New Anatomical Concepts regarding Pubic-Related Groin Pain: A Dissection Study.

OBJECTIVE: Pubis-related groin pain remains a difficult topic in orthopedic and sports medicine. A better understanding of the anatomy of the adductors and the pubic ligaments is necessary. The aim of this study is to map all the musculotendinous attachments to the pubic ligaments and to investigate in detail all the possible inter-adductor fusions. METHODS: The pubic symphyses were dissected in eight male and fourteen female embalmed cadavers (mean age 85 years), focusing on the fusion between the adductors, pubic ligaments, and musculotendinous attachments at the pubic ligaments. The 95% confidence intervals for the prevalence of the different conjoint tendons and tendon attachment to ligament were calculated. RESULTS: The presence of three types of conjoint tendons was found: adductor brevis and gracilis (AB/G) 90.9 [72.2 - 97.5]%; adductor brevis and adductor longus (AB/AL) 50.0 [30.7 - 69.3]%; adductor longus and gracilis (AL/G) 50.0 [30.7 - 69.3]%. The AL, AB and G were in every cadaver attached to the anterior pubic ligament (APL). 64% of the AB and 100% of the G were attached to the inferior pubic ligament (IPL). CONCLUSION: The proximal anatomy of the adductors is more complex than initially described. This study identified three possible conjoint tendons between the proximal adductors. The AB/G conjoint tendon was significantly more present than the AB/AL or AL/G conjoint tendon. The IPL has attachments only from the AB and G. Rectus Abdominis (RA) and AL were not attached to IPL. Mapping the musculotendinous attachments on the pubic ligaments creates more clarity on the pathophysiology of lesions in this area.

Does Orexin B-Binding Receptor 2 for Orexins Regulate Testicular and Epididymal Functions in Normal and Cryptorchid Dogs?

Orexins A (OXA) and B (OXB) and the receptors 1 (OX1R) and 2 (OX2R) for orexins are hypothalamic peptides found in several mammalian organs and participated to the control of a wide assortment of physiological and pathological functions. The distribution of OXA and OX1R has been extensively studied in the male gonad of mammals. Here, we examined the expression and localization of OXB and OX2R as well as their possible involvement in the regulation of testicular and epididymal functions, in healthy and cryptorchid dogs, employing some techniques such as immunohistochemistry, Western blotting, and real-time RT-PCR. In vitro tests were also carried out for evaluating the steroidogenic effect of OXB. OXB and OX2R were expressed in spermatocytes, spermatids, and Leydig cells in normal testis. Their localization was restricted to Sertoli and Leydig cells in cryptorchid conditions. OXB was found to be localized in all tracts of both normal and cryptorchid epididymis, whereas OX2R was found only in the caput. Because the small molecular weight of the peptides OXA and OXB, the expression of their precursor prepro-orexin (PPO), OX1R, and OX2R proteins and mRNAs were investigated by means of Western blot and real-time RT-PCR analyses, respectively, in all tested groups of. In particular, the mRNA level expression of all three genes was higher in cryptorchid dogs than in normal ones. In vitro tests demonstrated that OXB-by binding OX2R-is not involved in testicular steroidogenic processes. Therefore, the findings of this study might be the basis for further functional and molecular studies addressing the possible biochemical effects of OXB and OX2R in normal and pathological conditions of the male reproductive system.

New insights into the musculotendinous and ligamentous attachments at the pubic symphysis: A systematic review.

