Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy.

INTRODUCTION: Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury. METHODS: Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility. RESULTS: There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups. CONCLUSIONS: Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.

Effects of AIBL on Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum: an enzyme histochemical study.

OBJECTIVE: To explore the effect of AIBL on Oncomelania hupensis, the intermediate snail host of Schistosoma japonicum. METHODS: The enzyme histochemical profiles of cholinesterase, cytochrome oxidase, lactate dehydrogenase, nitric oxide synthase, and succinate dehydrogenase in the soft tissues of Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum, were analyzed before and after treatment with the active ingredient of Buddleia lindleyana (AIBL), a potent and safe plant molluscicide. RESULTS: Treatment with AIBL induced a notable decrease in the activities of the five enzymes (P<0.01). CONCLUSIONS: The results indicate that AIBL impairs the activities of the enzymes, thereby influencing the transfer of neurotransmitter and energy supply in Oncomelania hupensis and ultimately harming their various physiological functions, which are considered to cause death of the species.

[Reversible lupininin inhibitors of cholinesterases of mammalian blood and of optical ganglia of individuals of the commander squid Berryteuthis magister from different zones of species areal].

Arylsulfoesters and carbonic lupinin esters are studied for the first time as reversible inhibitors of mammalian blood cholinesterases. Studied in detail is sensitivity of cholinesterases to mono- and bislupinin inhibitors in Commander squid individuals from different habitation zones.

Evidence of nNOS and ChAT positive phenotypes in nervous ganglia of the retrostyloid space.

The cholinergic and nitrergic phenotypes in human fetal ganglia (inferior) of the glossopharyngeal and vagus nerves were overlooked in basic research. Lack of a positive neuronal NO synthase (nNOS) phenotype in the inferior vagal fetal ganglion was recently suggested to be an individually variable phenotype. Choline acetyltransferase (ChAT) was not evaluated previously in ontogenesis. We aimed to evaluate these phenotypes in human midterm fetuses. Samples from five specimens with gestational ages varying from 4 to 6 months were used. Immunohistochemistry for nNOS, ChAT, neurofilaments, and S100 protein was performed. Neuronal somata were positively stained for nNOS, ChAT and neurofilaments in the inferior glossopharyngeal and vagal ganglia. S100 protein distinctively labelled the satellite glial cells ensheating the respective neurons. In human midterm fetuses vagal and glossopharyngeal inferior ganglia are nitrergic and cholinergic. To evaluate a functional role of these phenotypes in ontogenesis, the specific anatomic circuits should be further checked. Differences in immune labelling should be evaluated by use of similar antibodies from different manufacturers.

Abelson family tyrosine kinases regulate the function of nicotinic acetylcholine receptors and nicotinic synapses on autonomic neurons.

Abelson family kinases (AFKs; Abl1, Abl2) are non-receptor tyrosine kinases (NRTKs) implicated in cancer, but they also have important physiological roles that include regulating synaptic structure and function. Recent studies using Abl-deficient mice and the antileukemia drug STI571 [imatinib mesylate (Gleevec); Novartis], which potently and selectively blocks Abl kinase activity, implicate AFKs in regulating presynaptic neurotransmitter release in hippocampus and postsynaptic clustering of nicotinic acetylcholine receptors (nAChRs) in muscle. Here, we tested whether AFKs are relevant for regulating nAChRs and nAChR-mediated synapses on autonomic neurons. AFK immunoreactivity was detected in ciliary ganglion (CG) lysates and neurons, and STI571 application blocked endogenous Abl tyrosine kinase activity. With similar potency, STI571 specifically reduced whole-cell current responses generated by both nicotinic receptor subtypes present on CG neurons (α3*- and α7-nAChRs) and lowered the frequency and amplitude of α3*-nAChR-mediated excitatory postsynaptic currents. Quantal analysis indicated that the synaptic perturbations were postsynaptic in origin, and confocal imaging experiments revealed they were unaccompanied by changes in nAChR clustering or alignment with presynaptic terminals. The results indicate that in autonomic neurons, Abl kinase activity normally supports postsynaptic nAChR function to sustain nAChR-mediated neurotransmission. Such consequences contrast with the influence of Abl kinase activity on presynaptic function and synaptic structure in hippocampus and muscle, respectively, demonstrating a cell-specific mechanism of action. Finally, because STI571 potently inhibits Abl kinase activity, the autonomic dysfunction side effects associated with its use as a chemotherapeutic agent may result from perturbed α3*- and/or α7-nAChR function.

