced locally in lymphoid follicles are probably responsible for selective effects on enterocyte differentiation.     

Time dependency of 1,25(OH)2D3 induction of calbindin mRNA and calbindin expression in chick enterocytes during their differentiation along the crypt-villus axis

Quantitative methods of in situ hybridization and immunocytochemistry have been used to measure 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) induction of calbindin mRNA and calbindin protein expressed in jejunal enterocytes at all points along the crypt-villus axis over a 24 h period. Small amounts of calbindin mRNA detected in vitamin D3 deficient (D-deficient) chick intestine increased rapidly to maximal values 8 h after hormone injection. The magnitude of this response was inversely related to age of enterocyte measured separately by injecting tritiated thymidine into D-deficient and 1,25(OH)2D3-injected birds. Enterocytes of all ages expressed small amounts of calbindin 3 h after hormone injection. This amount of calbindin then increased up to 24 h after hormone injection. Maximal calbindin expression took place in basal villus enterocytes. Later decrease in the ability of upper villus enterocytes to express calbindin was associated with a similar fall in calbindin mRNA expression. Previously it was suggested that inefficient translation to calbindin mRNA might take place in basal villus enterocytes 48 h after vitamin D injection. Present work using 1,25 (OH)2D3 shows that calbindin expression takes place at a constant rate during this early stage of enterocyte development. Secondary events limiting higher rates of calbindin synthesis in upper crypt and basal villus enterocytes remain to be identified.     

Toxoplasma Gondii: scanning electron microscope studies on the small intestine of infected and uninfected cats

The mucosal surfaces of villi from the small intestine of cats infected with Toxoplasma gondii were studied with the scanning electron microscope and compared with those from uninfected control cats. In uninfected cats villi were predominantly leaf shaped and were lined with ridges; goblet cell openings could be seen. The enterocytes had a hexagonal surface outline and were dome-shaped. Infected cats had both normal and abnormal villi. Injured villi were shortened and attained a broad leaf shape, often with blunt edges. Enterocytes containing oocysts were enlarged, and microvilli were resolvable only on these surfaces. Ruptured cells from which parasite discharge had occurred were seen. Oocysts were observed and possessed a smooth coat.     

The rhythm of mucus formation in the small intestinal epithelium of rats and mice]

The diurnal rhythm of mucose formation was studied in the epithelium of villi of the small intestine in rats and mice. The total amount of goblet enterocytes on a villus as well as their amount in the basal, medial and apical areas underwent changes during the day. The period of the greatest mucose formation was noted in the night time. The process of secretion of goblet enterocytes was found to be rhythmical and to alternate with predominant accumulation and producing secretion. The mutual shift of similar processes in the phase was noted at different levels of the villi. In the medial third of the villus the corresponding state is observed later than in the basal third and earlier than in the apical one. The agreement of these processes in time permits considering the villus as a system with circadian organization. The rhythmics of mucose formation in rats and mice has certain species peculiarities: in rats there is no difference in the distribution of goblet enterocytes along the length of the villi, but the diurnal rhythm of the changes of their total amount is more pronounced.     

Effects of genetic selection on growth rate and intestinal structure in the domestic fowl (Gallus domesticus)

1. Pieces of small intestine taken from chickens subjected previously to continuous selection, relaxed selection or no selection for rapid growth were used to estimate villus surface area and microvillus development to determine what effects genetic selection might have on factors controlling intestinal function. 2. Crypt size and the rates at which enterocytes migrated out of crypts were also measured, after injection of tritiated thymidine, to determine the time course of microvillus elongation. 3. Differences in growth rates measured between highly selected, relaxed selected or unselected birds were found to be correlated with parallel changes in villus surface area. Selection for growth did not change the density, dimensions or pattern of development of enterocyte microvilli. Microvilli did, however, produce a maximal 20-fold increase in villus surface area under all conditions. 4. Crypt size and enterocyte migration rates did not vary significantly between tissue taken from unselected and relaxed selected chickens. Tissue taken from highly selected birds had a crypt size and enterocyte migration rate 40% higher than values found for the other two groups of chickens. 5. The possibility that early genetic selection increased growth potential by uncoupling diet-induced changes on crypt hyperplasia from secondary effects on villus structure, and that later selection increased growth potential by increasing appetite, is discussed.     

