No description available.

Submucosal arterioles in the small intestine are the main point of control for gastrointestinal (GI) circulation as they are the final resistance vessels feeding the highly perfused mucosal layer. Ischemia can lead to pathophysiology of a variety of GI tissues. In chronic intestinal inflammation, alterations in blood flow have been purported to be involved in disease etiology. The aim of this study was to characterize purinergic neurotransmitter pathways involved in physiological submucosal arteriole diameter control by the enteric nervous system long vasodilatory reflex (LVD) and to establish a protocol to determine how inflammatory neural changes affect vasodilation in the small intestine. Following euthanasia, segments of small intestine were harvested from adult male guinea pigs and changes in nerve stimulated small intestine submucosal arteriole diameter were identified using videomicroscopy techniques; vessels were preconstricted and nicotinic cholinergic transmission was blocked with hexamethonium. Purinergic receptor antagonists were applied. Immunohistochemical analysis was conducted to identify P2Y1 receptors localization. In a subset of experiments sensory neuronal excitability was initiated using phorbol dibutyrate (PDBu) shown previously to induce hyperexcitability in the sensory neurons similar to changes found in intestinal inflammation. In these experiments, intestinal segments were placed into a novel dual chamber bath separated into two portions and PDBu was applied unilaterally. Blood vessel vasodilation was either abolished or decreased by both suramin (100 μM, n=6), a non-specific P2 purinergic antagonist. MRS 2179 (10 μM, n=5), a P2Y1 specific antagonist, also decreased vasodilation, which suggests that there is a purinergic neurotransmission component to the LVD mediated by P2 receptors, including the P2Y1 subtype. Immunohistochemistry identified P2Y1 receptor staining that was uniformly punctated in both the myenteric and submucosal plexuses but specific neuronal locations of the receptor could not be identified. Nerve stimulated vasodilation was not altered by application of PDBu suggesting that neuronal hypersensitivity did not modify vessel dilation. Taken together these data suggest that purinergic receptor pathways contribute to the LVD reflex under normal conditions however more experiments are still required to fully elucidate how these pathways are affected /altered by intestinal inflammation. / Thesis (Master, Physiology) -- Queen's University, 2012-05-30 11:38:43.324

The enteric nervous system (ENS) is commonly referred to as the ‘second brain’ due to its complex networks of neuronal cells. The abnormality of these neurons and/or their absence has been shown to play a fundamental role in diseases of both the ENS and the central nervous system. Accordingly, electrophysiological studies of the ENS and general understanding of how enteric neurons behave in the gastrointestinal tract are critical in the characterization of the pathophysiology of enteric and neurodevelopmental diseases. To date, studies on these aspects have been limited by the difficulty of culturing enteric neurons in-vitro, as well as by their poor adhesion properties. The primary objectives of this thesis are to develop strategies to investigate electrodynamics processes of enteric neurons and close in on their interactions with polymeric substrates, aiming at optimizing conventional experimental approaches and expanding the current knowledge and critical understanding of this elusive cell type. By capitalizing on a rapid and efficient culturing method developed by our group, different polymers were tested in order to assess their ability to promote adhesion of enteric neurons, as confirmed by immunofluorescence analysis. The most effective polymer resulting from this initial screening was then applied as a coating onto the glass surface of multichannel electrode arrays (MEAs) allowing for the analysis of neuron dynamics. While Matrigel® was the most effective at promoting both neuron adhesion and neurite outgrowth, it acted as an insulating material which prevented the MEA electrodes from picking up electrical signals. Therefore, we opted instead for laminin protein and poly-d-lysine immobilized on glass by polydopamine, to study the electrophysiology of the neurons. Of note, polydopamine was found to be critical in enhancing the stability of the protein coating and ensuring cellular viability. The same protein coating was also used to functionalize the surface of blends of poly(styrene) and poly(methyl methacrylate), which segregate when mixed to give rise to varying topographical features. These surfaces aimed at elucidating fundamental processes that dictate how neurons interact with surfaces when compared to smooth rigid surfaces (i.e. glass). Finally, the most effective surface for neuron adhesion was applied to study how chemotaxis influences neurite elongation and directionality. Enteric neurons were cultured onto both a linear concentration gradient of protein created using a microfluidic system and a uniform concentration profile to compare their response to chemical signals. In general, their motion was random and lacked directionality on the uniform protein surface. The neuronal response to the chemical gradient could not be evaluated to completion; however, this analysis still provided meaningful insight as a starting point for future studies. The results presented in this thesis serve as a significant stepping-stone for the improvement of the in-vitro study of the ENS and will be used to gain a deeper understanding of enteric diseases, ultimately contributing to the development of novel polymeric scaffolds for tissue-engineering applications.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Physiology. Title from title-page of electronic thesis. Bibliography: leaves 145-[160].

5-HT powerfully affects gastrointestinal function. However, the study of these effects is complicated because 5-HT from both mucosa and a subset of enteric neurons acts on multiple receptor subtypes in enteric tissues. The role of neural 5-HT has been difficult to isolate with current techniques. This thesis aimed to elucidate the role of 5-HT neurons in motility using anatomical and functional methods. In Chapter 2, confocal microscopy was used to examine over 95% of myenteric neurons in guinea pig jejunum, categorized neurochemically, to identify neurons that received anatomically-defined input from 5-HT interneurons. The data showed that cholinergic secretomotor neurons were strongly targeted by 5-HT interneurons. In another key finding, excitatory motor neurons were surrounded by 5-HT terminals; this could provide an anatomical substrate for the descending excitation reflex. Subgroups of ascending interneurons and neurons with immunoreactivity for NOS, were also targeted by 5-HT interneurons. Thus, subtypes of these neurons might act in separate reflex pathways. Despite strong physiological evidence for 5-HT inputs to AH/Dogiel type II neurons, few contacts were identified. In Chapter 3, the confocal microscopy survey was extended to the three other interneuron classes (VIP/NOS and SOM descending interneurons; calretinin ascending interneurons) of guinea pig small intestine. A high degree of convergence between the otherwise polarized ascending and descending interneuron pathways was identified.

