Elin Sand

Vasoactive intestinal peptide rescues cultured rat myenteric neurons from lipopolysaccharide induced cell death

UNLABELLED The role of the enteric nervous system in intestinal inflammation is not fully understood and the plethora of cellular activities concurrently ongoing in vivo renders intelligible studies difficult. In order to explore possible effects of bacterial lipopolysaccharide (LPS) on enteric neurons we utilised cultured myenteric neurons from rat small intestine. Exposure to LPS caused markedly reduced neuronal survival and increased neuronal expression of vasoactive intestinal peptide (VIP), while the expression of Toll-like receptor 4 (TLR4) was unchanged. TLR4 was expressed in approximately 35% of all myenteric neurons irrespective of if they were cultured in the presence or absence of LPS. In neurons cultured in medium, without LPS, 50% of all TLR4-immunoreactive neurons contained also VIP. Addition of LPS to the neuronal cultures markedly increased the proportion of TLR4-immunoreactive neurons also expressing VIP, while the proportion of TLR4 neurons devoid of VIP decreased. Simultaneous addition of LPS and VIP to the neuronal cultures resulted in a neuronal survival comparable to controls. CONCLUSIONS LPS recognition by myenteric neurons is mediated via TLR4 and causes neuronal cell death. Presence of VIP rescues the neurons from LPS-induced neurodegeneration.

Enteric Neuropathy Can Be Induced by High Fat Diet In Vivo and Palmitic Acid Exposure In Vitro

Objective Obese and/or diabetic patients have elevated levels of free fatty acids and increased susceptibility to gastrointestinal symptoms. Since the enteric nervous system is pivotal in regulating gastrointestinal functions alterations or neuropathy in the enteric neurons are suspected to occur in these conditions. Lipid induced intestinal changes, in particular on enteric neurons, were investigated in vitro and in vivo using primary cell culture and a high fat diet (HFD) mouse model. Design Mice were fed normal or HFD for 6 months. Intestines were analyzed for neuronal numbers, remodeling and lipid accumulation. Co-cultures of myenteric neurons, glia and muscle cells from rat small intestine, were treated with palmitic acid (PA) (0 – 10−3 M) and / or oleic acid (OA) (0 – 10−3 M), with or without modulators of intracellular lipid metabolism. Analyses were by immunocyto- and histochemistry. Results HFD caused substantial loss of myenteric neurons, leaving submucous neurons unaffected, and intramuscular lipid accumulation in ileum and colon. PA exposure in vitro resulted in neuronal shrinkage, chromatin condensation and a significant and concentration-dependent decrease in neuronal survival; OA exposure was neuroprotective. Carnitine palmitoyltransferase 1 inhibition, L-carnitine- or alpha lipoic acid supplementation all counteracted PA-induced neuronal loss. PA or OA alone both caused a significant and concentration-dependent loss of muscle cells in vitro. Simultaneous exposure of PA and OA promoted survival of muscle cells and increased intramuscular lipid droplet accumulation. PA exposure transformed glia from a stellate to a rounded phenotype but had no effect on their survival. Conclusions HFD and PA exposure are detrimental to myenteric neurons. Present results indicate excessive palmitoylcarnitine formation and exhausted L-carnitine stores leading to energy depletion, attenuated acetylcholine synthesis and oxidative stress to be main mechanisms behind PA-induced neuronal loss.High PA exposure is suggested to be a factor in causing diabetic neuropathy and gastrointestinal dysregulation.