OBJECTIVE: Collection and meta-analysis of all relevant anatomical studies related to the pubic symphysis to provide a state of the art review of its musculotendinous and ligamentous attachments from 2010 to date. METHODS: A systematic search of published literature databases (PubMed, Web of Science and Embase) was conducted according to the PRISMA guidelines from January 2010 up until now. All papers investigating the anatomy of the musculotendinous attachments of the pubis and the pubic ligaments were eligible. Methodological quality was assessed using the Quality Appraisal for Cadaveric Studies (QUACS scale). A narrative analysis approach was adopted to synthesize the findings. RESULTS: After screening and review of 1313 papers, a total of six studies investigating the anatomy of the pubic ligaments and tendons were included. Of the six articles included in this systematic review, five articles performed a macroscopic anatomical dissection, three articles performed a microscopic (histological) study, and one article combined microscopic examination with an MRI imaging examination. The anatomy of the pubic symphysis was examined in 76 anatomical cadavers (60 embalmed, 16 fresh frozen). In total 44 male cadavers (58%), 28 female cadavers (37%) and four cadavers whose gender was not stated were dissected. CONCLUSION: The age-old accepted concept of the fusion of the rectus abdominis with the adductor longus via the aponeurotic plate is outdated. New anatomical concepts like the pyramidalis-anterior pubic ligament-adductor longus complex (PLAC), recto-gracilis tendon, fusion of adductor brevis with gracilis, etc. are recently introduced. The awareness of anatomy and morphology of the pubic ligaments plays a significant role in understanding the diagnosis and treatment of groin pain.

Anatomical variations of the upper thoracic sympathetic chain: a review.

ABSTRACT OBJECTIVES: The objective of this study is to provide a thorough overview of the anatomical variations of the upper thoracic sympathetic trunk to improve clinical results of upper thoracic sympathectomy. In addition, this study strives for standardization of future studies regarding the anatomy of the upper thoracic sympathetic chain. METHODS: The Web of Science, PubMed and Google Scholar databases were searched using keywords, alone or combined, regarding the anatomy of the thoracic sympathetic chain. The search was limited to studies performed in humans. RESULTS: Fifteen studies were finally included. Cervicothoracic ganglion and nerve of Kuntz were present in 77% and 53%, respectively. The upper thoracic ganglia were predominantly located in their corresponding intercostal space with a relatively downwards shift at the lower thoracic levels. The right sympathetic trunk is prone to have more communicating rami then the left. The lower levels of ganglia tend to have more normal rami. No clear pattern was found concerning the presence of the ascending rami and there was a decrease in the number of descending rami as the chain runs caudally. The intercostal rami remain a rare anatomical variation. CONCLUSIONS: This study presents an overview of the anatomy of the upper thoracic sympathetic chain. Its results may guide upper thoracic sympathectomy to improve clinical results. This review also provides a baseline for future studies on anatomical variations of the thoracic sympathetic trunk. More uniform reporting is necessary to compare different anatomical studies.

Effect of TNBS-induced colitis on enteric neuronal subpopulations in adult zebrafish.

Inflammatory bowel disease (IBD) includes inflammation of the gastrointestinal (GI) tract and is characterized by periods of acute inflammation and remission. Therapeutic management of IBD is still problematic, because of incomplete understanding its pathogenesis. This study focuses on the effect of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis on changes in enteric neuronal subpopulations in adult zebrafish. These changes are suggested to be related to the altered neuro-immune interactions and GI motility, and in IBD pathogenesis. New insights into neuroplasticity will be instrumental in finding appropriate therapeutic treatments. TNBS was intraluminally administered in the distal intestine (DI) of anesthetized adult zebrafish. A histological time course of the intestinal inflammatory response was created to establish optimal TNBS concentration and acute inflammation phase. Using double immunolabelling on whole mounts, the effect of inflammation on neuronal populations was analyzed. Based on intestinal wall thickening, epithelial fold disruption, reduced goblet cell number, and eosinophil infiltration, our analysis indicated that the optimal TNBS concentration (320 mM in 25% ethanol) inducing non-lethal inflammation reached a peak at 6 hours post-induction. The inflammatory response returned to baseline values at 3 days post-induction. At the acute inflammation phase, no influence on the distribution or proportion of nitrergic neurons was observed, while only the proportion of cholinergic neurons was significantly reduced in the DI. The proportion of serotonergic neurons was significantly increased in the entire intestine during inflammation. This study describes a method of TNBS-induced colitis in the adult zebrafish. Given that the acute inflammation phase is accompanied by neuroplasticity comparable to changes observed in IBD patients, and the unique and versatile characteristics of the zebrafish, allows this model to be used alongside IBD animal models to unravel IBD pathology and to test new IBD therapies.

