Roles of pre-treatment time and junctional proteins in Caco-2 cell microparticle uptake.

Microparticle uptake in the small intestine is relevant to both the delivery of pharmaceutics and exposure to environmental pollutants. The Caco-2 enterocyte model is a useful tool to study the parameters that affect epithelial microparticle permeability and the mechanisms controlling them. The current study used this model to explore further the different effects of 10% ethanol v/v or ice on transepithelial resistance (TER), microparticle uptake and immunofluorescent labelling of intercellular junctions. The same exposure times for both treatments were used, rather than those shown in the literature to produce demonstrable changes induced by each. The effects of both pre-treatments were greater after 60 min than after 15 min. Ethanol pre-treatment for 60 min decreased TER, increased particle uptake and was associated with a disorganisation of tight and adhering junctional proteins. Pre-treatment with ice for 60 min however, increased TER, decreased particle uptake and was associated with concentration of intercellular junctional proteins in a more constrained manner. These findings on the effects of pre-treatment with ethanol or ice for 60 min suggest that the extent of uptake is influenced by changes in the distribution of intercellular junctional proteins.

Culture conditions and treatments affect Caco-2 characteristics and particle uptake.

Small intestinal microparticle uptake via a paracellular route is relevant to oral drug delivery and environmental pollution. In vitro investigation uses latex microparticle passage across a confluent Caco-2 cell epithelium. This paper examines the influence of culture conditions on transepithelial resistance (TER); cell dimensions from confocal microscopy; and number of particles below the epithelium. Variables investigated include level of initial TER; multiple TER measurements; involvement of medium; cell source; and pretreatment with ethanol or a range of temperatures. Data were collected after exposure to 2 microm latex particles for 5-120 min: sham groups were exposed to pretreatment but not particles. The results highlight the importance of very precise control of the experimental environment; confirm the pattern of sequential-TER increase/decrease in groups exposed only to particles and show accompanying increases in cell dimensions. Greater particle uptake was associated with ethanol-induced decreased TER, decreased cell height and increased intercellular spaces, similar to previous findings for external irradiation. Low temperatures raised TER but, despite this, cooling did not alter particle uptake. In conclusion, culture microenvironment and sham treatment are crucial considerations in studies of epithelial microparticle uptake in vitro.

Uptake of inert microparticles in normal and immune deficient mice.

Intestinal microparticle uptake is important for drug delivery, environmental pollution and multiple organ dysfunction syndrome. This paper explores further whether uptake occurs at mucosa associated lymphoid tissue (MALT) via the microfold (M) cells of Peyer's patch domes or through villous epithelium. It does this by comparing the results of exposure of either severe combined immunodeficient (SCID) mice (lacking MALT) or normal BALBc mice, to oral gavage with 2 microm fluorescent latex microparticles. At 5 and 30 min after gavage, full circumference samples along the small intestine were processed for fluorescence microscopy and microparticle numbers were collected for surface and tissue sites. Uptake occurred in both BALBc and SCID mice within 5 min of particle administration and increased further in the following 25 min. In BALBc mice, almost all particles (96%) are in non-MALT sites in MALT circumference samples, with very few at the domes: uptake was also substantial in entirely villous samples. In SCID mice, particle numbers were only slightly lower than those of the BALBc mice, and occurred exclusively by the villous route. These findings confirm that the villous uptake route must be considered when assessing the extent of the dose delivered following pharmaceutical or toxicological oral exposure to microparticles.

Parameters influencing intestinal epithelial permeability and microparticle uptake in vitro.

