A novel serotonin-containing tuft cell subpopulation in mouse intestine.

In this study, a novel subset of doublecortin-like kinase 1 (DCLK1)-immunoreactive (IR) tuft cells that also contain serotonin (5-hydroxytryptamine, 5HT) is described, in terms of their number, regional distribution, possible synthesis or reuptake of 5HT and proximity to 5-HT-containing enterochromaffin (EC) cells. The small intestine from C57BL/6J mice was divided into five segments while the large intestine was kept undivided. Double immunostaining was used to estimate numbers and topographic distribution of 5HT-IR (DCLK1/5HT) tuft cells and their possible expression of tryptophan hydroxylase (TPH) and serotonin transporter (SERT). Also, possible contacts between tuft cells and 5HT-IR EC cells were studied. In the small intestine, up to 80% of all tuft cells were identified as DCLK1/5HT-IR; in the large intestine, such cells were rare. The highest number of DCLK1/5HT-IR cells was found in the upper small intestine. The numbers of DCLK1/5HT-IR cells gradually decreased distally. DCLK1-IR tuft cells were not found to contain TPH, the rate-limiting enzyme in 5HT synthesis. SERT, the selective transporter for 5HT reuptake, could not convincingly be demonstrated in tuft cells. In villi and crypts, 3% and 10%, respectively, of all DCLK1-IR cells were in close proximity to EC cells. EC cells in close proximity to DCLK1-IR cells were, in villi and crypts, 3 and 8%, respectively. We conclude that DCLK1/5HT-IR cells constitute a novel subset of tuft cells that may have unique roles in the GI tract.

Tuft cells: Distribution and connections with nerves and endocrine cells in mouse intestine.

Tuft cells are gastrointestinal (GI) sensory cells recognized by their characteristic shape and their microvilli "tuft". Aims of the present study were to elucidate their regional distribution and spatial connections with satiety associated endocrine cells and nerve fibers throughout the intestinal tract. C57BL/6 J mice were used in the experiments. The small intestine was divided into five segments, and the large intestine was kept undivided. The segments were coiled into "Swiss rolls". Numbers and topographic distribution of tuft cells and possible contacts with endocrine cells and nerve fibers were estimated in the different segments, using immunocytochemistry. Tuft cells were found throughout the intestines; the highest number was in proximal small intestine. Five percent of tuft cells were found in close proximity to cholecystokinin-immunoreactive (IR) endocrine cells and up to 10% were in contact with peptide YY- and glucagon-like peptide-1-IR endocrine cells. Sixty percent of tuft cells in the small intestine and 40% in the large intestine were found in contact with nerve fibers. Calcitonin gene-related peptide-IR fibers constituted one-third of the fiber-contacts in the small intestine and two-thirds in the large intestine. These observations highlight the possibility of tuft cells as modulators of GI activities in response to luminal signaling.

Focal, but not global, cerebral ischaemia causes loss of myenteric neurons and upregulation of vasoactive intestinal peptide in mouse ileum.

Reduced blood flow to the brain induces cerebral ischaemia, potentially causing central injury and peripheral complications including gastrointestinal (GI) dysfunction. The pathophysiology behind GI symptoms is suspected to be neuropathy in the enteric nervous system (ENS), which is essential in regulating GI function. This study investigates if enteric neuropathy occurs after cerebral ischaemia, by analysing neuronal survival and relative numbers of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expressing neurons in mouse ileum after three types of cerebral ischaemia. Focal cerebral ischaemia, modelled by permanent middle cerebral artery occlusion (pMCAO) and global cerebral ischaemia, modelled with either transient occlusion of both common carotid arteries followed by reperfusion (GCIR) or chronic cerebral hypoperfusion (CCH) was performed on C56BL/6 mice. Sham-operated mice for each ischaemia model served as control. Ileum was collected after 1-17 weeks, depending on model, and analysed using morphometry and immunocytochemistry. For each group, intestinal mucosa and muscle layer thicknesses, neuronal numbers and relative proportions of neurons immunoreactive (IR) for nNOS or VIP were estimated. No alterations in mucosa or muscle layer thicknesses were noted in any of the groups. Loss of myenteric neurons and an increased number of VIP-IR submucous neurons were found in mouse ileum 7 days after pMCAO. None of the global ischaemia models showed any alterations in neuronal survival or relative numbers of VIP- and nNOS-IR neurons. We conclude that focal cerebral ischaemia and global cerebral ischaemia influence enteric neuronal survival differently. This is suggested to reflect differences in peripheral neuro-immune responses.

