Crypt Area Research Articles

Effects of Microplastic on the Gastrointestinal Tract and Gut microbiome of Sprague Dawley Rats

ABSTRACT Nowadays there is evidence that animals are exposed to microplastics (MPs) via food, however there is not much known about their effect on tissue of gastrointestinal tract and on gut microbiome. In this study, adult male rats were fed with polyethylene MPs, mixed with standard pellet food for 24 days at different concentrations ranging from 0 to 1000 μg/day. At the end of experiment, rats were euthanized and histopathological investigation using light and electron microscopy and the gut microbiome was analyzed using MiSeq 16S rRNA amplicon sequencing. Microscopic analysis revealed significant impact of MPs on the intestine, including visible changes to the crypt area as well as reduction in mucus secretion. Autophagic vacuoles were observed in the livers of the rats fed with MPs at 100 µg/day. Oxidative stress was evident in rats fed with 1000 µg/day of MPs, as indicated by the presence of myeloid bodies in both intestinal and hepatic cells. The gut microbiome was also affected by MPs. Although, no distinct clusters were formed when the bacterial communities in the different treatments were ordinated by multidimensional scaling (NMDS), the diversity indices including operational taxonomic unit (OTU) richness and Chao1 exhibited an increasing trend with increasing MPs concentration. Pearson correlations revealed a linear increase in the relative sequence abundance of Clostridia (R=0.036, P=0.003) with increasing MPs concentration, but not in the case of other bacterial groups,. Lactobacillus faecis was the most abundant OTU in the entire dataset, and its relative abundance decreased significantly with increasing MPs concentration. We conclude that exposure to MPs can disrupt cellular function and disturb microbiome balance, and in the event of prolonged exposure, organisms might experience unpredictable effects.

The staining results of early gastric cancer by indigo carmine chromoendoscopy associated with histological structure: a retrospective study

BackgroundAt present, conventional endoscopy and chromoendoscopy using indigo carmine (IC) is a very useful method to determine the demarcation line (DL) of early gastric cancer lesions, but it is not suitable for all lesions.AimsThis study aimed to determine the applicable conditions for IC chromoendoscopy.MethodsWe retrospectively evaluated 187 lesions in 181 patients who had an endoscopic diagnosis of EGC and were treated with endoscopic submucosal dissection (ESD). According to the existence of the DL between the lesion mucosa and normal mucosa with IC chromoendoscopy, the lesions were divided into two groups: clear group and unclear group. Clinicopathological characteristics were evaluated in each group. From January 2022 to March 2023, the postoperative pathological sections of 19 lesions (81 slices) in the clear group and 19 lesions (80 slices) in unclear group were scanned with high definition, and the crypt structure between the two groups was evaluated.ResultsThere was no significant difference in clinical factors between the clear group and unclear group. There were significant differences in crypt area, crypt length, and crypt opening diameter between the two groups. In the clear group, there were significant differences in crypt area, crypt length, and crypt opening diameter between the normal area and cancer area, but there was no significant difference in the unclear group.ConclusionsThe margins of lesions with fused or absent crypt structures, a small crypt area, a short crypt length, and a short crypt opening diameter can be easily determined with IC chromoendoscopy.

A Periodic Comparison of Harderian Gland in Henna Partridge (Alectoris chukar) According to Different Developmental Stages

Background: The present study aimed at determining histological structure and histochemical properties of the henna partridge (Alectoris chukar) Harderian gland periodically. Objectives: For this purpose, the Harderian glands of 12 (6 females + 6 males) healthy henna partridges aged 3 and 6 months were used. Methods: Totally removed Harderian gland tissue samples were kept in 10% neutral formolin for 36 hours and fixed. After washing, dehydrating, and polishing with known histological techniques, tissue pieces were blocked in paraffin. Results: We found that a thin connective tissue capsule surrounds the Harderian gland of the henna partridge and that the capsule sends septums into the gland and divides the gland into lobes and lobules. It was observed that the corpus glandulae consist of low or high prismatic epithelial cells. Within each lobe, there is a single main draining channel which has a very large lumen along with primary draining channels. Primary and main draining channel epithelial cells have a single-layered cuboidal structure. By Gordon-Sweet staining, it was found that reticular fiber bundles started from the connective tissue capsule that surrounded the organ and spread by branching. The reticular fiber bundles extended in thin strands to the periphery of the corpus glandulae. These reticular fiber bundles in the regions where the connective tissue septums widened are thicker and form a network. The reticular fiber bundles are seen in the basal part of the epithelial cells forming the main draining channel and especially in the crypt areas. Reticular fibers are also found around blood vessels. Strong Alcian blue (AB)-positive epithelial cells are observed in the corpus glandulae in AB pH=2.5 staining, but no periodic acid-Schiff (PAS)-positive epithelial cells are observed in PAS staining. While the majority of primary draining channel cells show a strong AB-positive reaction, a few cells showed a weak PAS-positive reaction. Regarding the main draining channel epithelium, goblet cells show a weak and strong AB-positive reaction in AB pH=2.5 staining method, respectively and a weak PAS-positive reaction in PAS staining method. Conclusion: In line with these findings, no significant difference was found in terms of general histological structure and histochemical properties of the 3- and 6-month-old henna partridge Harderian gland.

