Muscularis Externa Layer Research Articles

Histological and histochemical study of effect of dietary fibre in motility of stomach in male mice.

To investigate the relationship between dietary fibres and muscularis externa layer. The experimental study was conducted at the Anatomy Department of the College of Medicine, Al- Nahrain University, Iraq, from November 2020 to April 2021, and comprised adult healthy males. They were divided randomly into experimental group A and control group B. Group A was fed high-fibre diet, and group B was fed standard pellet diet. The tissue samples were harvested at day 30 post-surgery. The stomach samplings were placed in 10% neutral formalin for 24 hours to obtain paraffin sections for routine histological and immunohistochemical staining. The protein expression in stomach's smooth muscle of each group was detected by immunohistochemical staining. Data was analysed using SPSS 24. Of the 20 mice, 10(50%) were in each of the two groups. Group A exhibited significant weight-gain compared to group B (p≤0.01). There was significant reduction in the thickness of the muscularis layer in group A compared to group B (p≤0.01). Anoctamin 1 expression in group A was significantly lower than that in group B (p≤0.01). The stress induced by an unstable fibre diet significantly affected the stomach by decreasing the muscularis layer thickness and Anoctamin 1 expression in interstitial cells of Cajal.

Anoctamin 1 and c-Kit immunohistochemical study of interstitial cells of Cajal in the muscularis externa of human gastrointestinal tract.

Interstitial cells of Cajal (ICC) are widely distributed in human gastrointestinal (GI) tract, especially in the layer of muscularis externa between neurons and smooth muscles. They play a very important role of coordination of GI tract motility. The aims of this research were to study the morphology and distribution of ICC in the muscularis externa of the GI tract, using immunohistochemistry staining methods, to determine the distribution of immune reactivity of anoctamin 1 (Ano1) compared with c-Kit, and to determine if Ano1 is a reliable marker for ICC in human GI tract. Specimens from the wall of stomach, small intestine, and colon were taken from human cadavers and processed for histological and immunohistochemical study using c-Kit and Ano1 primary antibodies. Interstitial cells of Cajal appeared as bipolar cells, not forming network, in both the circular and longitudinal muscle layers, while in the myenteric area they appeared as multipolar interconnected cells. They were unevenly distributed in and between the muscle layers of the muscularis externa of human GI tract. They were more numerous in the stomach followed by the colon then the small intestine, and more numerous in the myenteric area followed by the circular muscle layer then the longitudinal muscle layer, in the three organs. Our results also showed that Ano1 is a more reliable marker for human ICC than c-Kit. Interstitial cells of Cajal differed in morphology and were unevenly distributed between muscle layers of muscularis externa and between different parts of human GI tract.

Histopathological dimensions differ between aganglionic and ganglionic bowel wall in children with Hirschsprung’s disease

BackgroundIn the validation of new imaging technology for children with Hirschsprung’s disease (HSCR), basic anatomical parameters of the bowel wall must be established specifically for this patient group.AimTo explore differences in histoanatomical layers of bowel wall, comparing ganglionic and aganglionic bowel walls, and to examine if the bowel wall thickness is linked to patient weight.MethodsThis was an observational study of bowel specimens from children weighing 0–10 kg, operated on consecutively during 2018–2020. Ganglionic and aganglionic bowel walls were measured in digitalized microscopy images from 10 sites per trans-sectional specimen and compared regarding the thickness of their histoanatomical layers.ResultsBowel walls were measured in 21 children. Full bowel wall thickness did not differ between aganglionic and ganglionic bowel (2.20 vs 2.04; p = 0.802) while weight at surgery correlated positively with both ganglionic and aganglionic bowel wall thickness (r = 0.688 and 0.849, respectively), and age at surgery with ganglionic bowel wall thickness (r = 0.517). In aganglionic segments, the muscularis externa layer was thicker compared to that in ganglionosis (0.45 vs 0.31 mm, p = 0.012) whereas the muscularis interna was thinner (0.45 vs 0.62 mm, p < 0.001). A diagnostic index was identified whereby a lower ratio of muscularis interna/externa thickness followed by a thinner muscularis interna differed between aganglionic and ganglionic bowel in all specimens.ConclusionThicknesses of the bowel wall’s muscle layers differ between aganglionic and ganglionic bowel walls in children with HSCR. These findings support a diagnostic index that could be validated for transfer to instant diagnostic imaging techniques.Level of evidenceDiagnostic: 3

