Immunocytochemical localization of hepatic fatty acid binding protein in the rat intestine: effect of fasting

Localization of hepatic fatty acid binding protein (h-FABP) in the small and large intestines of rats was studied by light and electron microscopic histochemistry using the peroxidase-antiperoxidase (PAP) method. In the small intestine of rats fed ad libitum, an intense FABP immunoreactivity was confined to the absorptive epithelial cells of the villi, but not of the crypts. The lowest margin of the immunoreactive cell sheet was closer to the crypts in the proximal than in the distal portions of the small intestine. In the large intestine, FABP immunoreactivity was present in the surface epithelial cells, with higher intensity in the proximal than in the distal portions of the intestine. After fasting rats for two days, many crypt cells exhibited intense immunoreactivity for h-FABP, resulting in an extension of the lowest margin of the immunoreactive cell sheet deep into the crypts. Such expansion of the immunoreactive cell population was reversed by refeeding the animals. With regard to the intracellular localization of immunoreactivity in the jejunum, the basolateral portion of the cytoplasm exhibited a more intense immunoreaction than the apical portion in the majority of immunoreactive cells lining the villi, whether the animals were fed or fasted. The immunoreactive products appeared in the cytoplasmic matrix without association with any subcellular structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired growth of small intestinal epithelium by adrenalectomy in weaning rats

Functional maturation of the small intestine occurs during the weaning period in rats. It is known that this development is facilitated by glucocorticoid. However, the effect of glucocorticoid on morphological development of small intestine has yet to be clarified. The present study evaluated the morphological development and cell proliferation of the small intestine in adrenalectomized (ADX) rat pups. To further understand the mechanism of glucocorticoid effects on intestinal development, we examined the localization of the glucocorticoid receptor in the small intestine. Microscopic analysis showed that growth of villi and crypts is age-dependent, and is significantly attenuated in ADX rats compared with sham-operated rats. BrdU-positive cells, i.e. proliferating cells, were primarily observed in crypt compartments and rapidly increased in number during the early weaning period. The increase in BrdU-positive cells could be attenuated by adrenalectomy. The morphological development of small intestine may be associated with increased proliferation of epithelial cells. On the other hand, glucocorticoid receptors were found in epithelial cells of the mid- and lower villi and not in crypts where BrdU-positive cells were localized. These results indicate that the growth of small intestine is attenuated by adrenalectomy, and that glucocorticoid indirectly acts on proliferation of epithelial cells during the weaning period.     

Ontogeny of hepatic fatty acid-binding protein immunoreactivity in human liver and intestinal tract

Liver and intestinal tissues of human fetuses at gestational ages between 6 and 30 weeks were immunostained with rabbit antibody against fatty acid-binding protein (FABP) isolated from rat liver, since the antibody crossreacted with human FABP of the hepatic type. FABP-immunoreactive hepatocytes were found in the liver as early as the 7th week of gestation, but not at the 6th week. The frequency of immunoreactive cells was about 80% throughout the gestational period examined. No immunoreactive cells other than hepatocytes were found in liver tissue. In the intestinal tract, ileal, colonic and vermiform appendicular FABP immunoreactivity was demonstrated at the 23rd week of gestation, and duodenal and jejunal immunoreactivity at the 26th week. Positive cells in the jejunum were very few at this stage, but numerous at the 30th week of gestation. The immunoreactive cells were primitive absorptive cells in intestinal villi, and no cryptic epithelial cells were positively immunostained. Thus, FABP immunoreactivity was considered to be a marker for hepatocytes at the early to late fetal stage, and for intestinal absorptive cells at mid- to late fetal stage.     