Effects of Letrozole Treatment and Vitamin C Supplementation on Morphology, Endoplasmic Reticulum Stress, Programmed Cell Death, and Oxidative Stress in the Small Intestine of Adult Male Rats.

Estrogens are hormones that play an important role in the digestive tract, including in men. Letrozole is an inhibitor of cytochrome P450 aromatase, an enzyme converting androgens to estrogens. The use of letrozole may cause oxidative stress and endoplasmic reticulum stress in the cells. Factors modulating cellular stress may include vitamin C. The purpose of this study was to examine whether letrozole and/or vitamin C supplementation can affect the morphology of the small intestine, the parameters of endoplasmic reticulum stress, programmed cell death markers, and oxidative damage. Three-month-old male rats were divided into four groups and treated with the following: (I) CTRL-water; (II) CTRL+C-L-ascorbic acid; (III) LET-letrozole; and (IV) LET+C-letrozole + L-ascorbic acid. The morphometrical measurements included epithelial thickness, crypt and lumen area, crypt perimeter, nuclei number in the crypt, and the cell size of crypts. The expression levels of PERK, caspase-3, and catalase were determined. Significant differences in the morphometrical measurements and immunoexpression were observed. This may indicate that chronic treatment with letrozole can affect morphology and induce ER stress, oxidative stress, and programmed cell death in the epithelial cells of the small intestine of adult male rats. Vitamin C supplementation exerts an effect on some parameters of the molecular processes.

Dietary polyamines promote intestinal adaptation in an experimental model of short bowel syndrome

Intestinal adaptation does not necessarily recover absorptive capacity in short bowel syndrome (SBS), sometimes resulting in intestinal failure-associated liver disease (IFALD). Additionally, its therapeutic options remain limited. Polyamines (spermidine and spermine) are known as one of the autophagy inducers and play important roles in promoting the weaning process; however, their impact on intestinal adaptation is unknown. The aim of this study was to investigate the impact of polyamines ingestion on adaptation and hepatic lipid metabolism in SBS. We performed resection of two-thirds of the small intestine in male Lewis rats as an SBS model. They were allocated into three groups and fed different polyamine content diets (0%, 0.01%, 0.1%) for 30 days. Polyamines were confirmed to distribute to remnant intestine, whole blood, and liver. Villous height and number of Ki-67-positive cells in the crypt area increased with the high polyamine diet. Polyamines increased secretory IgA and mucin content in feces, and enhanced tissue Claudin-3 expression. In contrast, polyamines augmented albumin synthesis, mitochondrial DNA copy number, and ATP storage in the liver. Moreover, polyamines promoted autophagy flux and activated AMP-activated protein kinase with suppression of lipogenic gene expression. Polyamines ingestion may provide a new therapeutic option for SBS with IFALD.

Induced acute hyperglycemia modifies the barrier function of the intestinal epithelium by tissue inflammation and tight junction disruption resulting in hydroelectrolytic secretion in an animal model.

