MRGPRX2 and Immediate Drug Hypersensitivity: Insights From Cultured Human Mast Cells.

BACKGROUND AND OBJECTIVES: Mast cell (MC) degranulation via activation of the Mas-related G protein-coupled receptor X2 (MRGPRX2) plays a key role in immediate drug hypersensitivity (IDH). However, data in humans are limited to observations in specific cell lines. Objective: To study the usefulness of silencing MRGPRX2 in human MCs with the aim of further unveiling the MRGPRX2 pathway in IDH. METHODS: MCs were cultured from CD34+ progenitor cells obtained from peripheral blood (PBCMCs) and incubated with substance P (as a positive control), rocuronium, moxifloxacin, morphine, or amoxicillin. Immunophenotyping of the cells included flow cytometry and microscopy analyses of the expression of CD117, CD203c, and MRGPRX2. Intracellular calcium was measured using Fluo-4. Degranulation was analyzed by quantifying CD63 expression. For MRGPRX2 silencing, MCs were electroporated with Dicer small interference RNAs. RESULTS: Incubation of MCs with substance P, morphine, and moxifloxacin increased intracellular calcium levels and triggered MC degranulation, which, for the drugs, is almost completely abolished by selective MRGPRX2 silencing. Despite an increase in intracellular calcium in MRGPRX2+ cells, incubation with nontoxic concentrations of rocuronium does not result in degranulation of PBCMCs. Amoxicillin has no effect on PBCMCs. CONCLUSION: The use of MRGPRX2 silencing in human MCs can provide important insights into the role of MRGPRX2 in the pathogenesis of IDH. As induction of calcium signals does not necessarily translate into a secretory response, measurement of the degranulation reaction seems more meaningful in the context of drug testing.

The risk for behavioural deficits is determined by the maternal immune response to prenatal immune challenge in a neurodevelopmental model.

BACKGROUND: Schizophrenia is a highly disabling psychiatric disorder with a proposed neurodevelopmental basis. One mechanism through which genetic and environmental risk factors might act is by triggering persistent brain inflammation, as evidenced by long-lasting neuro-immunological disturbances in patients. Our goal was to investigate whether microglia activation is a neurobiological correlate to the altered behaviour in the maternal immune activation (MIA) model, a well-validated animal model with relevance to schizophrenia. A recent observation in the MIA model is the differential maternal body weight response to the immune stimulus, correlated with a different behavioural outcome in the offspring. Although it is generally assumed that the differences in maternal weight response reflect differences in cytokine response, this has not been investigated so far. Our aim was to investigate whether (i) the maternal weight response to MIA reflects differences in the maternal cytokine response, (ii) the differential behavioural phenotype of the offspring extends to depressive symptoms such as anhedonia and (iii) there are changes in chronic microglia activation dependent on the behavioural phenotype. METHODS: Based on a dose-response study, MIA was induced in pregnant rats by injecting 4mg/kg Poly I:C at gestational day 15. Serum samples were collected to assess the amount of TNF-α in the maternal blood following MIA. MIA offspring were divided into weight loss (WL; n=14) and weight gain (WG; n=10) groups, depending on the maternal body weight response to Poly I:C. Adult offspring were behaviourally phenotyped for prepulse inhibition, locomotor activity with and without amphetamine and MK-801 challenge, and sucrose preference. Finally, microglia activation was scored on CD11b- and Iba1-immunohistochemically stained sections. RESULTS: Pregnant dams that lost weight following MIA showed increased levels of TNF-α compared to controls, unlike dams that gained weight following MIA. Poly I:C WL offspring showed the most severe behavioural outcome. Poly I:C WG offspring, on the other hand, did not show clear behavioural deficits. Most interestingly a reduced sucrose preference indicative of anhedonia was found in Poly I:C WL but not Poly I:C WG offspring compared to controls. Finally, there were no significant differences in microglia activation scores between any of the investigated groups. CONCLUSIONS: The individual maternal immune response to MIA is an important determinant of the behavioural outcome in offspring, including negative symptoms such as anhedonia. We failed to find any significant difference in the level of microglia activation between Poly I:C WL, Poly I:C WG and control offspring.

Annular dark-field transmission electron microscopy for low contrast materials.