The impact of prolonged fasting during aestivation on the structure of the small intestine in the green-striped burrowing frog, Cyclorana alboguttata

The effects of short‐term fasting and prolonged fasting during aestivation on the morphology of the proximal small intestine and associated organs were investigated in the green‐striped burrowing frog, Cyclorana alboguttata (Anura: Hylidae). Animals were fasted for 1 week while active or for 3–9 months during aestivation. Short‐duration fasting (1 week) had little effect on the morphology of the small intestine, whilst prolonged fasting during aestivation induced marked enteropathy including reductions in intestinal mass, length and diameter, longitudinal fold height and tunica muscularis thickness. Enterocyte morphology was also affected markedly by prolonged fasting: enterocyte cross‐sectional area and microvillous height were reduced during aestivation, intercellular spaces were visibly reduced and the prevalence of lymphocytes amongst enterocytes was increased. Mitochondria and nuclei were also affected by 9 months of aestivation with major disruptions to mitochondrial cristae and increased clumping of nuclear material and increased infolding of the nuclear envelope. The present study demonstrates that the intestine of an aestivating frog responds to prolonged food deprivation during aestivation by reducing in size, presumably to reduce the energy expenditure of the organ.     

Iron regulatory protein as an endogenous sensor of iron in rat intestinal mucosa. Possible implications for the regulation of iron absorption.

Duodenal enterocytes adjust intestinal iron absorption to the body's state of iron repletion. Here we tested how iron supply from the blood modulates the RNA-binding activity of iron regulatory proteins (IRP-1 and IRP-2) in immature duodenal rat enterocytes, and whether the modulation is compatible with the hypothesis that IRPs, in turn, may regulate the expression of iron transport proteins in maturating enterocytes during migration to the villus tips. Tissue uptake of parenterally applied 59Fe along the duodenal crypt-villus axis was compared to local IRP-1 and IRP-2 activity and to duodenal 59Fe transport capacity 12 h, 48 h, and 72 h after intravenous iron administration to iron-deficient rats. IRP-1 and IRP-2 activity was significantly increased in iron-deficiency. 59Fe administrated from the blood side was almost exclusively taken up by crypt enterocytes. Accordingly, the activity of IRP-1 decreased at this site 12 h after parenteral iron administration, but remained high at the villus tips. After 48 h the bulk of 59Fe containing enterocytes had migrated to the villus tips. Correspondingly, IRP-1 activity was decreased at duodenal villus tips after 48 h. IRP-2 activity also tended to decrease, though the change was statistically not significant. IRP-2 activity remained significantly higher at duodenal villus tips than in crypts, even after 72 h. Intestinal iron absorption capacity decreased with the same delay as IRP-1 activity after intravenous iron administration. In the ileum 59Fe uptake from the blood and IRP activity showed no significant difference between crypt and villus region. Luminal administration of iron decreased duodenal IRP-1 and IRP-2 activity at tips and crypts within 2 h. Thus, recently absorbed iron becomes available to cytosolic IRP during its passage through the enterocyte. Our results are compatible with a role of IRPs in gearing the expression of intestinal iron transporters in the duodenal brushborder to the body's state of iron repletion.     

Cell proliferation in chicken intestinal epithelium occurs both in the crypt and along the villus

The location of cell proliferation and differentiation in chicken small intestinal epithelium was examined using immunostaining, measurement of DNA synthesis and brush-border enzyme activities. Chicken enterocytes were removed sequentially from the villus using a modification of the Weiser (1973) method. Alkaline phosphatase activity was relatively constant along the villus tip-crypt axis but decreased in the crypt fractions, whereas sucrase and maltase activities showed higher activity in the upper half of the villus and lower activity in the lower half of the villus and in the crypt. Immunostaining of proliferating cell nuclear antigen indicated the presence of proliferating cells both in the crypt and along the villus, including some activity in the upper portion; the crypt region exhibited a significantly higher number of proliferating cells. Labelled thymidine incorporation into cell fractions after 2 h incubation exhibited a similar pattern of proliferation, with the most active region observed in the crypt and proliferation activity decreasing along the villus. However, some activity was found in the upper half of the villus. After 17 h incubation, cells from the middle region of the villi showed greater proliferation ability than the 2 h incubation. These results indicate that, unlike mammals, chicken enterocyte proliferation is not localized only in the crypt region, and that the site of enterocyte differentiation is not precisely localized. Accepted: 22 January 1998     