The hypothesis that, in vivo in situ, villous uptake of 2 microm latex microparticles involves changes at enterocyte tight junctions (TJs) was tested using Caco-2 cells on porous membranes. Epithelial permeability was measured by transepithelial resistance (TER) and particle numbers in surface, intraepithelial and sub-epithelial compartments by microscopy. Apical particle or medium addition initially closed TJs, but this was subsequently reversed in particle-treated groups. Peristaltic onward movement of a bolus was simulated by removing apical particles after an exposure period and leaving the remaining particles to interact with the epithelium: this produced marked TJ loosening during the interaction period. For particle exposure groups, the early similarity with particle numbers in vivo taken up in young adult rats became less marked with time, although bolus removal counteracted this tendency. The TJ response to vasoactive intestinal polypeptide (VIP) was time-dependent. Adsorbed and intraepithelial particle numbers increased with particle exposure time; epithelial-associated microparticle aggregation varied with treatment and submembranous particles were seen in all groups. Correlation between TER changes and particle numbers suggests TJ loosening may be important in microparticle uptake. This Caco-2 model gives epithelial particle numbers that approximate well to published figures for microparticle uptake in vivo and allows effective microenvironmental manipulation.

Factors influencing intestinal microparticle uptake in vivo.

The aim of this study is to compare microparticle uptake in animals of different ages, gender and species and at different time points. The 2mum latex/in vivo in situ model uses the observation of animal responses or post-mortem changes and also particle identification by fluorescence microscopy in nine sequential intestinal segments and secondary sites. The wide size range of animals studied requires particle numbers in tissue compartments to be related to intestinal tissue section area through a circumference measurement. Area under the curve (AUC) data for particles in intestinal tissue are plotted against measurements of intestinal length, allowing comparisons to be made across different ages and species and between males and females. The percentage uptake of administered dose and particle numbers in macerated tissue are also reported. Some parameters, in particular species, do not appear to affect the extent of microparticle uptake, which ranges from 0.12 to 0.32% of the administered dose. Particle uptake does, however, vary with age, being significantly greater in young adult males (7 weeks) than in younger (3 weeks) and older (17 and 52 weeks) age groups. It is concluded that age is more important in determining the extent of uptake than gender or species.

Effects of radiation damage on intestinal morphology.

The current flow of papers on intestinal structure, radiation science, and intestinal radiation response is reflected in the contents of this review. Multiparameter findings and changes in compartments, cells, or subcellular structure all contribute to the overall profile of the response. The well-recognized changes in proliferation, vessels, and fibrogenesis are accompanied by alterations in other compartments, such as neuroendocrine or immune components of the intestinal wall. The responses at the molecular level, such as in levels of hormones, cytokines, or neurotransmitters, are of fundamental importance. The intestine responds to localized radiation, or to changes in other organs that influence its structure or function: some structural parameters respond differently to different radiation schedules. Apart from radiation conditions, factors affecting the outcome include the pathophysiology of the irradiated subject and accompanying treatment or intervention. More progress in understanding the overall responses is expected in the next few years.

Radiation effects in the small bowel of the diabetic mouse.

PURPOSE: Irradiation of the small intestine in the mouse induces damaging structural alterations to the architecture of the enteric mucosa. There is growing interest in the possible relevance of underlying additional pathology when appreciating the total response of tissues to irradiation. The possibility that small intestinal mucosal abnormalities in the streptozotocin-induced diabetic mouse may exacerbate radiation-induced injury was tested by examining the combined effects of the two treatments. MATERIALS AND METHODS: Streptozotocin-diabetic and -non-diabetic mice were exposed to 10 Gy abdominal X-radiation. Profiles of mucosal epithelial cell populations were quantified and comparisons with corresponding groups of unirradiated mice made on the third day post-irradiation. RESULTS: The histological appearances of the small intestinal mucosa were similar in both groups of irradiated mice, but the numbers of profiles of crypts and of columnar, goblet, Paneth and entero-endocrine cells were depressed in these groups when compared with values in corresponding groups of unirradiated mice. However, the expression of radiation damage in the diabetic mouse was less severe than in the non-diabetic mouse, particularly in the jejunum where the changes attendant on the onset of diabetes were most marked. CONCLUSION: These findings suggest that the response of mouse to radiation may be moderated by the presence of this type of pathophysiology. However, there is no evidence that the damage produced by streptozotocin-induced diabetes and radiation is additive.