The host defense peptide LL-37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils.

The human host defense peptide, LL-37, is an important player in the first line of defense against invading microorganisms. LL-37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL-37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL-37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL-37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL-37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL-37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL-37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL-37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL-37. For the first time, we demonstrate hCAP18/LL-37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL-37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL-37 content of whole saliva.

Galectin-3 causes enteric neuronal loss in mice after left sided permanent middle cerebral artery occlusion, a model of stroke.

In addition to brain injury stroke patients often suffer gastrointestinal complications. Neuroimmune interactions involving galectin-3, released from microglia in the brain, mediates the post-stroke pro-inflammatory response. We investigated possible consequences of stroke on the enteric nervous system and the involvement of galectin-3. We show that permanent middle cerebral artery occlusion (pMCAO) induces loss of enteric neurons in ileum and colon in galectin-3(+/+), but not in galectin-3(-/-), mice. In vitro we show that serum from galectin-3(+/+), but not from galectin-3(-/-), mice subjected to pMCAO, caused loss of C57BL/6J myenteric neurons, while myenteric neurons derived from TLR4(-/-) mice were unaffected. Further purified galectin-3 (10(-6) M) caused loss of cultured C57BL/6J myenteric neurons. Inhibitors of transforming growth factor ß-activated kinase 1 (TAK1) or AMP activated kinase (AMPK) counteracted both the purified galectin-3 and the galectin-3(+/+) pMCAO serum-induced loss in vitro. Combined we show that stroke (pMCAO) triggers central and peripheral galectin-3 release causing enteric neuronal loss through a TLR4 mediated mechanism involving TAK1 and AMPK. Galectin-3 is suggested a target for treatment of post-stroke complications.

Vitamin D-induced up-regulation of human keratinocyte cathelicidin anti-microbial peptide expression involves retinoid X receptor α.

The biologically active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25D3), has been reported to positively regulate the human cathelicidin anti-microbial peptide (CAMP) gene coding for LL-37, but the mechanisms are not completely understood. We have determined the expression of CAMP, vitamin D receptor (VDR), and the retinoid X receptor (RXR) isoforms in human skin and gingival tissue biopsies and investigated the signaling pathways involved in 1,25D3-induced upregulation of CAMP. Human skin and gingival biopsies exhibited few VDR-immunoreactive cells within the stratum basale, whereas rat colon enterocytes (positive control) possessed abundant VDR immunoreactivity. Nuclear VDR immunoreactivity was demonstrated in human skin keratinocytes (HaCaT cells). Gene analysis revealed that human skin biopsies expressed higher levels of both CAMP and RXRα mRNA than human gingival biopsies, whereas VDR and RXRß transcript levels were similar in skin and gingiva. In HaCaT cells, treatment with 1,25D3 (5 nM and 1 µM) for 4 and 24 h up-regulated CAMP mRNA several fold, and treatment with 1,25D3 for 24 h increased protein expression of the pro-form of LL-37 (hCAP-18) by about 13 times. The 1,25D3-evoked stimulation of HaCaT CAMP expression was associated with attenuated VDR mRNA and protein expression. Treatment with RXRα short interfering RNA reversed the 1,25D3-induced CAMP expression in HaCaT cells, showing that RXRα is involved in the up-regulation of CAMP by 1,25D3. We conclude that the 1,25D3-evoked stimulation of CAMP expression in human skin keratinocytes is dependent on RXRα but is not associated with the up-regulation of VDR expression.