Stress activated protein kinases, JNKs and p38 MAPK, are differentially activated in ganglia and heart of land snail Helix lucorum (L.) during seasonal hibernation and arousal.

The present work aimed to investigate the phosphorylation and hence activation of stress activated protein kinases, p38 MAPK and JNKs in the tissues of the snail Helix lucorum during seasonal hibernation. Snails were put in large glass boxes, which were placed outdoors so that they were exposed to natural conditions of light and temperature. Phosphorylation and hence activation of JNKs and p38 MAPK was determined in both heart and ganglia. Deep hibernation caused significant increases in the levels of the phosphorylated form of JNK and p38-MAPK in both heart and ganglia. Phosphorylation of JNK remained elevated in the ganglia or increased after a transient drop in the heart, when the snails were prepared for arousal. In addition, phosphorylation of p38-MAPK was further increased in the heart during this period. These data support the conclusion that MAPK signalling cascade might contribute in the physiological and biochemical remodelling in the tissues of land snails during hibernation and upon preparation for arousal.

[Effect of temperature stress on activities of nitric oxide synthase and tyrosine hydroxylase in the central nervous system of bivalve molluscs].

Effects of temperature stress on nitric oxide synthase (NOS) and tyrosine hydroxylase (TH) activities in CNS of two species of bivalve molluscs Mizuchopecten yessoensis and Chlamys farreri nipponensis (Pectinidae) were studied using NADPH-diaphorase histochemistry and ummunocytochemistry. General and specific peculiarities in distribution and relative content ofNOS- and TH-positive neurons in nervous ganglia were revealed in norm and under stress at 30 degrees C for 10, 30, and 60 min. The initial stress stage (for 10 min) has been shown to be accompanied by an increase of the relative content of TH-positive neurons in some CNS areas of both mollusc species. In Chlamys farreri nipponensis under normal conditions, the presence of NOS in the CNS and its significant activation under temperature stress might have possibly been an important neuroprotective component of stress reaction in some mollusc species.

Correlation of enteric NADPH-d positive cell counts with the duration of incubation period in NADPH-d histochemistry.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) staining can be used in the enteric nervous system to determine nitrergic neuronal counts, critical in motility disorders such as intestinal neuronal dysplasia and hypoganglionosis. The reported incubation periods of specimens with NADPH-d staining solution has varied from 2 to 24 h. The aim of this study is to investigate the impact of the incubation period on the overall NADPH-d positive cell counts in porcine rectal submucosal plexus. The submucosal plexus of rectal specimens from 12-week-old pigs (n = 5) were studied. Conventional frozen sections were used to identify nitrergic neurons while whole-mount preparations were used to quantify the effect of prolonged duration of incubation on positively identified ganglion cells with NADPH-d histochemistry. The same submucosal ganglia on the conventional sections, and a minimum of 12 ganglia per whole-mount preparation specimen were photographed sequentially at 2, 6, and 24 h and used to count the number of nitrergic cells per ganglion. The same staining solution was used throughout the experiment. Results were analysed using a one-way ANOVA test. Prolonged incubation with the staining solution revealed new NADPH-d positive cells in the ganglia on the conventional sections. The total number of neurons counted in the 12 adjacent ganglia in the whole-mount specimens was 180 +/- 55, the mean neuronal cell per ganglion was 15 +/- 8 after 2 h of incubation. This increased to 357 +/- 17, and to 29 +/- 12 after 6 h (p < 0.05). A further increase was observed of 515 +/- 19 and 43 +/- 17 after 24 h (p < 0.05). When the photomicrographs were retrospectively analysed, not even the outline of the neuronal cells that stained with prolonged incubation was evident at the earlier time points. NADPH-d positive cell counts increase in proportion to the duration of incubation in NADPH-d histochemistry. Comparative studies attempting to quantify nitrergic cell counts in dysmotility disorders must take into account the variability in NADPH-d positive cell count associated with prolonged incubation in NADPH-d histochemistry.