Mas-related G protein-coupled receptor C11 (Mrgprc11) induces visceral hypersensitivity in the mouse colon: A novel target in gut nociception?

BACKGROUND: Visceral hypersensitivity, an important cause of abdominal pain in disorders such as IBD and IBS, presents with a poorly understood pathophysiology and limited treatment options. Several members of the Mas-related G protein-coupled receptor family (Mrgprs) have become promising targets in pain research. The potential link between the murine Mrgpr C11 (Mrgprc11) and gut nociception is currently uninvestigated. Therefore, we explored the expression and functional role of Mrgprc11 in the gut nociceptive innervation. METHODS: Mrgprc11 expression was evaluated in DRG neurons innervating the mouse colon using in situ hybridization and immunohistochemistry. Visceromotor responses to colorectal distension (CRD) assessed the effect of the Mrgprc11 agonist, BAM(8-22), on colonic pain sensitivity in healthy mice. Moreover, we determined pERK1/2-immunoreactivity in the thoracolumbar spinal cord after noxious CRD. Finally, from a translational point of view, we looked for expression of the human counterpart of Mrgprc11, MRGPRX1, in human thoracolumbar DRGs. KEY RESULTS: In situ hybridization and immunohistochemistry revealed Mrgprc11 expression in colonic DRG neurons. Intracolonic administration of BAM(8-22) significantly increased colonic pain sensitivity in an Mrgprc11-dependent manner, and led to a significantly increased degree of neuronal activation in the splanchnic spinal cord upon noxious stimulation. Furthermore, MRGPRX1 expression was also detected in human thoracolumbar DRG neurons. CONCLUSIONS & INFERENCES: Our findings established a novel function for Mrgprc11 in the gut nociceptive innervation and propose the receptor as a new player in visceral hypersensitivity. Given the presence of MRGPRX1 in human DRG neurons, our study warrants future research on its therapeutic potential in abdominal pain disorders.

In situ proximity of CX3CR1-positive mononuclear phagocytes and VIP-ergic nerve fibers suggests VIP-ergic immunomodulation in the mouse ileum.

Being continuously exposed to a plethora of antigens ranging from food antigens to potential pathogenic organisms, the gastrointestinal (GI) tract harbors the largest collection of immune cells in the mammalian body. This immune system has to maintain a delicate balance between mounting an active immune response and maintaining tolerance. The GI tract is also home to an elaborate intrinsic nervous system, the enteric nervous system (ENS). Various in vitro studies of neuro-immune communication have suggested that vasoactive intestinal peptide (VIP), an important GI neurotransmitter, modulates mononuclear phagocytes (MNPs), i.e., dendritic cells and macrophages. Using a combined approach of reverse transcription plus the polymerase chain reaction, immunofluorescence, three-dimensional maximum intensity projections and immunoelectron microscopy, we investigate the interaction between the enteric innervation and MNPs in the ileal lamina propria (LP). We demonstrate that VIP-ergic fibers of the ENS lie adjacent to CX3CR1+ MNPs and that VPAC1 is constitutively expressed on ileal CX3CR1+ cells in the LP of the mouse. We also identify, for the first time, CX3CR1+ immune cells in the LP at the ultrastructural level. Our data thus reveal the in situ presence of the molecular components that are necessary for a VIP-mediated neuro-immune interaction between the ENS and CX3CR1-expressing immune cells in the LP of the ileum.

The effect of a Le Fort I incision on nose and upper lip dynamics: Unraveling the mystery of the "Le Fort I lip".

