Collagen detection in histological sections and its quantitative estimation by computer-aided image analysis represent important procedures to assess tissue localization and distribution of connective fibers. Different histochemical approaches have been proposed to detect and quantify collagen deposition in paraffin slices with different degrees of satisfaction. The present study was performed to compare the qualitative and quantitative efficiency of three histochemical methods available for collagen staining in paraffin sections of colon. van Gieson, Sirius Red and Sirius Red/Fast Green stainings were carried out for collagen detection and quantitative estimation by morphometric image analysis in colonic specimens from normal rats or animals with 2,4-dinitrobenzenesulfonic acid (DNBS) induced colitis. Haematoxylin/eosin staining was carried out to assess tissue morphology and histopathological lesions. Among the three investigated methods, Sirius Red/Fast Green staining allowed to best highlight well-defined red-stained collagen fibers and to obtain the highest quantitative results by morphometric image analysis in both normal and inflamed colon. Collagen fibers, which stood out against the green-stained non-collagen components, could be clearly appreciated, even in their thinner networks, within all layers of normal or inflamed colonic wall. The present study provides evidence that, as compared with Sirius Red alone or van Gieson staining, the Sirius Red/Fast Green method is the most sensitive, in terms of both qualitative and quantitative evaluation of collagen fibers, in paraffin sections of both normal and inflamed colon.

Essential hypertensive patients show a reduced nitric oxide availability secondary to oxidative stress generation in peripheral microcirculation. Cyclooxygenase (COX) contributes to reduce nitric oxide availability. We assessed the possible vascular sources of oxidative stress, including COX-1, COX-2, and nicotinamide adenine dinucleotide phosphate oxidase, as determinants of endothelial dysfunction in small arteries isolated from essential hypertensive patients or normotensive controls. Small arteries were dissected after subcutaneous fat biopsies and evaluated on a pressurized micromyograph. Endothelium-dependent vasodilation was assessed by acetylcholine, repeated under NG-nitro- l -arginine methyl ester, SC-560 (COX-1 inhibitor), DuP-697 (COX-2 inhibitor), ascorbic acid, or the nicotinamide adenine dinucleotide phosphate oxidase inhibitors apocynin or diphenylene iodonium. Vascular oxidative stress generation (fluorescent dihydroethidium), COX-1 and COX-2 expression (Western blot), and localization (immunohistochemistry) were also assessed. In controls, response to acetylcholine was blunted by NG-nitro- l -arginine methyl ester ( P <0.001) and unmodified by SC-560, DuP-697, or ascorbic acid. In hypertensive patients, relaxation to acetylcholine was blunted, resistant to NG-nitro- l -arginine methyl ester or SC-560, and enhanced ( P <0.01) by DuP-697, apocynin, or diphenylene iodonium ( P <0.05). Furthermore, in hypertensive patients, response to acetylcholine was normalized by ascorbic acid or apocynin+DuP-697. Intravascular oxidative stress generation was enhanced in hypertensive patients, decreased ( P <0.01) by DuP-697, partly attenuated by apocynin or diphenylene iodonium, and prevented by ascorbic acid. Enhanced COX-2 expression and localization in the vascular media of hypertensive patients were also detected. In small resistance arteries of essential hypertensive patients, COX-2 is overexpressed and reduces nitric oxide availability. COX-2 represents a major source of oxidative stress generation, whereas nicotinamide adenine dinucleotide phosphate oxidase plays a minor, but significant, role in promoting superoxide generation.

Ulcerative colitis (UC) is an inflammatory bowel disease with alterations of colonic motility, which influence clinical symptoms. Although morpho‐functional abnormalities in the enteric nervous system have been suggested, in UC patients scarce attention has been paid to possible changes in the cells that control colonic motility, including myenteric neurons, glial cells and interstitial cells of Cajal (ICC). This study evaluated the neural‐glial components of myenteric ganglia and ICC in the colonic neuromuscular compartment of UC patients by quantitative immunohistochemical analysis. Full‐thickness archival samples of the left colon were collected from 10 patients with UC (5 males, 5 females; age range 45–62 years) who underwent elective bowel resection. The colonic neuromuscular compartment was evaluated immunohistochemically in paraffin cross‐sections. The distribution and number of neurons, glial cells and ICC were assessed by anti‐HuC/D, ‐S100β and ‐c‐Kit antibodies, respectively. Data were compared with findings on archival samples of normal left colon from 10 sex‐ and age‐matched control patients, who underwent surgery for uncomplicated colon cancer. Compared to controls, patients with UC showed: (i) reduced density of myenteric HuC/D⁺ neurons and S100β⁺ glial cells, with a loss over 61% and 38%, respectively, and increased glial cell/neuron ratio; (ii) ICC decrease in the whole neuromuscular compartment. The quantitative variations of myenteric neuro‐glial cells and ICC indicate considerable alterations of the colonic neuromuscular compartment in the setting of mucosal inflammation associated with UC, and provide a morphological basis for better understanding the motor abnormalities often observed in UC patients.

