Intrinsic cholinergic innervation in the human sigmoid colon revealed using CLARITY, three-dimensional (3D) imaging, and a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum.

BACKGROUND: We previously reported the specificity of a novel anti-human peripheral choline acetyltransferase (hpChAT) antiserum for immunostaining of cholinergic neuronal cell bodies and fibers in the human colon. In this study, we investigate 3D architecture of intrinsic cholinergic innervation in the human sigmoid colon and the relationship with nitrergic neurons in the enteric plexus. METHODS: We developed a modified CLARITY tissue technique applicable for clearing human sigmoid colon specimens and immunostaining with hpChAT antiserum and co-labeling with neuronal nitric oxide synthase (nNOS) antibody. The Z-stack confocal images were processed for 3D reconstruction/segmentation/digital tracing and computational quantitation by Imaris 9.2 and 9.5. KEY RESULTS: In the mucosa, a local micro-neuronal network formed of hpChAT-ir fibers and a few neuronal cell bodies were digitally assembled. Three layers of submucosal plexuses were displayed in 3D structure that were interconnected by hpChAT-ir fiber bundles and hpChAT-ir neurons were rarely co-labeled by nNOS. In the myenteric plexus, 30.1% of hpChAT-ir somas including Dogiel type I and II were co-labeled by nNOS and 3 classes of hpChAT-ir nerve fiber strands were visualized in 3D images and videos. The density and intensity values of hpChAT-ir fibers in 3D structure were significantly higher in the circular than in the longitudinal layer. CONCLUSIONS AND INFERENCES: The intrinsic cholinergic innervation in the human sigmoid colon was demonstrated layer by layer for the first time in 3D microstructures. This may open a new venue to assess the structure-function relationships and pathological alterations in colonic diseases.

Is intestinal oxidative stress involved in patients with compensated liver cirrhosis?

BACKGROUND: Liver cirrhosis is associated with intestinal epithelial barrier dysfunction, which may be affected by oxidative stress. Studies in cirrhotic rats provided evidence for intestinal oxidative stress, but studies in cirrhotic patients are scarce. We have shown intestinal barrier dysfunction in patients with compensated cirrhosis. AIM: The present study aimed to investigate whether oxidative stress occurs in the intestinal mucosa of compensated cirrhotic patients and may contribute to barrier dysfunction. MATERIAL AND METHODS: Oxidative stress was studied in duodenal and sigmoid biopsies from 15 cirrhotic patients and 22 controls by analyzing transcription of genes involved in glutathione and uric acid metabolism using quantitative real-time polymerase chain reaction. Protein levels of glutathione and glutathione disulphide were measured and the glutathione/glutathione disulphide ratio was calculated as marker of oxidative stress. In addition, intestinal myeloperoxidase and fecal calprotectin were determined. RESULTS: Gene transcription of glutathione synthetase and glutathione reductase were significantly different in duodenal and sigmoid biopsies of cirrhotic patients vs. controls, but no alterations were found for other genes nor for glutathione, glutathione disulphide, glutathione/glutathione disulphide ratio and intestinal myeloperoxidase and fecal calprotectin concentrations. CONCLUSION: This study did not find indications for oxidative stress and low-grade inflammation in the small and large intestine of stable compensated cirrhotic patients. Although these preliminary findings need further validation, we found intestinal oxidative stress not to be a major mechanism contributing to epithelial barrier dysfunction in patients with compensated cirrhosis.

Surgical diverticulitis is not associated with defects in the expression of wound healing genes.

PURPOSE: The development of diverticuli may represent defects in collagen vascular tissue integrity possibly from a genetic predisposition. We evaluated the tissue expression of wound healing genes in sigmoid tissue from youthful patients undergoing surgery for diverticulitis and thus would more likely suffer from a genetic predisposition (SD mean age 39 ± 0.9) versus controls in the form of patients over the age of 50 (mean age 52.9 ± 10.5 years) without evidence of diverticular disease. METHODS: The mRNA expression of 84 genes associated with the extracellular matrix, cellular adhesion, growth factors, inflammatory cytokines, and signal transduction was evaluated in 16 SD and 15 control tissues using a Qiagen Wound Healing Array. Vitronectin, the gene protein with the highest potential significance on raw analysis, was further investigated using a Taqman assay with an additional 11 SD (total n = 27) and four control (total n = 19) samples. Statistics were by Student's t and Mann-Whitney tests with Bonferroni correction. RESULTS: No significant differences in mRNA expression between the SD and control tissue in the 84 measured genes were demonstrated after correction. Vitronectin mRNA expression was downregulated 2.7-fold in SD tissue vs. tissue from non-neoplastic control patients (p = 0.001 raw/0.08 corrected). However, on vitronectin TaqMan analysis, no difference in expression was seen in SD vs. all controls or in all subset comparisons. CONCLUSIONS: The lack of significant alteration in mRNA expression of traditionally associated wound healing genes/proteins in young SD patients suggests that such genes play a minor role in the genetic predisposition to youthful diverticulitis.

