Histochemical and biochemical analysis of collagen content in formalin-fixed, paraffin embedded colonic samples

Collagen is the most abundant structural protein and extracellular matrix component in mammals. In the colon, collagen fibres reside in all the major sublayers; namely, the mucosa, submucosa, muscularis externa and the serosa. Methods to quantify collagen content in formalin-fixed, paraffin-embedded (FFPE) stained sections are required and image analysis offers a technique by which the spatial distribution and localisation of collagen fibres can be easily measured. This laboratory protocol was developed from established techniques using FFPE colon. Human colonic samples embedded transversally in paraffin wax were serially sectioned and stained with either Masson's trichrome (MT) or Picrosirius red (PSR). Quantitation estimation of collagen content in each sublayer was performed via ImageJ processing. Hydroxyproline content was quantified using a rapid and sensitive assay in sectioned tissue. Either MT or PSR staining followed by morphometric image analysis via ImageJ provided equally appreciable quantitative results. Moreso, analysis of hydroxyproline content in our samples indicate that this protocol could be useful in retrospective studies for FFPE samples. This laboratory protocol provides a systematic and reproducible method that can be utilized to accurately assess collagen content in individual sublayers of the colonic wall as well as detection of overall hydroxyproline content in FFPE specimens

Stable U(IV) Complexes Form at High-Affinity Mineral Surface Sites

Uranium (U) poses a significant contamination hazard to soils, sediments, and groundwater due to its extensive use for energy production. Despite advances in modeling the risks of this toxic and radioactive element, lack of information about the mechanisms controlling U transport hinders further improvements, particularly in reducing environments where UIV predominates. Here we establish that mineral surfaces can stabilize the majority of U as adsorbed UIV species following reduction of UVI. Using X-ray absorption spectroscopy and electron imaging analysis, we find that at low surface loading, UIV forms inner-sphere complexes with two metal oxides, TiO2 (rutile) and Fe3O4 (magnetite) (at <1.3 U nm–2 and <0.037 U nm–2, respectively). The uraninite (UO2) form of UIV predominates only at higher surface loading. UIV–TiO2 complexes remain stable for at least 12 months, and UIV–Fe3O4 complexes remain stable for at least 4 months, under anoxic conditions. Adsorbed UIV results from UVI reduction by FeII or by the reduced electron shuttle AH2QDS, suggesting that both abiotic and biotic reduction pathways can produce stable UIV–mineral complexes in the subsurface. The observed control of high-affinity mineral surface sites on UIV speciation helps explain the presence of nonuraninite UIV in sediments and has important implications for U transport modeling

Protective Intestinal Effects of Pituitary Adenylate Cyclase Activating Polypeptide

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide widely distributed throughout the body, including the gastrointestinal tract. Several effects have been described in human and animal intestines. Among others, PACAP infl uences secretion of intestinal glands, blood fl ow, and smooth muscle contraction. PACAP is a well-known cytoprotective peptide with strong anti-apoptotic, anti-infl ammatory, and antioxidant effects. The present review gives an overview of the intestinal protective actions of this neuropeptide. Exogenous PACAP treatment was protective in a rat model of small bowel autotransplantation. Radioimmunoassay (RIA) analysis of the intestinal tissue showed that endogenous PACAP levels gradually decreased with longer-lasting ischemic periods, prevented by PACAP addition. PACAP counteracted deleterious effects of ischemia on oxidative stress markers and cytokines. Another series of experiments investigated the role of endogenous PACAP in intestines in PACAP knockout (KO) mice. Warm ischemia–reperfusion injury and cold preservation models showed that the lack of PACAP caused a higher vulnerability against ischemic periods. Changes were more severe in PACAP KO mice at all examined time points. This fi nding was supported by increased levels of oxidative stress markers and decreased expression of antioxidant molecules. PACAP was proven to be protective not only in ischemic but also in infl ammatory bowel diseases. A recent study showed that PACAP treatment prolonged survival of Toxoplasma gondii infected mice suffering from acute ileitis and was able to reduce the ileal expression of proinfl ammatory cytokines. We completed the present review with recent clinical results obtained in patients suffering from infl ammatory bowel diseases. It was found that PACAP levels were altered depending on the activity, type of the disease, and antibiotic therapy, suggesting its probable role in infl ammatory events of the intestine

Serotonin and GI Disorders: An Update on Clinical and Experimental Studies

The gastrointestinal (GI) tract is the largest producer of serotonin (5-hydroxytryptamine (5-HT)) in the body, and as such it is intimately connected with GI function and physiology. 5-HT produced by enterochromaffin (EC) cells is an important enteric mucosal signaling molecule and has been implicated in a number of GI diseases, including inflammatory bowel disease and functional disorders such as irritable bowel syndrome. This review will focus on what is known of basic 5-HT physiology and also on the emerging evidence for its novel role in activation of immune response and inflammation in the gut. Utilizing pubmed.gov, search terms such as “5-HT,” “EC cell,” and “colitis,” as well as pertinent reviews, were used to develop a brief overview of EC cell biology and the association between 5-HT and various GI disorders. It is the aim of this review to provide the readers with an update on EC cell biology and current understanding on the role of 5-HT in GI disorders specifically in inflammatory conditions

Total collagen content and distribution is increased in human colon during advancing age.

BACKGROUND: The effect of ageing on total collagen content of human colon has been poorly investigated. The aim of this study was to determine if ageing altered total collagen content and distribution in the human colon. METHODS: Macroscopically normal ascending colon was obtained at surgery from cancer patients (n = 31) without diagnosis of diverticular disease or inflammatory bowel disease. Masson's trichrome and Picrosirius red stains were employed to identify the total collagen content and distribution within the sublayers of the colonic wall for adult (22-60 years; 6 males, 6 females) and elderly (70 - 91years; 6 males, 4 female) patients. A hydroxyproline assay evaluated the total collagen concentration for adult (30-64 years; 9 male, 6 female) and elderly (66-91 years; 8 male, 8 female) patients. KEY RESULTS: Histological studies showed that the percentage mean intensity of total collagen staining in the mucosa, submucosa and muscularis externa was, respectively, 14(1.9) %, 74(3.2) % and 12(1.5) % in the adult ascending colon. Compared with the adults, the total collagen fibres content was increased in the submucosa (mean intensity; 163.1 ± 11.1 vs. 124.5 ± 7.8; P < 0.05) and muscularis externa (42.5 ± 8.0 vs. 20.6 ± 2.8; P < 0.01) of the elderly patients. There was no change in collagen content of the mucosa. The total collagen concentration was increased in the elderly by 16%. Sex-related differences were not found, and data were combined for analysis. CONCLUSIONS: Greater total collagen content was found in the submucosa and muscularis externa of the elderly human male and female colon. These changes may contribute to a possible loss of function with ageing