Variations in cell and structure populations along the length of murine small intestine.

Previous workers have reported a range of structural variations occurring along the length of small intestine. These studies have concentrated on the major intestinal components with little information available on the intersite variation of other members of a highly heterogeneous population. Using resin histology, the current study has collected data per circumference for all definable structural features within the murine intestinal wall, along with measurements for epithelial, muscle, nerve and connective tissue areas, and villous height and cryptal depth. Seven different sites along the length of the small intestine were examined. Following statistical comparisons a number of features exhibited no intersite variation; these were numbers of villi, submucosal vessels, myenteric nerve plexus profiles, inner muscle nuclei and apoptotic bodies as well as measurements for tissue areas and cryptal depth. Variations were seen between sites for crypts, enterocytes, villous and cryptal stromal cells, cryptal goblet cells, cryptal non-secretory epithelial cells, Paneth cells, endocrine cells, intra-epithelial lymphocytes, submucosal nerve plexus profiles, outer muscle nuclei and mitotic figures. A reduced villous height was observed caudally. Certain correlations between villous height/crypt number and constituent parameters have been noted. The results provide a complete description of how each definable structural feature within the gut wall varies at regular intervals along the length of the small intestine in C57 BL mice. A number of previously unreported variations have been described. The work provides a comprehensive data bank for future intestinal investigations.

Dietary fibre and intestinal microflora: effects on intestinal morphometry and crypt branching.

BACKGROUND: Fermentable dietary fibre has many effects on the gastrointestinal tract. One is to alter epithelial crypt cell proliferation, especially in the colon. A discrepancy between epithelial cell production rates and intestinal weights has been noted previously: crypt cell production rates only increase if bacterial fermentation occurs, but intestinal wet weight can increase in the same animals without bacterial fermentation of fibre. AIMS: To quantify intestinal cell populations in order to resolve the above paradox. METHODS: Conventional and germ-free rats were fed fibre-free or fibre supplemented diets and their intestines were quantified by morphometry. RESULTS: There was evidence of fibre associated muscle hypertrophy in the colon, but the main effect of fibre was an increase in the number of crypts per circumference and also the number of branched crypts in the proximal colon in both groups. There was also a large increase in the number of branched crypts in the mid colon of the germ-free rats (both fibre-free and fibre supplemented). Fibre had a direct (bacteria independent) effect on goblet cells in the small intestine and a direct effect on the goblet cells in the colon, which was attenuated by the presence of bacteria. There was a notable decline in the number of enteroendocrine cells in the small intestine of the germ-free animals. CONCLUSIONS: Fibre has several direct and indirect effects on the gut. In the proximal colon it can directly increase the number of crypts present. This provides a means for increasing intestinal mass in addition to intestinal crypt cell production.

Acute and protracted radiation effects on small intestinal morphological parameters.

PURPOSE: To compare the responses of small intestinal morphological parameters after acute and protracted doses of radiation. MATERIALS AND METHODS: Male C57BL6 mice were examined 6, 24 and 72 h after whole body gamma-irradiation, given either as an acute 5 Gy dose, or as a protracted (continuous) dose of 20 cGy per day for 25 days to a total dose of 5 Gy. Many different structural parameters at both the light microscopical and ultrastructural levels were assessed quantitatively. RESULTS: At different time points following both schedules there were changes in the number of villous enterocytes, goblet cells, lamina propria cells and mitotic figures. Ultrastructural changes occurred in the epithelium. Many of the parameters that showed changes following the protracted schedule appeared to be returning to normal within 3 days of the cessation of radiation, a finding which was in contrast with the acute dose. The protracted schedule produced increases in the number of Paneth cells and in the length of enterocyte microvilli. CONCLUSIONS: Many of the responses that occurred after the protracted schedule suggest that adaptive mechanisms may be being triggered following persistent exposure to radiation.