Comparison of electroacupuncture and moxibustion on brain-gut function in patients with diarrhea-predominant irritable bowel syndrome: A randomized controlled trial.

OBJECTIVE: To compare the effects of electroacupuncture (EA) and moxibustion therapies on patients with diarrhea-predominant irritable bowel syndrome (D-IBS). METHODS: A total of 60 D-IBS patients were randomly allocated to the EA group (30 cases) and moxibustion group (30 cases). Before and after treatment, the gastrointestinal symptoms and psychological symptoms were scored by Visual Analogue Scale, Bristol Stool Form Scale, Hamilton Anxiety Rating Scale (HAMA), and Hamilton Depression Rating Scale (HAMD); the expressions of 5-hydroxytryptamine (5-HT), 5-HT3 receptor (5-HT3R), and 5-HT4 receptor (5-HT4R) in the sigmoid mucosal tissue were measured by immunohistochemical staining. Additionally, the effects on the functional brain areas of the anterior cingulate cortex (ACC), insular cortex (IC) and prefrontal cortex (PFC) were observed by functional magnetic resonance imaging. RESULTS: Compared with before treatment, both EA and moxibustion groups reported significant improvements in abdominal pain and abdominal bloating after treatment (P<0.01 or P<0.05). The moxibustion group reported greater improvements in defecation emergency, defecation frequency, and stool feature than the EA group (P<0.01). Both HAMA and HAMD scores were significantly decreased in the moxibustion group than in the EA group (P<0.01). Both groups demonstrated significantly reduced expressions of 5-HT, 5-HT3R and 5-HT4R in the colonic mucosa after treatment (P<0.01), with a greater reduction of 5-HT in the moxibustion group (P<0.05). Finally, decreased activated voxel values were observed in the left IC, right IC and PFC brain regions of patients in the moxibustion group under stimulation with 150 mL colorectal distension after treatment (P<0.05 or P<0.01), while in the EA group only PFC area demonstrated a reduction (P<0.05). CONCLUSION: Moxibustion can significantly improve the symptoms of D-IBS, suggesting that moxibustion may be a more effective therapy than EA for D-IBS patients.

Elastofibromatous change of the intestine: report of four lesions from three patients with review of the literature.

Elastofibromatous change, also referred to as elastofibromatous polyp or elastofibroma, has been extremely rarely described in the gastrointestinal tract. This lesion is characterized histopathologically by an excessive accumulation of elastic fibers occasionally with a fibrous component involving the submucosa and/or muscularis mucosae of the gastrointestinal tract. Herein, we report four additional lesions of the intestine and review the clinicopathological features of this rare lesion. Three patients (76-, 72-, and 52-year-old males) were detected with polypoid lesions in the jejunum, transverse and sigmoid colons, and sigmoid colon, respectively. All four lesions showed fundamentally the same histopathological and immunohistochemical features. The polypoid lesions were covered by non-neoplastic epithelium, and degenerated and truncated elastic fibers occasionally with a fibrous component had accumulated in the submucosa and/or muscularis mucosae. The characteristic feature was the elastofibromatous change centered around collections of elastotic submucosal vessels. Desmin-positive degenerative ruptured smooth muscle fibers were scattered within the elastic fibers in the submucosa. Our analyses of the clinicopathological features of the previously reported 32 cases of elastofibromatous change of the gastrointestinal tract as well as the present cases demonstrated that this type of lesion is most commonly found in the colon or rectum (29 cases), males, and middle-aged to elderly persons. Although the pathogenesis remains unclear, the convincing hypothesis that this lesion represents elastic degeneration of submucosal vessels by previous persistent vascular injury has been proposed. The collections of degenerative elastotic vascular walls may have an important role in the development of this lesion.

Palmitoylethanolamide improves colon inflammation through an enteric glia/toll like receptor 4-dependent PPAR-α activation.