Gene transfer of neuronal nitric oxide synthase into intracardiac Ganglia reverses vagal impairment in hypertensive rats.

Hypertension is associated with reduced cardiac vagal activity and decreased atrial guanylate cyclase and cGMP levels. Neuronal production of NO facilitates cardiac parasympathetic transmission, although oxidative stress caused by hypertension may disrupt this pathway. We tested the hypothesis that peripheral vagal responsiveness is attenuated in the spontaneously hypertensive rat (SHR) because of impaired NO-cGMP signaling and that gene transfer of neuronal NO synthase (nNOS) into cholinergic intracardiac ganglia can restore neural function. Cardiac vagal heart rate responses in the isolated SHR atrial/right vagus preparation were significantly attenuated compared with age-matched normotensive Wistar-Kyoto rats. [(3)H] acetylcholine release was also significantly lower in the SHR. The NO donor, sodium nitroprusside, augmented vagal responses to nerve stimulation and [(3)H] acetylcholine release in the Wistar-Kyoto rat, whereas the soluble guanylate cyclase inhibitor 1H-(1,2,4)oxadiazolo(4,3-a)quinoxaline-1-one attenuated [(3)H] acetylcholine release in Wistar-Kyoto atria. No effects of sodium nitroprusside or 1H-(1,2,4)oxadiazolo(4,3-a)quinoxaline-1-one were seen in the SHR during nerve stimulation. In contrast, SHR atria were hyperresponsive to carbachol-induced bradycardia, with elevated production of atrial cGMP. After gene transfer of adenoviral nNOS into the right atrium, vagal responsiveness in vivo was significantly increased in the SHR compared with transfection with adenoviral enhanced green fluorescent protein. Atrial nNOS activity was increased after gene transfer of adenoviral nNOS, as was expression of alpha(1)-soluble guanylate cyclase in both groups compared with adenoviral enhanced green fluorescent protein. In conclusion, a significant component of cardiac vagal dysfunction in hypertension is attributed to an impairment of the postganglionic presynaptic NO-cGMP pathway and that overexpression of nNOS can reverse this neural phenotype.

Partial urethral obstruction enhances NADPH-diaphorase activity in the monkey (Macaca fascicularis) bladder: light and electron microscopic studies.

The effect of partially obstructing the urethra on the nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) bladder was examined by light and electron microscopy. Partial urethral ligation was done in adult male monkeys. The animals were sacrificed 2, 4 weeks after partial urethral obstruction. This was compared to controls (normal and sham operated). Urethral obstructed animals were observed to have increased urinary frequency and decreased urinary flow rate. Two weeks after urethral obstruction, the overall NADPH-d activity in the intramural ganglia of the bladder base was enhanced compared to control animals. The frequency of intensely stained NADPH-d positive neurons was increased compared to the control animals. About one-third of intensely stained NADPH-d positive neurons appeared to undergo degenerative changes. At 4 weeks after urethral obstruction, a wide occurrence of NADPH-d positive neurons in advanced stages of degeneration in the bladder base was observed. Cellular debris was strewn among normal looking ganglion cells and along the nerve processes. The proportion of intensely stained NADPH-d positive neurons was relatively lower than the controls. The total number of NADPH-d positive neurons and the nerve fibres in the entire bladder was significantly reduced when compared to control animals. Electron microscopy showed some NADPH-d activity in intramural ganglion cells in 2 weeks after partial urethral obstruction. NADPH-d reaction product (formazan) was deposited on the membranes of the rough endoplasmic reticulum, and the outer membranes of some mitochondria in the intramural neuron. At 4 weeks after urethral obstruction, NADPH-d was present in the membrane of the mitochondria and some mitochondria appeared swollen with disrupted cristae. Present results show that NADPH-d activity in neurons of the intramural ganglia of the monkey (Macaca fascicularis) urinary bladder was increased after two weeks and reduced after 4 weeks of partial urethral obstruction. It is speculated that the increased NADPH-d activity associated with partial urethral obstruction would lead to neuronal damage and death, which may contribute to detrusor overactivity. However, it warrants further investigation to understand the mechanism of neuronal cell death after partial urethral obstruction.

