Anthony Krantis

Nitric oxide synthase distribution in the rat intestine: A histochemical analysis

BACKGROUND Nitric oxide is an inhibitory transmitter of nonadrenergic, noncholinergic neurons and is purported to be an endothelium-derived relaxant-type factor in the mammalian gut. This study aimed to provide a complete report on the distribution of NO synthase in the rat small and large intestine. METHODS NO synthase was visualized histochemically through this enzymes reduced nicotinamide adenine dinucleotide phosphate diaphorase activity and the distribution of staining within the gut wall. RESULTS The presence of NO synthase activity in myenteric neurons and their efferents to the circular muscle was confirmed. The largest proportion of stained cells per ganglion was found in the ileum, and the smallest proportion was in the colon. Stained neural elements were also found within the submucosa throughout the intestine. Stained cells within the myenteric and submucous nerve plexi displayed both type I and type II morphologies, with the latter being more numerous. In addition to neural staining, submucosal arterioles showed a regular pattern of small patches of staining unrelated to any perivascular innervation. CONCLUSIONS These findings indicate an extensive neural and vascular localization of NO generation potential throughout the wall of the rat intestine, thus providing a structural basis for the functional diversity of NO.

Use of L-Arginine in the Treatment of Experimental Necrotizing Enterocolitis

UNLABELLED Although multifactorial in etiology, prematurity and feeding are two of the most common risk factors associated with necrotizing enterocolitis (NEC). To understand the pathogenesis of NEC, the complex interaction between intestinal contents and clearing mechanisms in the immature human gut must be elucidated. Nitric oxide (NO) is a proposed mediator of nonadrenergic noncholinergic neural inhibition, causing relaxation in the gut. In addition to its role as a neuroeffector substance, studies suggest that endogenous formation of NO maintains intestinal mucosal integrity, protecting the gut from blood-borne toxins and tissue-destructive mediators. Thus, NO has a dual role in both gut smooth muscle relaxation and mucosal protection. Because two of the primary risk factors in the development of NEC are prematurity (as it relates to gut dysmotility) and enteral feeding (as it relates to mucosal damage by intraluminal substrate), the authors chose to investigate the role of NO in the pathogenesis of NEC induced by intraluminal injection of acidified casein solution in neonatal piglets. METHODS Having confirmed the consistent induction of NEC both macroscopically and histologically with this model (n = 32), the following were undertaken. Neonatal piglets (< 3 days old) were laparotomized, and intestinal loops were created from the terminal ileum to the proximal colon. The loops were injected with acidified casein solution and separated by saline-injected control loops. When the abdomen was closed, a continuous peripheral intravenous infusion of L-arginine, an NO synthase substrate (600 mg/kg/h [n = 6]), or N-omega-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor (20 mg/kg/h [n = 6]), was begun. Gut segments were harvested 3 hours later and processed for evaluation of the extent of necrosis. RESULTS Macroscopically, the L-NAME-treated group showed areas of hemorrhagic necrosis in the NEC-induced loops. The L-arginine-treated group had greatly diminished or virtually absent lesions. H&E-stained sections were graded microscopically, using a scale from 0 to 4, ranging from intact villi (grade 0) to transmural necrosis (grade 4). In the untreated NEC group, intestinal damage in the acidified casein loops was exhibited by areas of necrosis (extending, in some cases, transmurally), submucosal edema, and inflammatory cell infiltrate (average grade, 3.5). In the L-NAME-treated group, the intestinal damage was similar to that of the NEC-induced group (average grade, 3.5), but also presented with marked hemorrhagic congestion. In the L-arginine group, NEC-induced tissue damage was greatly attenuated, with necrosis limited primarily to the villus tips (average grade, 1). Nevertheless, inflammatory cell infiltrate and mild submucosal edema were still present. CONCLUSION Continuous intravenous infusion with the NO synthase substrate L-arginine markedly attenuates intestinal injury in this neonatal piglet model of NEC. Intravenous administration of the NO synthase inhibitor L-NAME causes hemorrhagic congestion of the gut wall. Based on these findings, the authors propose that treatment with the amino acid L-arginine should be considered as a potential therapeutic modality for NEC.

