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Background and Aim. Obesity is a metabolic disorder with an increasing incidence in Western countries and childhood. It is characterized by low-grade systemic inflammation and several comorbidities, including alterations of gastrointestinal (GI) functions, which impact negatively on patients’ quality of life. There is currently limited information on the morpho-functional features of the GI tract in obese subjects. Of note, the intestinal barrier function has been found to be altered in obese subjects, even before the occurrence of body weight increase [1]. In this light, the present study was carried out to assess, in a mouse model of diet-induced obesity, whether high fat diet (HFD) is associated with morphological alterations of the colonic mucosal barrier. Methods. C57BL/6 mice (n=5/group) were fed with standard diet (SD, 18% calories from fat) or HFD (60% calories from fat). After 8 weeks, body weight, and levels of blood cholesterol, triglycerides and glucose were evaluated. Malondialdehyde (MDA, colorimetric assay), IL-1β and IL-6 levels (ELISA assays) were examined in colonic tissues. Morphological features of colonic mucosal structures (lining epithelial cells, goblet cells, inflammatory infiltrates and enteric glia) were examined by histochemistry and immunohistochemistry. Results. HFD mice displayed significant differences at both molecular and histomorphological level, as compared with SD animals: increased body weight and blood metabolic indexes; increased MDA, IL-1β and IL-6 levels in colonic tissues; altered pattern of claudin-1 expression along with upregulation of transmembrane 16A protein and induced nitric oxide synthase in the enteric epithelium facing the lumen; increased proliferation rate of crypts; altered composition of goblet cell mucous; mucosal gliosis and infiltrates with mixed inflammatory cells. Conclusions. After 8 weeks, HFD intake led to significant alterations of systemic metabolic indexes, colonic tissue inflammation, and colonic mucosal barrier in obese mice, as compared with controls. Morphological studies can be useful to allow the characterization of histopathological patterns of colonic wall remodelling and inflammation underlying bowel motor dysfunctions associated with obesity., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Inflammatory bowel disease (IBD) are chronic and progressive pathologies associated with invalidating abdominal symptoms and increasing incidence in Western countries. Although fibrostenosis is a rare event in ulcerative colitis [UC], it may evolve to fibrosis in the later stages (1, 2). In the present study we examined the histopathological remodeling of the colonic wall from short- and long-lasting (SL and LL) UC patients. Full-thickness left colonic surgical samples were obtained from non-stenotic SL (≤ 3 years) and LL (≥ 10 years) UC patients with a severe exacerbation of colitis and active disease, without clinical signs of fibrostenosis. Collagen and elastic fibres, vascular networks and parameters of fibrosis have been evaluated by histochemistry, immunohistochemistry, confocal immunofluorescence and/or western blot. For comparison, normal colonic control samples from subjects who underwent surgery for uncomplicated colon cancer and without IBD and diverticular disease, were considered.Both SL- and LL-UC showed a thickening in tunica muscularis and activation of transmural neovessels, with proliferating CD105-positive endothelial cells and activated nestin-positive pericytes, as compared with controls. The colonic wall of LL-UC displayed a significant increase in collagen deposition and fibrotic markers (type I and III collagen, fibronectin, vimentin, RhoA), an enhancement of proliferation (PCNA), a decrease in elastic fibres, together with a fibrotic rearrangement of the tunica muscularis smooth muscle cells. In conclusion, a significant full-thickness remodeling of the colonic wall has been documented in the colonic tissues from both SL-UC and LL-UC, as compared to controls: transmural fibrotic thickening and angiogenesis were found in both UC groups, together with a cellular fibrotic/proliferative switch in the tunica muscularis which occurs in the later stages. These histopathological remodeling may contribute to the reduced elastic properties and tissue stretch capability of the wall, thereby impairing the occurrence of a normal motor activity in these subjects., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Background and Aim. Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by motor and non-motor clinical signs, among which gastrointestinal disturbances represent relevant manifestations [1]. Nevertheless, the morphological alterations associated with intestinal dysfunctions in PD have been barely investigated [2]. The present study was aimed at investigating the remodelling of colonic wall in a rat model of PD with central dopaminergic denervation by intra-nigral injection of the neuro-toxin 6-hydroxydopamine (6-OHDA). Methods. Histopathological analysis of the whole colonic wall was performed 4 and 8 weeks after central 6-OHDA injection. Inflammatory infiltrates, collagen deposition as well as the remodelling of intestinal epithelial barrier and tunica muscularis were examined by microscopic techniques (histochemistry/immunohistochemistry/confocal immunofluorescence). Results. Colonic tissue from 8-week 6-OHDA rats were significantly altered, as compared with controls. The tunica mucosa showed: eosinophil infiltration; altered lining epithelium (reduced claudin-1 and transmembrane 16A protein expression) and goblet cells (increased mucus expression); enhanced glial fibrillar acid protein-positive cells and vimentin-positive fibroblast-like cells. Along with transmural collagen deposition, significant changes were observed also in the tunica muscularis: reduced expression of alpha-smooth muscle actin/desmin and increased proliferation index in smooth muscle cells; increased vimentin expression and proliferative phenotype in myenteric ganglia. Conclusions. A full-thickness structural remodelling occurs in the colon of PD rats 8 weeks after central dopaminergic denervation; the main changes include an alteration of the colonic epithelial barrier along with the activation of the mucosal defence and fibrotic switch of the colonic wall. Overall, these findings suggest that: a) early histological modifications occur in the colon of rats with experimental PD at both mucosal and muscular level; b) these changes and the fibrotic alterations might contribute to bowel motor dysfunctions associated with PD., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Parkinson’s disease (PD) is a degenerative neurological disorder, which is often associated with gastrointestinal disturbances (e.g., constipation and defecatory dysfunctions), whose mechanisms are still unknown [1]. Recently, an inflammatory pathogenesis has been proposed to explain these colonic disorders, but low literature are available. This study aims to analyze whether the central dopaminergic denervation, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), can alter the morphological arrangement of colon in rats. Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Histological, histochemical and immunohistochemical analysis were carried out on formalin-fixed, paraffin-embedded colonic samples in order to evaluate: histology, inflammatory cells (eosinophils and mast cells) and collagen fibers in the whole wall; glial fibrillary acidic protein (GFAP), immunoperoxidase, alpha-smooth muscle actin (alpha-SMA) and vimentin immunofluorescence by confocal microscopy. Malondialdehyde (MDA, colorimetric assay), TNF and IL-1β (ELISA assay) levels were also examined. 6-OHDA-induced nigrostriatal denervation was associated with the following histopathological changes observed in the colonic wall: eosinophil and mast cell infiltration, collagen deposition, activation of myenteric glial cells (GFAP+), increased vimentin immunostaining associated with alpha-SMA decrease in the tunica muscularis, enhanced colonic tissue levels of MDA, TNF and IL-1β. On the basis of the present results it is possible to conclude that the induction of central nigrostriatal dopaminergic denervation is followed by inflammation and fibrotic rearrangement of the colonic wall., Italian Journal of Anatomy and Embryology, Vol. 121, No. 1 (Supplement) 2016

Background. Intestinal fibrosis is a common complication of inflammatory bowel diseases, affecting patients with both Crohn’s disease and ulcerative colitis (UC). Of note, the progression of intestinal fibrosis has been recently considered to depend on distinct processes from those involved in inflammation [1]. In this context, angiogen- esis is currently regarded as a good candidate of active gut disease, closely related to fibrogenesis [2]. Therefore, studies on the multifactorial pathways promoting these processes are needed for understanding the pathophysiology of fibrosis, and there- by identifying anti-fibrogenic therapies. Aim. The present study was performed to evaluate the distribution of fibrotic tissue, the behaviour of smooth muscle cells and the presence of neovessels in the colon of UC patients. Patients and Methods. Full- thickness left colonic samples were studied, from patients with established and phar- macologically unresponsive UC for the following