Long­term follow­up of buserelin­induced enteric neuropathy in rats.

A few patients have been shown to develop severe abdominal pain and gastrointestinal dysmotility during treatment with gonadotropin­releasing hormone (GnRH) analogs. A rat model of enteric neuropathy has been developed by administration of the GnRH analog buserelin to rats. Loss of enteric neurons and ganglioneuritis throughout the gastrointestinal tract has been described, without other histopathological changes. The aim of the present study was to investigate the long­term effects of this rat model on body weight, and on morphology and inflammatory changes in the gastrointestinal tract. Rats were administered subcutaneous injections of buserelin or saline once daily for 5 days and allowed to recover for 3 weeks. This regimen was repeated four times. The rats were weighed weekly and were sacrificed 16 weeks after the fourth treatment. The bowel wall was measured by morphometry, and the presence of enteric neurons, mast cells, eosinophils and T­lymphocytes was evaluated. Buserelin­treated rats were shown to have a lower body weight at sacrifice, as compared with the controls (P<0.05). Compared with controls, buserelin treatment caused loss of myenteric neurons in the ileum and colon (P<0.01), a thinner circular muscle layer in ileum (P<0.05) and longitudinal muscle layer in colon (P<0.05), increased number of eosinophils in the submucosa of the ileum (P<0.05), and an increased number of T­lymphocytes in the submucosa and circular muscle layer of the fundus (P<0.01 and P<0.05, respectively) and circular muscle layer of the colon (P<0.05). Mast cells were equally distributed in the two groups. Thus, long­term follow­up of buserelin­induced enteric neuropathy reveals reduced body weight, loss of myenteric neurons, thinning of muscle layers, and increased numbers of eosinophils and T­lymphocytes in the gastrointestinal tract.

Buserelin treatment to rats causes enteric neurodegeneration with moderate effects on CRF-immunoreactive neurons and Enterobacteriaceae in colon, and in acetylcholine-mediated permeability in ileum.

BACKGROUND: The gonadotropin-releasing hormone (GnRH) analog buserelin causes enteric neuronal loss. Acute stress or injection of corticotropin-releasing factor (CRF) affects motility, secretion, and barrier function of the gastrointestinal tract. The aim of the study was to characterize the CRF immunoreactivity in enteric neurons after buserelin treatment, and to evaluate possible effects of enteric neuropathy on gut microbiota, intestinal permeability, and stress response behavior. RESULTS: Sixty rats were given buserelin (20 µg) or saline subcutaneously for 5 days, repeated four times with 3 weeks in-between. At the study end, enteric neuronal density, enteric expression of CRF, gut microbial composition, and plasma levels of adrenocorticotropic hormone (ACTH) and CRF were analyzed. Intestinal permeability was examined in Ussing chambers and the reaction to stressful events was measured by behavior tests. Buserelin treatment reduced the number of neurons along the entire gastrointestinal tract, with increased relative numbers of CRF-immunoreactive submucosal and myenteric neurons in colon (p < 0.05 and p < 0.01, respectively). The overall microbial diversity and relative abundance did not differ between groups, but Enterobacteriaceae was decreased in colon in buserelin-treated rats (p = 0.020). Basal intestinal permeability did not differ between groups, whereas carbachol stimulation increased ileum permeability in controls (p < 0.05), but not in buserelin-treated rats. Buserelin did not affect stress behavior. CONCLUSIONS: Although buserelin treatment leads to enteric neuronal loss along the gastrointestinal tract with an increased percentage of CRF-immunoreactive neurons in colon, the physiology is well preserved, with modest effects on colon microbiota and absence of carbachol-induced permeability in ileum as the only observed changes.

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.

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.

Enhanced colonic tumorigenesis in alkaline sphingomyelinase (NPP7) knockout mice.