Number and ultrastructure of epithelial cells in crypts and villi along the streptozotocin-diabetic small intestine: a quantitative study on the effects of insulin and aldose reductase inhibition

This study has quantified the effects of insulin treatment with and without aldose reductase inhibitor (ponalrestat) on intestinal epithelial cell morphology in streptozotocin-diabetic rats. Epithelial volumes, villous and microvillous surface areas and mean volumes of cells (and their nuclei) in crypts and villi were estimated in each of four segments and in the entire intestine. We derived total numbers of cells, quantified the ultrastructural features of average cells and explored variation along the intestine and between experimental groups. In crypts, insulin and ponalrestat had significant effects on cell number (reduced towards normal values) and size (volume and apex area increased beyond normal values). There were interaction effects between insulin and ponalrestat for cell volume and apex area (insulin producing more exaggerated effects when given without ponalrestat). On villi, insulin and ponalrestat returned cell numbers towards normal values but neither treatment normalised cell size or the number and area of microvilli per cell. Indeed, ponalrestat increased microvillous number and area beyond values found in untreated diabetic animals. Again, there were interaction effects between insulin and ponalrestat. Patterns of segmental variation seen in crypts of normal rats (values tending to be higher in proximal or mid-intestinal regions) were not preserved, and only some of the segmental differences seen on villi (higher values at proximal or mid-intestinal sites) were maintained during therapy. Apart from reducing the abnormally high numbers of cells in untreated diabetic rats, these results show that insulin and ponalrestat treatment fail to restitute epithelial cell morphology in the small intestines of experimental diabetic rats.     

Temporal differentiation and migration of substance P, serotonin, and secretin immunoreactive enteroendocrine cells in the mouse proximal small intestine.

Precise spatial interrelationships exist between substance P, serotonin, and secretin containing enteroendocrine cells in the gastrointestinal tract of mice. In the proximal small intestine these products are coexpressed in various combinations in single enteroendocrine cells along the crypt to villus axis in a pattern that suggests the sequential expression of substance P, serotonin, and secretin. In this report we use bromodeoxyuridine (BrdU) and multilabeling immunohistochemistry to define the temporal and spatial interrelationships between substance P, serotonin, and secretin immunoreactive cells in the mouse proximal small intestine. Our findings demonstrate the sequential expression of substance P, serotonin, and secretin in a population of upwardly migrating enteroendocrine cells and, furthermore, identify a population of crypt associated cells coexpressing substance P and serotonin that fails to traverse this pathway. The lack of secretin immunoreactive cells in the crypts suggests that local factors present in the crypts and/or on villi regulate secretin expression. The combined use of BrdU and multilabeling immunohistochemistry provides a method for defining enteroendocrine cell differentiation pathways throughout the gastrointestinal tract.     

ONTOGENY OF HEPATIC FATTY ACID-BINDING PROTEIN IMMUNOREACTIVITY IN HUMAN LIVER and INTESTINAL TRACT

Liver and intestinal tissues of human fetuses at gestational ages between 6 and 30 weeks were immunostained with rabbit antibody against fatty acid-binding protein (FABP) isolated from rat liver, since the antibody crossreacted with human FABP of the hepatic type. FABP-immunoreactive hepatocytes were found in the liver as early as the 7th week of gestation, but not at the 6th week. The frequency of immunoreactive cells was about 80% throughout the gestational period examined. No immunoreactive cells other than hepatocytes were found in liver tissue. In the intestinal tract, ileal, colonic and vermiform appendicular FABP immunoreactivity was demonstrated at the 23rd week of gestation, and duodenal and jejunal immunoreactivity at the 26th week. Positive cells in the jejunum were very few at this stage, but numerous at the 30th week of gestation. The immunoreactive cells were primitive absorptive cells in intestinal villi, and no cryptic epithelial cells were positively immunostained. Thus, FABP immunoreactivity was considered to be a marker for hepatocytes at the early to late fetal stage, and for intestinal absorptive cells at mid- to late fetal stage. ACTA PATHOL JPN 38: 979∼987, 1988.     