Sodium Benzoate (NaB) Induced Impairment of the Functions of Duodenal Visceral Smooth Muscle (VSM) ex vivo in Rat

Sodium benzoate (NaB) is a versatile food additive used to preserve packaged foods and beverages. Thus, people are unknowingly consuming it above the WHO recommended daily intake limit which draws our attention to experimenting with the effects of NaB on contractile functions of visceral smooth muscle (VSM) of the small intestine, the primary target organ exposed to NaB during digestion and absorption of food, in the albino rat model. We have observed a significant (p&lt;0.05) increase in amplitude and decrease in the frequency of the contraction of the duodenum, the initial part of the small intestine, in dose and duration response manner in NaB exposed groups of rats compared to control contractions. This result indicates that NaB probably impairs the contraction of duodenal VSM by promoting the activity of myenteric cholinergic and/or the activity of adrenergic and/or nitrergic efferents. We have also found, a significant (p&lt;0.05) decrease in the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR) and an increase in the level of malondialdehyde (MDA) in duodenal VSM homogenate of groups of exposed rats compared to control. These results suggest that NaB probably impairs the contractile function of VSM by altering the functions of myenteric intrinsic efferents by producing oxidative stress in VSM and/or in intrinsic neurons. Further, we have found significant degenerative lesions and altered tissue architecture in the muscularis externa layer of the wall of the duodenum in NaB exposed hematoxylin and eosin stained duodenal tissue sections. These findings reveal that NaB might impair the contractile function of duodenal VSM by inducing the oxidative stress induced degenerations of the architecture of muscle at tissue and cellular levels. Hence, it is concluded that NaB impairs the contraction of duodenal VSM probably by inducing the cytoarchitectural degenerations of VSM structure and/or altering the functions of myenteric intrinsic efferent through oxidative stress.

Toxoplasma gondii infection impairs the colonic motility of rats due to loss of myenteric neurons.

Toxoplasma gondii infection causes intestinal inflammation and diarrhea indicating possible intestinal motor dysfunction. Anatomical studies have shown alterations in the colonic myenteric plexus, but it is unknown whether this impacts motility and therefore whether motility is a target for treatment. We determined whether colonic coordinated movements are compromised by toxoplasmic infection and how it is associated with anatomical changes. Male Wistar rats were evaluated at 6, 12, 24, 48, and 72hours and 30days postinfection (dpi) and controls. Infected rats received orally 5×103 sporulated oocysts of strain ME-49 (genotype II) of Tgondii. The colon was collected for anatomical analysis (including the myenteric plexus immunolabeled with HuC/D, nNOS, and ChAT) and motility analysis in vitro (conventional manometry). Fecal output was measured daily. At 12hours postinfection, Tgondii caused hypertrophy of the muscularis externa layer of the distal colon. There was loss of total, nitrergic, and cholinergic myenteric neurons in the proximal colon at 30day postinfection (dpi); however, only loss of cholinergic neurons was found in the distal colon. Contractile complexes in the middle and distal colon were longer in duration in infected animals, which was associated with slower migration of the colonic motor complex. However, gastrointestinal transit time and fecal pellet output remained unchanged during the Tgondii infection. Toxoplasma gondii caused myenteric neuronal loss in the proximal and distal colon and altered the motility pattern in the middle and distal colon to a more propulsive phenotype.

Histopathological Evaluation of Zebrafish (Danio rerio) Intestinal Tissue After Imidacloprid Exposure

The use of pesticides has been increasing day by day in order to increase productivity in agriculture. Pesticides which used in the agricultural area mix into the aquatic ecosystem through rains and groundwater etc. and it threatens the life of aquatic organisms. Nowadays, one of the most used pesticide group is neonicotinoids. Imidacloprid is the most well known in this group. In this study, it was aimed to observe the histopathological effects of imidacloprid in zebrafish intestinal tissue. Adult zebrafish individuals were administered imidacloprid in concentrations of 9.5, 19 and 38 mg/L for 5 days. When tissues are examined under light microscopy, deterioration of villi morphology, degeneration in brush border structure of epithelial cells, hyperplasia in Goblet cells and enterocytes due to inflammation, enlargement and disintegration in lamina propria were detected. In addition, hypertrophy of the smooth muscle cells and thickening in the muscularis externa layer were observed. Dysplasia was detected in the small intestine tissues in samples belonging to the highest concentration group.