Intestinal alkaline sphingomyelinase (alk-SMase) generates ceramide and inactivates platelet-activating factor (PAF) and was previously suggested to have anticancer properties. The direct evidence is still lacking. We studied colonic tumorigenesis in alk-SMase knockout (KO) mice. Formation of aberrant crypt foci (ACF) was examined after azoxymethane (AOM) injection. Tumor was induced by AOM alone, a conventional AOM/dextran sulfate sodium (DSS) treatment, and an enhanced AOM/DSS method. ß-Catenin was determined by immunohistochemistry, PAF levels by ELISA, and sphingomyelin metabolites by mass spectrometry. Without treatment, spontaneous tumorigenesis was not identified but the intestinal mucosa appeared thicker in KO than in wild-type (WT) littermates. AOM alone induced more ACF in KO mice but no tumors 28 weeks after injection. However, combination of AOM/DSS treatments induced colonic tumors and the incidence was significantly higher in KO than in WT mice. By the enhanced AOM/DSS method, tumor number per mouse increased 4.5 times and tumor size 1.8 times in KO compared with WT mice. Although all tumors were adenomas in WT mice, 32% were adenocarcinomas in KO mice. Compared with WT mice, cytosol expression of ß-catenin was significantly increased and nuclear translocation in tumors was more pronounced in KO mice. Lipid analysis showed decreased ceramide in small intestine and increased sphingosine-1-phosphate (S1P) in both small intestine and colon in nontreated KO mice. PAF levels in feces were significantly higher in the KO mice after AOM/DSS treatment. In conclusion, lack of alk-SMase markedly increases AOM/DSS-induced colonic tumorigenesis associated with decreased ceramide and increased S1P and PAF levels.

Structural and functional consequences of buserelin-induced enteric neuropathy in rat.

BACKGROUND: Women treated with gonadotropin-releasing hormone (GnRH) analogs may develop enteric neuropathy and dysmotility. Administration of a GnRH analog to rats leads to similar degenerative neuropathy and ganglioneuritis. The aim of this study on rat was to evaluate the early GnRH-induced enteric neuropathy in terms of distribution of neuronal subpopulations and gastrointestinal (GI) function. METHODS: Forty rats were given the GnRH analog buserelin (20 µg, 1 mg/ml) or saline subcutaneously, once daily for 5 days, followed by 3 weeks of recovery, representing one treatment session. Two weeks after the fourth treatment session, the animals were tested for GI transit time and galactose absorption, and fecal weight and fat content was analyzed. After sacrifice, enteric neuronal subpopulations were analyzed. Blood samples were analyzed for zonulin and antibodies against GnRH and luteinizing hormone, and their receptors. RESULTS: Buserelin treatment transiently increased the body weight after 5 and 9 weeks (p < 0.001). Increased estradiol in plasma and thickened uterine muscle layers indicate high estrogen activity. The numbers of both submucous and myenteric neurons were reduced by 27%-61% in ileum and colon. The relative numbers of neurons containing calcitonin gene-related peptide (CGRP), cocaine- and amphetamine-related transcript (CART), galanin, gastrin-releasing peptide (GRP), neuropeptide Y (NPY), nitric oxide synthase (NOS), serotonin, substance P (SP), vasoactive intestinal peptide (VIP) or vesicular acetylcholine transporter (VAchT), and their nerve fiber density, were unchanged after buserelin treatment, but the relative number of submucous neurons containing somatostatin tended to be increased (p = 0.062). The feces weight decreased in buserelin-treated rats (p < 0.01), whereas feces fat content increased (p < 0.05), compared to control rats. Total GI transit time, galactose absorption, zonulin levels in plasma, and antibody titers in serum were unaffected by buserelin treatment. CONCLUSIONS: A marked enteric neuronal loss with modest effects on GI function is found after buserelin treatment. Increased feces fat content is suggested an early sign of dysfunction.

Lipopolysaccharide-induced loss of cultured rat myenteric neurons - role of AMP-activated protein kinase.