Thesis (M. Phil.)--University of Hong Kong, 2007. / Also available in print.

Diss. (sammanfattning) Göteborg : Göteborgs universitet , 2007. / Härtill 4 uppsatser.

The Endocannabinoid system has been found in the gastrointestinal tract, where it plays an important role in gut under both physiological and pathological conditions. Although the major effects of cannabinoids in the gut are mediated through effects on enteric neurons, the role of cannabinoids in the enteric nervous system is poorly understood. In the present study, we have used the primary cultures of myenteric ganglia and a newly developed fetal enteric neuronal cell line to identify whether the endocannabinoid, anandamide, affects ganglionic and neuronal survival and the pathways involved. Anandamide had a biphasic effect on ganglionic survival, increasing survival at low concentrations (1nM-0.1uM) and decreasing survival at high concentrations (1-10uM). Maximal survival (68% increase in number of ganglia surviving) occurred at 0.1uM and the ED50 was 3nM. This effect on promoting survival was inhibited by the CB1 antagonist AM251 (1uM) and by AraC (10uM), but not the CB2 antagonist AM630 (1uM). AM630 (1uM) significantly blocked the decreased survival induced by high concentration anandamide (10uM). The enteric glia was involved in anandamide-induced ganglion survival. Anandamide had no effect on the number of neurons/ganglion in the presence of enteric glia, but decreased the number of neurons/ganglion by 15-20% in absence of enteric glia. This effect was partially reversed by CB1 antagonist, AM251 (1uM) (20%-145% at 1nM-10uM) and by CB2 antagonist AM630 (1uM) (40%-185% at 1nM-10uM). In the fetal enteric neural cell line (IM-FEN), anandamide decreased enteric neuronal survival in a concentration-dependent manner at both 39 and 33 degree (11-45% and 10-22%decrease in survival at 1nM-10uM, respectively). Coculture of astrocytes with the enteric neuronal cells was not able to reverse anandamide-mediated neuronal death. Immunocytochemistry and western blot confirmed that the presence of both CB1 and CB2 receptors in enteric neurons (primary cultures and IM-FEN) and glia (primary cultures). In addition, the PLC-beta inhibitor U73122 (1uM) inhibited anandamide induced ganglia survival significantly. Anandamide also induced increased expression of phospho-P44/42MAPK (13-48% at 1nM-10uM) and phospho-AKT (1-28% at 1 nM-10uM) in IM-FEN. We conclude that anandamide has a differential effect on survival of enteric ganglia and neurons. It promotes ganglionic and neuronal survival by CB1 receptors in the presence of glia and this involves the PLC-beta pathway. Conversely, anandamide promotes neuron death in absence of glia as a result of effects on both the MAPK and PI-3K/AKT pathways. Since the endocannabinoid system is upregulated in inflammatory bowel diseases, these effects may play a role in the pathogenesis of the response to inflammation as well as the recovery and reinnervation of the gut following the acute phase of inflammation. The further significance of this work could contribute to developing new therapeutic methods for treatment of inflammatory bowel disease and related symptoms in clinic practice.

Inflammatory bowel disease causes structural and functional alterations in the enteric nervous system (ENS). Since the onset of intestinal inflammation involves the activation of resident immune cells as well as rapid influx of infiltrating cells, we proposed that changes in the ENS are a result of the release of toxic inflammatory factors. We hypothesized that early damage to the ENS in inflammation is caused by harmful levels of nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) found in immune cells. This was assessed in the 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-model of colitis in rats. Large increases in infiltrating granulocytes, particularly neutrophils and blood-derived monocytes were found in the muscularis layers adjacent to the ENS. A rapid increase in iNOS immunoreactivity in the muscularis regions during early stages of inflammation (6 – 24 hr) was observed. Whether high NO levels generated by chemical donors could be toxic to neurons was tested in a co-culture model of myenteric neurons, smooth muscle and glia enzymatically isolated from neonatal rats. Exposure of co-cultures to NO for 48 hr resulted in significant, concentration dependent decrease in neuron survival. We then developed a model that permitted the direct study of immune cell interactions with myenteric neurons. Myenteric neurons were co-cultured with activated peritoneal immune cells that expressed iNOS and generated high NO levels (49 + 6.2µM) for 48 hr. This caused significant neuronal death, reducing neuron number by 19 + 5%, and disruption of axons. Pre-treatment of immune cells with a selective iNOS-inhibitor, L-NIL resulted in neuron numbers that were not significantly different from control (96 + 2%) suggesting that NO played a central role in mediating the damaging effects of immune cells. Lastly, when direct contact between immune cells and neurons was prevented in the previous experiment through use of trans-wells, unanticipated neurotrophic effects were observed. Increased axon outgrowth (282 + 57%) was detected in addition to loss of the neurotoxic effects in spite of similar experimental conditions. We concluded that proximity and contact plays an important role in determining the nature of immune cell mediated alterations in enteric neurons. / Thesis (Master, Physiology) -- Queen's University, 2009-11-30 10:09:38.384