MONOSODIUM GLUTAMATE POTENTIATES THE CONTRACTION OF THE VISCERAL SMOOTH MUSCLE OF DUODENUM BY AUGMENTING THE ACTIVITY OF INTRINSIC CHOLINERGIC EFFERENTS, INDUCING OXIDATIVE STRESS AND PROLIFERATING SMOOTH MUSCLE CELLS

Objective: The objective of the present study was to examine the effects of monosodium glutamate (MSG) on the contraction of visceral smooth muscle (VSM) of the duodenum in a rat model to understand the MSG-induced impairment of the function of the small intestine.&#x0D; Methods: Male albino rats of Charles Foster strain were exposed with MSG at three different dosages (632, 1264, and 2528 mg/kg BW/day) for 30-day duration. The records of the contraction of the duodenum were achieved with isotonic transducer (IT-2245) coupled with RMS-Polyrite D by our standard laboratory protocol.&#x0D; Results: We have observed potentiation of contraction of duodenum ex vivo dose-dependently in MSG exposed groups of rats compared to control. Furthermore, the enzymatic activity of acetylcholinesterase (AChE) in VSM tissue homogenate and expression of AChE protein in fixed duodenal muscle cell layers have been decreased in a dosage response manner comparing to control rats. We have found a significant decrease in the activities of some antioxidant enzymes such as Cu-Zn superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione-s-transferase, and increase in the level of malondialdehyde in MSG exposed VSM tissue homogenate of the duodenum. We have also observed thickening of muscularis externa layer and increase in the number of muscle cells in circular and longitudinal muscle layers of the duodenal wall in transverse duodenal wall sections stained with eosin-hematoxylin.&#x0D; Conclusion: MSG potentiates the contraction of VSM of duodenum by augmenting the activity of intrinsic cholinergic efferents predominantly, and inducing oxidative stress and proliferating smooth muscle cells.

Impairment of rat oesophageal muscle contractility associated with experimental non-erosive oesophageal mucosal damage.

What is the central question of this study? Is the responsiveness of isolated segments of the rat oesophagus to contractile or relaxant stimuli susceptible to acute luminal exposure of the oesophagus to an acid solution that contains pepsin and bile salt? What is the main finding and its importance? The study reveals that luminal acidity is an important factor that disrupts barrier function in the oesophagus to allow the diffusion of noxious agents, such as bile acid, that alter the contractile status of the oesophageal body, even in the absence of inflammation. We investigated whether the experimental simulation of duodenogastro-oesophageal reflux alters the contractile responsiveness of rat oesophageal strips. After 30min of luminal exposure to a solution at acid pH that contained pepsin and taurodeoxycholic acid, isolated strips of the rat oesophagus and gastro-oesophageal junction were subjected to contractile or relaxing stimuli. Acid challenge decreased the responsiveness of oesophageal strips to contractile stimulation, especially in oesophageal preparations that were mounted following the circular orientation of the muscularis externa layer. The contractility of longitudinal preparations of the rat oesophagus appeared less susceptible to the deleterious effects of acid challenge. In contrast, the responsiveness of ring-like preparations from the gastro-oesophageal junction to contractile stimulation was unaltered by acid challenge. Taurodeoxycholic acid decreased the responsiveness of circular oesophageal preparations to KCl, an effect that was exacerbated by luminal acidity. On the contrary, although the relaxant ability of the rat oesophagus did not change, acid challenge increased the relaxant efficacy of sodium nitroprusside and isoprenaline in strips of the gastro-oesophageal junction. A significant decrease in transepithelial electrical resistance was seen when the oesophageal mucosa was challenged at pH1 but not at pH4. Treatment with alginate blunted the deleterious effects of acid challenge on transepithelial electrical resistance and the responsiveness of oesophageal preparations to KCl. The present findings support the notion that luminal acidity is an important factor that disrupts barrier function in the oesophagus to allow the diffusion of noxious agents, such as bile acid, that alter the contractile status of the oesophagus.