Histochemical localization of nitric oxide-synthesizing neurons and vascular sites in the guinea-pig intestine

Laminar preparations of fixed segments of the guinea-pig intestine were examined for nitric oxide synthase activity using reduced nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium salt as substrates. Under conditions specific for detecting nitric oxide synthase-related diaphorase activity, a subpopulation of neural elements in the myenteric plexus, deep muscular plexus and submucosa were intensely stained. Intensely stained nerve fibres were distributed throughout the meshworks of the myenteric plexus and its innervation of the circular muscle, and in the submucosa within Henles plexus. Intensely stained nerve cells and their processes were evident in most myenteric ganglia but were rare in ganglia of Henles plexus. Stained ganglion cells comprised types I, II and VI of the morphologically defined enteric nerve cells. Stained neural elements were increasingly prevalent within successively more caudal segments of the intestine. In addition to neuronal staining, arterioles of the submucosal vascular network displayed distinct, punctate patches of staining distributed over their surface. Perivascular nerve fibre staining was absent. These results show nitric oxide synthase activity to be present within neurons and fibres of the major enteric nerve layers and within submucosal blood vessels throughout the guinea-pig small and large intestine.

An Intraluminal Model of Necrotizing Enterocolitis in the Developing Neonatal Piglet

UNLABELLED The most common risk factors for the development of necrotizing enterocolitis (NEC) are prematurity and enteral feeding. Most models of NEC involve ischemic insult resulting in generalized necrosis, different from the classical ileocecal predilection of NEC. This anatomic predisposition is explained by dysmotility of immature gut, leading to bacterial overgrowth in the terminal ileum and colon. Infant formula containing lactose as the sole carbohydrate source overwhelms partially developed lactase activity, allowing enteric bacteria to ferment excess carbohydrate to short-chain fatty acids, decreasing intraluminal gut pH and predisposing to mucosal injury. Impaired clearance of intraluminal contents exacerbates this effect. In the present study the authors used a model of NEC, originally developed in rabbits and based on analysis of intestinal contents of NEC babies, modified and adapted here to neonatal piglets, the gastrointestinal tract of which more closely resembles the human neonate. METHOD Piglets < 3 days old and 2 weeks old were laparotomized. Loops created from the distal ileum to the proximal colon were injected with isoosmolar acidified casein solution or 0.9% saline. Segments were harvested 3 hours later, sectioned for H&E, and graded from 0 (intact villi) to 4 (transmural necrosis). RESULTS Acidified casein-induced damage included areas of necrosis, submucosal edema, inflammatory cell infiltrate, and lymphatic distension. In younger animals, lesions were more pronounced (3.25 +/- 0.13 for the < 3-day-old v 2.43 +/- 0.14 for the 2-week-old piglets; P < .005). CONCLUSION The authors believe that this piglet NEC model most closely approximates human NEC because it incorporates two of the most common risk factors: dysmotility (by creating intestinal loops) and enteral feeding (by intraluminal injection of acidified casein).

GABA-related actions in isolated in vitro preparations of the rat small intestine.

Longitudinal organ bath preparations of the rat duodenum, jejunum and ileum were tested for their responsiveness to GABA-receptor agonists. The GABAA-receptor agonists, GABA and 3APS, induced non-adrenergic, non-cholinergic relaxations and/or contractions, although the magnitude and type of response varied depending upon the region tested. All regions relaxed to applied GABA or 3APS, however the jejunum and ileum also responded with cholinergic contractions. These relaxant and contractile actions were neurogenic and sensitive to blockade by the GABA antagonists bicuculline or picrotoxinin, and desensitization to either agonist. The GABAB-receptor agonist baclofen, caused a reduction in electrically evoked cholinergic contractions. These inhibitory actions of baclofen were insensitive to bicuculline or picrotoxinin. Taken together, these results show that GABA-ergic actions in the rat small intestine are mediated by two pharmacologically distinct neural receptor populations, the GABAA and GABAB sites, the distribution and sensitivity of which differ along the length of the small intestine.

Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons.

BACKGROUND Carbon monoxide (CO), like nitric oxide (NO), is a putative gaseous neurotransmitter. CO is produced by the enzyme heme oxygenase (HO) acting on a family of heme-containing compounds. Two isomers of HO have been characterized (HO-1, HO-2). In the CNS and in peripheral ganglia HO-2 occurs in a majority of neurons. NO and CO function as transmitters of enteric neurons but the relative distribution of enteric neurons utilizing these gaseous transmitters is unknown in rodent. We have studied the distribution of HO-2 immunoreactivity and NO synthase (NOS) activity within the rat ileum. METHODS Tissue sections and primary neuronal cell cultures were incubated with a HO-2 specific antibody, and then assessed or reprocessed for NOS activity using NADPH-dependent diaphorase staining. RESULTS HO-2 immunoreactivity was expressed in subpopulations of myenteric and submucosal neurons. Approximately 45% of the ganglion cells in tissue section were HO-2 positive. This was similar in proportion to those found to stain for NOS activity, and 10% of HO-2 positive neurons also contained NOS. HO-2 immunoreactivity was also found in epithelial cells within the villi, and in interstitial cells around the myenteric plexus and within the smooth muscle. In culture, the distribution and colocalisation of HO-2 and NOS positive neurons was similar to that in tissue sections. We identified labelled neurons as either Dogiel Type I or II; only Type II cells colocalized NOS and HO-2. CONCLUSION Neurons, endocrine-like cells and interstitial cells with the capacity for CO production are distributed throughout the ileum and some neurons have the capacity to synthesize both NO and CO as gaseous messengers.

Distribution of the NPY receptor subtype Y1 within human colon: Evidence for NPY targeting a subpopulation of nitrergic neurons

Neuropeptide Y is a neurotransmitter in both the central nervous system and the enteric nervous system. Neuropeptide Y receptors have been demonstrated by in situ hybridization and ligand binding techniques to be present in both of these systems. In this study we report on the distribution of the Y1 isoform of the neuropeptide Y receptor (YY1) in human colon using an antibody raised against the Y1 receptor. This method permits greater resolution in determining the distribution of the receptor and provides the opportunity to study neurotransmitter markers in relationship to the Y1 receptor. Y1 receptor immunoreactivity was localized within ganglionic neurons and axons of the myenteric and submucosal nerve networks, axons within the muscularis mucosae, longitudinal and circular smooth muscle layers, sympathetic nerve fibers around blood vessels and within scattered cells in the mucosa and basal cells of the crypts. Neuropeptide Y/Y1 double staining showed that the peptide and its Y1 receptor subtype were often colocalized within ganglion cells of Henles plexus in the submucosa. Thus, Y1 may act as an autoreceptor within the colonic gut wall. Nitric oxide synthase was found within most neurons of the myenteric plexus which displayed Y1-receptor immunoreactivity but this correlation was not seen in the submucosa. Instead, the colocalization of nitric oxide synthase and Y1-immunoreactivity was extremely low. These results indicate a striking difference in the Y1 Neuropeptide Y activation of nitrergic mechanisms within the myenteric and submucosal nerve networks.

Effects of oral exposure to naturally-occurring and synthetic deoxynivalenol congeners on proinflammatory cytokine and chemokine mRNA expression in the mouse