Immunofluorescence studies on the pathogenesis of intestinal chlamydial infections in calves

Newborn calves were exposed orally to the chlamydial agent of bovine polyarthritis. The chlamydial infection in the gastrointestinal tract was traced by reisolation of the agent and by fluorescent-antibody techniques. Absorption of the fluorescein isothiocyanate (FITC)-labeled antiserum with bovine fetal intestinal tissue powder eliminated effectively the nonspecific fluorescence of eosinophilic granules in intestinal tissue sections. Cells of the eosinophilic series were observed in great numbers in the gastrointestinal tract of inoculated and normal calves. Although chlamydial agents could be reisolated from mucosal scrapings of abomasum and duodenum for 5 days after inoculation, specific fluorescence was not observed in these gastrointestinal portions. As the chlamydial infection progressed, it localized in the mucosal epithelial cells of the jejunum and ileum. Fluorescing chlamydial inclusions were observed most consistently in the cytoplasm of mucosal epithelial cells on the tips of the jejunal and ileal villi. The inclusions were located between the nucleus and the free border of the epithelial cells. In the deeper parts of the villi, the inclusions in the epithelial cells were situated frequently between the nucleus and the basement membrane of the mucosal lamina propria. In calves examined 7 days after inoculation, fluorescing chlamydial inclusions were seen in the cells of the crypts and the mucosal lamina propria of the lower portions of the small intestine. Chlamydial infection of cells in the intestinal interstitium reflected a process of systemic invasion.     

Isolated crypts form spheres prior to full intestinal differentiation when grown as xenografts: an in vivo model for the study of intestinal differentiation and crypt neogenesis, and for the abnormal crypt architecture of juvenile polyposis coli

We describe a model system in which single crypts, isolated from newborn rats, were embedded in a type I collagen gel and subcutaneously grafted to the flanks of nude mice, whereupon they underwent full intestinal morphogenesis. Small fragments of small intestine and colon were incubated with the divalent cation chelator EDTA, resulting in the release of crypts and villi. Released crypts were then suspended sparsely in type I collagen gel. Segments of gel containing a single crypt were grafted subcutaneously into a nude mouse. Grafts were harvested at weekly intervals. By 2 days, the mouth of the crypts had joined to seal the crypt and, within 1 week, the structure ballooned to form a spherical cystic structure lined by flattened epithelial cells showing no evidence of cytodifferentiation. After 2 weeks, host stromal cells had invaded the collagen and settled around this spherical crypt. At points where stromal cells appeared in contact with the crypt, the epithelium exhibited a more columnar phenotype. By 4 weeks, the 'crypt sphere' was surrounded by stroma expressing alpha-smooth muscle actin and, at this time, multiple buds appeared that gave rise to new crypts. By 5 weeks, villi had formed and cell lineages associated with the small intestine and colon were present; the original single crypt had transformed into a functional intestinal unit. Therefore, we have shown that a single crypt has the potential to grow, give rise to other crypts and dependent structures such as villi. This model has considerable potential for use in gene transfer experiments in the study of intestinal differentiation, and for the analysis of crypt neogenesis via crypt fission. Moreover, the appearances showed a close resemblance to those seen in juvenile polyposis syndrome (JPS), where the budding and fission of single crypts isolated by stromal overgrowth offers an alternative explanation for the histogenesis of JPS.     

GnRH免疫活性细胞和神经在大鼠胃肠胰系统分布的初步研究

本研究用免疫金银法。对促性腺激素释放激素(GnRH)免疫活性细胞和神经在大鼠胃肠胰系统的分布,进行了研究。在胃、小肠、大肠和胰腺有GnRH免疫反应阳性的上皮细胞,这些细胞的顶端到达腺腔面或器官腔面,属于开放型的内分泌细胞。在胃和小肠的肌间神经丛、粘膜下神经丛和浆膜下,有GnRH免疫活性神经细胞。在小肠的粘膜下层和固有层,有GnRH免疫活性神经纤维。这些结果提示,GnRH对消化生理可能有重要的意义。     

Distribution of diazo-positive (argentaffin) cells in the small intestine of rats of various ages