Imaging soft matter by transmission electron microscopy (TEM) is anything but straightforward. Recently, interest has grown in developing alternative imaging modes that generate contrast without additional staining. Here, we present a dark-field TEM technique based on the use of an annular objective aperture. Our experiments demonstrate an increase in both contrast and signal-to-noise ratio in comparison to conventional bright-field TEM. The proposed technique is easy to implement and offers an alternative imaging mode to investigate soft matter.

Functional interactions between residues in the S1, S4, and S5 domains of Kv2.1.

The voltage-gated potassium channel subunit Kv2.1 forms heterotetrameric channels with the silent subunit Kv6.4. Chimeric Kv2.1 channels containing a single transmembrane segment from Kv6.4 have been shown to be functional. However, a Kv2.1 chimera containing both S1 and S5 from Kv6.4 was not functional. Back mutation of individual residues in this chimera (to the Kv2.1 counterpart) identified four positions that were critical for functionality: A200V and A203T in S1, and T343M and P347S in S5. To test for possible interactions in Kv2.1, we used substitutions with charged residues and tryptophan for the outermost pair 203/347. Combinations of substitutions with opposite charges at both T203 and S347 were tolerated but resulted in channels with altered gating kinetics, as did the combination of negatively charged aspartate substitutions. Double mutant cycle analysis with these mutants indicated that both residues are energetically coupled. In contrast, replacing both residues with a positively charged lysine together (T203K + S347K) was not tolerated and resulted in a folding or trafficking deficiency. The nonfunctionality of the T203K + S347K mutation could be restored by introducing the R300E mutation in the S4 segment of the voltage sensor. These results indicate that these specific S1, S4, and S5 residues are in close proximity and interact with each other in the functional channel, but are also important determinants for Kv2.1 channel maturation. These data support the view of an anchoring interaction between S1 and S5, but indicate that this interaction surface is more extensive than previously proposed.

Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

TRPV1 receptors on unmyelinated C-fibres mediate colitis-induced sensitization of pelvic afferent nerve fibres in rats.

Patients with inflammatory bowel disease often suffer from gastrointestinal motility and sensitivity disorders. The aim of the current study was to investigate the role of transient receptor potential of the vanilloid type 1 (TRPV1) receptors in the pathophysiology of colitis-induced pelvic afferent nerve sensitization. Trinitrobenzene sulphate (TNBS) colitis (7.5 mg, 30% ethanol) was induced in Wistar rats 72 h prior to the experiment. Single-fibre recordings were made from pelvic nerve afferents in the decentralized S1 dorsal root. Fibres responding to colorectal distension (CRD) were identified in controls and rats with TNBS colitis. The effect of the TRPV1 antagonist N-(4-tertiarybutylphenyl)-4-(3-chlorophyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 0.25-5 mg kg(-1)) or its vehicle (hydroxypropyl-beta-cyclodextrin) was tested on the afferent response to repetitive distensions (60 mmHg). Immunocytochemical staining of TRPV1 and NF200, a marker for A-fibre neurons, was performed in the dorsal root ganglia L6-S1. TNBS colitis significantly increased the response to colorectal distension of pelvic afferent C-fibres. BCTC did not significantly affect the C-fibre response in controls, but normalized the sensitized response in rats with colitis. TNBS colitis increased the spontaneous activity of C-fibres, an effect which was insensitive to administration of BCTC. TNBS colitis had no effect on Adelta-fibres, nor was their activity modulated by BCTC. TNBS colitis caused an immunocytochemical up-regulation of TRPV1 receptors in the cell bodies of pelvic afferent NF200 negative neurons. TRPV1 signalling mediates the colitis-induced sensitization of pelvic afferent C-fibres to CRD, while Adelta-fibres are neither sensitized by colitis nor affected by TRPV1 inhibition.

Interactions between cell death induced by statins and 7-ketocholesterol in rabbit aorta smooth muscle cells.