OBJECTIVE: Intestinal barrier function is vital for homeostasis. Conditions where the mucosal barrier is compromised lead to increased plasma content of lipopolysaccharide (LPS). LPS acts on Toll-like receptor 4 (TLR4) and initiates cellular inflammatory responses. TLR4 receptors have been identified on enteric neurons and LPS exposure causes neuronal loss, counteracted by vasoactive intestinal peptide (VIP), by unknown mechanisms. In addition AMP activated protein kinase (AMPK) stimulation causes loss of enteric neurons. This study investigated a possible role of AMPK activation in LPS-induced neuronal loss. DESIGN: Primary cultures of myenteric neurons isolated from rat small intestine were used. Cultures were treated with LPS (0.2-20 µg/mL) with and without TAK1-inhibitor (5Z)-7-Oxozeaenol (10-6 M) or AMPK inhibitor compound C (10-5 M). AMPK-induced neuronal loss was verified treating cultures with three different AMPK activators, AICAR (10-4-3×10-3 M), metformin (0.2-20 µg/mL) and A-769662 (10-5-3×10-4 M) with or without the presence of compound C (10-5 M). Upstream activation of AMPK-induced neuronal loss was tested by treating cultures with AICAR (10-3 M) in the presence of TAK1 inhibitor (5Z)-7-Oxozeaenol (10-6 M). Neuronal survival and relative numbers of neurons immunoreactive (IR) for VIP were evaluated using immunocytochemistry. RESULTS: LPS caused a concentration dependent loss of neurons. All AMPK activators induced loss of myenteric neurons in a concentration dependent manner. LPS-, AICAR- and metformin-,but not A-769662-, induced neuronal losses were inhibited by presence of compound C. LPS, AICAR or metformin exposure increased the relative number of VIP-IR neurons; co-treatment with (5Z)-7-Oxozeaenol or compound C reversed the relative increase in VIP-IR neurons induced by LPS. (5Z)-7-Oxozeaenol, compound C or A-769662 did not per se change neuronal survival or relative numbers of VIP-IR neurons. CONCLUSION: AMPK activation mimics LPS-induced loss of cultured myenteric neurons and LPS-induced neuronal loss is counteracted by TAK1 and AMPK inhibition. This suggests enteric neuroimmune interactions involving AMPK regulation.

The enteric nervous system of P2Y13 receptor null mice is resistant against high-fat-diet- and palmitic-acid-induced neuronal loss.

Gastrointestinal symptoms have a major impact on the quality of life and are becoming more prevalent in the western population. The enteric nervous system (ENS) is pivotal in regulating gastrointestinal functions. Purinergic neurotransmission conveys a range of short and long-term cellular effects. This study investigated the role of the ADP-sensitive P2Y13 receptor in lipid-induced enteric neuropathy. Littermate P2Y13 (+/+) and P2Y13 (-/-) mice were fed with either a normal diet (ND) or high-fat diet (HFD) for 6 months. The intestines were analysed for morphological changes as well as neuronal numbers and relative numbers of vasoactive intestinal peptide (VIP)- and neuronal nitric oxide synthase (nNOS)-containing neurons. Primary cultures of myenteric neurons from the small intestine of P2Y13 (+/+) or P2Y13 (-/-) mice were exposed to palmitic acid (PA), the P2Y13 receptor agonist 2meSADP and the antagonist MRS2211. Neuronal survival and relative number of VIP-containing neurons were analysed. In P2Y13 (+/+), but not in P2Y13 (-/-) mice, HFD caused a significant loss of myenteric neurons in both ileum and colon. In colon, the relative numbers of VIP-containing submucous neurons were significantly lower in the P2Y13 (-/-) mice compared with P2Y13 (+/+) mice. The relative numbers of nNOS-containing submucous colonic neurons increased in P2Y13 (+/+) HFD mice. HFD also caused ileal mucosal thinning in P2Y13 (+/+) and P2Y13 (-/-) mice, compared to ND fed mice. In vitro PA exposure caused loss of myenteric neurons from P2Y13 (+/+) mice while neurons from P2Y13 (-/-) mice were unaffected. Presence of MRS2211 prevented PA-induced neuronal loss in cultures from P2Y13 (+/+) mice. 2meSADP caused no change in survival of cultured neurons. P2Y13 receptor activation is of crucial importance in mediating the HFD- and PA-induced myenteric neuronal loss in mice. In addition, the results indicate a constitutive activation of enteric neuronal apoptosis by way of P2Y13 receptor stimulation.