In unit lengths of longitudinally cut histological sections of the rat small intestine, the number of diazo-positive argentaffin cells was determined along with the number of other epithelial cells. From these results, the frequency of argentaffin cells among the epithelial cells was calculated. The number, as well as the frequency, was maximal in the proximal duodenum and progressively decreased to a minimum in the mid-intestine. Thereafter, it increased progressively caudally to reach a second maximum in the terminal ileum. The frequency in the terminal ileum was almost as high as in the proximal duodenum. When calculated for the duodenum of various age groups, it decreased from eight to two argentaffin cells per 1000 epithelial cells from newborn to adult ages respectively. It was calculated furthermore that the total number of diazo-positive argentaffin cells in the rat small intestine should be around six million. Considering that the argentaffin cells are renewed about every four days (Ferreira and Leblond, ′71), about 1.5 million of them should be formed as well as exfoliated daily. The cells in the villi were stained more intensely than in the crypts, indicating that the argentaffin cells accumulate granules as they migrate from the crypts toward the villus tips, where indeed, intensely stained exfoliating argentaffin cells were occasionally observed. It is suggested that the exfoliation of argentaffin cells full of granules may be a mode of secretion for 5-hydroxytryptamine into the intestinal lumen.     

Immunohistochemical analysis of neural cell adhesion molecule (N-CAM) and pan-cadherin in the small intestine of intrauterine growth-retarded newborn rats caused by maternal protein malnutrition

Cell-cell and cell-matrix adhesion molecules play an important role in morphogenesis, cell signaling and are involved in cell proliferation, cell death, cell polarization and differentiation. In the present study, we investigated N-CAM and pan-cadherin expression in small intestine of intrauterine growth-retarded (IUGR) newborn rats after maternal protein malnutrition during pregnancy. N-CAM and pan-cadherin immunostaining was increased in small intestine of IUGR newborn rats. This increase was evident in epithelial cells on villi, lamina propria, submucosa, muscularis mucosa and muscularis externa. The increase in numbers of villous N-CAM-positive and pan-cadherin-positive epithelial cells was statistically significant (p < 0.001). In most areas, crypts did not show any immunopositive epithelial cells or showed decreased expression of the adhesion molecules. Although the villous height was decreased in IUGR rats, the decrease was statistically not significant. Expression and recruitment of N-CAM and pan-cadherin in small intestine of newborn IUGR rats may indicate a direct or indirect involvement of adhesion mechanisms or signaling in the growth retardation process.     

Morphological effects of a large single dose of cycloheximide on the intestinal epithelium of the rat

Adult male rats received 15 mg/kg cycloheximide and the subsequent morphological effects at three and six hours after injection were evaluated using histometry, light and electron microscopy, histological demonstration of terminal web and acid phosphatase, and radioautography with tritiated thymidine. Rapid atrophy of the villi took place, progressing from the villus tip by premature exfoliation of epithelial cells. The crypts also diminished by random exfoliation of many crypt cells and by partial or complete disintegration. Mitosis and epithelial cell migration were absent. By six hours, the area occupied by the villi and the crypts per unit length of histological section was decreased by about 70–90% in most of the small intestine but only by about 40–60% in the duodenum and the terminal ileum. In the upper half of the villi, the epithelium was strongly positive for acid phosphatase and contained large numbers of round bodies resembling primary lysosomes. In the lower half, the microvillous border and terminal web were found to be disrupted. Animals receiving only 5 mg/kg cycloheximide also showed the atrophy of villi and crypts, and the round bodies resembling lysosomes. Evidence from several sources has indicated that protein synthesis in normal villus epithelial cells subsides toward the villus tip and becomes minimal at exfoliation. At exfoliation, proteins responsible for epithelial cohesion probably fail because they are no longer replenished. Cycloheximide appears to accelerate this process.     

Distribution of fibroblast growth factor in cultured dorsal root ganglion neurons and Schwann cells. I. Localization during maturationin vitro