OBJECTIVE: Enteric glia activation has been reported to amplify intestinal inflammation via the enteroglial-specific S100B protein. This neurotrophin promotes macrophage recruitment in the mucosa, amplify colonic inflammation and interacts with toll-like receptors (TLR). Molecules inhibiting S100B-driven enteric activation might mitigate the course of ulcerative colitis (UC). This study aims to investigate the effects of palmitoylethanolammide (PEA), a drug able to counteract astroglial activation in the central nervous system, on intestinal inflammation, in humans and mice. DESIGN: Mouse models of dextran sodium sulphate (DSS)-induced colitis, colonic biopsies deriving from UC patients and primary cultures of mouse and human enteric glial cells (EGC), have been used to assess the effects of PEA, alone or in the presence of specific PPARα or PPARγ antagonists, on: macroscopic signs of UC (DAI score, colon length, spleen weight, macrophages/neutrophils infiltration); the expression and release of proinflammatory markers typical of UC; TLR pathway in EGCs. RESULTS: PEA treatment improves all macroscopic signs of UC and decreases the expression and release of all the proinflammatory markers tested. PEA anti-inflammatory effects are mediated by the selective targeting of the S100B/TLR4 axis on ECG, causing a downstream inhibition of nuclear factor kappa B (NF-kB)-dependent inflammation. Antagonists at PPARα, but not PPARγ, abolished PEA effects, in mice and in humans. CONCLUSIONS: Because of its lack of toxicity, its ability in reducing inflammation and its selective PPARα action, PEA might be an innovative molecule to broaden pharmacological strategies against UC.

Evaluation of WGA and Concanavalin A (Con A) lectin as biomarkers of hepatosplenic schistosomiasis in human biopsies with no evidence of egg-granuloma system.

INTRODUCTION: Colonic lesions are predominant in patients with schistosomiasis. However, carbohydrate alterations in colonic schistosomiasis remain unclear. Lectin-ligands allow us to identify changes in the saccharide patterns of cells. METHODS: Biopsies of descending and rectosigmoid colon of patients were submitted to WGA and Con A lectin histochemistry. RESULTS: WGA stained stroma and gland cells of descending colon and rectosigmoid tissues in a granular strong cytoplasmatic pattern in schistosomiasis specimens differing from normal control and Con A failing to recognize all samples analyzed. CONCLUSIONS: WGA ligands are expressed differently in patients with hepatosplenic schistosomiasis and no evidence of egg-granuloma system.

A comparative study of extra- and intraluminal sigmoid colonic tonometry to detect colonic hypoperfusion after operation for abdominal aortic aneurysm.

OBJECTIVES: There is no ideal method to monitor colonic perfusion after abdominal aortic aneurysm (AAA) repair. The aim was to evaluate extraluminal sigmoid colon tonometry, comparing with the established intraluminal method. METHODS: Eighteen patients were monitored with both methods, 10 after elective and eight after ruptured AAA repair. One tonometric catheter was placed inside the sigmoid colon (intraluminal) and another extraluminally in close contact with the serosa of the sigmoid colon (extraluminal). Intra- and extraluminal partial pressure of carbon dioxide (pCO(2)) were measured every 10 min during 48 h postoperatively, 1536 simultaneous measurements. Intraluminal pH (pHi) and extraluminal pH (pHe) were calculated, and intra-abdominal pressure (IAP) was measured, every 4 h. Colonic ischaemia was defined as pHi ≤ 7.1. RESULTS: Mean pHi was 7.18 ± 0.11 and mean pHe was 7.28 ± 0.09. With a pHe cut-off value of ≤ 7.2, the sensitivity and specificity to detect colonic ischaemia were 95% and 95%, respectively. Accuracy was 95% and the positive and negative predictive values 0.80 and 0.99, respectively. The positive likelihood ratio was 19 and the negative likelihood ratio 0.05. CONCLUSION: Extraluminal tonometry may serve as a screening test: A pHe-value < 7.2 indicates suspected colonic ischaemia, meriting further investigation. It was not able to evaluate the severity of ischaemia.

Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine.