Mast cells reduce survival of myenteric neurons in culture

Mast cell-nerve interactions play a key role in intestinal inflammation and irritable bowel disease. Loss of enteric neurons has been reported in inflammatory conditions but the contribution of mast cells in this event is unknown. To study neuronal survival and plasticity of myenteric neurons in contact with mast cells a co-culture system using myenteric neurons from rat small intestine and peritoneal mast cells was set up. Dissociated myenteric neurons were cultured for 4 days before addition of mast cells isolated by peritoneal lavage. Neuronal survival and expression of vasoactive intestinal peptide (VIP) and nitric oxide synthase (NOS) were studied by immunocytochemistry and neuronal cell counting. Myenteric neurons cultured without mast cells were used to study the rate of neuronal survival after the addition of various mast cell mediators, proteinase-activated receptor(2) (PAR(2)) agonist, VIP or corticosteroid. A striking mast cell-induced neuronal cell death was found after co-culturing. It was counteracted by the addition of mast cell stabiliser doxantrazole, protease inhibitors, PAR(2) antagonist FSLLRY-amide, corticosteroid or VIP. In myenteric neurons cultured without mast cells the PAR(2) agonist SLIGRL-amide, prostaglandin D(2) and interleukin (IL) 6 reduced neuronal survival while histamine, serotonin, heparin, IL1beta and tumour necrosis factor alpha had no effect; corticosteroid and VIP enhanced neuronal survival. The relative numbers of VIP-, but not NOS-expressing myenteric neurons increased after co-culturing. Mast cell-induced neuronal cell death is suggested to be mediated via PAR(2) activation, IL6 and prostaglandin D(2). Corticosteroid and VIP are neuroprotective and able to prevent cell death of myenteric neurons in co-culture.

Glucagon-like peptides 1 and 2 and vasoactive intestinal peptide are neuroprotective on cultured and mast cell co-cultured rat myenteric neurons

BackgroundNeuropathy is believed to be a common feature of functional and inflammatory intestinal diseases. Vasoactive intestinal peptide (VIP) is an acknowledged neuroprotective agent in peripheral, including enteric, and central neurons. The proglucagon-like hormones glucagon-like peptide 1 and 2 (GLP1 and GLP2) belong to the secretin/glucagon/VIP superfamily of peptides and GLP1 and GLP2 receptors are expressed in enteric neurons. Possible neuroprotective effects of these peptides were investigated in the present study.MethodsGLP1, GLP2 and VIP were added to cultured myenteric neurons from rat small intestine or to co-cultures of myenteric neurons and rat peritoneal mast cells. Receptor selectivity was tested by the simultaneous presence of a GLP1 receptor antagonist (exendin (9-39) amide) or a VIP receptor antagonist (hybrid of neurotensin 6-11 and VIP 7-28). Neuronal survival was examined using immunocytochemistry and cell counting.ResultsGLP1, GLP2 and VIP significantly and concentration-dependently enhanced neuronal survival. In addition the peptides efficiently counteracted mast cell-induced neuronal cell death in a concentration-dependent manner. Exendin(9-39)amide reversed GLP1-induced neuroprotection while GLP2- and VIP-induced enhanced neuronal survival were unaffected. The VIP receptor antagonist reversed GLP1- and VIP-induced neuroprotection while the GLP2-induced effect on neuronal survival was unaffected.ConclusionsBy activating separate receptors VIP, GLP1 and GLP2 elicit neuroprotective effects on rat myenteric neurons cultured with or without mast cells. This implies a powerful therapeutic potential of these peptides in enteric neuropathies with a broad spectrum of applications from autoimmunity to functional disorders.

Expression and distribution of GnRH, LH, and FSH and their receptors in gastrointestinal tract of man and rat

BACKGROUND Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) regulate the reproductive axis. Their analogs have been found to influence gastrointestinal activity and enteric neuronal survival. The aims of the study were to investigate expression and cellular distribution of GnRH, LH, and FSH and their receptors in human and rat gastrointestinal tract. METHODS Bioinformatic analysis of publicly available microarray gene expression data and Real-Time PCR mRNA quantification were used to study mRNA expression levels of hormones and receptors in human intestinal tissue. Full-thickness sections of human ileum and colon, and rat stomach, ileum, and colon, were used for immunocytochemistry. Antibodies against human neuronal protein HuC/D (HuC/D) were used as general neuronal marker. LH and FSH, and GnRH-, LH-, and FSH receptor immunoreactive (IR) neurons were evaluated. RESULTS GnRH1 mRNA was detected in both small and large intestine, whereas GnRH2 was mainly expressed in small intestine. Approximately 20% of both submucous and myenteric neurons displayed LH receptor immunoreactivity in human ileum and colon. In rat, 4%-9% of all enteric neurons in fundus and ileum, and 13% of submucous neurons and 21% of myenteric neurons in colon were LH receptor-IR. Neither mRNA (man) nor the fully expressed proteins (man and rat) of LH and FSH, or GnRH and FSH receptors, could be detected. CONCLUSIONS GnRH1 and GnRH2 mRNA are expressed in human intestine. LH receptor-IR enteric neurons are found along the entire gastrointestinal tract in both man and rat.