Diabetic-metabolic syndrome (MetS-D) has a high prevalence worldwide, in which an association with the rupture of the intestinal epithelium barrier function (IEBF) has been pointed out, but the functional and morphological properties are still not well understood. This study aimed to evaluate the impact of acute hyperglycemia diabetes on intestinal tight junction proteins, metabolic failure, intestinal ion and water transports, and IEBF parameters. Diabetes was induced in male Rattus norvegicus (200-310 g) with 0.5 mL of streptozotocin (70 mg/kg). Glycemic and clinical parameters were evaluated every 7 days, and intestinal parameters were evaluated on the 14th day. The MetS-D animals showed a clinical pattern of hyperglycemia, with increases in the area of villi and crypts, lactulose:mannitol ratio, myeloperoxidase (MPO) activity, and intestinal tissue concentrations of malondialdehyde (MDA), but showed a reduction in reduced glutathione (GSH) when these parameters were compared to the control. The MetS-D group had increased secretion of Na+, K+, Cl-, and water compared to the control group in ileal tissue. Furthermore, we observed a reduction in mRNA transcript of claudin-2, claudin-15, and NHE3 and increases of SGLT-1 and ZO-1 in the MetS-D group. These results showed that MetS-D triggered intestinal tissue inflammation, oxidative stress, complex alterations in gene regulatory protein transcriptions of intestinal transporters and tight junctions, damaging the IEBF and causing hydroelectrolyte secretion.

Standardized Quantification of Mast Cells in the Gastrointestinal Tract in Adults

Current data on the normal quantity of mast cells throughout the adult gastrointestinal tract are limited in several domains. These include microanatomic localization of mast cells, standardization of staining and counting methods, and reporting of microscope field of view. To address this lack of reliable reference ranges to facilitate the study of and diagnosis of emerging mast cell-mediated diseases. We examined biopsies obtained from the esophagus, stomach, duodenum, and colon from an unselected cohort. Mean and peak mast cell density were determined on slides stained for tryptase and CD117, and were expressed per high power field (hpf) and surface area (mm2), thus deriving reference ranges (average ± 2 SDs). For the most common hpf surface area (0.238 mm2), upper limits of the derived reference ranges for average/peak mast cells were 0.15/3.67 (esophagus, tryptase), 0.70/5.98 (esophagus, CD117), 22.56/35.30 (stomach, tryptase), 31.32/53.10 (stomach, CD117), 30.28/49.77 (duodenal crypts, tryptase), 41.96/65.26 (duodenal crypts, CD117), 4.98/11.56 (duodenal villi, tryptase), 8.38/14.17 (duodenal villi, CD117), 26.58/41.08 (colon, tryptase), and 35.57/57.92 (colon, CD117). Interobserver variability was moderate to good. There was significant correlation between average and peak mast cell counts. These data help standardize mast cell reference ranges throughout the gastrointestinal tract in adults, which can be used to determine whether abnormal levels of mast cells are present in patients with suspected mast cell-mediated disease. Our data show that the commonly used cutoff of 20 mast cells per hpf irrespective of the gastrointestinal tract segment is an underestimate of an appropriate cutoff in stomach, duodenum (crypt area), and colon.

A Deep Learning-Based Approach to Estimate Paneth Cell Granule Area in Celiac Disease.

Changes in Paneth cell numbers can be associated with chronic inflammatory diseases of the gastrointestinal tract. So far, no consensus has been achieved on the number of Paneth cells and their relevance to celiac disease (CD). To compare crypt and Paneth cell granule areas between patients with CD and those without CD (non-CD) using an artificial intelligence-based solution. Hematoxylin-eosin-stained sections of duodenal biopsies from 349 patients at the McGill University Health Centre were analyzed. Of these, 185 had a history of CD and 164 were controls. Slides were digitized, and NoCodeSeg, a code-free workflow using open-source software (QuPath, DeepMIB), was implemented to train deep learning models to segment crypts and Paneth cell granules. The total area of the entire analyzed tissue, epithelium, crypts, and Paneth cell granules was documented for all slides, and comparisons were performed. A mean intersection-over-union score of 88.76% and 91.30% was achieved for crypt areas and Paneth cell granule segmentations, respectively. On normalization to total tissue area, the crypt to total tissue area in CD was increased and the Paneth cell granule area to total tissue area decreased when compared to non-CD controls. Crypt hyperplasia was confirmed in CD compared to non-CD controls. The area of Paneth cell granules, an indirect measure of Paneth cell function, decreased with increasing severity of CD. More importantly, our study analyzed complete hematoxylin-eosin slide sections using an efficient and easy to use coding-free artificial intelligence workflow.

