Increased serotonin transporter immunoreactivity intensity in the ileum of patients with irritable bowel disease.

Irritable bowel syndrome (IBS) is a common chronic gastrointestinal disorder, which represents an economic burden to society and considerably reduces the quality of life of patients. In a previous study, the density of serotonin cells in the ileum of IBS patients was lower compared with control subjects. The present study aimed to further investigate the immunoreactivity intensity of serotonin and serotonin­selective reuptake transporter (SERT) in the ileum of IBS patients. A total of 98 patients (77 females and 21 males; mean age, 35 years; range, 18­66 years), which fulfilled Rome III Criteria for IBS, were included in the study. This cohort included 35 patients with diarrhoea­predominant (IBS­D), 31 patients with mixed diarrhoea and constipation (M­IBS) and 32 patients with constipation­predominant (IBS­C) symptoms. A total of 27 subjects were included as controls (16 females and 11 males; mean age, 52 years; range, 20­69 years). Ileal biopsy specimens were immunostained using the avidin­biotin (ABC) complex method for serotonin and SERT. The immunoreactivity intensity was quantified by computerised image analysis using Olympus cellSens imaging software. No statistical difference of serotonin immunoreactivity intensity was identified in multiple comparisons between controls, IBS­total, IBS­D, IBS­M and IBS­C. The SERT immunoreactivity intensity was significantly increased in IBS patients as compared with controls, regardless of the subtype. It was concluded that the increase in ileal epithelial content of SERT increases the intracellular uptake of serotonin and its degradation in the gut epithelial cells and consequently decreases the availability of serotonin within the gut mucosa. The low availability of serotonin at its receptors occurred in all IBS subtypes. This may indicate that this abnormality is associated with a common symptom in all IBS subtypes, namely abdominal pain/discomfort. Serotonin acts upon sensory neurons in the submucosal and myenteric ganglia, as well as in the spinal cord, which is in agreement with this hypothesis.

Serotonin and serotonin transporter in the rectum of patients with irritable bowel disease.

Irritable bowel syndrome (IBS) is a common chronic gastrointestinal disorder, which considerably reduces the quality of life of patients and represents an economic burden to society. In previous studies, the density of serotonin­expressing cells in the rectum of IBS patients did not differ from that of control subjects. The present study was undertaken to investigate the immunoreactivity intensity of serotonin and serotonin­selective reuptake transporter (SERT) in the rectum of IBS patients. A cohort of 50 patients with IBS (41 females and 9 males) were included in the study. Thirty patients had diarrhoea (IBS­D) and 20 had constipation (IBS­C) as the predominant symptom. Twenty­seven subjects were included as controls (19 females and 8 males). Rectal biopsy specimens were immunostained using the avidin­biotin complex method for serotonin and SERT. The immunoreactivity intensity was quantified by computerised image analysis using Olympus cell Sens imaging software. There was no statistical difference of serotonin immunoreactivity intensity in multiple comparisons between controls, IBS­total, IBS­D and IBS­C. Dunn's post test did not reveal any statistical differences among the four groups. There was a significant statistical difference in multiple comparisons between controls, IBS­total, IBS­D and IBS­C regarding the SERT immunoreactivity intensity. SERT immunoreactivity intensity of IBS­total, IBS­D and IBS­C differed significantly from that of controls. It was concluded that the reduced rectal SERT in the IBS patients could be one of the factors contribu-ting to the development of both diarrhoea and constipation in these patients, and that the increasing body of evidence of a genetic abnormality involving SERT underlines the importance of the role of SERT in the pathophysiology of IBS.

Evaluation of the usefulness of colonoscopy with mucosal biopsies in the follow-up of TNBS-induced colitis in rats.

Animal models are required for research regarding the pathogenesis and efficacy of anti-inflammatory agents in inflammatory bowel disease (IBD). Trinitrobenzene sulfonic acid (TNBS)-induced colitis closely mimics Crohn's disease. The present study was undertaken in order to determine the reliability of following the inflammatory course of TNBS-induced colitis using colonoscopy together with biopsy samples obtained during the examination. In this study we used 20 adult male Wistar rats, with a mean weight of 201.9 g. The rats were divided into two groups, control and TNBS, with ten rats in each group. Following the induction of TNBS colitis, the rats underwent colonoscopy with mucosal biopsies. At the end of the experiment, the rats were sacrificed and whole-wall colonic samples were obtained. The degree of inflammation was assessed endoscopically, macroscopically and microscopically. There was no significant change in the body weight of the control group but significant weight loss was observed in the TNBS group. Examination of the control group did not reveal any inflammation. Severe colitis was observed in the TNBS-induced colitis rats, as assessed endoscopically, macroscopically and microscopically. The endoscopic inflammation score obtained through colonoscopy examinations correlated with that obtained macroscopically, and those obtained microscopically from the whole-wall colon and biopsy samples collected during the colonoscopy. Moreover, the inflammation scores obtained from the whole-wall colon and biopsy samples collected during colonoscopy correlated markedly. In conclusion, colonoscopy is a reliable method for following up the course of inflammation in experimentally induced colitis. Although biopsy samples collected during colonoscopies may be used to assess the degree of inflammation, whole-wall samples are superior in this regard.