BACKGROUND AND PURPOSE: 7-Ketocholesterol, an oxysterol present in atherosclerotic lesions, induces smooth muscle cell (SMC) death, thereby destabilizing plaques. Statins protect patients from myocardial infarction, though they induce SMC apoptosis. We investigated whether statins and 7-ketocholesterol exerted additive cell death effects. EXPERIMENTAL APPROACH: Cultured rabbit aorta SMCs (passage 2-6) were exposed to 7-ketocholesterol with or without fluvastatin, simvastatin or pravastatin. Uptake of neutral red (NR), monolayer protein, cleavage of the pan-caspase substrate Asp-Glu-Val-Asp-rhodamine110, cell morphology (light and electron microscopy) and processing of microtubule-associated protein 1 light chain 3 (LC3, immunoblot) were determined. KEY RESULTS: NR uptake declined upon 18 h exposure to 25 microM 7-ketocholesterol (-41+/-3%, n=13), 100 microM fluvastatin (-59%) or 30-100 microM simvastatin (-28 to -74%). Oxysterol and high statin concentrations exerted additive effects, but lower concentrations (fluvastatin 10-30 microM, simvastatin 1-10 microM) partly reversed viability loss. 7-Ketocholesterol caused intense cytoplasmic vacuolization, processing of LC3-I to LC3-II, but little caspase activation (increase 29.5%). Fluvastatin (10-100 microM, 70-545% increase) and simvastatin (3-100 microM 43-322% increase) induced caspase activation without LC3 processing, but failed to activate caspases in 7-ketocholesterol-treated SMCs. Pravastatin up to 100 microM was always inactive. CONCLUSIONS AND IMPLICATIONS: 7-Ketocholesterol caused SMC death, mainly via autophagic vesicle formation with LC3 processing, whereas lipophilic statins evoked SMC apoptosis. Cell death following 7-ketocholesterol and low statin concentrations were not additive, presumably because the autophagic process interfered with statin-induced caspase activation. This further illustrates that drug effects in normal SMCs are not necessarily predictive for activities in atherosclerotic settings.

FABP4 blocker attenuates colonic hypomotility and modulates white adipose tissue-derived hormone levels in mouse models mimicking constipation-predominant IBS.

BACKGROUND: The role of fatty acid binding protein 4 (FABP4) in lower gastrointestinal (GI) motility is unknown. We aimed to verify the effect of inhibition of FABP4 on GI transit in vivo, and to determine the expression of FABP4 in mouse and human tissues. METHODS: Fatty acid binding protein 4 inhibitor, BMS309403, was administered acutely or chronically for 6 and 13 consecutive days and its effect on GI transit was assessed in physiological conditions and in loperamide-induced constipation. Intracellular recordings were made to examine the effects of BMS309403 on colonic excitatory and inhibitory junction potentials. Abdominal pain was evaluated using behavioral pain response. Localization and expression of selected adipokines were determined in the mouse colon and serum using immunohistochemistry and Enzyme-Linked ImmunoSorbent Assay respectively. mRNA expression of FABP4 and selected adipokines in colonic and serum samples from irritable bowel syndrome (IBS) patients and control group were assessed. KEY RESULTS: Acute injection of BMS309403 significantly increased GI motility and reversed inhibitory effect of loperamide. BMS309403 did not change colonic membrane potentials. Chronic treatment with BMS309403 increased the number of pain-induced behaviors. In the mouse serum, level of resistin was significantly decreased after acute administration; no changes in adiponectin level were detected. In the human serum, level of adiponectin and resistin, but not of FABP4, were significantly elevated in patients with constipation-IBS (IBS-C). FABP4 mRNA expression was significantly downregulated in the human colon in IBS-C. CONCLUSIONS AND INFERENCES: Fatty acid binding protein 4 may be involved in IBS pathogenesis and become a novel target in the treatment of constipation-related diseases.

Inhibitory purinergic P2 receptor characterisation in rat distal colon.

The aim of this study was to characterise the P2 receptors involved in purinergic relaxant responses in rat distal colon circular muscle. Concentration-response curves for purinergic agonists were constructed on methacholine-precontracted circular muscle strips of rat distal colon in the absence and presence of the nerve blocker TTX and the ecto-nucleotidase inhibitor ARL67156. The effects of the P2 receptor antagonists RB2, PPADS, suramin, MRS2179 and NF279, the NO-synthase inhibitor L-NAME and the small conductance K(+) channel blocker apamin were investigated. The localisation of the different P2 receptors was examined immunocytochemically. Immunocytochemistry demonstrated the expression of P2Y(1), P2Y(6) and P2X(1) receptors on smooth muscle cells and P2Y(2), P2Y(12), P2X(2) and P2X(3) receptors in the myenteric plexus; almost a quarter of the P2Y(2)-immunopositive neurons co-expressed nNOS. The P2X-selective agonist alphabetameATP and the P2Y-selective agonist ADPbetaS were the most potent relaxants; their effects were abolished by apamin. The effect of ADPbetaS was antagonised by the P2Y(1)-selective antagonist MRS2179 pointing to interaction with the muscular P2Y(1)-receptors. The relaxant effect of alphabetameATP was partially reduced by TTX and concentration-dependently antagonised by PPADS, suramin, RB2 and the P2X(1)-selective antagonist NF279; this correlates with an interaction with neuronal P2X(3) and muscular P2X(1) receptors. UTP was the least potent agonist; its effect was markedly increased by ARL67156, nearly abolished by TTX and reduced by L-NAME. This points to interaction with the neuronal P2Y(2)-receptors inducing relaxation, at least partially, by NO release.