BACKGROUND: Myenteric ganglia are key-structures for the control of intestinal motility and their mRNA expression profiles might be altered under pathological conditions. A drawback of conventional RT-PCR from full-thickness specimens is that gene expression analysis is based on heterogeneously composed tissues. To overcome this problem, laser microdissection combined with real-time RT-PCR can be used to detect and quantify low levels of gene expression in isolated enteric ganglia. METHODS: Fresh unfixed full-thickness specimens of sigmoid colon were obtained from patients (n = 8) with diseases unrelated to intestinal motility disorders. 10 microm cryo-sections were mounted on membrane-coated slides and ultra-rapidly stained with toluidine blue. Myenteric ganglia were isolated by laser microdissection and catapulting for mRNA isolation. Real-time RT-PCR was performed for selected growth factors, neurotransmitter receptors and specific cell type markers. KEY RESULTS: Collection of 0.5 mm(2) of ganglionic tissue was sufficient to obtain positive RT-PCR results. Collection of 4 mm(2) resulted in ct-values allowing a reliable quantitative comparison of gene expression levels. mRNA analysis revealed that neurotrophic growth factor, neurotrophin-3, serotonin receptor 3A, PGP 9.5 and S100 beta are specifically expressed in myenteric ganglia of the human colon. CONCLUSIONS & INFERENCES: Laser microdissection combined with real-time RT-PCR is a novel technique to reliably detect and quantify site-specific expression of low-abundance mRNAs (e.g. growth factors, neurotransmitter receptors) related to the human enteric nervous system. This technical approach expands the spectrum of available tools to characterize enteric neuropathologies underlying human gastrointestinal motility disorders at the molecular biological level.

Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis.

Decreased sodium (Na(+)), chloride (Cl(-)), and water absorption, and increased potassium (K(+)) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na(+) and Cl(-) absorption are becoming clearer, but the mechanism of increased K(+) secretion is unknown. Human colon is normally a K(+) secretory epithelium, making it likely that K(+) channels are expressed in the luminal (apical) membrane. Based on the assumption that these K(+) channels resembled the high conductance luminal K(+) (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel alpha-subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel alpha-subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel alpha-subunit protein and mRNA expression (evaluated by western blotting and real-time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible beta-subunits (beta(1-4)), the beta(1)- and beta(3)-subunits were dominant. Voltage-dependent, barium-inhibitable, luminal K(+) channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K(+) losses in ulcerative colitis, and possibly other diseases associated with altered colonic K(+) transport, may reflect wider expression of luminal BK channels along the crypt axis.

Endogenous gamma-hydroxybutyric acid is in the rat, mouse and human gastrointestinal tract.

By using Gas Chromatography-Mass Spectrometry high concentrations of endogenous gamma-hydroxybutyric acid (GHB) have been demonstrated in the rat and mouse gastrointestinal tract, including stomach, small intestine and colon-rectum. GHB concentrations were many folds higher than those present in the brain. High GHB concentrations have been also found in the human operatory specimen of sigmoid colon. Since GHB administration has been found to modify gastrointestinal motility via GABA(B) receptors, the present results suggest that endogenous GHB might be involved in the GABA(B) receptor-mediated control of gastrointestinal function.

Disparate surgical margin lengths of colorectal resection specimens between in vivo and in vitro measurements. The effects of surgical resection and formalin fixation on organ shrinkage.

We noticed almost routine disparate results in margin lengths when colorectal specimens are measured in vivo by the surgeon and in vitro by the pathologist. We studied 26 sigmoid and rectum specimens to document the amount of organ shrinkage after surgical removal and fixation. Each specimen had a 5.0-cm segment at each end of the specimen marked by serosal sutures before vascular devitalization. The segments were measured after the specimen sat unfixed for 10 to 20 minutes and after 12 to 18 hours of formalin fixation. The segments shrank to a median length of 3.0 cm (40% of the in vivo length) after 10 to 20 minutes and an additional 0.85 cm, to a median length of 2.15 cm, after fixation. Overall after fixation, the segments shrank 57% of the in vivo length. Approximately 70% of the shrinkage occurred during the first 10 to 20 minutes after removal, and the remaining 30% occurred after fixation. For optimal accuracy, margin distance must be obtained immediately after surgical removal. Once the specimen has been removed for several minutes, the difference between unfixed and fixed margin lengths is 30%. A correction factor of approximately 2x should be applied when interpreting the margin length.

Two-dimensional electrophoretic study of normal colon mucosa and colorectal cancer.

Two-dimensional gel electrophoresis has been used to analyze polypeptide composition of specimens of human normal colon mucosa and colorectal carcinomas. Immobilized pH gradient in the first dimension improved the separation and enabled us to analyze proteins from whole tissue samples with high reproducibility. Polypeptides were separated within sigmoidal 3.5-10 pH gradient and range of mol. weight 15-200 KD. In global, protein patterns of normal and malignant tissue seemed to be close to each other. We attempted to combine two-dimensional electrophoretic separation with the immunoblotting technique using patient sera to identify possible tumor antigens. From a wealth of spots on silver stained protein maps of adjacent normal colon mucosa or tumor tissue, only few spots gave positive reaction. These spots might be classified into two groups: 1) common for normal colon mucosa and tumor, 2) specific for tumor tissue. The extent of patient's antibody reaction with antigens appeared to correspond to urinary neopterin level, an index of immune activation.