Colonoscopy with mucosal biopsies in young rats: a model for experimental gastroenterology.

Endoscopy is an important tool in the diagnosis, follow-up and management of several gastrointestinal diseases. In experimental studies, the course and progression of gastrointestinal lesions are followed by sacrificing animals at specific time intervals and the disease is usually assessed postmortem. This approach has several disadvantages, including the requirement of a large number of experimental animals and the inability to evaluate and follow the progression of the same lesion. The present study tested the feasibility of performing a total colonoscopy with mucosal biopsies in small, young rats. Eighteen adult male Wistar rats with an average bodyweight of 194 g (range, 166-232 g) were used. Three bowel preparation regimes with different fasting times and doses of sodium-picosulfate and magnesium citrate were examined. A video gastroscope with a 4.9 mm outer diameter, 210/120˚ up/down tip deflection, 103 cm working length, 140˚ view field and a 2 mm working channel (GIF-N180, Olympus, Tokyo, Japan) was used. The rats were anesthetized by inhalation of isoflurane prior to and during colonoscopy. Biopsies were obtained using biopsy forceps through the endoscope working channel. The animals were allowed to recover and were monitored for ~1 h, following the procedure. Sections from the biopsy samples taken during colonoscopy and colon tissue obtained during postmortem laparotomy were stained with hematoxylin and eosin. The optimum results were obtained with the bowel preparation that involved rats fasting for 24 h and the administration of 1 and 2 ml of sodium-picosulfate/magnesium citrate at 24 and 12 h prior to colonoscopy, respectively. Colonoscopy was performed with minor complications, with the only difficulty arising when maneuvering the endoscope past the hepatic flexure. This was achieved by manual rotation of the endoscope shift to the right and left of a two-way endoscope. Taking biopsies with the forceps at full jaw length and swing resulted in perforation, which may be avoided by obtaining biopsies with the outer tip of the biopsy forceps.

Reduced chromogranin A cell density in the ileum of patients with irritable bowel syndrome.

Irritable bowel syndrome (IBS) is a common disorder that considerably reduces the quality of life and productivity of patients. Chromogranin A (CgA) is a common marker for endocrine cells. CgA cell density has been reported to be reduced in the duodenum and colon of IBS patients. This study was undertaken to investigate CgA cell density in the ileum of these patients. The study involved 98 patients with IBS, according to the Rome III Criteria (77 females and 21 males, with an average age of 35 years). In total, 35 patients had diarrhoea-predominant symptoms (IBS-D), 32 had constipation-predominant symptoms (IBS-C), and 31 had a mixture of both diarrhoea and constipation (IBS-M). In this study, 27 subjects were used as controls (16 females and 11 males, with an average age of 52 years). Colonoscopies were performed on the patients and controls and biopsies were obtained from the ileum. Sections were immunostained with the avidin-biotin complex (ABC) for CgA and quantified using computerized image analysis. The CgA density in the controls was 63.2±4.4 (mean ± SEM), for all IBS patients it was 28.6±2.1, for IBS-D it was 28.8±3.4, for IBS-M it was 26.5±3.9 and for IBS-C it was 30.3±3.7. There was a statistically significant difference between the controls and all IBS patients (IBS-D, IBS-M and IBS-C; P<0.0001 for all). The present study showed that CgA cell density in the ileum of IBS patients was reduced, regardless of subtype. Thus, it appears that there is endocrine cell depletion in both the small and large intestine of IBS patients, whereas IBS is normally considered to be a functional condition without any detectable abnormalities. The present finding lends support to the suggestion that IBS is caused by a biological abnormality, and intestinal CgA cell density may be used as a biological marker for the diagnosis of IBS.

Automated tracking of nanoparticle-labeled melanoma cells improves the predictive power of a brain metastasis model.

Biologic and therapeutic advances in melanoma brain metastasis are hampered by the paucity of reproducible and predictive animal models. In this work, we developed a robust model of brain metastasis that empowers quantitative tracking of cellular dissemination and tumor progression. Human melanoma cells labeled with superparamagnetic iron oxide nanoparticles (SPION) were injected into the left cardiac ventricle of mice and visualized by MRI. We showed that SPION exposure did not affect viability, growth, or migration in multiple cell lines across several in vitro assays. Moreover, labeling did not impose changes in cell-cycle distribution or apoptosis. In vivo, several SPION-positive cell lines displayed similar cerebral imaging and histologic features. MRI-based automated quantification of labeled cells in the brain showed a sigmoid association between metastasis frequency and doses of inoculated cells. Validation of this fully automated quantification showed a strong correlation with manual signal registration (r(2) = 0.921, P < 0.001) and incidence of brain metastases (r(2) = 0.708, P < 0.001). Metastasis formation resembled the pattern seen in humans and was unaffected by SPION labeling (histology; tumor count, P = 0.686; survival, P = 0.547). In summary, we present here a highly reproducible animal model that can improve the predictive value of mechanistic and therapeutic studies of melanoma brain metastasis.