Summary The distribution of acidic and basic fibroblast growth factor in co-cultures of dorsal root ganglion neurons and Schwann cells was examined as a function of time in culture. After two daysin vitro, the cytoplasm of the neuronal cell bodies demonstrated both acidic and basic FGF immunoreactivity, whereas the cytoplasm of the neuntes was not immunoreactive. Schwann cells, in contrast, exhibited both acidic and basic fibroblast growth factor cytoplasmic immunoreactivity. After two days in culture, immunoreactivity was not detected on the plasma membrane surface of either the neurons or the Schwann cells. By 10 daysin vitro, fibroblast growth factor immunoreactivity was observed in the cytoplasm of the most proximal portion of some, but not all, neuntes but was unchanged in Schwann cells. At 20 daysin vitro, immunoreactivity was still restricted to the intracellular compartment of both Schwann cells and neurons. Acidic fibroblast growth factor was primarily localized to the cytoplasm of Schwann cells, neuron cell bodies and along the entire length of the neurites. In contrast, basic fibroblast growth factor was predominantly localized to the nuclei of Schwann cells and small to medium size neurons. In many cases, the nucleolar region demonstrated the most intense basic fibroblast growth factor. The cytoplasm of the neurites was also immunoreactive for basic fibroblast growth factor. At 30 daysin vitro the intracellular distribution of fibroblast growth factor immunoreactivity was similar to that observed at 20 days. However, both acidic and basic fibroblast growth factor were detected on the surface of the neurites. In contast, no fibroblast growth factor immunoreactivity was detected at the Schwann cell surface at any time point examined. The distribution of fibroblast growth factor in Schwann cells cultured by themselves was similar to that of Schwann cells co-cultured with neurons after 20 daysin vitro. Both Schwann cells and dorsal root ganglia exhibited increased fibroblast growth factor immunoreactivity with increased time in culture and an increased expression of basic fibroblast growth factor in the nucleus. Of particular interest was the appearance of fibroblast growth factor on the surface of neurites after 30 daysin vitro where it could function to modulate neuron-glial cell interactions.     

The development of the chicken small intestine: a scanning electron microscopy study

The surface pattern of the small intestine of the chicken was studied using SEM in stages ranging from 11th day of foetal development to 60 days of post-natal life. The definite villi of the small intestine were preceded by the development of the previllous ridges. The villi were finger like and of unequal length during incubation. After hatching, gradually, the villi were longer, well formed with furrows along their sides. Respectively, the crypts, being present at late incubation, increased with age. Columnar epithelial cells with dense microvilli lined the luminal surface of the intestine with obvious goblet cells openings among them.     

Prenatal and postnatal development of the small intestine in the insectivore Suncus murinus

The development of the small intestine in the insectivore Suncus murinus was noted during the period from 21 days' gestation to 20 clays after birth. At 21 days of gestation, the proximal small intestine exhibited the beginning of villus formation, whereas the distal small intestine preserved the stratified epithelium. Stratified epithelium in the distal small intestine changed into a single layer by 24 days' gestation. At 26 day's gestation, each epithelial cell was immature; but by 28 days mature-looking epithelial colls were found. The shape of the villi changed from cuboid to columnar during the same period. The connective-tissue cores of the villi began to develop at 7 days after birth in the proximal small intestine and at 15 days after birth in the distal small intestine. Crypts appeared at 15 days after birth. Endocytosis of epithelial cells took place at 28 days of gestation. In the proximal small intestine, supranuclear vesicle clusters were observed first at birth; they began to decrease both in number and size at 10 days' gestation and then disappeared completely by 20 days after birth. In the distal small intestine, large supra-nuclear vacuoles were observed first at 28 days of gestation. Although these vacuoles invariably were found up to 15 days after birth, they also disappeared completely by 20 days. Epithelial cells showed a structure similar to those of the adult after weaning.     

Developmental expression and distribution of sheep beta-defensin-2

The aim of this study was to define the ontogeny of sheep beta-defensin-2 (SBD-2) mRNA and peptide in selected tissues of fetal, neonatal and adult sheep by real-time PCR and immunohistochemistry, respectively. Fetal and neonatal lambs had significantly greater SBD-2 tissue distribution than adult sheep. For all ages, the intestines had consistent SBD-2 mRNA expression while extra intestinal expression was sporadic and weak. In adult sheep, SBD-2 mRNA levels decreased from the jejunum caudally to the rectum and a pooled sample from all age groups showed a similar tendency. SBD-2 immunoreactive cells were predominantly in the crypts and base of villi in the small intestine and in a modest number of glands in the large intestine. Interestingly, ileal follicle-associated epithelium lacked detectable SBD-2 immunoreactivity. SBD-2 mRNA and peptide expression are greatest in the intestinal tract and tissue distribution progressively decreases with maturity.     