Ornithine decarboxylase and polyamines in colorectal neoplasia and mucosa.

Ornithine decarboxylase (ODC) and polyamines are intimately involved in normal cellular proliferation and are likely to play a role in carcinogenesis. ODC activity and polyamine content were measured in tissue samples obtained during colonoscopy from 48 benign neoplastic polyps (20 tubular adenomas; 28 villous adenomas), 18 cancers (including 5 malignant polyps), and adjacent mucosa. ODC activity in polyp and cancer tissue specimens was higher than in adjacent mucosa in 75 and 83% of pairs, respectively. Similarly, putrescine, spermidine, and spermine contents were higher in the majority of polyps and cancers compared to adjacent mucosa. ODC activity and polyamine content in colonic mucosa from 10 patients without a history of colorectal neoplasia were not different from adjacent mucosal values in the patients with neoplasia. In conclusion, ODC and polyamines are elevated in the majority of colorectal neoplasms, but amounts in normal mucosa do not differentiate between patients with cancer, benign neoplastic polyps, and normal subjects.

A technique for segmental rectal and colonic perfusion in humans.

To enable a better characterization of pathophysiologic processes in colon and rectum, we have developed a perfusion technique for collection of soluble substances and cells from standardized intestinal segments. A tube with balloons attached to its outer wall was endoscopically introduced into the rectum and sigmoid colon, and its position ascertained fluoroscopically. The balloons delimited two segments, one in the sigmoid colon and one in the rectum. The segments were simultaneously perfused by a buffer at 37 degrees C. After a 30-min rinsing period, perfusate and cells were collected at 20-min intervals. Of 51 attempted perfusions, 45 were successfully completed. Recovered volumes equaled those infused. Leakage from the proximal intestine to the segments was negligible. In 18 healthy volunteers, the mean perfusate concentration from the rectal segment was 57.5 (27.5-120.2) mg/L for albumin, 1.3 (1.0-1.7) micrograms/L for eosinophil cationic protein, 5.1 (2.8-9.5) ng/L for prostaglandin E2, and 61.7 (41.7-89.1) micrograms/L for hyaluronan, and all values were lower in the sigmoid segment. Steady state conditions were achieved from the second perfusion period. The perfusate contained 4-80 x 10(4) cells, more than 95% of which were epithelial cells. The technique is safe, has a good subject compliance, and seems to be a valuable tool in investigations on quantitative release of soluble substances and cells in, e.g., colorectal inflammation.

Spinal and intestinal levels of substance P, calcitonin gene-related peptide and vasoactive intestinal polypeptide following perendoscopic injection of formalin in rat colonic wall.

Acute inflammation of the colonic wall was induced by perendoscopic injection of formalin in rats. As compared to control animals (no endoscopy, no injection), the procedure was followed by a marked reduction of immunoreactive substance P, calcitonin gene-related peptide and vasoactive intestinal peptide concentrations in rectosigmoid wall. Tissue substance P concentration in the spinal cord, at the level of afferent projection, increased at the same time. The three peptides tested are thus likely to be involved in the pathophysiology of acute intestinal inflammation. In addition, substance P may play a role in the transmission of nociceptive signals from the inflamed colonic segment.

Tissue-type plasminogen activator of colonic mucosa in ulcerative colitis. Evidence of endothelium-derived fibrinolytic activation.

Microvascular endothelial alterations are thought to be a crucial step for development of hemorrhagic changes in various pathological states. In this study, we determined the activity and amount of tissue-type plasminogen activator (t-PA) in the biopsy specimens from sigmoid colon of patients with ulcerative colitis to evaluate endothelial alterations and vascular changes of permeability. The results of this investigation revealed that mucosal amount of t-PA in the active stage of ulcerative colitis was two- to threefold higher than in healthy controls, while t-PA levels in plasma samples showed no remarkable differences among the groups. Increased t-PA activity appeared to correlate well to the degree of inflammation of colonic mucosa, and t-PA amount was still increased in the inactive stage. The present study indicates that t-PA determination in colonic biopsy specimens may be useful for the evaluation of clinical activity of ulcerative colitis in terms of the mucosal microvascular endothelial changes.