INTRODUCTION: Postoperative flattening of the upper lip with loss of lip pout and down turning of the corners of the mouth is often seen after Le Fort I surgery. We aim to determine which facial muscles are involved in this phenomenon to update the literature on this subject. METHODS: In 6 cadavers, a unilateral Le Fort I incision was executed. After removal of the skin, all individual facial muscles were identified and submitted to bilateral tactile traction, comparing incised sides with non-incised sides. CONCLUSION: All the components of the deep layer of the modiolus alae nasi (transverse part of the nasalis muscle and the myrtiformis muscle) and the deep layer of the midface musculature (levator anguli oris muscle) were transected by the Le Fort I incision. After performing the incision, the majority of the depressor septi nasi is intact. Further, the superficial layer of the midface musculature is intact but it loses tension because of its connection to the deep layer. This study suggests the importance of correctly suturing the deep muscular layers to maintain the 3-dimensional facial contour. Moreover, in this cadaver study, we attempt to predict the functional consequences on the impairment of facial mimics related to the Le Fort I incision.

Identification and putative roles of distinct subtypes of intestinal dendritic cells in neuroimmune communication: what can be learned from other organ systems?

The gastrointestinal (GI) tract, just like the skin and the airways, is constantly exposed to both harmless and pathogenic organisms and hence requires a tightly regulated immune homeostasis to function properly. A central role in the regulation of this balance is played by the dendritic cells (DCs), a heterogeneous population of antigen-presenting cells that can be further divided into distinct subsets with different functions depending on the tissue they reside in. In recent years, the DC population in the lamina propria (LP) of the intestine has emerged as a key player in immune surveillance. Given the extensive innervation of the GI mucosa, these DC subsets possibly are also regulated by interactions with neuronal components. Current knowledge, be it still fragmentary, indicates that dysregulation of this neuroimmune communication leads to the onset of pathological disorders. The present review article deals with the identification and interaction of distinct subtypes of mouse intestinal LP DCs with elements of the enteric nervous system (ENS) in normal and inflammatory conditions. Furthermore, the question is addressed whether any parallels can be drawn between intestinal LP DCs and DCs residing in the skin and lung in order to gain a better insight into common or clearly distinct mechanistic pathways and the possible impact of the mucosal components in the microenvironment. The exact way in which the ENS is serving its immunomodulatory roles in the GI tract is still largely unknown, although there are significant indications for a crosstalk between LP DCs and components of the ENS. This review clearly shows that in the three different organ systems the same neurotransmitters (i.e., SP, CGRP, and VIP) reoccur, serving similar functions. Mechanistic lessons learned from other organ systems, such as the skin and lung, may be of substantial help in further exploring the nature of the neuroimmune communication between GI innervation and LP DCs.

Expression of corticotropin-releasing factor and urocortins in the normal and Schistosoma mansoni-infected mouse ileum.

Corticotropin-releasing factor (CRF) and urocortins (UCNs) are important ligands in the CRF signaling pathways, which are most known for their role in the hypothalamic-pituitary-adrenal stress axis. However, peripheral CRF signaling also has profound effects on gastrointestinal functions. Although the murine animal model is highly relevant for the exploration of this complexly balanced pathway via genetic manipulation, little is known about the expression of CRF and UCNs in the mouse intestine. This study aims to investigate the cellular localization of CRF and UCNs in the ileum and to explore whether and how this cellular expression is altered in conditions of intestinal Schistosoma mansoni-induced inflammation. The results show a distinct expression pattern for the different CRF receptor ligands in the ileum. CRF was located in nerve fibers and stromal cells. All UCNs were expressed in polymorphonuclear leukocytes. Furthermore, UCN2 and UCN3 were found in the musculature. During acute schistosomiasis, UCN1 showed an increased immunoreactivity in blood vessels and UCN3 was de novo expressed mainly in submucous neurons. Typical features of S. mansoni-inflamed ileum, such as nerve fiber sprouting, muscle layer thickening and granuloma formation thus all have an impact on the CRF signaling pathways. In conclusion, we outline for the first time the expression of CRF signaling ligands in the mouse ileum; our results point to important changes of this signaling system in S. mansoni-induced intestinal inflammation, which warrants further functional investigation with specific focus on CRF2, given the exclusive binding of UCN2 and UCN3 to this receptor.