Exploring Vitamin B12 Deficiency in Sleeve Gastrectomy from a Histological Study of a Cadaveric Stomach and Ileum

ABSTRACTIntroduction: Vitamin B12 deficiency is more commonly found among patients who have undergone Roux-en-Y gastric bypass (RYGB) as compared to those with post-sleeve gastrectomies (SG). The major difference between SG and RYGB is that the latter greatly bypasses the stomach whereas the former simply reduces the gastric volume. Purpose: The aim of this article was to study the stomach and the distal ileum histologically in a cadaver with SG to explain the higher rate of incidences of vitamin B12 deficiency seen in patients post-RYGB relative to patients post-SG. Since the stomach is the major variable in these two procedures, we hypothesize that it has the ability to regenerate and increase its surface area to compensate for the loss of its volume in SG patients. Material and Methods: Tissue biopsies and hematoxylin and eosin stains were performed from various anatomical locations of the GI tract, specifically the gastric fundus, body, and antrum, and from the distal ileum of the small intestine of a cadaver with SG and another without SG (control). Results: Compared with the control, the SG cadaver's gastric tissue biopsies were significant for chronic gastritis and hypertrophy of the muscularis externa layer. More importantly, parietal cell hyperplasia and deeper mucosal glands were also noted in the SG cadaver supporting the hypothesis. Conclusions: The compensatory role of an intact stomach, given its ability to regenerate parietal cells and increase its numbers in the gastric fundus and body, can be better appreciated in a gastric-sparing procedure such as SG versus RYGB in terms of limiting vitamin B12 deficiencies.

Exploring Vitamin B12 Deficiency in Sleeve Gastrectomy from a Histological Study of a Cadaveric Stomach and Ileum

Introduction: Vitamin B12 Deficiency is more commonly found amongst patients who have undergone Roux-en- Y Gastric Bypasses (RYGB) as compared to those post-Sleeve Gastrectomies (SG). The major difference between SG and RYGB is that the latter greatly bypasses the stomach, whereas the former simply reduces the gastric volume. Purpose: The aim of this paper is to study the stomach and distal ileum, histologically, in a cadaver with SG to explain the higher rate of incidences of B12 deficiency seen in patient’s post-RYGB relative to patient’s post-SG. Since the stomach is the major variable in the two procedures, we hypothesize that it has the ability to regenerate and increase its surface area to compensate for the loss of its volume in SG patients. Material and Methods: Tissue biopsies and Hematoxylin and Eosin stains were performed from various anatomical locations of the GI tract, specifically, the fundus, the body, and the antrum of the stomach and from the distal ileum of the small intestine of a cadaver with SG and one without (control). Results: Compared to the control, the SG cadaver's gastric tissue biopsies were significant for chronic gastritis and hypertrophy of the muscularis externa layer. More importantly, parietal cell hyperplasia and deeper mucosal glands were also noted in the SG cadaver supporting the hypothesis. Conclusion: The compensatory role of an intact stomach, given its ability to regenerate parietal cells and increase its number in the gastric fundus and body, can be better appreciated in a gastric-sparing procedure such as SG versus RYGB in terms of limiting B12 deficiencies.

Simultaneous follow-up of mouse colon lesions by colonoscopy and endoluminal ultrasound biomicroscopy