Cortical bone responses to 2G hypergravity in growing rats.

BACKGROUND: Rat cortical bone adaptation to chronic hypergravity (2G) was studied using young growing male Wistar rats (60 d). METHODS: Animals (10 rats) were subjected to chronic hypergravity (14 d) in order to understand the plastic nature of bone under a constant hypergravity stress using a special rodent habitat that was attached to a 12-ft. radius centrifuge. Also, an equal number of stationary controls were housed in a rodent vivarium containing identical cages that were used for centrifugation. After 14 d of centrifugation, femur bones were excised and prepared for morphological and biochemical measurements. RESULTS: Results showed that 2G had significantly shortened the femurs (3%) and reduced the cortical bone area (13%). In particular, hypergravity induced significant reductions in the thicknesses of cortical bone at the anterior (13%) and medial regions (15%) of the mid-diaphysis. However, femoral bone density, collagen and calcium concentrations were unaltered. The content of mature, stable bone collagen cross-links hydroxylysylpyridinoline (HP), lysylpyridinoline (LP), were significantly greater in bones from centrifuged animals. CONCLUSION: Our findings suggest that short term exposure to 2G does not enhance bone formation or induce changes in cortical bone composition, or alter specific gravity. These data also suggest that bone maturation as reflected by collagen cross-linking is upregulated. However, it is undetermined at this time whether the enhanced content of mature bone collagen in the centrifuged rats is a result of either an increased rate of cross-linking or reduction in the degradation of "older collagen."

A morphological study of the enteric mucosal epithelium in the streptozotocin-diabetic mouse.

In the acutely diabetic rat, the polyphagia-induced increase in the weight of the small intestine is associated with reported increases in mucosal mass. Whereas, some of the individual mucosal components in the rat have been studied, comparable information for the acutely streptozotocin-diabetic mouse is lacking. A detailed morphological comparison of the epithelium of the small intestinal mucosa in control and untreated streptozotocin-diabetic mice was therefore undertaken. Samples from three small intestinal sites were examined by light and scanning electron microscopy and quantitative data obtained from histological sections. Although the morphological appearance of the small intestine in acutely diabetic mice was similar in many respects to literature accounts for the diabetic rat, infestation with filamentous microorganisms was present in the jejunum and ileum. The quantitative data showed that these sites also contained distorted villi, fewer crypt profiles, more goblet and Paneth cell profiles and a smaller epithelial volume in comparison to controls. These findings may represent differences between the rat and mouse models of streptozotocin-induced diabetes mellitus.

Gastrointestinal uptake and translocation of microparticles in the streptozotocin-diabetic rat.

Uptake and translocation of particulates across the mucosal barrier of the gastrointestinal (GI) tract is now generally recognised but the effect of pathophysiologically induced changes on this process is less well established. This study evaluated the effect of diabetes mellitus on GI absorption of particles, comparing particle localisation and particle loading in different microanatomical sites of the primary organ (small intestine) and possible particle translocation pathways to selected secondary organs (mesenteric lymph nodes, liver, spleen) in normal and streptozotocin-induced diabetic animals. Fluorescent polystyrene latex particles (approximately 2 microns diameter) were fed orally to young adult Sprague-Dawley rats and quantitative bulk tissue and morphological techniques used to chart particle transit across the small intestine to secondary organs 0.5 h postadministration. In the normal animal, epifluorescence and confocal laser scanning microscopy provided confirmatory evidence for particle absorption within the primary organ and transport to other sites in the body. By contrast, in the diabetic animal, particle translocation and peripheral distribution were reduced with approximately 30% decrease in particle loading in the epithelial/nonepithelial tissue compartments. This could be a consequence of gastric retention and altered intestinal motility and permeability which are known to be associated with diabetes.

Surface features of small-intestinal mucosa in childhood diarrheal disorders.