Nitric oxide selectively depletes macrophages in atherosclerotic plaques via induction of endoplasmic reticulum stress.

BACKGROUND AND PURPOSE: Macrophages in atherosclerotic plaques have a tremendous impact on atherogenesis and plaque destabilization. We previously demonstrated that treatment of plaques in cholesterol-fed rabbits with the nitric oxide (NO) donor molsidomine preferentially eliminates macrophages, thereby favouring features of plaque stability. In this study, we investigated the underlying mechanism. EXPERIMENTAL APPROACH: Macrophages and smooth muscle cells (SMCs) were treated in vitro with the NO donors, spermine NONOate or S-nitroso-N-acetylpenicillamine (SNAP) as well as with the well-known endoplasmic reticulum (ER) stress inducers thapsigargin, tunicamycin, dithiothreitol or brefeldin A. Cell viability was analysed by Neutral Red viability assays. Cleavage of caspase-3, DNA fragmentation and ultrastructural changes were examined to characterize the type of macrophage death. Induction of ER stress was evaluated by measuring C/EBP homologous protein (CHOP) expression, phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a), splicing of X-box binding protein 1 (XBP1) and inhibition of protein synthesis. KEY RESULTS: Macrophages and SMCs treated with spermine NONOate or SNAP showed several signs of ER stress, including upregulation of CHOP expression, hyperphosphorylation of eIF2 alpha, inhibition of de novo protein synthesis and splicing of XBP1 mRNA. These effects were similar in macrophages and SMCs, yet only macrophages underwent apoptosis. Plaques from molsidomine-treated atherosclerotic rabbits showed a 2.7-fold increase in CHOP expression as compared to placebo. Beside NO, selective induction of macrophage death could be initiated with thapsigargin and tunicamycin. CONCLUSIONS AND IMPLICATIONS: Induction of ER stress explains selective depletion of macrophages in atherosclerotic plaques by a NO donor, probably via inhibition of protein synthesis.

Effect of intestinal inflammation on the cell-specific expression of somatostatin receptor subtypes in the murine ileum.

Despite our knowledge of somatostatin (SOM) in gastrointestinal functions, little information is available on the SOM receptors (SSTRs) mediating these effects. This study focussed on the expression of SSTRs in non-inflamed and Schistosoma mansoni-infected murine ileum using immunocytochemistry, reverse transcriptase (RT)-PCR and quantitative real time RT-PCR (qPCR). In the non-inflamed ileum, SSTRs showed a widespread, cell-type specific expression pattern. For instance, SSTR2A immunoreactivity was detected in a minor population of submucous but not myenteric glial cells. In the inflamed ileum, significant changes in the expression pattern of SSTRs occurred, with SSTR1 and SSTR3 expression on mucosal mast cells (MMCs) and mucosal nerve fibres. SSTR4-immunoreactive nerve fibres were detected in granulomas and the lamina propria. qPCR experiments indicated significantly increased mRNA levels for SOM, SSTR1 and SSTR3 in inflamed ileum. This study reveals that SSTRs are expressed in specific cell types in murine ileum. Expression of SSTR1 and SSTR3 on MMCs and increased density of SOM-expressing nerve fibres in the lamina propria during inflammation, support the hypothesis that SOM is implicated in the physiological control of MMCs during intestinal inflammation. Evidence is provided that in mouse mainly SSTR1, SSTR3 and SSTR4 are involved in the somatostatinergic inflammatory effects during intestinal schistosomiasis.

Age-related characteristics of the neurotransmitter composition of neurons in the stellate ganglion.