Comprehensive characteristics of rectal morphology in chickens with different types of autonomous regulation

The influence of typological features of autonomous regulation on the dimensions, structure, and blood supply of the rectum in chickens has been studied. The experimental group consisted of clinically healthy adult chickens of the Isa-Brown breed, divided into two groups using electrocardiography and variation pulsometry methods: chickens with sympathetic-tonic regulation and chickens with sympathetic-normotonic regulation. In each group, the length and diameter of the rectum, the branching of its arterial vessels, and the microscopic structure of the intestinal wall were examined. The corresponding artery provides the blood supply to the rectum, the branching of which showed no significant differences between the poultry groups. The peculiarities of autonomous regulation significantly influence the morphological structure of the intestinal wall’s mucous and muscular membranes. The serous membrane does not respond to these features. Chickens with sympathetic-tonic regulation (ST chickens) are characterized by a greater thickness of all indicated membranes and, consequently, the entire intestinal wall. Chickens with sympathetic-normotonic regulation (ST-NT chickens) have a greater thickness only in the muscular plate of the mucous membrane and its longitudinal layer. Regarding the quantity of collagen and elastic fibers, ST chickens dominate the mucous membrane’s crypt area, while ST-NT chickens dominate the muscular membrane. However, the density of connective tissue fibers in the mucous membrane is much lower than in the muscular one. The quantity and distribution of plasma cells have similar patterns in both groups. Specific morphometric compensation between two functionally related indicators is also an important aspect. In this case, larger values of one indicator are combined with smaller values of another. For example, a smaller diameter of the rectum in ST chickens is associated with its greater length; a smaller number of goblet cells on one villus corresponds to a larger area of their secretory section; a larger area of the nerve node of the intramuscular nerve plexus is combined with a smaller number of these nodes per 1 cm² of the intestinal wall. Thus, the structure of the rectum in chickens, adapting to the corresponding type of autonomous regulation, maintains the balance at the level of individual membranes and the entire intestinal wall.

L-serine improves lipid profile, performance, carcass weight and intestinal parameters in feed restricted broiler chickens during the hot-dry season.

The study evaluated effects of L-serine on lipid profile, performance, carcass weight and small intestinal parameters in heat-stressed broiler chickens subjected to feed restriction. Broiler chickens were divided into four groups, comprising 30 each. Group 1, feed restriction (FR); Group 2, feed restriction + L-serine (200mg/kg) (FR + L-serine); Group 3, ad libitum (AL); Group 4, ad libitum + L-serine (200mg/kg) (AL + L-serine). L-serine was administered orally from days 1 to 14, and feed restriction was performed on days 7-14. Serum harvested from blood samples on days 21, 28 and 35 was evaluated for lipid profile. Feed and water intake, live weight gain, organ and carcass weight were measured. At 35days old, broiler chickens (n = 7) per group were sacrificed to evaluate small intestinal morphology. Temperature-humidity index in the pen (30.88 ± 0.81) was above thermoneutral zone, indicating that chickens were subjected to heat stress. Concentrations of low-density lipoprotein, total cholesterol and total triglycerides were lower (p < 0.05), while higher concentration of high-density lipoprotein was recorded in L-serine groups than in the controls. Feed intake and live weight gain on day 35 in L-serine groups were higher (p < 0.05) than in controls. In L-serine groups, liver, spleen, pancreas and heart weight were higher, but abdominal fat was lower than in FR and AL groups. Villus height:crypt height ratio and area of villus surface were highest in L-serine groups than any other group. In conclusion, L-serine decreased low-density lipoprotein, increased feed intake, live weight, organ and carcass weight, villus height:crypt height ratio and villus surface area.

Does the crypt: tooth ratio in the developing tooth correlate with chronological age in children?—a novel, radiographic, cross-sectional pilot study using ImageJ tools

BackgroundThe bony cavity known as the crypt encloses the developing tooth, the size of which is influenced by the tooth movements during various phases of tooth eruption. The radiographic observations have revealed that the size of the crypt decreases as the root formation continues. The present study was conducted to assess the relationship between the crypt to tooth ratio (CTR) and the chronological age in children in the age range of 4 to 16 years using their digital panoramic radiographs (OPGs). The ImageJ tools were used to calculate the area of the bony crypt and the area of the developing mandibular second molar from 145 OPGs. The ratio between the area of the crypt and the area of the calcified portion of the tooth was obtained and compared with the calendric age (CA) to develop a regression model for age estimation.ResultsThere was a significant negative correlation between the chronological age and the CTR values (r= −0.898, p<0.001) in all the age groups. The derived regression formula was age = 17.192–3.855 (CTR) with an r2 value of 0.807 and a standard error of estimate (SEE) being 1.385. The derived formula was tested using 52 OPGs from a different set of patients in the same age group. The mean chronological age of the test sample was 9.57 years (± 3.42), their mean estimated age (EA) was 8.50 years (± 3.25), and the difference was statistically significant (p<0.001). A difference of more than 1 year was observed in 69% of boys and 54% of girls.ConclusionsThis method is an attempt to correlate the crypt to tooth ratio of developing the second molar tooth with the chronological age. A large sample study is required to validate this novel technique of age estimation in children.