Parkinson’s disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1β (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR. In colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1β levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype. The results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility.

Angiotensin II induces endothelial dysfunction by reducing NO availability and increasing reactive oxygen species. We assessed whether cyclooxygenase (COX)-1 or COX-2 participate in the angiotensin II–induced endothelial dysfunction in murine mesenteric small arteries and examined the role of reduced nicotinamide-adenine dinucleotide phosphate–dependent reactive oxygen species production. Mice received angiotensin II (600 ng/kg per minute, SC), saline (controls), angiotensin II + apocynin (reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, 2.5 mg/day), or apocynin alone for 2 weeks. Endothelial function of mesenteric arteries was assessed by pressurized myograph. In controls, acetylcholine-induced relaxation was inhibited byN^G-monomethyl-l-arginine and unaffected by DFU (COX-2 inhibitor), SC-560 (COX-1 inhibitor), or ascorbic acid. In angiotensin II–infused animals, the attenuated response to acetylcholine was less sensitive toN^G-monomethyl-l-arginine, unaffected by DFU, and enhanced by SC-560 and, similarly, by SQ-29548, a thromboxane–prostanoid receptor antagonist. Moreover, response to acetylcholine was unchanged by ozagrel, a thromboxane synthase inhibitor, and normalized by ascorbic acid. Apocynin prevented the angiotensin II–induced vascular dysfunctions. In angiotensin II–infused mice, RT-PCR analysis showed a significant COX-2 downregulation, whereas COX-1 expression was upregulated. These changes were unaffected by apocynin. Modulation of COX isoform by angiotensin II was also documented by immunohistochemistry. In small mesenteric vessels, the reduced NO availability and oxidant excess, which characterize endothelial dysfunction secondary to angiotensin II, are associated with a reduced COX-2 and an increased COX-1 function and expression. Angiotensin II causes an oxidative stress–independent COX-1 overexpression, whereas angiotensin II–mediated oxidant excess production stimulates COX-1 activity to produce a contracting prostanoid endowed with agonist activity on thromboxane–prostanoid receptors.

Parkinson’s disease is frequently associated with gastrointestinal symptoms, mostly represented by constipation and defecatory dysfunctions. This study examined the impact of central dopaminergic denervation, induced by injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle, on distal colonic excitatory cholinergic neuromotor activity in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. In vivo colonic transit was evaluated by radiologic assay. Electrically induced and carbachol-induced cholinergic contractions were recorded in vitro from longitudinal and circular muscle colonic preparations, whereas acetylcholine levels were assayed in the incubation media. Choline acetyltransferase (ChAT), HuC/D (pan-neuronal marker), muscarinic M2 and M3 receptors were assessed by immunohistochemistry or western blot assay. As compared with control rats, at week 4, 6-OHDA–treated animals displayed the following changes: decreased in vivo colonic transit rate, impaired electrically evoked neurogenic cholinergic contractions, enhanced carbachol-induced contractions, decreased basal and electrically stimulated acetylcholine release from colonic tissues, decreased ChAT immunopositivity in the neuromuscular layer, unchanged density of HuC/D immunoreactive myenteric neurons, and increased expression of colonic muscarinic M2 and M3 receptors. The majority of such alterations were also detected at week 8 post 6-OHDA injection. These findings indicate that central nigrostriatal dopaminergic denervation is associated with an impaired excitatory neurotransmission characterized by a loss of myenteric neuronal ChAT positivity and decrease in acetylcholine release, resulting in a dysregulated smooth muscle motor activity, which likely contributes to the concomitant decrease in colonic transit rate.