The neurotransmitter composition of neurons in the stellate ganglion of rats of different ages (neonatal, 10, 20, 30, and 60 days) was studied by an immunocytochemical method using double labeling. Most neurons in rat pups of all age groups contained tyrosine hydroxylase. Most choline acetyltransferase-positive neurocytes in neonatal and 10-day-old rat pups were also tyrosine hydroxylase-positive. Only occasional cells in 30-and 60-day rat pups contained both of these enzymes. There were increases in the proportions of cells containing tyrosine hydroxylase and neuropeptide Y from birth to all time points of the study. In addition, there was a decrease in the proportion of somatostatin-positive neurons. The proportions of VIP-positive cells and choline acetyltransferase-containing neurons increased to age 10 days and then decreased. Somatostatin-positive neurons in all rat pups were small cells, while those containing choline acetyltransferase were large. Maturation of the neurotransmitter set in the rat stellate ganglion was complete by the end of the second month of life.

Highly selective CB2 receptor agonist A836339 has gastroprotective effect on experimentally induced gastric ulcers in mice.

Cannabinoid type 2 (CB2) receptors are distributed in central and peripheral tissues, including immunocytes and the gastrointestinal (GI) tract, suggesting that CB2 receptor agonists represent potential therapeutics in GI inflammatory states. In this study, we investigated the effect of highly selective CB2 agonist, A836339, on the development of gastric lesions. We used two models of gastric ulcer (GU) induced by ethanol (EtOH) and diclofenac. To confirm the involvement of CB2 receptors, a selective CB2 antagonist, AM630 was used. Clinical parameters for gastroprotection were assessed based on inhibition of the gastric lesion area. To investigate the anti-inflammatory effect of A836339, the expression of TNF-α and IL-1ß was assessed. To establish the mechanism of gastroprotective action, catalase (CAT), superoxide dismutase (SOD) activity and H2O2 and glutathione (GSH) levels were measured. Moreover, expression of CB2 and cyclooxygenase-2 (COX-2) was characterized using immunohistochemistry (IHC). A836339 reduced ulcer index in a dose-dependent manner in both EtOH- and diclofenac-induced GU models. This effect was reversed by the CB2 antagonist AM630. Administration of A836339 reduced TNF-α and IL-1ß levels in gastric tissue. Furthermore, A836339 exhibited potent anti-oxidant activity, as demonstrated by reduced H2O2 levels and increased CAT and SOD activities. IHC studies revealed a co-localization of CB2 receptors and COX-2 in the gastric tissue. Activation of CB2 receptors exhibited gastroprotective effect through enhancement of anti-oxidative pathways in the stomach. Activation of CB2 receptors may thus become a novel therapeutic approach in the treatment of GU.

G protein-coupled estrogen receptor and estrogen receptor ligands regulate colonic motility and visceral pain.

BACKGROUND: Diarrhea-predominant irritable bowel syndrome (IBS-D) is a functional gastrointestinal (GI) disorder, which occurs more frequently in women than men. The aim of our study was to determine the role of activation of classical estrogen receptors (ER) and novel membrane receptor, G protein-coupled estrogen receptor (GPER) in human and mouse tissue and to assess the possible cross talk between these receptors in the GI tract. METHODS: Immunohistochemistry was used to determine the expression of GPER in human and mouse intestines. The effect of G-1, a GPER selective agonist, and estradiol, a non-selective ER agonist, on muscle contractility was characterized in isolated preparations of the human and mouse colon. To characterize the effect of G-1 and estradiol in vivo, colonic bead expulsion test was performed. G-1 and estradiol activity on the visceral pain signaling was assessed in the mustard oil-induced abdominal pain model. KEY RESULTS: GPER is expressed in the human colon and in the mouse colon and ileum. G-1 and estradiol inhibited muscle contractility in vitro in human and mouse colon. G-1 or estradiol administered intravenously at the dose of 20 mg/kg significantly prolonged the time to bead expulsion in females. Moreover, G-1 prolonged the time to bead expulsion and inhibited GI hypermotility in both genders. The injection of G-1 or estradiol resulted in a significant reduction in the number of pain-induced behaviors in mice. CONCLUSIONS AND INFERENCES: GPER and ER receptors are involved in the regulation of GI motility and visceral pain. Both may thus constitute an important pharmacological target in the IBS-D therapy.

Targeting fatty acid amide hydrolase and transient receptor potential vanilloid-1 simultaneously to modulate colonic motility and visceral sensation in the mouse: A pharmacological intervention with N-arachidonoyl-serotonin (AA-5-HT).