The foodborne mycotoxin deoxynivalenol (DON) induces a ribotoxic stress response in mononuclear phagocytes that mediate aberrant multi-organ upregulation of TNF-α, interleukins and chemokines in experimental animals. While other DON congeners also exist as food contaminants or pharmacologically-active derivatives, it is not known how these compounds affect expression of these cytokine genes in vivo. To address this gap, we compared in mice the acute effects of oral DON exposure to that of seven relevant congeners on splenic expression of representative cytokine mRNAs after 2 and 6h. Congeners included the 8-ketotrichothecenes 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), nivalenol (NIV), the plant metabolite DON-3-glucoside (D3G) and two synthetic DON derivatives with novel satiety-inducing properties (EN139528 and EN139544). DON markedly induced transient upregulation of TNF-α IL-1β, IL-6, CXCL-2, CCL-2 and CCL-7 mRNA expressions. The two ADONs also evoked mRNA expression of these genes but to a relatively lesser extent. FX induced more persistent responses than the other DON congeners and, compared to DON, was: 1) more potent in inducing IL-1β mRNA, 2) approximately equipotent in the induction of TNF-α and CCL-2 mRNAs, and 3) less potent at upregulating IL-6, CXCL-2, and CCL-2 mRNAs. EN139528s effects were similar to NIV, the least potent 8-ketotrichothecene, while D3G and EN139544 were largely incapable of eliciting cytokine or chemokine mRNA responses. Taken together, the results presented herein provide important new insights into the potential of naturally-occurring and synthetic DON congeners to elicit aberrant mRNA upregulation of cytokines associated with acute and chronic trichothecene toxicity.

Immunopositive GABAergic neural sites display nitric oxide synthase-related NADPH diaphorase activity in the human colon

In the enteric nervous system, gamma-aminobutyric acid (GABA) is a transmitter of interneurons which are proposed to innervate excitatory and inhibitory motor neurons. Nitric oxide (NO) is a putative transmitter of enteric inhibitory motor nerves targeted by GABA. In addition, NO is synthesized by a variety of enteric nerves throughout the gut wall indicative of its potential to be a transmitter of other nerve types, including interneurons. We sought to determine if some populations of nitrergic neurons are interneurons in human infant colon. As enteric neural GABA is exclusive to interneurons, colocalization with NO synthase-related NADPH diaphorase was examined. GABA-transaminase (GABA-T) immunohistochemistry was used to identify GABAergic neurons and a histochemical protocol was used as a marker of neuronal NO synthase-related NADPH diaphorase activity in enteric layers. GABA-T immunoreactive neurons were seen in the ganglionated nerve networks of the myenteric and submucosal layers. GABA-T immunoreactive fibres were also present in the longitudinal and circular muscle layers. A subpopulation of GABA-T immunoreactive neurons within both the myenteric and submucosal ganglia express NO synthase-related activity. This colocalization extends further to a subpopulation of fibers within the muscle layers. These findings strongly suggest that in addition to its role in inhibitory motor neurons, NO may also be a transmitter of enteric interneurons.

Neurochemical characterization and distribution of enteric GABAergic neurons and nerve fibres in the human colon

GABA, somatostatin and enkephalin are neurotransmitters of enteric interneurons and comprise part of the intrinsic neural circuits regulating peristalsis. Within the relaxation phase of reflex peristalsis, nitric oxide (NO) is released by inhibitory motor neurons and perhaps enteric interneurons as well. Previously, we identified by GABA transaminase (GABA-T) immunohistochemistry, a subpopulation of GABAergic interneurons in the human colon which also contain NO synthase activity and hence produce NO. In this study, we have examined further the capacity for cotransmission within the GABAergic innervation in human colon. The expression of two important neuropeptides within GABAergic neurons was determined by combined double-labelled immunocytochemistry using antibodies for GABA-T, enkephalin and somatostatin, together with the demonstration of NO synthase-related NADPH diaphorase staining in cryosectioned colon. Both neuropeptides were found in GABAergic neurons of the colon. The evidence presented herein confirms the colocalization of NO synthase activity and GABA-T immunoreactivity in subpopulations of enteric neurons and further allows the neurochemical classification of GABAergic neurons of the human colon into three subsets: (i) neurons colocalizing somatostatin-like immunoreactivity representing about 40% of the GABAergic neurons, (ii) neurons colocalizing enkephalin-like immunoreactivity, about 9% of the GABAergic neurons and (iii) neurons colocalizing NO synthase activity, about 23% of the GABAergic neurons. This division of GABAergic interneurons into distinct subpopulations of neuropeptide or NO synthase containing cells is consistent with and provides an anatomical correlate for the pharmacology of these transmitters and the pattern of transmitter release during reflex peristalsis.