An unusual case of aortic valve obstruction.

During a dissection class for anatomy, a white lipoid mass was found in the ascending aorta, which was partly attached to the wall and filled the sinuses ofValsalva and almost fitting as a cast. This mass prevented full opening of the mobile aortic valve leaflets, thereby causing an obstruction. Microscopic analysis revealed fibres and presence of polymorphonuclear white blood cells. It seems reasonable to assume that this mass has formed in the last weeks or months of the life of this subject, which is much quicker than for calcified aortic valve stenosis. Therefore, signs and symptoms of aortic obstruction might have been missed or misinterpreted. In case of timely detection during life, diagnostic imaging and therapeutic approach can be challenging.

Expression of neuropeptides and anoctamin 1 in the embryonic and adult zebrafish intestine, revealing neuronal subpopulations and ICC-like cells.

This immunohistochemical study in zebrafish aims to extend the neurochemical characterization of enteric neuronal subpopulations and to validate a marker for identification of interstitial cells of Cajal (ICC). The expression of neuropeptides and anoctamin 1 (Ano1), a selective ICC marker in mammals, was analyzed in both embryonic and adult intestine. Neuropeptides were present from 3 days postfertilization (dpf). At 3 dpf, galanin-positive nerve fibers were found in the proximal intestine, while calcitonin gene-related peptide (CGRP)- and substance P-expressing fibers appeared in the distal intestine. At 5 dpf, immunoreactive fibers were present along the entire intestinal length, indicating a well-developed peptidergic innervation at the onset of feeding. In the adult intestine, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), galanin, CGRP and substance P were detected in nerve fibers. Colchicine pretreatment enhanced only VIP and PACAP immunoreactivity. VIP and PACAP were coexpressed in enteric neurons. Colocalization stainings revealed three neuronal subpopulations expressing VIP and PACAP: a nitrergic noncholinergic subpopulation, a serotonergic subpopulation and a subpopulation expressing no other markers. Ano1-immunostaining revealed a 3-dimensional network in the adult intestine containing multipolar cells at the myenteric plexus and bipolar cells interspersed between circular smooth muscle cells. Ano1 immunoreactivity first appeared at 3 dpf, indicative of the onset of proliferation of ICC-like cells. It is shown that the Ano1 antiserum is a selective marker of ICC-like cells in the zebrafish intestine. Finally, it is hypothesized that ICC-like cells mediate the spontaneous regular activity of the embryonic intestine.

Transient receptor potential vanilloid type 1 channel (TRPV1) immunolocalization in the murine enteric nervous system is affected by the targeted C-terminal epitope of the applied antibody.

The expression of transient receptor potential vanilloid type 1 channel (TRPV1) in the enteric nervous system is still the subject of debate. Although a number of studies have reported that TRPV1 is limited to extrinsic afferent fibers, other studies argue for an intrinsic expression of TRPV1. In the present study, reverse transcriptase PCR was employed to establish the expression of TRPV1 mRNA throughout the gastrointestinal tract. Using two antibodies directed against different epitopes of TRPV1, we were able to show at the protein level that the observed distribution pattern of TRPV1 is dependent on the antibody used in the immunohistochemical staining. A first antibody indeed mainly stained neuronal fibers, whereas a second antibody exclusively stained perikarya of enteric neurons throughout the mouse gastrointestinal tract. We argue that these different distribution patterns are due to the antibodies discriminating between different modulated forms of TRPV1 that influence the recognition of the targeted immunogen and as such distinguish intracellular from plasmalemmal forms of TRPV1. Our study is the first to directly compare these two antibodies within the same species and in identical conditions. Our observations underline that detailed knowledge of the epitope that is recognized by the antibodies employed in immunohistochemical procedures is a prerequisite for correctly interpreting experimental results.