Testing the hypothesis that crypt size determines the rate of enterocyte development in neonatal mice

Pieces of mid-jejunum taken from 7-9 day old mice have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis to test the hypothesis that enterocyte development of structure is directly determined by the physical characteristics of the intestinal crypt. Parallel measurements of enterocyte migration rate were carried out using tritiated thymidine to determine the time course of microvillus elongation in neonatal mice. Microvillus length approximately doubled during early enterocyte migration from the crypt base to the lower part of the villus. Enterocyte migration rate was only 0.9 micron/hr at this stage of development, a value considerably less than that found in adult intestine. Results plotting the time dependency of microvillus elongation were fitted by a logistic curve giving a maximal rate for microvillus growth of 0.004 micron/hr. The corresponding estimate of crypt depth was 35 microns. Both these values are considerably less than those found in adult intestine. These results provide strong support for the general hypothesis that some factor associated with the physical length of the crypt, called crypt factor or CF, is directly responsible for controlling the way enterocytes organize subsequent structural differentiation of their surface membranes.     

Histological and ultrastructural studies of the marsupial Didelphis albiventris Peyer's patches

Differing from the studied Eutheria the white belly opossum Peyer's patches do not present a conspicuous dome. M cells are located in the inner layer of bilaminal formed at the bottom of the villi. A great variation in the morphology of M cells was observed. The enterocytes located at the epithelial inner layer may present endocytic vesicles, and the microvilli are shorter than the microvilli of enterocytes lining the small intestine. As these morphological aspects have been described to exist in the enterocytes of the lactent opossum small intestine it was surmised that the opossum Peyer's patches special epithelium could represent the persistence in adult animals of a cellular pattern established before the intestinal maturation had occurred.     

Modulation of xanthine dehydrogenase and oxidase activities during the hormonal induction of vaginal epithelial differentiation in ovariectomized mice

In situ hybridization and immunocytochemical techniques have been used to examine the distribution of vitamin-D-induced calbindin mRNA and calbindin protein in enterocytes lining the crypts and villi of chicken small intestine. Basal villus enterocytes contained approximately twice as much calbindin but over three times as much calbindin mRNA compared to values found in basal crypt and upper villus enterocytes, all values being measured 2 days after vitamin D injection into D-deficient chickens. Virtually no calbindin mRNA was detected in tissues taken from control D-deficient birds. Direct proportionality found between calbindin mRNA and calbindin content in enterocytes of basal crypt, mid and upper villus suggests pre-translational control over calbindin synthesis. The implications of possible inefficient translation of calbindin mRNA in basal villus enterocytes are discussed. Present methods of analysis provide a novel way to study mechanisms controlling gene expression throughout the whole process of enterocyte differentiation.     

Retinoblastoma protein (pRb), but not p107 or p130, is required for maintenance of enterocyte quiescence and differentiation in small intestine

The function of retinoblastoma protein (pRb) in the regulation of small intestine epithelial cell homeostasis has been challenged by several groups using various promoter-based Cre transgenic mouse lines. Interestingly, different pRb deletion systems yield dramatically disparate small intestinal phenotypes. These findings confound the function of pRb in this dynamic tissue. In this study, Villin-Cre transgenic mice were crossed with Rb (flox/flox) mice to conditionally delete pRb protein in small intestine enterocytes. We discovered a novel hyperplasia phenotype as well as ectopic cell cycle reentry within villus enterocytes in the small intestine. This phenotype was not seen in other pRb family member (p107 or p130) null mice. Using a newly developed crypt/villus isolation method, we uncovered that expression of pRb was undetectable, whereas proliferating cell nuclear antigen, p107, cyclin E, cyclin D3, Cdk2, and Cdc2 were dramatically increased in pRb-deficient villus cells. Cyclin A, cyclin D1, cyclin D2, and Cdk4/6 expression was not affected by absent pRb expression. pRb-deficient villus cells appeared capable of progressing to mitosis but with higher rates of apoptosis. However, the cycling villus enterocytes were not completely differentiated as gauged by significant reduction of intestinal fatty acid-binding protein expression. In summary, pRb, but not p107 or p130, is required for maintaining the postmitotic villus cell in quiescence, governing the expression of cell cycle regulatory proteins, and completing of absorptive enterocyte differentiation in the small intestine.     