To evaluate the potential use of colonoscopy and endoluminal ultrasonic biomicroscopy (eUBM) to track the progression of mouse colonic lesions. Ten mice were treated with a single azoxymethane intraperitoneal injection (week 1) followed by seven days of a dextran sulfate sodium treatment in their drinking water (week 2) to induce inflammation-associated colon tumors. eUBM was performed simultaneously with colonoscopy at weeks 13, 17-20 and 21. A 3.6-F diameter 40 MHz mini-probe catheter was used for eUBM imaging. The ultrasound mini-probe catheter was inserted into the accessory channel of a pediatric flexible bronchofiberscope, allowing simultaneous acquisition of colonoscopic and eUBM images. During image acquisition, the mice were anesthetized with isoflurane and kept in a supine position over a stainless steel heated surgical waterbed at 37 °C. Both eUBM and colonoscopic images were captured and stored when a lesion was detected by colonoscopy or when the eUBM image revealed a modified colon wall anatomy. During the procedure, the colon was irrigated with water that was injected through a flush port on the mini-probe catheter and that acted as the ultrasound coupling medium between the transducer and the colon wall. Once the acquisition of the last eUBM/colonoscopy section for each animal was completed, the colons were fixed, paraffin-embedded, and stained with hematoxylin and eosin. Colon images acquired at the first time-point for each mouse were compared with subsequent eUBM/colonoscopic images of the same sites obtained in the following acquisitions to evaluate lesion progression. All 10 mice had eUBM and colonoscopic images acquired at week 13 (the first time-point). Two animals died immediately after the first imaging acquisition and, consequently, only 8 mice were subjected to the second eUBM/colonoscopy imaging acquisition (at the second time-point). Due to the advanced stage of colonic tumorigenesis, 5 animals died after the second time-point image acquisition, and thus, only three were subjected to the third eUBM/colonoscopy imaging acquisition (the third time-point). eUBM was able to detect the four layers in healthy segments of colon: the mucosa (the first hyperechoic layer moving away from the mini-probe axis), followed by the muscularis mucosae (hypoechoic), the submucosa (the second hyperechoic layer) and the muscularis externa (the second hypoechoic layer). Hypoechoic regions between the mucosa and the muscularis externa layers represented lymphoid infiltrates, as confirmed by the corresponding histological images. Pedunculated tumors were represented by hyperechoic masses in the mucosa layer. Among the lesions that decreased in size between the first and third time-points, one of the lesions changed from a mucosal hyperplasia with ulceration at the top to a mucosal hyperplasia with lymphoid infiltrate and, finally, to small signs of mucosal hyperplasia and lymphoid infiltrate. In this case, while lesion regression and modification were observable in the eUBM images, colonoscopy was only able to detect the lesion at the first and second time-points, without the capacity to demonstrate the presence of lymphoid infiltrate. Regarding the lesions that increased in size, one of them started as a small elevation in the mucosa layer and progressed to a pedunculated tumor. In this case, while eUBM imaging revealed the lesion at the first time-point, colonoscopy was only able to detect it at the second time-point. All colonic lesions (tumors, lymphoid infiltrate and mucosal thickening) were identified by eUBM, while colonoscopy identified just 76% of them. Colonoscopy identified all of the colonic tumors but failed to diagnose lymphoid infiltrates and increased mucosal thickness and failed to differentiate lymphoid infiltrates from small adenomas. During the observation period, most of the lesions (approximately 67%) increased in size, approximately 14% remained unchanged, and 19% regressed. Combining eUBM with colonoscopy improves the diagnosis and the follow-up of mouse colonic lesions, adding transmural assessment of the bowel wall.

Morphology of the developing muscularis externa in the mouse esophagus

Muscularis externa of mouse esophagus is composed of two skeletal muscle layers in the adult. But less attention is paid to the histogenesis of the muscularis externa of the esophagus, and controversies still exist about the developmental process and the spatio-temporal expression characteristics of muscle-specific proteins during the development of esophageal muscularis externa. To further probe into the developmental pattern of muscularis externa of the mouse esophagus and the expression characteristics of different muscle-specific proteins, immunohistochemical and terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP)-digoxigenin nick-end labeling apoptotic staining methods are used to investigate the expression patterns of different muscle-specific proteins and to elucidate the relationship of these protein expressions with the development of muscularis externa of the mouse esophagus. Thus, an understanding of the developing esophageal muscularis externa may be important for developing therapeutic strategies for the treatment of human esophagus diseases. Serial sections of mouse embryos from embryonic day (ED) 12 to ED18, and full-length esophagi from postnatal first to 5th day were stained with monoclonal antibodies against α-smooth muscle actin (α-SMA), α-sarcomerical actin (α-SCA), desmin, and monoclonal anti-skeletal myosin (MHC), while apoptosis was determined using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labeling assay. The expression of α-SMA was started at ED12. During the development of ED14-ED15, α-SMA positive cells were seen extending from the walls of left three, four, and six arch arteries toward the dorsal wall of esophagus. Stronger expression of α-SCA and desmin could be detected at ED14 and ED15, expression intensity in caudal segment and inner layer was stained stronger than that of cranial segment and outer layer, but after ED16, strong expression of α-SCA and desmin was found in the outer layer of muscularis externa. Expression of MHC was first detected in the outer layer of cranial segment of muscularis externa at ED17. At ED18, MHC had extended to the level of thyroid gland, staining intensity in the outer layer and cranial segment was stronger than that of inner layer and caudal segment. One to five days after birth, the thickness of the esophageal muscle layer was obviously increased. Most of the muscle cells in the cranial segment of esophagus showed strong expression of α-SCA and clear cross striations at higher magnification. With progression toward the caudal segment, expression intensity of α-SCA became weaker, but the expression intensity of desmin was the same at different levels of esophagus. The muscle fibers were arranged densely with high expression of MHC in the cranial segment. During the development of esophageal muscularis externa, few apoptotic cells were observed. α-SMA, α-SCA, desmin, and MHC show different expression patterns. The differentiation of outer layer of esophageal muscularis externa is quicker than that of inner layer, and the caudal segment is quicker than that of the cranial segment. Besides, apoptosis may not participate in the development of esophageal muscularis externa. The smooth muscle cells from arch arteries may participate in the development of esophageal muscularis externa.