The pathophysiology of diarrhea, especially in the otherwise healthy child, is still poorly understood. The aim of this study was to use the scanning electron microscope (SEM) to examine the surface of the jejunal mucosa of children with chronic nonspecific diarrhea (CNSD) (n = 9) and to compare the findings with specimens obtained from children with (n = 21) and without (n = 11) other gastrointestinal diseases. Light microscopy of the specimens from children with CNSD was normal. However, SEM showed the presence of bacterial colonization with predominantly coccoid organisms in 100% of cases. This colonization was associated with loss of glycocalyx and clumping of the microvilli. The children with celiac disease (n = 9) all showed characteristic appearances with light microscopy, but only one had bacterial colonization on SEM. The surface features of specimens from children with other gastrointestinal disorders (food intolerance, postenteritis syndrome, protracted diarrhea of infancy, and immune deficiency states) were very similar to those from the CNSD group. Bacteria were visible on 89% of specimens, and in half of these cases the organisms were bacilli. SEM of specimens from children with no gastrointestinal disease (ages 11-107 months) suggested an increased density of villi/unit area with advancing age. Bacteria were present in only two cases and did not include bacilli. The findings suggest that bacterial colonization of the surface of the small intestine is common in children with several gastrointestinal diseases and may play a part in their pathogenesis. Routine SEM examination of jejunal biopsies provides information not available from standard light microscopy, which may be relevant to the treatment of children with chronic diarrhea.

Early intestinal microparticle uptake in the rat.

This time-course study investigates the early uptake and passage of microparticles across the intestinal mucosa. Single intraoral doses of fluorescent latex particles, 1.82 microns in diameter, were administered to young adult male, nonfasted rats at a dose of 1.88 x 10(9) particles. Peyer's patch regions and mesenteric lymph nodes were collected at 5, 15 and 30 min time points for both bulk tissue and morphological analyses. Particles were found at all experimental time points in macerated intestinal and nodal specimens: particle numbers were higher in proximal than in distal intestine at all time points despite the fact that particle numbers in distal Peyer's patch regions increased with time. Particle numbers in mesenteric lymph nodes also increased with time after administration. Detailed morphological data for several intestinal and nodal tissue compartments showed substantial early uptake of particles by villous epithelium, including goblet cells, but low involvement of follicle-associated cells. The distribution of particles in the lymph nodes confirmed that translocation occurred to all nodal compartments. These studies give confirmatory evidence that uptake and translocation of microparticles may take place as early as 5 min after administration and suggest that intestinal region may influence uptake.

The effect of size on uptake of orally administered latex microparticles in the small intestine and transport to mesenteric lymph nodes.

PURPOSE: The present study examines the relationship between size and particle transit across the mucosal barrier of the gastrointestinal tract to other sites of the body. The extent of particle uptake with increasing size, the tissue distribution and cut-off points for 2-20 microns particles is investigated. METHODS: An established fluorescent latex particle-young adult rat model is used and particle numbers in small intestine and mesenteric lymph nodes, 0.5 h post administration, counted by fluorescence microscopy in bulk tissue specimens and cryosections. RESULTS: Bulk tissue analysis provides evidence for the presence of particles of all sizes in the Peyer's patch regions, but only for 2 microns particles in the nodal tissues. Microscopy establishes uptake of both 2 and 6 microns particles in most intestinal and nodal tissue sites and compartments. By contrast, uptake of the larger particles is much reduced. CONCLUSIONS: Although more of the smaller (2 microns) particles are taken up, particularly by epithelial tissues, the 6 microns size appears more efficient in terms of volume translocated to lymph nodes. This could have implications in the therapeutic use of particles for drug and vaccine delivery and for radiation safety.

Morphometric analysis of the small intestinal epithelium in the indomethacin-treated mouse.