parameters: collagen and elastic fib- ers by histochemistry; fibrotic and profibrotic factors [type 1 and 3 collagens, elastin, fibronectin, vimentin, alpha-smooth muscle actin (α-SMA), proliferating nuclear anti- gen (PCNA), RhoA] by immunohistochemistry and western blot; vascular networks [CD31, CD105, nestin] by confocal microscopy immunofluorescence. Results. A sig- nificant increase in collagen fibers and decrease in elastin content were detected in the colon from UC patients as compared with controls. The increment of type 1 and 3 collagens, fibronectin, vimentin, PCNA and RhoA expression was associated with alpha-SMA decrease in the tunica muscularis of UC colon. A relevant rearrangement of vascular networks was observed in the fibrotic tunica muscularis, with neovessels displaying both proliferating CD105+ endothelial cells and activated nestin+ pericytes. Conclusion. The present data show that a significant muscle and vascular remodel- ling occurs in inflamed colonic tissues from UC patients, suggesting that, under these conditions, smooth muscle cells and vascular cells may be involved in fibrogenic pro- cesses by cell transition to mesenchymal phenotype., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

The pathogenesis of diverticular disease (DD) seems to be a result of a complex interaction between exposure to a low-fibre diet, possible genetic influence, coexistence of other bowel disease and impact of medicine use. These conditions may lead to alterations in colonic pressure and motility [1]. To date, histopatological studies in the field of disturbances in intestinal motility have been mainly focused on the enteric nervous system and interstitial cells of Cajal, which have been found to be both variously affected in different conditions of gut dysmotility. By contrast, although smooth muscle cells (SMCs) are well recognized as the final effectors of the enteric neuromuscular units and have been extensively studied by electrophysiological experiments in colonic motility dysfunction, scarce attention has been paid to the molecular abnormalities of enteric musculature in gut dysmotility, and, in particular, data on SMCs in DD are fairly lacking. Accordingly, the aim of the present study was to evaluate the expression patterns of molecules involved in the contractile functions of SMCs within the colonic tunica muscularis from patients with DD. In particular, we examined the expression of the following molecules by immunohistochemistry and image analysis: Cx26 and Cx43, which are prominent components of gap junctions in human colonic SMCs [2], and an array of signaling molecules, which are known to regulate the functions of gap junctions and the contractile activity of SMCs [3,4,5]. The immunohistochemical analysis revealed significant abnormalities in DD samples, concerning both the expression and distribution patterns of most of the investigated molecular factors. Indeed, Cx26, Cx43, PKCps and RhoA appeared to be markedly deranged in all DD colonic samples compared to normal ones, except for α-SMA and pS368-Cx43. The present results provide the first evidence of an altered pattern of factors involved in smooth muscle contractility at level of tunica muscularis SMCs of DD patients. These abnormalities may contribute to the altered motility function, which characterize the colonic tunica muscularis of patients with DD., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

Inflammation-driven tissue remodelling may develop to fibrotic rearrangement. With regard to inflammatory bowel diseases, fibrotic remodelling has been evaluated in Crohn’s disease, while little attention to such processes has been paid to ulcerative colitis (UC) [1]. The present study evaluated the distribution of connective tissue and angiogenesis in colon of patients with UC, paying particular attention to the tonaca muscularis, which is poorly considered in histopathological studies. Full-thickness left colonic samples were obtained from 10 patients with established, severe and pharmacologically unresponsive UC, who underwent bowel resection. Routine histology, histochemistry and immunohistochemistry were conducted in paraffin cross-sections. The distribution of collagen and elastic fibers was evaluated and quantified by both histochemical (Van Gieson, orcein, Verroheff staining) and immunohistochemical (anti-collagen I and III, anti-elastin) assays. The vascular network pattern was studied by anti-CD31 and nestin immunostaining. For comparison, the same evaluations were performed in healthy colonic control samples obtained from 10 subjects, who underwent surgery for uncomplicated colon cancer. A significant increase in collagen fibers and a decrease in