Applicability of the Ussing Chamber Technique to Permeability Determinations in Functionally Distinct Regions of the Gastrointestinal Tract in the Rat

Background: Ussing chambers are commonly utilized for in vitro investigations into gastrointestinal permeability. However, their sensitivity and applicability to the small intestine have not been well characterized. Methods: In order to investigate the effects of experimentally induced damage and the relative contribution of the mucosa and muscularis externa layers to transmural permeability in the small intestine, stomach and colon, normal rat intestinal tissues were mounted in Ussing chambers with or without removal of the muscularis externa or mucosal layers. Gastric tissues were damaged in vivo by exposure to indomethacin (100 r mg r kg m 1 ), while ileal tissues were damaged in vitro by 0.4 r M NaCl. Tissue damage was assessed histologically, while permeability parameters included conductance (G), potential difference (PD) and mucosal to serosal flux of horseradish peroxidase (HRP). Results: Damage localized to the tissue edges (edge damage) accounted for 25%-50% of the exposed epithelial length in the ileum, while less than 20% of stomach and colon epithelium was affected by edge damage. In the damaged stomach, a 20% reduction in epithelialization was accompanied by increases in G ( P r < r 0.001) and HRP ( P r < r 0.01) flux. Removal of the muscularis externa did not affect mucosal viability in the undamaged ileum or colon although HRP flux in the colon, but not ileum, was increased ( P r < r 0.01). Removal of the ileal mucosa produced increases in G and HRP flux, while PD was maintained. Conclusion: We conclude that the Ussing chamber technique is suitable for application to studies of gastric and colonic permeability in rats. However, owing to the prevalence and extent of edge damage in the small intestine, we would caution against the use of this technique for permeability studies in this region of the gastrointestinal tract in the rat.

Total antioxidant capacity is reduced in Crohn’s disease

In previous work we identified depletion of the micronutrient glutathione in colon from patients with Crohn’s disease. Since there are several linked intestinal antioxidant systems, we hypothesized that there would be global alteration of the tissue content of antioxidants in Crohn’s disease. We therefore examined total antioxidant capacity and lipid hydroperoxide levels in colon from non-obstructed neoplasia and acquired megacolon as non-inflammatory controls and in colon from Crohn’s disease. Total antioxidant capacity in the mucosal-submucosal layer was decreased in Crohn’s disease (pANOVA = .02) and there was a strong trend toward decreased total antioxidant capacity in the muscularis externa layer from Crohn’s disease ( p = .06). There were trends toward increased lipid hydroperoxide levels in Crohn’s disease. This study supports the presence of a generalized consumption of tissue antioxidants in Crohn’s disease. The clinical response to nutritional therapy in patients with Crohn’s disease may be related to repletion of tissue antioxidants.

IL-12 gene transfer alters gut physiology and host immunity in nematode-infected mice.

Immune responses elicited by nematode parasite infections are characterized by T helper 2 (Th2) cell induction. The immunologic basis for changes in intestinal physiology accompanying nematode infection is poorly understood. This study examined whether worm expulsion and associated goblet cell hyperplasia and muscle contractility share a similar immune basis by shifting the response from Th2 to Th1 using interleukin-12 (IL-12) overexpression. We used a single administration of recombinant adenovirus vector expressing IL-12 (Ad5IL-12) in Trichinella spiralis-infected mice. Ad5IL-12 administered 1 day after infection prolonged worm survival and inhibited infection-induced muscle hypercontractility and goblet cell hyperplasia. This was correlated with upregulated interferon-gamma (IFN-gamma) expression and downregulated IL-13 expression in the muscularis externa layer. We also observed increased IFN-gamma production and decreased IL-4 and IL-13 production from in vitro stimulated spleen and mesenteric lymph node cells of infected Ad5IL-12-treated mice. These results indicate that transfer and overexpression of the IL-12 gene during Th2-based nematode infection shifts the immune response toward Th1 and delays worm expulsion. Moreover, the immune response shift abrogated the physiological responses to infection, attenuating both muscle hypercontractility and goblet cell hyperplasia. These findings strongly indicate that worm expulsion, muscle hypercontractility, and goblet cell hyperplasia share a common immunologic basis and may be causally linked.