Luminal and systemic signals trigger intestinal adaptation in the juvenile python

Juvenile pythons undergo large rapid upregulation of intestinal mass and intestinal transporter activities upon feeding. Because it is also easy to do surgery on pythons and to maintain them in the laboratory, we used a python model to examine signals and agents for intestinal adaptation. We surgically isolated the middle third of the small intestine from enteric continuity, leaving its mesenteric nerve and vascular supply intact. Intestinal continuity was restored by an end-to-end anastomosis between the proximal and distal thirds. Within 24 h of the snake's feeding, the reanastomosed proximal and distal segments (receiving luminal nutrients) had upregulated amino acid and glucose uptakes by up to 15-fold, had doubled intestinal mass, and thereby soon achieved total nutrient uptake capacities equal to those of the normal fed full-length intestine. At this time, however, the isolated middle segment, receiving no luminal nutrients, experienced no changes from the fasted state in either nutrient uptakes or in morphology. By 3 days postfeeding, the isolated middle segment had upregulated nutrient uptakes to the same levels as the reanastomosed proximal and distal segments, but it still lacked any appreciable morphological response. These contrasting results for the reanastomosed intestine and for the isolated middle segment suggest that luminal nutrients and/or pancreatic biliary secretions are the agents triggering rapid upregulation of transporters and of intestinal mass and that systemic nerve or hormonal signals later trigger transporter regulation but no trophic response.     

Comparative analysis of SGLT1 and GLUT2 transporters distribution in rat small-intestine enterocytes and Caco-2 cells during hexose absorption

The distribution of SGLT1 and GLUT2 hexose transporters has been evaluated in enterocytes of an isolated loop of the small intestine and Caco-2 cell culture after absorption of hexoses at their high and low concentrations. The SGLT1 transporter was found to be located in enterocytes along the edge of the intestinal villus. The GLUT2 transporter after loading with high hexose concentrations is located in the apical part of enterocytes. In culture, Caco-2 cells form a characteristic of enterocytes microvilli and the cell junction complex. During the incubation of the culture in solutions of glucose and galactose, the absorption of these sugars from the incubation medium was observed. The SGLT1 transporter in the Caco-2 cells is located in the apical and perinuclear enterocyte parts and is organized in globules. After loading with hexoses at low concentrations, the GLUT2 transporter is in the basal cell area. The Caco-2 cell culture can serve a model for studying the transport of sugar in the intestinal epithelium.     

A comprehensive description of brush border membrane development applying to enterocytes taken from a wide variety of mammalian species

Pieces of small intestine taken from rabbit, rat, mouse, guinea-pig, hamster and pig have been used to determine microvillus length in enterocytes located at different points along the crypt-villus axis. Thymidine labelling has also been used to convert measurements of enterocyte position into age of enterocyte. Microvillus lengths showed lower and upper plateaux (a and a + c respectively) with fairly rapid transition from one to the other defined by an exponential coefficient b. The mid-point of elongation (m) usually occurred within 60 micron of the crypt-villus junction. Correlations were found to exist between a and m and between a, m and the depth of the intestinal crypt. Values of b and bc/4, the maximal rate of microvillus elongation, were also found to be correlated with the size of the crypt. None of these parameters were related in any way to the villus height or enterocyte turnover time. The possibility that some factor associated with the physical size of the crypt might be exerting positional and temporal control over the subsequent structural differentiation of enterocytes is discussed.     

Positional dependence of enterocyte membrane potential in hamster and rabbit enterocytes

A technique is described allowing microelectrode impalement of enterocytes located at known positions along intestinal villi from rabbits and hamsters. Using this technique a 5 mV hyperpolarization in membrane potential is shown to occur as enterocytes migrate over the basal third of intestinal villi. The villus structure of the hamster ileum is similar to the rabbit, but the enterocyte lifespan in these two tissues differs considerably (enterocyte migration rates of 17.6 and 6.3 microns hr-1 for hamster and rabbit respectively). A correlation was found between the position an enterocyte occupied on the crypt-villus axis and the developmental state of the membrane potential. No such correlation existed when making comparisons on a time basis. These results are discussed both in terms of what is now known concerning different aspects of enterocyte development and in relation to what type of control mechanism might be generally responsible for initiating differentiation in this tissue.     