BACKGROUND: Endocannabinoid anandamide (AEA) inhibits intestinal motility and visceral pain, but it may also be proalgesic through transient receptor potential vanilloid-1 (TRPV1). AEA is degraded by fatty acid amide hydrolase (FAAH). This study explored whether dual inhibition of FAAH and TRPV1 reduces diarrhea and abdominal pain. METHODS: Immunostaining was performed on myenteric plexus of the mouse colon. The effects of the dual FAAH/TRPV1 inhibitor AA-5-HT on electrically induced contractility, excitatory junction potential (EJP) and fast (f) and slow (s) inhibitory junction potentials (IJP) in the mouse colon, colonic propulsion and visceromotor response (VMR) to rectal distension were studied. The colonic levels of endocannabinoids and fatty acid amides were measured. KEY RESULTS: CB1-positive neurons exhibited TRPV1; only some TRPV1 positive neurons did not express CB1. CB1 and FAAH did not colocalize. AA-5-HT (100 nM-10 µM) decreased colonic contractility by ~60%; this effect was abolished by TRPV1 antagonist 5'-IRTX, but not by CB1 antagonist, SR141716. AA-5-HT (1 µM-10 µM) inhibited EJP by ~30% and IJPs by ~50%. The effects of AA-5-HT on junction potentials were reversed by SR141716 and 5`-IRTX. AA-5-HT (20 mg/kg; i.p.) inhibited colonic propulsion by ~30%; SR141716 but not 5`-IRTX reversed this effect. AA-5-HT decreased VMR by ~50%-60%; these effects were not blocked by SR141716 or 5`-IRTX. AA-5-HT increased AEA in the colon. CONCLUSIONS AND INFERENCES: The effects of AA-5-HT on visceral sensation and colonic motility are differentially mediated by CB1, TRPV1 and non-CB1/TRPV1 mechanisms, possibly reflecting the distinct neuromodulatory roles of endocannabinoid and endovanilloid FAAH substrates in the mouse intestine.

Immunocytochemical characteristics of neurons in the stellate ganglion of the sympathetic trunk in mice during postnatal ontogenesis.

The neurotransmitter composition of neurons in the stellate ganglion of mice of different ages (neonatal and aged 10, 20, 30, and 60 days) was studied using an immunocytochemical method. Most of the neurons in the mice of these age groups contained tyrosine hydroxylase (TH). Most choline acetyltransferase (CAT)-positive neurons in neonatal and 10-day mice were also TH-positive. From birth and at all subsequent ages, there were increases in the proportion of cells contained TH and neuropeptide Y. The proportion of neurons containing vasoactive intestinal peptide and CAT increased to 10 days of life and then decreased. Somatostatin-and galanin-positive neurons were absent from all animals. Maturation of the set of neurotransmitters in stellate ganglia was complete by age two months.

Proof-of-concept: neonatal intravenous injection of adeno-associated virus vectors results in successful transduction of myenteric and submucosal neurons in the mouse small and large intestine.

BACKGROUND: Despite the success of viral vector technology in the transduction of the central nervous system in both preclinical research and gene therapy, its potential in neurogastroenterological research remains largely unexploited. This study asked whether and to what extent myenteric and submucosal neurons in the ileum and distal colon of the mouse were transduced after neonatal systemic delivery of recombinant adeno-associated viral vectors (AAVs). METHODS: Mice were intravenously injected at postnatal day one with AAV pseudotypes AAV8 or AAV9 carrying a cassette encoding enhanced green fluorescent protein (eGFP) as a reporter under the control of a cytomegalovirus promoter. At postnatal day 35, transduction of the myenteric and submucosal plexuses of the ileum and distal colon was evaluated in whole-mount preparations, using immunohistochemistry to neurochemically identify transduced enteric neurons. KEY RESULTS: The pseudotypes AAV8 and AAV9 showed equal potential in transducing the enteric nervous system (ENS), with 25-30% of the neurons expressing eGFP. However, the percentage of eGFP-expressing colonic submucosal neurons was significantly lower. Neurochemical analysis showed that all enteric neuron subtypes, but not glia, expressed the reporter protein. Intrinsic sensory neurons were most efficiently transduced as nearly 80% of calcitonin gene-related peptide-positive neurons expressed the transgene. CONCLUSIONS & INFERENCES: The pseudotypes AAV8 and AAV9 can be employed for gene delivery to both the myenteric and the submucosal plexus, although the transduction efficiency in the latter is region-dependent. These findings open perspectives for novel preclinical applications aimed at manipulating and imaging the ENS in the short term, and in gene therapy in the longer term.