Glutathione levels in chronic inflammatory disorders of the human colon

Glutathione depletion has been described in tissues obtained from several chronic diseases. Increased free radical production by inflammatory cells occurs in inflammatory bowel disease. We hypothesized that this could induce depletion of gut antioxidants. In this study, we examined the potential relationship between chronic inflammation and colonic glutathione levels. Using a validated assay, glutathione levels were determined in the mucosal-submucosal layer and the muscularis externa layer in surgical colonic specimens from 26 patients with ulcerative colitis, 14 patients with Crohn's colitis, and 10 patients who underwent partial colectomy for non-obstructive neoplasia. Inflammation was graded histologically. Glutathione levels were decreased in the muscularis externa and in the mucosal-submucosal layers from both ulcerative colitis and Crohn's colitis (both p<0.05). There were parallel declines of glutathione levels in the muscularis externa layer compared to the mucosal-submucosal layer from individual colonic specimens. In ulcerative colitis, glutathione levels were reduced in histologically active disease compared to inactive disease (in the mucosal-submucosal layers: Mean±SEM were 214±68 nmol/g wet tissue and 808±30, respectively; in the muscularis externa layers: 333±97 and 890±340; both p<0.05). In Crohn's colitis, there were no significant differences between histologically active and inactive disease (in the mucosal-submucosal layers: 114±53 and 461±206; in the muscularis externa layers: 105±59 and 553±211; both p>0.05). This study provides evidence that chronic inflammatory disorders of the colon are associated with glutathione depletion. In ulcerative colitis, there was a relationship between the severity of inflammation and glutathione depletion. By contrast, this relationship was not significant in Crohn's colitis. The results suggest that there could be a primary defect in glutathione production in Crohn's colitis, or a difference in the relative levels of free radical production by inflammatory cells present in these two disorders of colonic inflammation.

Immunocytes and abnormal gastrointestinal motor activity during ileitis in dogs.

Infiltration of specific immunocytes and stimulation of abnormal gastrointestinal motor activity during ileal inflammation induced by mucosal exposure to ethanol and acetic acid were investigated in 17 dogs. Ileal inflammation significantly increased the frequency of giant migrating contractions (GMCs) and decreased the frequency of migrating motor complexes (MMCs). The frequency of retrograde giant contractions (RGCs) increased only on the day of ethanol and acetic acid treatment. Diarrhea, urgency of defecation, and apparent abdominal discomfort were related to the increased frequency of GMCs. Ileal inflammation also prolonged the duration of postprandial MMC disruption. Histological and immunohistochemical findings indicated transmural inflammation with marked increase in polymorphonuclear cells in the lamina propria and muscularis externa layers. Myeloperoxidase activity increased severalfold in both layers. Cells containing interleukin-2 receptor (IL-2R) increased in the lamina propria. Other immunocytes, such as B and T lymphocytes, dendritic cells, and human leukocyte antigen DR-1 (HLADR)-positive cells, did not exhibit a significant increase in the inflamed ileum compared with the normal proximal jejunum. We conclude that stimulation of GMCs may be the major motility marker of intestinal inflammation.

Fractionated irradiation alters enteric neuroendocrine products.