Expression and distribution patterns of Mas-related gene receptor subtypes A-H in the mouse intestine: inflammation-induced changes.

Mas-related gene (Mrg) receptors constitute a subfamily of G protein-coupled receptors that are implicated in nociception, and are as such considered potential targets for pain therapies. Furthermore, some Mrgs have been suggested to play roles in the regulation of inflammatory responses to non-immunological activation of mast cells and in mast cell-neuron communication. Except for MrgD, E and F, whose changed expression has been revealed during inflammation in the mouse intestine in our earlier studies, information concerning the remaining cloned mouse Mrg subtypes in the gastrointestinal tract during (patho) physiological conditions is lacking. Therefore, the present study aimed at identifying the presence and putative function of these remaining cloned Mrg subtypes (n = 19) in the (inflamed) mouse intestine. Using reverse transcriptase-PCR, quantitative-PCR and multiple immunofluorescence staining with commercial and newly custom-developed antibodies, we compared the ileum and the related dorsal root ganglia (DRG) of non-inflamed mice with those of two models of intestinal inflammation, i.e., intestinal schistosomiasis and 2,4,6-trinitrobenzene sulfonic acid-induced ileitis. In the non-inflamed ileum and DRG, the majority of the Mrg subtypes examined were sparsely expressed, showing a neuron-specific expression pattern. However, significant changes in the expression patterns of multiple Mrg subtypes were observed in the inflamed ileum; for instance, MrgA4, MrgB2and MrgB8 were expressed in a clearly increased number of enteric sensory neurons and in nerve fibers in the lamina propria, while de novo expression of MrgB10 was observed in enteric sensory neurons and in newly recruited mucosal mast cells (MMCs). The MrgB10 expressing MMCs were found to be in close contact with nerve fibers in the lamina propria. This is the first report on the expression of all cloned Mrg receptor subtypes in the (inflamed) mouse intestine. The observed changes in the expression and cellular localization of the Mrg subtypes suggest that these receptors are involved in the mediation of primary afferent responses, mast cell responses, and in neuroimmune communication during intestinal inflammation.

Whole-genome microarray analysis and functional characterization reveal distinct gene expression profiles and patterns in two mouse models of ileal inflammation.

BACKGROUND: Although a number of intestinal inflammatory conditions pertain to the ileum, whole-genome gene expression analyses in animal models of ileal inflammation are lacking to date. Therefore, we aimed to identify and characterize alterations in gene expression in the acutely inflamed ileum of two murine models of intestinal inflammation, namely intestinal schistosomiasis and TNBS-induced ileitis, compared to healthy controls. To this end, we used whole-genome microarrays, followed by bioinformatics analyses to detect over-represented Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology categories. RESULTS: Following screening of almost all known mouse genes and transcripts represented on the array, intestinal schistosomiasis and TNBS-induced ileitis yielded 207 and 1417 differentially expressed genes, respectively, with only 30 overlapping concordantly changed genes. Functional category groups consisting of complement and coagulation cascades, extracellular matrix (ECM)-receptor interaction, Fc epsilon receptor I signaling pathways and protein activation cascade, cell adhesion categories were over-represented in the differential gene list of intestinal schistosomiasis. Antigen processing and presentation, cell adhesion molecules, ABC transporters, Toll-like receptor signaling pathways and response to chemical stimulus categories were over-represented in the differential gene list of TNBS-induced ileitis. Although cytokine-cytokine receptor interaction, intestinal immune network for IgA production, focal adhesion pathways and immune, inflammatory and defense response categories were over-represented in the differential gene lists of both inflammation models, the vast majority of the associated genes and changes were unique to each model. CONCLUSIONS: This study characterized two models of ileal inflammation at a whole-genome level and outlined distinct gene expression profiles and patterns in the two models. The results indicate that intestinal schistosomiasis involves Th2 responses, complement activation, protein activation and enhanced ECM turnover, while TNBS-induced ileitis involves Th17 responses, defective antigen processing and presentation and altered Toll-like receptor-mediated responses. Signs of an impaired epithelial barrier are apparent in both inflammation models. Furthermore, the comprehensive differential gene list and functional groups provided by this study constitute an interesting starting point to explore new targets and extended functional networks dealing with small bowel inflammation.