Lymphocyte subsets in the small intestine of piglets fed with probiotic and zinc: a qualitative and quantitative micro-anatomical study.

Piglet mortality is a real concern to the pig farmers. The major cause is due to the late maturation of the immune system and dietary changes in postweaned piglets. The potential role of probiotic and zinc in the stimulation of the immune system is well established. Hence, the present study was undertaken to evaluate alterations of T and B cells in the small intestine after dietary inclusion of probiotic and zinc in pre and post-weaned piglets. A total of 18 healthy Large White Yorkshire (LWY) piglets, irrespective of sex obtained from 3 litters at the age-group of 20, 30 and 60 days. They were divided into a control group fed with basal diet and a treatment group fed with probiotic and zinc supplement along with the basal diet, consisting of three animals in each group. The piglets were weaned at 28 days of age. After sacrificing the animals at day 20, 30 and 60 from both the groups, the abdominal cavity was opened and small intestinal tissue samples were collected, processed and stained by indirect immunofluorescence technique. The slides were evaluated under the fluorescent light microscope. The data were statistically analysed. The different T and B cell subsets were recorded in the lining epithelium, core of villus, crypt area of lamina propria and Peyer's patch area. The number of CD4+, CD8+, IgA+ and IgM+ cells was higher in the treated piglets than the control group of animals, irrespective of segments of intestine and age-group. It can be concluded that the dietary supplementation of probiotic and zinc was found to be good additives as they can stimulate the immune response in piglets, especially during the critical early post-weaning period.

Dietary Yeast Cell Wall Improves Growth Performance and Prevents of Diarrhea of Weaned Pigs by Enhancing Gut Health and Anti-Inflammatory Immune Responses

Simple SummaryPost-weaning stress can substantially affect performance of weaned pigs as well as overall pig production, and thus, a practical approach is needed to improve their performance by alleviating the stress that can cause intestinal barrier dysfunction of weaned pigs. There are potential ways to solve the concern in swine production, but dietary yeast cell wall in weaner diets may be one possible solution. The results of the present study suggest that dietary yeast cell wall improves growth performance of weaned pigs by enhancing gut health and provide its potential mechanism.Dietary yeast cell wall products (YCW) are recognized as a feed additive due to multifunctional benefits by the biological response modulators. Thus, this study was conducted to verify a potential advantage of YCW for improving growth performance, nutrient digestibility, immune responses, and intestinal health and microbiota of weaned pigs. A total of 112 weaned pigs (7.99 ± 1.10 kg of body weight; 28 days old) were arbitrarily allocated to two experimental treatments with eight pigs (four barrows and four gilts) per pen and seven replicate pens per treatment in a completely randomized block design (block = BW and sex): (1) a basal diet based on corn and soybean meal (CON) and (2) CON + 0.05% YCW. The experimental period was for 4 weeks. There were no differences in final body weight, average daily feed intake, and gain-to-feed ratio between dietary treatments. In contrast, pigs fed YCW had higher average daily gain (p = 0.088) and apparent ileal digestibility of DM (p < 0.05) and energy (p = 0.052) and lower diarrhea frequency (p = 0.083) than those fed control diet (CON). Pigs fed YCW also had a higher (p < 0.05) ratio between villus height and crypt depth, villus width and area, and goblet cell counts in the duodenum and/or jejunum than those fed CON. Dietary YCW decreased (p < 0.05) serum TNF-α and IL–1β of weaned pigs on day 7 and 14, respectively, compared with CON. Furthermore, pigs fed YCW had higher (p < 0.05) ileal gene expression of claudin family, occludin, MUC1, INF-γ, and IL-6 and lower (p < 0.05) that of TNF-α than those fed CON. Lastly, there were no differences in the relative abundance of bacteria at the phylum level between CON and YCW. However, dietary YCW increased (p < 0.05) the relative abundance of genera Prevotella and Roseburia compared with CON. This study provided that dietary YCW improved growth rate, nutritional digestibility, and intestinal health and modified immune responses and intestinal microbiota of weaned pigs.