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.

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.

Gonadotropin-releasing hormone analog buserelin causes neuronal loss in rat gastrointestinal tract.

Gonadotropin-releasing hormone (GnRH) analogs are given to women undergoing in vitro fertilization. Case reports describing the development of chronic intestinal pseudo-obstruction and auto-antibodies against GnRH after such treatment suggest a strong association between intestinal dysfunction and GnRH analogs. No experimental model for studying such a relationship is currently at hand. Our main goal was to investigate possible enteric neurodegeneration and titers of GnRH antibodies in response to repeated administration of the GnRH analog buserelin in rat. Rats were treated for 1-4 sessions with daily subcutaneous injections of buserelin or saline for 5 days, followed by 3 weeks of recovery. Buserelin treatment caused significant loss of submucous and myenteric neurons in the fundus, ileum, and colon. The loss of enteric neurons can, at least partly, be explained by increased apoptosis. No GnRH- or GnRH-receptor-immunoreactive (IR) enteric neurons but numerous luteinizing hormone (LH)-receptor-IR neurons were detected. After buserelin treatment, the relative number of enteric LH-receptor-IR neurons decreased, whereas that of nitric-oxide-synthase-IR neurons increased. No intestinal inflammation or increased levels of circulating interleukins/cytokines were noted in response to buserelin treatment. Serum GnRH antibody titers were undetectable or extremely low in all rats. Thus, repeated administrations of buserelin induce neurodegeneration in rat gastrointestinal tract, possibly by way of LH-receptor hyperactivation. The present findings suggest that enteric neurodegenerative effects of GnRH analog treatment in man can be mimicked in rat. However, in contrast to man, no production of GnRH auto-antibodies has been noted in rat.

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

BACKGROUND: Neuropathy 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. METHODS: GLP1, 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. RESULTS: GLP1, 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. CONCLUSIONS: By 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.

Intimal hyperplasia in balloon dilated coronary arteries is reduced by local delivery of the NO donor, SIN-1 via a cGMP-dependent pathway.

BACKGROUND: To elucidate the mechanism by which local delivery of 3-morpholino-sydnonimine (SIN-1) affects intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA). METHODS: Porcine coronary arteries were treated with PTCA and immediately afterwards locally treated for 5 minutes, with a selective cytosolic guanylate cyclase inhibitor, 1 H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ) + SIN-1 or only SIN-1 using a drug delivery-balloon. Arteries were angiographically depicted, morphologically evaluated and analyzed after one and eight weeks for actin, myosin and intermediate filaments (IF) and nitric oxide synthase (NOS) contents. RESULTS: Luminal diameter after PCI in arteries treated with SIN-1 alone and corrected for age-growth was significantly larger as compared to ODQ + SIN-1 or to controls (p < 0.01). IF/actin ratio after one week in SIN-1 treated segments was not different compared to untreated segments, but was significantly reduced compared to ODQ + SIN-1 treated vessels (p < 0.05). Expression of endothelial NADPH diaphorase activity was significantly lower in untreated segments and in SIN-1 treated segments compared to controls and SIN-1 + ODQ treated arteries (p < 0.01). Restenosis index (p < 0.01) and intimal hyperplasia (p < 0.01) were significantly reduced while the residual lumen was increased (p < 0.01) in SIN-1 segments compared to controls and ODQ + SIN-1 treated vessels. CONCLUSIONS: After PTCA local delivery of high concentrations of the NO donor SIN-1 for 5 minutes inhibited injury induced neointimal hyperplasia. This favorable effect was abolished by inhibition of guanylyl cyclase indicating mediation of a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent pathway. The momentary events at the time of injury play crucial role in the ensuring development of intimal hyperplasia.

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.