The effect of inflammation on the expression and distribution of the MAS-related gene receptors MrgE and MrgF in the murine ileum.

The MAS-related gene (Mrg) receptor MrgE has been suggested to be expressed at all tissue levels involved in pain sensation and to influence the expression of another Mrg receptor, MrgF. Given the knowledge on the role of the enteric nervous system (ENS) in sensation, and the plasticity of enteric neurons during intestinal inflammation, it can be hypothesized that MrgE is expressed in enteric neurons, and that MrgE and MrgF change expression in intestinal inflammatory conditions. Therefore, we aimed to reveal the expression details of MrgE and MrgF in the murine ileum in normal and inflamed conditions. Using reverse transcriptase-PCR, quantitative-PCR and immunohistochemistry, we compared the ileum of non-inflamed control mice with that of two models of intestinal inflammation, i.e. intestinal schistosomiasis and chemically induced ileitis. MrgE and MrgF mRNAs were detected in control and inflamed conditions. MrgE and MrgF mRNAs showed a trend towards downregulation during intestinal schistosomiasis and a significant reduction during ileitis. MrgE and MrgF receptors were expressed in distinct enteric neuronal subpopulations, such as the sensory, secretomotor and vasodilator neurons, and in nerve fibres in the tunica muscularis and lamina propria of control and inflamed ileum. Only a minor proportion of enteric neurons co-expressed MrgE and MrgF. The number of enteric neurons expressing MrgE and MrgF receptors was significantly reduced during intestinal schistosomiasis and ileitis. This is the first report on the expression of MrgE and MrgF in the ENS in (patho)physiological conditions. The expression of MrgE and MrgF in enteric neurons was negatively affected by inflammation.

The DFNA5 gene, responsible for hearing loss and involved in cancer, encodes a novel apoptosis-inducing protein.

DFNA5 was first identified as a gene causing autosomal dominant hearing loss (HL). Different mutations have been found, all exerting a highly specific gain-of-function effect, in which skipping of exon 8 causes the HL. Later reports revealed the involvement of the gene in different types of cancer. Epigenetic silencing of DFNA5 in a large percentage of gastric, colorectal and breast tumors and p53-dependent transcriptional activity have been reported, concluding that DFNA5 acts as a tumor suppressor gene in different frequent types of cancer. Despite these data, the molecular function of DFNA5 has not been investigated properly. Previous transfection studies with mutant DFNA5 in yeast and in mammalian cells showed a toxic effect of the mutant protein, which was not seen after transfection of the wild-type protein. Here, we demonstrate that DFNA5 is composed of two domains, separated by a hinge region. The first region induces apoptosis when transfected in HEK293T cells, the second region masks and probably regulates this apoptosis inducing capability. Moreover, the involvement of DFNA5 in apoptosis-related pathways in a physiological setting was demonstrated through gene expression microarray analysis using Dfna5 knockout mice. In view of its important role in carcinogenesis, this finding is expected to lead to new insights on the role of apoptosis in many types of cancer. In addition, it provides a new line of evidence supporting an important role for apoptosis in monogenic and complex forms of HL.

Neurochemical coding of enteric neurons in adult and embryonic zebrafish (Danio rerio).