P397 Response to biologics in IBD patients assessed by Computerized image analysis of Probe Based Confocal Laser Endomicroscopy with molecular labeling

Abstract Background The increase in therapeutic choices in inflammatory bowel diseases (IBD) imposed the identification of personalized therapeutic strategy. Confocal laser endomicroscopy (CLE) is a new endoscopic tool developed to obtain virtual in vivo histology. This study aimed to identify CLE in vivo and ex vivo features predictive of response for patients starting biologics. Methods We performed a prospective observational study at a single tertiary referral centre. 29 IBD patients (14 ulcerative colitis-UC and 15 Crohn’s Disease-CD) underwent colonoscopy with CLE before and 12-14 weeks after starting anti -TNF or 16 weeks after anti α4β7 integrin. CLE parameters analyzed were: crypt distribution along the mucosal surface, crypt area (CA), eccentricity, diameter, inter-cryptic distance (ICD), vessel tortuosity (VT), fluorescein leakage through the colonic mucosa (FLCM). Ex-vivo binding activity of fluorescein labelled biologics on corresponding biopsies was also assessed. Mosaicism reconstruction of CLE images were analyzed using a dedicated software algorithm (CellvizioViewer, Mauna Kea Technologies, ParisFrance). A dedicated Graphical User Interface was designed to enable a semiautomated analysis. Results After treatment, VT resulted significantly changed in overall IBD population (p&amp;lt;0.05), FLCM were reduced in UC patients (p&amp;lt;0.05), whilst CA, eccentricity and ICD decreased in CD patients (p&amp;lt; 0.05). The univariate regression analysis identified FLCM as the most accurate parameter for predicting responsiveness (AUROC) 83%, accuracy 83%, positive predictive value-PPV 94% and negative predictive value-NPV 57%). FLCM and ICD were the best discriminants in responders Vs non responders in UC patients (AUROC 85%, accuracy 85%, PPV 100% and NPV 71%); whilst VT, CA and ICD in CD patients (AUROC 95%, 86% and 83%; accuracy 90%, 90% and 88%; PPV 100%, 100% and 86%; and NPV 75%, 75% and 100%, respectively). Ex vivo confocal imaging revealed that UC patients, especially those responders, had higher basal fluorescent intensity signals with a significant reduction after biological treatment (p&amp;lt; 0.05), whereas in CD patients no significant change was found. An increased mucosal binding to the fluorescent labelled biological agent was associated to a higher likelihood of response to the treatment; this effect was higher in UC patients (AUROC 81%, accuracy 77%, PPV 100%, NPV 63%) compared to CD patients (AUROC 64%, accuracy 79%, PPV 80%, NPV 50%). Conclusion FLCM and ICD were the best discriminants of response in UC, while VT, CA and ICD in CD. A higher mucosal binding to a biological agent before treatment was observed in responders UC patients but not in CD patients.

Intestinal epithelial replacement by transplantation of cultured murine and human cells into the small intestine.

Adult intestinal epithelial stem cells are a promising resource for treatment of intestinal epithelial disorders that cause intestinal failure and for intestinal tissue engineering. We developed two different animal models to study the implantation of cultured murine and human intestinal epithelial cells in the less differentiated “spheroid” state and the more differentiated “enteroid” state into the denuded small intestine of mice. Engraftment of donor cells could not be achieved while the recipient intestine remained in continuity. However, we were able to demonstrate successful implantation of murine and human epithelial cells when the graft segment was in a bypassed loop of jejunum. Implantation of donor cells occurred in a random fashion in villus and crypt areas. Engraftment was observed in 75% of recipients for murine and 36% of recipients for human cells. Engrafted spheroid cells differentiated into the full complement of intestinal epithelial cells. These findings demonstrate for the first time successful engraftment into the small bowel which is optimized in a bypassed loop surgical model.

Influence of the type of autonomic tone on the volume of the mucous membrane of the small intestine of laying hens