Corticotropin releasing factor-Distribution in rat intestine and role in neuroprotection

UNLABELLED Aims of the present study were to describe the distribution of corticotropin releasing factor (CRF) immunoreactivity in rat small and large intestines, to quantify the percentage of CRF-immunoreactive (CRF-IR) enteric neurons, to reveal possible CRF immunoreactivity in cultured myenteric neurons from rat ileum and to examine if additions of CRF, urocortin 1 (Ucn1), CRF antagonist or vasoactive intestinal peptide (VIP) affect neuronal survival in vitro. Co-localization of CRF- and VIP-immunoreactivity was examined, as well as a possible interplay between CRF and VIP in neuroprotection. Further we wanted to elucidate if mast cells affect neuronal survival via CRF signaling. Networks of CRF-containing nerve cell bodies and fibers were detected in rat intestine. CRF-IR neurons contained to a high degree also VIP. A low number of cultured myenteric neurons was CRF-IR. CRF, Ucn1 or CRF-antagonist did not promote neuronal survival of cultured myenteric neurons, while VIP significantly enhanced neuronal survival. Simultaneous presence of CRF attenuated the VIP mediated increase in neuronal survival. Co-culturing neurons and mast cells resulted in a marked reduction in neuronal survival, not executed via CRF signaling pathways. CONCLUSION CRF is present in enteric neurons and counteracts the neuroprotective effect of VIP in vitro.

Severe gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs

Abstract Background. Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. Methods. Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skåne University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. Results. Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. Conclusion. A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs.

Ganglioneuritis is common in rats with enteric neuropathy due to buserelin treatment.

• The aim was to further characterize the enteric histopathological changes in a rat model.

Luteinizing hormone receptors are expressed in rat myenteric neurons and mediate neuronal loss.

BACKGROUND Clinical observations have suggested repeated gonadotropin-releasing hormone (GnRH) exposure to cause intestinal dysfunction and loss of enteric neurons. This has been further studied and confirmed in a rat in vivo model involving iterated GnRH treatments. Mechanisms behind are enigmatic since no GnRH receptors are found to be expressed in enteric neurons neither in man nor rat. Both species, however, harbor substantial subpopulations of luteinizing hormone (LH) receptor-immunoreactive myenteric neurons which suggests that intestinal GnRH-induced neuropathy may be mediated by LH release. AIMS To reveal if exposures of GnRH or LH to rat myenteric neurons in vitro cause neuronal loss. METHODS Primary cultured adult rat myenteric neurons were exposed to single or repeated treatments of the GnRH analog buserelin or the LH analog lutrotropin alpha, and neuronal survival was determined by cell counting. Possible presence of GnRH- or LH receptor -immunoreactive neurons was determined by immunocytochemistry. RESULTS Exposure to the LH, but not the GnRH, analog caused significantly reduced neuronal survival. LH, but not GnRH, receptors were found to be expressed on cultured myenteric neurons. CONCLUSION Myenteric neurons express LH receptors in vitro and LH exposure causes reduced neuronal survival. This suggests that GnRH-induced enteric neuropathy in vivo is mediated by way of LH release and activation of enteric neuronal LH receptors.

Characterization of Gastric Mucosa Biopsies Reveals Alterations in Huntington's Disease.

Weight loss is an important complication of Huntington’s disease (HD), however the mechanism for weight loss in HD is not entirely understood. Mutant huntingtin is expressed in the gastrointestinal (GI) tract and, in HD mice, mutant huntingtin inclusions are found within the enteric nervous system along the GI tract. A reduction of neuropeptides, decreased mucosal thickness and villus length, as well as gut motility impairment, have also been shown in HD mice. We therefore set out to study gastric mucosa of patients with HD, looking for abnormalities of mucosal cells using immunohistochemistry. In order to investigate possible histological differences related to gastric acid production, we evaluated the cell density of acid producing parietal cells, as well as gastrin producing cells (the endocrine cell controlling parietal cell function). In addition, we looked at chief cells and somatostatin-containing cells. In gastric mucosa from HD subjects, compared to control subject biopsies, a reduced expression of gastrin (a marker of G cells) was found. This is in line with previous HD mouse studies showing reduction of GI tract neuropeptides.