Changes in the small intestine of Schistosoma mansoni-infected mice fed a high-fat diet

The consumption of a high-fat diet modifies both the morphology of the small intestine and experimentally tested effects of schistosomiasis mansoni. However, whether a schistosomiasis infection associated with a high-fat diet causes injury to the small intestine has never been investigated. Mice were fed either a high-fat or a standard-fat diet for 6 months and were then infected with Schistosoma mansoni cercariae. Physical characteristics of the intestinal tissue (mucosal thickness, small intestinal villi length and height, and abundance of goblet cells and enterocytes on the villous surface) and the distribution of granulomas along the intestinal segments and their developmental stage were measured at the time of sacrifice (9 or 17 weeks post-infection). The group fed a high-fat diet exhibited different granuloma stages, whereas the control group possessed only exudative granulomas. The chronically infected mice fed a high-fat diet exhibited higher granuloma and egg numbers than the acutely infected group. Exudative, exudative/exudative-productive and exudative-productive granulomas were present irrespective of diet. Computer-aided morphometric analysis confirmed that villus length, villus width, muscular height and submucosal height of the duodenal and jejunal segments were affected by diet and infection. In conclusion, a high-fat diet and infection had a significant impact on the small intestine morphology and morphometry among the animals tested.     

Respiratory consequences of feeding in the snake Python molorus

Snakes can ingest large meals and exhibit marked increases in metabolic rate during digestion. Because postprandial oxygen consumption in some snakes may surpass that attained during exercise, studies of digestion offers an alternative avenue to understand the cardio-respiratory responses to elevated metabolic rate in reptiles. The effects of feeding on metabolic rate, arterial oxygen levels, and arterial acid-base status in the snake Python molorus are described. Four snakes (180-250 g) were cannulated in the dorsal aorta and blood samples were obtained during 72 h following ingestion of a meal (rat pups) exceeding 20% of body weight. Oxygen consumption increased from a fasting value of 1.71 +/- 0.08 to 5.54 +/- 0.42 ml kg-1 min-1 at 48 h following feeding, and the respiratory gas exchange ratio increased from 0.67 +/- 0.02 to a maximum of 0.92 +/- 0.03 at 32 h. Plasma lactate was always less than 0.5 mM, so the postprandial increase in metabolic rate was met by aerobic respiration. In fasting animals, arterial PO2 was 66 +/- 4 mmHg and haemoglobin-O2 saturation was 92 +/- 3%; similar values were recorded during digestion, but haematocrit decreased from 15.8 +/- 1.0 to 9.8 +/- 0.8 due to repeated blood sampling. Plasma [HCO3-] increased from a fasting level of 19.3 +/- 0.8 to 25.8 +/- 1.0 mmol l-1 at 24 h after feeding. However, because arterial PCO2 increased from 21.1 +/- 0.5 to 27.9 +/- 1.4 mmHg, there was no significant change in arterial pH from the fasting value of 7.52 +/- 0.01. Acid-base status returned to pre-feeding levels at 72 h following feeding. The increased arterial PCO2 is most likely explained by a reduction in ventilation relative to metabolism, but we predict that lung PO2 does not decrease below 115 mmHg. Although ingestion of large meals is associated with large metabolic changes in pythons, the attendant changes in blood gases are relatively small. In particular, the small changes in plasma [HCO3-] and stable pH show that pythons respond very differently to digestion than alligators where very large alkaline tides have been observed. It is unclear why pythons and alligators differ in the magnitude of their responses, but given these interspecific differences it seems worthwhile to describe arterial blood gases during digestion in other species of ectothermic vertebrates.     

Intestinal villus structure contributes to even shedding of epithelial cells

In the intestinal epithelium, proliferated epithelial cells ascend the crypts and villi and shed at the villus tips into the gut lumen. In this study, we theoretically investigate the roles of the villi on cell turnover. We present a stochastic model that focuses on the duration over which cells migrate the shortest paths between the crypt orifices and the villus tips, where shedding cells are randomly chosen from among those older than the shortest-path cell migration times. By extending the length of the shortest path to delay cell shedding, the finger-like shape of the villus would tightly regulate shedding-cell ages compared with flat surfaces and shorter projections; the villus allows epithelial cells to shed at around the same age, which limits them from shedding early or staying in the epithelium for long periods. Computational simulations of cell dynamics agreed well with the predictions. We also examine various mechanical conditions of cells and confirm that coordinated collective cell migration supports the predictions. These results suggest the important roles of the villi in homeostatic maintenance of the small intestine, and we discuss the applicability of our approach to other tissues with collective cell movement.