Comparative toxic potential of market formulation of two organophosphate pesticides in transgenic Drosophila melanogaster (hsp70-lacZ).

This study investigated the working hypothesis that two widely used organophosphate pesticides; Nuvan and Dimecron, exert toxic effects in Drosophila. Transgenic D. melanogaster (hsp70-lacZ) was used as a model for assaying stress gene expression and AchE activity as an endpoint for toxicity and also to evaluate whether stress gene expression is sufficient to protect against toxic insult of the chemicals and to prevent tissue damage. The study was extended to investigate the effect of the pesticides on the life cycle and reproduction of the organism. The study showed that Nuvan affected emergence of the exposed flies more drastically than Dimecron and the effect was lethal at the highest tested concentration (0.075 ppm). While Nuvan at 0.0075 and 0.015 ppm concentrations affected reproduction of the flies significantly, the effect of Dimecron was significant only at 0.015 and 0.075 ppm. Nuvan-exposed third-instar larvae exhibited a 1.2-fold to 1.5-fold greater hsp70 expression compared to Dimecron at concentrations ranging from 0.0075 to 0.075 ppm following 12 and 18 h exposure. While maximum expression of hsp70 was observed in Nuvan-exposed third-instar larval tissues following 18 h exposure at 0.075 ppm, Dimecron at the same dietary concentration induced a maximum expression of hsp70 following 24 h exposure. Further, concomitant with a significant induction of hsp70, significant inhibition of AchE was observed following chemical exposure and temperature shock. Concurrent with a significant decline in hsp70 expression in Nuvan-exposed larvae after 48 h at 0.075 ppm, tissue damage was evident. Dimecron-exposed larvae exhibited a plateau in hsp70 induction even after 48 h exposure and moderate tissue damage was observed in these larvae. The present study suggests that Nuvan is more cytotoxic than Dimecron in transgenic Drosophila melanogaster.

Sensitivity of adenylyl cyclase signaling system of the mollusk Anodonta cygnea ganglions to serotonin and adrenergic agonists.

For the first time, the adenylyl cyclase system (ACS) sensitive to biogenic amines in the mollusk Anodonta cygnea ganglions was revealed and characterized. Serotonin and isoproterenol stimulated AC activity and GTP-binding activity of heterotrimeric G-proteins. The AC-stimulating action of serotonin and isoproterenol was blocked by cyproheptadine and adrenergic antagonists, respectively. Using synthetic C-terminal peptides of G-protein alpha-subunit, it was shown that the biogenic amines realized their action on the ACS through different G-protein types: serotonin and isoproterenol activate G(s)-protein, while adrenaline preferably activates G(i)-protein.

[Age-dependent changes of morphometric and histochemical characteristics of neurocytes in different ganglia of albino rats]. / Vozrastnye preobrazovaniia morfometricheskikh i gistokhimicheskikh kharakteristik neirotsitov razlichnykh gangliev u belykh krys.