elastin content were detected in the UC inflamed colon, as compared with controls. In particular, enhanced collagen deposition (mainly collagen type III) were found at level of submucosa, and tonaca muscularis within the longitudinal muscle (mainly along the serosal side) and circular muscle layer, and in perivascular connective tissue. By contrast, elastic fibers were significantly reduced throughout the whole muscle compartment, with particular regard for the myenteric ridge. Microvessel density was significantly higher in both submucosa and tonaca muscularis of colonic samples from UC patients compared with those from healthy control individuals. The present findings indicate that a significant tissue remodelling occurs in the inflamed colonic wall in patients with UC, also at the level of muscle layers. This rearrangement of the connective fibers and vascular network, together with the known alterations affecting the myenteric neurons and interstitial cells of Cajal, may contribute to the development of enteric dysmotility, and, accordingly, to the serious digestive symptoms which afflict patients with UC., Italian Journal of Anatomy and Embryology, Vol 117, No 2 (Supplement) 2012

The term gastrointestinal neuromuscular disease comprises a heterogeneous group of chronic conditions associated with impaired bowel motility. Gastrointestinal motor dysfunctions, differening for etiopathogenic mechanisms, pathologic lesions, and region of gut involvement (e.g., irritable bowel syndrome, slow transit constipation, inflammatory bowel disease, diverticular disease), represent a relevant matter for public health: in fact, they are very common, can be disabling, and induce major social and economic burdens. These motor disturbances are presumed or proven to arise as a result of dysfunctional enteric neuromuscular apparatus set up by myenteric ganglionic cells, interstitial cells of Cajal and smooth muscle cells of the muscularis propria. Despite the presence of intestinal dysmotility in the clinical phenotype of these patients, scarce attention has been paid to the morphological arrangements of the enteric neuromuscular apparatus. Furthermore, standards for histopathological reports remain relatively neglected resulting in significant differences in applied methodologies which confound the reliable delineation of normality and, as a consequence, the specificity of particular pathological changes for disease. Thus, in order to overcome the lack or heterogeneity of current data, to get normative data and delineation of abnormality, careful morphological examinations and development of standardized procedures are particularly required in the field of gastrointestinal neuromuscular pathology, as recently suggested., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Diverticular disease (DD) is one of the most common diseases related to the gastrointestinal tract in Western countries. It has been postulated that abnormal colonic motility including increased overall motility, abnormal response to a physiologic stimulus and retropropagation of mass movement, predisposes to the formation of pulsion diverticula in the segments of the bowel containing the diverticula. Intestinal motility is regulated by complex interactions among smooth muscle cells (SMCs) of muscularis propria, enteric nerve endings and interstitial cells of Cajal (ICC). ICC are emerging as potential colonic pacemaker cells which modulate neuroenteric transmission by connecting SMC with varicosities of myenteric neuron axons. In colonic SMCs, transmenbrane channels rich in connexin (Cx), in particular Cx43, contribute to intercellular gap junctions, which ensure coordinated motor responses to nerve inputs.There is evidence that SMCs motility and gap junction permeability are regulated by the GTPase RhoA, an emerging key modulator of SMCs phenotype. The aim of the present study was to evaluate the Cx43 and RhoA expression in SMCs, as well as ICC density in colonic specimens from patients affected by diverticular disease (DD). Immunohistochemistry for Cx43, RhoA and c-kit and image analysis were used to examine muscularis propria of surgical whole thickness colonic samples from DD patients. Semiquantitative analysis of DD colonic specimens displayed a marked decrease in Cx43 and RhoA expression in SMCs. There was also a reduced ICC density in myenteric ganglia (ICC-MY), circular (ICC-CM) and longitudinal (ICC-LM) muscle, as compared to controls. Overall, it is suggested that abnormalities in gap junctions and RhoA expression in SMCs, together with a reduced density of muscular ICC, account for the colonic dismotility occurring in DD., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011