The connection between the separate structural parts of the mucous membrane of the small intestine of laying hens and the typological features of the autonomic tone were investigated. The studies were conducted on adult chickens of the cross-breed "Isa-Brown", which were divided into two groups: sympathicotonic chickens and sympathico-normotonic chickens, by the methods of electrocardiography and variation-pulsometry research. In the small intestine of the poultry of each group, the linear dimensions were determined first, and then the volume of the entire mucous membrane and its separate parts (villi, crypt, muscular plate), as well as the volume of connective tissue fibers in the crypt region. Research has shown that the villi account for ¾ of the volume of the entire mucous membrane. The volume of villi in the entire small intestine is more important in sympathico-normotonic chickens. This same poultry group has an advantage in the indicators of the volume of the muscular plate, but only in the duodenum and jejunum. In indicators of crypt volume, it turned out to be quite the opposite of the relationship with the typology of autonomous influences. In all studied intestines, the higher values of this indicator belong to the sympathotonic chickens. However, in terms of the volume of the entire mucous membrane, larger values still correspond to sympathico-normotonic chickens. Sympathicotonic chickens are inferior to them in the duodenum – at 1,005 mm3, in the jejunum – at 2,699 mm3 and in the ileum – only 78 mm3. Investigating the structure of the villi, we established that the volume of their epithelium in all three sections of the small intestine has higher values in the sympathico-normotonic chickens, and the larger volume of the lamina propria of the villi in the sympathicotonic chickens. At the same time, the connection with the type of autonomic tone is reflected in the ratio between the two layers of villi. In all three sections of the small intestine of sympathicotonic chickens, the ratio between the epithelium and the lamina propria was, on average, 65%/35%, or 2/1. In sympathico-normotonic chickens, this ratio varied and, on average, equalled 75%/25%, or 3/1. The increase in the tone of sympathetic centers contributes to the growth of volume both as the total amount of fibers of connective tissue, and also as separate elastic fibers in the area of crypt. According to the data,indicators of the sympathico-normotonic chickens are inferior to those of sympathicotonic chickens in all of the small intestine. It was also found that in both groups of poultry the volume of elastic fibers was approximately half the volume of all fibers of the connective tissue of the duodenum. In other sections of the small intestine, this figure was reduced to about ⅓. Consequently, the typological features of the autonomic tone, providing various trophic-regulatory effects on the mucous membrane of the small intestine, cause the formation of differences in its volume.

Comparison of two methods for determination of intestinal villus to crypt ratios and documentation of early age-associated ratio changes in broiler chickens , ,

The determination of intestinal villus to crypt ratios (VCR) is a common method utilized to evaluate effects of various diet regimens on gut microanatomy and for the histologic quantification of intestinal responses to disease processes. Two methods for the determination of small intestinal VCR were compared in early age chickens. A standard method for VCR determination based on 10 villus and crypt length measurements in the jejunal region of the small intestine was employed for the calculation of villus to crypt length ratio (VCLR). That method was compared to a new approach based on a single histomorphometric determination of the crypt and total mucosal areas using image analysis software. Subtraction of the crypt area from the total area provided the villus area and allowed for the subsequent calculation of villus to crypt area ratio (VCAR). At 4 and 18 h posthatch, VCLR was higher than that of VCAR, but there was no significant difference between VCLR and VCAR at 0 h (hatch) and at 168 h (d 7) posthatch. Nevertheless, the pattern of age-associated changes for VCLR and VCAR were comparable throughout the early posthatch period. Furthermore, the new method used in determining VCAR is subject to less human error, allows for an appreciable reduction in the number of measurements required, and facilitates a larger intestinal segment evaluation. Standard linear measurements require the selection of variable numbers of villi and crypts, whereas the area method only requires selection of a single region that incorporates numerous villi and crypts of variable sizes in providing a less subjective approach. This is particularly advantageous in studies on intestinal disease conditions resulting in marked multifocal variation in villus stature. This study further documented age-associated changes occurring in the VCR of the small intestine during the early posthatch period. Across the 2 methods used for VCR determination, a major and highly significant reduction in the VCR was observed to occur between 18 h and 168 h posthatch.

The Sodium/Hydrogen Exchanger 2 (Slc9a2/NHE2) is Involved in the Differentiation of Colonic Intestinal Epithelial Cells