To obtain a clearer understanding of the changes which are induced in the small intestine of the mouse by an ulcerogenic dose of indomethacin, a quantitative analysis of the nonulcerated small intestinal mucosa was performed in mice that were given 2 injections of indomethacin at a dose of 85 mg/kg body weight. At 20 h after the administration of the drug, values were obtained for epithelial volume, whole crypt number, and for the number of profiles of columnar, Paneth, entero-endocrine and goblet cells and cryptal mitotic figures in the small intestine. Comparison of the values obtained from indomethacin-treated mice with those from control mice showed that there were fewer whole crypts and a reduced epithelial volume in the jejunum and ileum in indomethacin-treated mice. The numbers of columnar and Paneth cell profiles and of mitotic figures were significantly greater in the jejunal and ileal crypts in indomethacin-treated mice than in controls. These findings suggest that the administration of high-dose indomethacin in the mouse leads to crypt losses and increased mitotic activity in the nonulcerated parts of the small intestine.

Uptake of yeast (Saccharomyces boulardii) in normal and rotavirus treated intestine.

BACKGROUND: There has recently been a growing interest in the use of the non-pathogenic yeast Saccharomyces boulardii, in the treatment of gastrointestinal disorders, including diarrhoea. The full effects of administration of the yeast are not fully understood. AIMS: To investigate the morphological effects of inoculated S boulardii on mouse intestinal villi, both in control animals and those treated with rotavirus. METHODS: Seven day old BALB/c seronegative mice were intubated with either rotavirus (30 microliters orally) or S boulardii (1.5 g/kg) or both rotavirus and S boulardii administered together. Control animals were given saline only. Animals were killed by decapitation 48 hours post-treatment. The middle region of the small intestine was studied using light microscopy and transmission and scanning electron microscopy, including backscattered electron imaging. RESULTS: Animals treated with rotavirus with or without S boulardii developed severe diarrhoea and showed morphological villous changes such as stromal separation and increased epithelial vacuolation. Specimens treated with S boulardii contained yeast particles within the mucosal tissues. CONCLUSION: The administration of S boulardii did not influence the changes produced by rotavirus, but yeast particles appeared to be taken up by the villous mucosa, with the predominant route apparently being uptake between adjacent epithelial cells.

The effect of graft-bed irradiation on the healing of rat skin grafts.

This study explores the possible side effects on healing skin grafts of irradiation, commonly used intraoperatively following surgical tumor removal. The experimental model involved the delivery of a single 10-Gy dose of electron radiation to the recipient bed of a skin wound, followed by attachment of a full thickness rat skin autograft. Skin graft repair was assessed by light microscopy, immunohistochemistry, and transmission electron microscopy over a 3-week period for grafted and grafted-irradiated groups. Graft-bed irradiation reduced fibrinogen, fibrin, and fibronectin deposition in the wound. It also produced brief changes in the extent of both re-epithelialization and granulation tissue formation, and reduced the diameter of collagen fibrils in the granulation tissue. Despite these changes, the results suggest that graft-bed irradiation only delays the healing process, producing no serious clinical complications at the time points studied.

Morphological profiles of neutron and X-irradiated small intestine.

This paper describes the response of mouse small intestine, at several time points after treatment with neutron or X-irradiation, using doses expected to give similar effects in terms of crypt/microcolony survival. Using resin histology, the effects of radiation on the numbers of duodenal cell types and measurements of tissue areas were assessed. The results for individual parameters and for an estimate of overall damage are given in a data display, which summarises the morphological profile of the organ after both types of radiation. Damage and recovery were seen for many of the parameters studied but there was no standard response pattern applicable for all parameters. In particular, the response of individual crypt cell types could not be predicted from knowledge of the change in crypt numbers. With regard to the holistic response of the gut, neutron irradiation appeared to have caused more damage and produced more early effects than the X-irradiation. More specifically, neutron treatment led to more damage to the neuromuscular components of the wall, while X-irradiation produced early vascular changes.