The aim of this study was to obtain the normative data on the age-dependent transformation of morphometric and histochemical characteristics of neurocytes in different ganglia in albino rats. Cell cross-sectional area, activities of cholinesterase (demonstrated with thioacetic acid method) monoamine oxidase (demonstrated with Glenner method) were measured in neurocytes of stellate, spinal, trigeminal and gastric ganglia in rats aged 2 to 360 days. Measurements were made with the help of "Bioscan" videoanalyzer. Informational analysis was used for the evaluation of the degree of maturation of neurocyte systems. General features, age- and organ-related peculiarities of morphometric and enzyme-histochemical characteristics were established for neurocytes of different ganglia, as well as a heterochronism of their definitive state attainment. The time of stabilization for neurocytes of stellate and I thoracic spinal ganglia was the age of 60 days, for those of trigeminal ganglion and intramural gastric ganglia -90 and 120 days, respectively. By this time, neurocyte systems turned from a determined state into a probabilistic-determined one, this transformation being considered as a population stabilization.

The effect of chronic bladder outlet obstruction on neuronal nitric oxide synthase expression in the intramural ganglia of the guinea pig bladder.

PURPOSE: We examined the effect of chronic partial outlet obstruction on expression of neuronal nitric oxide synthase (nNOS) in the intramural ganglion cells of the guinea pig bladder. MATERIALS AND METHODS: Partial urethral ligation was done in young male guinea pigs. The animals were sacrificed 2, 4, 6, 8 and 12 weeks after partial outlet obstruction and nNOS immunohistochemistry was done in the intramural neurons of the bladder. This was compared to controls (normal and sham operated). In addition, the mRNA expression of nNOS in bladders of 4-week sham operated and operated animals was also investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. RESULTS: Two weeks after urethral obstruction a decrease in the number of nNOS positive intramural neurons was detected. This decrease was most drastic at 4 weeks. Cell counting showed a 60.6% decrease in the number of nNOS positive neurons compared to controls. Some neurons appeared to undergo degenerative changes, such as irregular outline, vacuolation and lysis. At 6 weeks the number of nNOS positive neurons increased from the nadir level at 4 weeks and the increase was sustained until 12 weeks, when the number of nNOS positive neurons was almost at the level of controls. Quantitative reverse transcriptase-polymerase chain reaction also showed 42.4% down-regulation of nNOS expression 4 weeks after obstruction comparing with sham operation. CONCLUSIONS: Partial urethral ligation resulted in an initial decrease in nNOS positive neurons, which have been due to actual neuronal loss and/or enzyme down-regulation. This may be attributable to regional hypoxia as a result of decreased blood flow consequent to high intravesical pressure created by partial ligation. The decrease in nNOS expression followed by a compensatory increase in nNOS positive neurons also suggests an attempt or mechanism to up-regulate nitric oxide bioactivity following increased bladder outlet resistance.

Blocked MAP kinase activity selectively enhances neurotrophic growth responses.

Bone morphogenetic proteins (BMPs) 4 and 6 as well as MEK inhibitors PD98059 and U0126 potentiate neurotrophin 3 (NT3)- and neurturin (NTN)-induced neurite outgrowth and survival of peripheral neurons from the E9 chicken embryo. Preexposure to BMP4 or PD98059 was sufficient to prime the potentiation of subsequently added NT3. Phosphorylation of Erk2, induced by NT3, was reduced by MEK inhibition but unaffected by BMP signaling. Real-time PCR showed that neither BMP stimulation nor MEK inhibition increased Trk receptor expression and that the BMP-induced genes Smad6 and Id1 were not upregulated by PD98059. In contrast, both MEK inhibition and BMP signaling suppressed transcription of the serum-response element (SRE)-driven Egr1 gene. A reporter assay using NGF-stimulated PC12 cells demonstrated that MEK/Erk/Elk-driven transcriptional activity was inhibited by Smad1/5 and by PD98059. Thus, suppression of SRE-controlled transcription represents a likely convergence point for pathways regulating neurotrophic responses.