The zebrafish mutant lessen: an experimental model for congenital enteric neuropathies.

BACKGROUND: Congenital enteric neuropathies of the distal intestine (CEN) are characterized by the partial or complete absence of enteric neurons. Over the last decade, zebrafish has emerged as a leading model organism in experimental research. Our aim was to demonstrate that the mutant zebrafish, lessen, expressing CEN characteristics, is an equally valuable animal model alongside mammalian models for CEN, by studying its enteric phenotype. METHODS: The effect of the lessen mutation on the development of the enteric nervous system (ENS), interstitial cells of Cajal (ICC), and intestinal motility in each intestinal region of mutant and wild-type (wt) zebrafish embryos at 3-6 dpf, was analyzed by immunofluorescent detection of neurochemical markers and motility assays. KEY RESULTS: Development of intestinal motility in the mutant was delayed and the majority of the observed contractions were disturbed. A significant disturbance in ENS development resulted in a distal intestine that was almost free of neuronal elements, in reduced neuronal density in the proximal and mid-intestine, and in a defect in the expression of neurochemical markers. Furthermore, markedly disturbed development of ICC gave rise to a less dense network of ICC. CONCLUSIONS & INFERENCES: The observed alterations in intestinal motility, intrinsic innervation and ICC network of the mutant in comparison with the wt zebrafish, are similar to those seen in the oligo- and aganglionic regions of the intestine of CEN patients. It is concluded that the zebrafish mutant lessen is an appropriate animal model to investigate CEN.

Thickness distribution of fresh eardrums of cat obtained with confocal microscopy.

The aim of this study was to measure the spatial thickness distribution of the cat tympanic membrane (TM), a very thin, virtually transparent and delicate biological membrane. Axial fluorescence images taken perpendicular through isolated TM were recorded for five different cats using confocal laser scanning microscopy. Thickness was measured on the cross-section of the membranes in the axial images. A correction for focal shift due to refractive-index mismatch was applied. Similar thickness distributions were obtained in all measured samples (n = 9). The pars tensa had a rather constant thickness in the central region between the annulus and manubrium. The thickness increased steeply toward the peripheral rim. Thickness was smallest in the inferior region, with values ranging between 5.5 and 9 microm in the central part and up to 50 microm near the annulus. More superiorly, thickness was slightly higher, up to 20 microm, between the annulus and manubrium. The anterior part was thicker than the posterior side. These findings are strongly different from a current value in the literature. Our data allow a more precise representation of the eardrum in mathematical models, which are a prerequisite for a better understanding of middle-ear mechanics. The optical sectioning technique of the confocal microscope did not result in any preparation artifacts and was therefore also used to quantify shrinkage due to preparation of histological sections of TMs.

Role of oxidative stress in the pathogenesis of septic ileus in mice.

We investigated the role of oxidative stress in the pathogenesis of septic ileus. Sepsis was induced by intraperitoneal (i.p.) injection of lipopolysaccharides (LPS, 20 mg kg(-1)) in mice. The effect of two i.p. injections of superoxide dismutase [polyethylene glycol (PEG)-SOD, 4000 U kg(-1)] and catalase (PEG-CAT, 15,000 U kg(-1)) was investigated on gastric emptying, intestinal transit and total nitrite plasma concentrations. We also performed immunohistochemical experiments on gastric and ileal tissue. LPS significantly delayed gastric emptying and intestinal transit while plasma nitrite levels increased. Polyethylene glycol (PEG)-SOD reversed the endotoxin-induced delay in gastric emptying and improved the delay in intestinal transit without effect on plasma nitrite levels. PEG-CAT slightly improved the delay in gastric emptying without effect on intestinal transit. Immunohistochemistry showed the presence of nitrotyrosine (NT) and 4-hydroxy-2-nonenal (HNE) in the gastric and ileal mucosa of LPS-treated mice. Treatment with PEG-SOD or PEG-CAT of LPS mice diminished the presence of NT or HNE in both tissues. In addition, LPS induced a significant increase in inducible nitric oxide synthase (iNOS)-positive residential macrophages in the external musculature of stomach and ileum, which significantly decreased after PEG-SOD or PEG-CAT treatment. The present results support a role for oxidative and nitrosative stress in the pathogenesis of septic ileus in mice.