Radiation profoundly alters the contractile activity of the small intestine and colon. We hypothesized that some motor changes of the gut might be secondary to impaired neural input to smooth muscle or abnormal release of gut endocrine peptides. The density of products within peptidergic and cholinergic nerves and gut endocrine cells was estimated in six normal controls and six dogs who had received 1500 cGy in six equal fractions of 250 cGy. Choline acetyltransferase, acetylcholinesterase, vasoactive intestinal peptide (VIP), substance P, peptide YY (PYY), and motilin were measured in tissue specimens divided into mucosal-submucosal (MS) and muscularis externa (ME) layers. Tissue samples were obtained from the duodenum, jejunum, ileum, and proximal and distal colon. In addition, serum levels of motilin and PYY were determined before and during the administration of 1500 cGy in four separate dogs instrumented to record upper gut contractile activity. Intrinsic cholinergic activity as estimated by choline acetyltransferase activity was unchanged, while acetylcholinesterase activity increased in the MS layers of distal small bowel and colon. VIP was increased in the MS layers of jejunum and proximal colon as well as in the ME layers the jejunum and ileum. By contrast, substance P increased in the jejunal and proximal colonic MS layers and in the ME layers of the jejunum and ileum. Duodenal and jejunal motilin levels markedly decreased after radiation exposure, while serum motilin levels continued to cycle at a decreased peak level with migrating motor complexes. Colonic PYY remained unchanged but serum PYY levels decreased after irradiation. Increased neuronal synthesis and inhibition of neurotransmitter release are potential explanations for elevated tissue concentrations of VIP, substance P, and acetylcholinesterase. There appeared to be differences in the sensitivity of gut endocrine cells to irradiation. Changes in gut regulatory peptides and cholinergic enzyme activity occur with fractionated doses of abdominal irradiation, while the same schedule of irradiation produces striking changes in the canine small intestinal and colonic motor activity. It is therefore likely that alterations of contractile events may be produced by changes in gut neuroendocrine products.

Quantitative and qualitative analysis of the extracellular matrix protein, laminin, in Hirschsprung's disease

Previous immunohistochemical studies have shown an abnormal distribution of extracellular matrix (ECM) proteins, including laminin, in the smooth muscle layer of muscularis externa in Hirschsprung's disease (HD) bowel. These findings supported the hypothesis that an abnormal ECM microenvironment may be responsible for the failure of migration and/or development of the neural crest cells in the gut in HD. In order to determine the cause of the abnormality in laminin distribution, solid-phase enzyme-linked immunosorbent assays and immunoblots were used to quantitate the ECM protein laminin and characterize its subunits, respectively, in extracts of the dissected smooth muscle layer of the muscularis externa. In the aganglionic bowel, laminin (median concentration, 32.4 ng/mg of tissue) was found to be present in significantly greater quantity than in both the normoganglionic bowel of the same specimen (median, 17.2 ng/mg, P ≤ .05) and the normal bowel of age-matched controls (median, 9.7 ng/mg, P ≤ .05). Laminin concentration was also found to be significantly higher in normoganglionic HD bowel (median, 17.2 ng/mg) than in age-matched control specimens (median, 10.8 ng/mg, P ≤ .05). No difference was observed in the subunit composition of laminin in HD and control extracts analysed by immunoblot after polyacrylamide gel electrophoresis. This study demonstrates a quantitative abnormality of laminin in the bowel in HD, supporting the hypothesis that “abnormal microenvironment” may have a role in the pathogenesis of HD.

Decreased colonic peptide histidine-methionine in idiopathic inflammatory bowel diseases.

The sequence for peptide histidine-methionine is present within the same preprohormone as vasoactive intestinal polypeptide. Since our previous study using radioimmunoassay had demonstrated significantly decreased colonic concentrations of vasoactive intestinal polypeptide in ulcerative colitis and Crohn's colitis compared to normal colon, we determined the distribution and quantitation of peptide histidine-methionine. Fresh surgical specimens were dissected into mucosal-submucosal and muscularis externa layers prior to acid extraction and specific radioimmunoassay. One immunoreactive species that appeared to coelute with peptide histidine-methionine was separated by reverse-phase high-performance liquid chromatography. Mucosal-submucosal concentrations of peptide histidine-methionine were significantly decreased in ulcerative colitis and Crohn's colitis, compared to those in normal colon. In normal ileum and colon, linear correlation analysis showed no relationship between patient age and tissue concentrations of peptide histidine-methionine. However, a parallel decrease in molar concentrations of peptide histidine-methionine and vasoactive intestinal polypeptide in ulcerative colitis and Crohn's colitis was demonstrated by linear correlation analysis. These results are consistent with the hypothesis that peptide histidine-methionine and vasoactive intestinal polypeptide are colocalized within the same neural structures that have been altered in the idiopathic inflammatory bowel diseases.