Pituitary adenylate cyclase-activating peptide (PACAP) immunoreactivity distribution in the small intestine of the adult New Hampshire chicken

We conducted a study in which we demonstrated by means of immunoperoxidase and immunofluorescence methods the presence of pituitary adenylate cyclase-activating peptide 38 (PACAP-38) immunoreactivity in the small intestine of adult New Hampshire chickens and its co-localization with VIP. In particular we describe for the first time the presence of PACAP-positive cells in the epithelium of crypts and villi. Using double immunostaining, we observed that these two peptides were widely co-localized in the nerve structures of duodenum and jejunum with the exception of the ileum, where we noticed a faint co-localization regarding the nerve fibers of the lamina propria of the villi. Furthermore, the two peptides were occasionally co-stored in the epithelial cells of the mucosa. Our findings suggest that in the chicken small intestine, PACAP can be considered, not only as a neuromodulator released by nerve elements, but also as a gut hormone secreted by endocrine cells, and it appears likely to have a role in the regulation of important intestinal physiological functions.     

Influence of starvation and refeeding on mucosal size and epithelial renewal in the rat small intestine

Adult male rats were fasted for 0 (controls), three, five and seven days; a group was refed for one day after six days of starvation. Histological samples were taken from five regions along the length of the small intestine. The sizes of the villi, crypts and mitotic pool were estimated by measuring the number of epithelial cells per villus and crypt section and the number of mitotic figures per crypt section. Additional studies were carried out using colchicine for estimating mitotic time and methotrexate for inhibiting mitosis. All three parameters decreased progressively during starvation; the decrease in villus size was most pronounced in the duodenum and gradually less distalward. Refeeding increased the mitotic pool in every region; crypt size did not increase and villus size increased slightly in duodenum and jejunum only. When refeeding was combined with mitotic inhibition, the cell population of the crypts became depleted by 30–40% without change in villus size; thus, renewal appeared to continue by crypt cells migrating to the villi. Mitosis in the crypts is used for epithelial renewal in the adult intestine. The calculated turnover time of the epithelium was longer than normal and similar in every stage of starvation. Refeeding appeared to stimulate renewal. Since villus size changed somewhat independently from mitotic activity, the involvement of a separate mechanism controlling villus size was indicated.     

Scanning electron microscopic studies on the subepithelial tissue of the gastrointestinal mucosa of the rat

The three-dimensional architecture of the subepithelial tissue of the gastric, the small and the large intestinal mucosa of the rat was observed by scanning electron microscopy (SEM) after removal of cellular elements through prolonged osmication followed by ultrasonication (the method by Highison and Low, 1982), or by cell-maceration with a low temperature NaOH solution (the method by Ohtani, 1987). The basal lamina was exposed by the former method, and the collagenous fibrous sheet immediately under the basal lamina was disclosed by the latter. The surface of the subepithelial tissue is grossly smooth in the pyloric gland and crypts of the small and the large intestine. However, in the fundic glands of the stomach, the surface structure of the subepithelial tissue greatly differs according to glandular location. In the pit of the fundic glands, the surface of both the basal lamina and the sub-basal laminar fibrous sheet is smooth. In the neck region, however, shallow round depressions are seen. The most striking feature of the subepithelial tissue of the fundic glands is the presence of numerous hemispherical concavities in the middle and basal regions of the glands which harbor parietal cells. On the surface of the small intestinal villi, the basal lamina is elevated by the underlying capillary network and forms a meshwork of ridges that surround shallow basins in which numerous round fenestrations 1-5 microns in diameter are seen. The fibrous sheath of the marginal arteriole is observed as a cord-like protuberance on the apical margin of the villi; this suggests its role in the maintenance of the structural integrity of the villi. In the large intestine, well defined round fenestrations are clearly seen, mainly distributed on the upper third of the crypts, and continuing to the lamina propria.