Although the morphology and development of the zebrafish enteric nervous system have been extensively studied, the precise neurochemical coding of enteric neurons and their proportional enteric distribution are currently not known. By using immunohistochemistry, we determined the proportional expression and coexpression of neurochemical markers in the embryonic and adult zebrafish intestine. Tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), and pituitary adenylate cyclase-activating peptide (PACAP) were observed only in nerve fibers, whereas other markers were also detected in neuronal cell bodies. Calretinin and calbindin had similar distributions. In embryos, all markers, except for choline acetyltransferase (ChAT) and TH, were present from 72 hours postfertilization. Nitrergic neurons, evenly distributed and remaining constant in time, constituted the major neuronal subpopulation. The neuronal proportions of the other markers increased during development and were characterized by regional differences. In the adult, all markers examined were expressed in the enteric nervous system. A large percentage of enteric neurons displayed calbindin and calretinin, and serotonin was the only marker showing significant distribution differences in the three intestinal regions. Colocalization studies showed that serotonin was not coexpressed with any of the other markers. At least five neuronal subpopulations were determined: a serotonergic, a nitrergic noncholinergic, two cholinergic nonnitrergic subpopulations along with one subpopulation expressing both ChAT and neuronal nitric oxide synthase. Analysis of nerve fibers revealed that nitrergic neurons coexpress VIP and PACAP, and that nitrergic neurons innervate the tunica muscularis, whereas serotonergic and cholinergic nonnitrergic neurons innervate the lamina propria and the tunica muscularis.

Chronic alcohol consumption affects gastrointestinal motility and reduces the proportion of neuronal NOS-immunoreactive myenteric neurons in the murine jejunum.

Alcohol consumption interferes with gastrointestinal transit causing symptoms in alcoholic patients. Nitric oxide (NO), synthesized by neuronal nitric oxide synthase (nNOS) plays an important role in the control of gastrointestinal motility. Our aim was to investigate whether chronic alcohol intake in a murine model induces gastrointestinal motility disturbances and affects the nitrergic myenteric neurons in the stomach and jejunum. Gastric emptying, small intestinal transit and geometric centre were measured in vivo after intragastric gavage of Evans blue. Nitrergic relaxations to electrical field stimulation (EFS) and exogenous NO were recorded in jejunal muscle strips in vitro. The proportion of nNOS-immunopositive myenteric neurons was assessed using PGP9.5 and nNOS immunostaining. After chronic alcohol consumption, gastric emptying and small intestinal transit were delayed compared with control mice, and the nitrergic nerve-mediated relaxations to EFS in the jejunum were decreased, whereas relaxations to exogenous NO did not differ. The proportion of nNOS-immunoreactive neurons did not change in the stomach, whereas in the jejunum the percentage decreased from 33% to 27% (P < 0.001) after chronic alcohol intake. The total number of myenteric neurons remained unchanged. These results suggest that chronic alcohol consumption disturbs gastric and small intestinal motility in vivo and in vitro and is associated with a decrease in the proportion of nNOS-immunoreactive myenteric neurons in the murine jejunum.

Current perspective of pathophysiological and interventional effects on endothelial progenitor cell biology: focus on PI3K/AKT/eNOS pathway.

For more than a decade, endothelial progenitor cells (EPCs) have been implicated in cardiovascular homeostasis. EPCs are believed to reside within the bone marrow in close contact with surrounding stromal cells, and, under stimulation of pro-inflammatory cytokines, EPCs are mobilized out of the bone marrow. Hereafter circulating EPCs home to peripheral tissues, undergoing further proliferation and differentiation. Under certain pathophysiologic conditions this process seems to be blunted, resulting in a reduced capacity of EPCs to engage in vasculogenesis at sites of endothelial injury or tissue ischemia. In this review, we focus on the effects of traditional cardiovascular risk factors on EPC biology and we explore whether pharmacological, dietary and lifestyle interventions can favorably restore EPC mobilization, differentiation, homing and angiogenic properties. Because the PI3K/Akt/eNOS pathway plays a pivotal role in the process of EPC mobilization, migration and homing, we specifically emphasize the involvement of PI3K, Akt and eNOS in EPC biology under these different (patho)physiologic conditions. (Pre)clinically used drugs or lifestyle interventions that have been shown to ameliorate EPC biology are reviewed. These treatment strategies remain attractive targets to restore the regenerative capacity of EPCs in cardiovascular diseases.