BackgroundBoth sodium/hydrogen exchanger 2 and 3 (NHE2 and NHE3) isoforms of the Slc9 family Na+/H+ exchangers are expressed in the luminal membrane of the intestinal enterocytes, but in contrast to the deletion of NHE3, the deletion of NHE2 does not result in diarrhea, hyperaldosteronism, or reduced survival in mice.Aim and methodsTo further analyze the physiological role of NHE2 in the intestinal epithelial cells, we fluorometrically measured the intracellular pH (pHi) along the colonic cryptal axis of NHE2−/− mice, and in the intestinal epithelial Caco 2Bbe (C2Bbe) after stable lentiviral sh‐mediated NHE2 silencing. RNA and protein expression were assessed by qPCR, Western analysis and immunohistochemistry, and proliferative, migratory, and functional features of colonic cells in vivo and in vitro were assessed by enzymatic and functional assays.ResultsSteady‐state pHI was significantly decreased in C2Bbe/shNHE2 compared to empty‐vector transfected C2Bbe cells. In the colonic crypts, the pHi in the cryptal base was significantly lower than in the surface region, and the acidic zone along the crypt axis was significantly longer in colonic crypts from NHE2−/− mice. NHE2−/− colonic crypts were elongated with a much longer proliferation zone but with less proliferating cells per crypt area. Cell proliferation in C2Bbe/shNHE2 cells was reduced compared to empty‐vector transfected C2Bbe. The expression and activity of alkaline phosphatase (an enterocyte differentiation marker), was significantly reduced in NHE2−/− colonic mucosa and in C2Bbe/shNHE2 cells. However, the number of goblet cells and mucin 2 (Muc2) expression was increased in the NHE2−/− compared to the WT colon, accompanied by formation of a thicker mucus layer. The shift from absorptive to secretory differentiation program was accompanied by a decrease in Hes1 expression, as a downstream transcription factor of Notch signaling that supports the development of absorptive enterocytes.ConclusionsThe results suggest that NHE2 expression and/or function is activated when the intestinal cells emerge from the stem cell niche, and that this is essential for the establishment of the pHi gradient along the colonic crypt axis. Its activity facilitates enterocyte proliferation and differentiation along the crypt axis.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gastrointestinal microphysiological systems.

Gastrointestinal diseases are a significant health care and economic burden. Prevention and treatment of these diseases have been limited by the available human biologic models. Microphysiological systems comprise organ-specific human cultures that recapitulate many structural, biological, and functional properties of the organ in smaller scale including aspects of flow, shear stress and chemical gradients. The development of intestinal microphysiological system platforms represents a critical component in improving our understanding, prevention, and treatment of gastrointestinal diseases. This minireview discusses: shortcomings of classical cell culture models of the gastrointestinal tract; human intestinal enteroids as a new model and their advantages compared to cell lines; why intestinal microphysiological systems are needed; potential functional uses of intestinal microphysiological systems in areas of drug development and modeling acute and chronic diseases; and current challenges in the development of intestinal microphysiological systems. Impact statement The development of a gastrointestinal MPS has the potential to facilitate the understanding of GI physiology. An ultimate goal is the integration of the intestinal MPS with other organ MPS. The development and characterization of nontransformed human intestinal cultures for use in MPS have progressed significantly since the inception of the MPS program in 2012, and these cultures are a key component of advancing MPS. Continued efforts are needed to optimize MPS to comprehensively and accurately recapitulate the complexity of the intestinal epithelium within intestinal tissue. These systems will need to include peristalsis, flow, and oxygen gradients, with incorporation of vascular, immune, and nerve cells. Regional cellular organization of crypt and villus areas will also be necessary to better model complete intestinal structure.

Monocytes/Macrophages Mobilization Orchestrate Neovascularization after Localized Colorectal Irradiation.

In patients undergoing radiotherapy for cancer, radiation dose to healthy tissue can occur, causing microvascular damage. Monocytes that have been shown to promote tissue revascularization comprise the subsets: CD11b+Ly6G-7/4hi/monocyteshi and CD11b+Ly6G-7/4lo/monocyteslo. We hypothesized that monocytes were implicated in postirradiation blood vessel formation. C57Bl6 mice underwent localized colorectal irradiation and were sacrificed at different times after exposure. Bone marrow, spleen, blood and colon were collected. Fourteen days postirradiation, colons expressed proangiogenic actors and adhesion molecules. Monocyteshi, which were the main subset of infiltrating monocytes, mobilized to the blood from spleen and bone marrow, peaking at day 14 postirradiation, and were associated with lymphocyte Th1 polarization. At day 28 postirradiation, angiographic score and capillary density increased by ∼1.8-fold, and then returned to nonirradiated levels at day 60. Clodronate-mediated depletion of circulating monocytes prior to irradiation resulted in a ∼1.4-fold decrease in angiographic score and capillary density compared to the nontreated control. Histological analysis of the colon in clodronate-treated mice revealed a massive decrease of macrophage and lymphocyte infiltration as well as reduced collagen deposition in crypt area at day 21. However, late depletion of monocytes from day 25 postirradiation had no effect on fibrotic process. These findings demonstrate a central role for monocyte/macrophage activation in the orchestration of a neovascularization mechanism after localized colorectal irradiation.