Fast evaluation of intraoperative biopsies for ganglia in Hirschsprung's disease.

PURPOSE: The most important diagnostic features of Hirschsprung's disease (HD) are the combination of aganglionosis and hypertrophic nerve bundles. Acetylcholinesterase staining is widely used for diagnosis of HD as it identifies hypertrophic nerves in both diagnostic and intraoperative biopsies. The main drawback of this method is in the identification of ganglia. It has been suggested that the combination of this method together with another histochemical marker would be a superior diagnostic tool. Hematoxylin and eosin is still the diagnostic measure of choice for identifying ganglia in many centers, although it presents a persistent diagnostic challenge for pathologists trying to rapidly and accurately interpret the frozen biopsies that guide intraoperative decision making. METHODS: Therefore, we sought to develop a fast, intraoperative immunohistochemical protocol for visualization of ganglia and nerves in HD specimens that can be used in conjunction with these other tools. RESULTS: With the use of acetone fixation and immunofluorescence staining with antibodies to neurofilament 68 and tubulin, ganglia in sections of full thickness and suction biopsies could be visualized in only 10 minutes. This protocol facilitated the identification of ganglia in hematoxylin and eosin-stained adjacent sections and also identified hypertrophic nerve trunks. CONCLUSION: This method should significantly enable the identification of ganglia in suction and full thickness biopsies.

In vitro activity of uva-ursi against cytochrome P450 isoenzymes and P-glycoprotein.

Some natural health products (NHPs) affect drug metabolism enzymes and transport proteins, potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of uva-ursi (Arctostaphylos uva-ursi) on cytochrome P450 isozyme (3A4, 3A5, 3A7, 2C19, and 19)-mediated metabolism and P-glycoprotein (P-gp) transport. Three bulk and 2 capsulated uva-ursi samples were obtained from commercial outlets. The capsules were batched, and herbal samples were ground to a common consistency. Aqueous and methanol extracts were freshly prepared. Cytochrome P450 isozyme-mediated metabolism was determined by using in vitro bioassays. P-gp transport function was determined by using a rhodamine 123 (Rh123) uptake test in human (THP-1) monocytes and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myricitrin, and isoquercetin. A large variation was observed in the biomarkers found between the bulk and capsulated samples. Our data indicate that both the aqueous and methanol extracts of all 5 uva-ursi products showed high cytochrome P450 isozyme inhibition, with the exception of the methanol extracts against cytochromes P3A4 and P19, which had low to moderate activity. The aqueous extracts of uva-ursi showed an inhibitory effect on Rh123 efflux by P-gp at 1 h and an inductive effect at 18 h for both cell lines. Our results show that the uva-ursi herbal products tested here have pharmacological properties, including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine whether these effects are clinically significant.

Analysis of Fusarium graminearum mycotoxins in different biological matrices by LC/MS.

The purpose of this study was to develop an LC/MS assay to accurately detect three mycotoxins produced by Fusarium graminearum in various matrices. Using different LC conditions, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) were detected in four different matrices (fungal liquid cultures, maize grain, insect larvae and pig serum). The sensitivity of MS detection allowed us to detect concentrations as low as 8 ppb of DON and 12 ppb of ZEN. A very small quantity of matrix was therefore necessary for successful analysis of these toxins and a variety of experimental situations were successfully investigated using this technique. Production of 15-ADON and butenolide was monitored in a liquid culture of F. graminearum under controlled conditions. Using simple extraction procedures, the differential accumulation of DON and 15-ADON was followed in inoculated maize genotypes varying in susceptibility to F. graminearum. Toxicokinetic studies were carried out with maize insect pests reared continually on artificial diets containing ZEN and suggested that larvae may possess the ability to degrade ZEN. Finally, persistence of DON was assessed in pigs fed diet supplemented with DON, results indicated that DON accumulates quickly in pig blood and then levels decline progressively for 12 hours thereafter. The LC/MS study reported here is very useful and flexible for the detection of these mycotoxins in different media and at very low concentrations.

In vitro inhibition of human cytochrome P450-mediated metabolism of marker substrates by natural products.

Spices, herbal and black teas, and soybean products were analyzed for their capacity to inhibit in vitro metabolism of drug marker substrates by human cytochrome P-450 (CYP) isoforms. Inhibition of drug metabolism was determined using aliquots or infusions from these products in a fluorescence-detection assay. Aliquots and infusions of all natural product categories inhibited 3A4 metabolism to some extent. Of the 26 aliquots from teas and spices further tested with 2C9, 2C19 and 2D6, many demonstrated significant inhibitory activity on the metabolism mediated by these isoforms. Black teas and herbal tea mixtures were generally more inhibitory than single-entity herbal teas. Spices and single-entity herbal teas showed species-specific isoform inhibition with sage, thyme, cloves, St John's Wort and goldenseal having the highest activity against several isoforms. Seven soybean varieties tested, as well as daidzein and genistein isolated from soybean, were found to inhibit 3A4-mediated metabolism. Genistein was found to inhibit 3A7- but not 3A5-mediated metabolism of the marker substrate. Assessment of the in vitro CYP inhibition potential for these natural products has important implications for predicting the likelihood of natural product-drug interactions if these products are taken concomitantly.

An in vitro evaluation of human cytochrome P450 3A4 and P-glycoprotein inhibition by garlic.

PURPOSE: Garlic has been used as a flavouring agent, traditional medicine, and functional food to improve physical or mental well-being. Garlic and garlic products generally have been regarded as safe but a number of conflicting reports in the literature and confounding factors make it difficult to unequivocally establish the clinical efficacy and safety of these products either alone or in the presence of therapeutic products. A preliminary study was undertaken with fresh garlic and garlic products using the major cDNA-expressed human cytochrome P-450 isozymes associated with the metabolism of HIV/AIDS drugs, and purified P-glycoprotein (P-gp) cell membranes to ascertain the risk potential for generating interactions with therapeutic products. METHODS: A broad screening was undertaken with 10 garlic products (aged, odourless, oil, freeze-dried) and 3 varieties of fresh garlic bulbs (common, Elephant and Chinese), all purchased from local outlets, to examine their potential to affect human cytochrome P-450 2C9*1, 2C9*2, 2C19, 2D6, 3A4, 3A5 and 3A7 mediated-metabolism of marker substrates using an in vitro fluorometric microtiter plate assay. Four garlic products were screened for their potential to interact with P-gp using an in vitro colourmetric ATPase assay. RESULTS: Extracts of fresh garlic, different brands and lots of odourless garlic and representative samples of garlic oil, freeze dried garlic, and aged garlic exhibited an inhibitory effect on cytochrome P450 2C9*1, 2C19, 3A4, 3A5 and 3A7 mediated metabolism of a marker substrate. The activity of 2D6 mediated-metabolism was generally unaffected by garlic. Extracts of the fresh garlic stimulated CYP2C9*2 metabolism of the marker substrate. With the extracts tested, garlic had very low to moderate P-gp interaction as compared with the positive control verapamil. CONCLUSIONS: Our in vitro findings demonstrate that garlic components can affect cytochrome P-450 2C, 2D and 3A mediated-metabolism of the isoforms studied. The safety and efficacy of conventional therapeutic products metabolized by the affected isozymes, particularly those with a narrow therapeutic index, taken concomitantly with garlic needs to be examined further under clinical settings.

Altered nitric oxide production in the premature gut may increase susceptibility to intestinal damage in necrotizing enterocolitis.

BACKGROUND/PURPOSE: Nitric oxide (NO) mediates enteric smooth muscle relaxation and mucosal protection. The authors have identified an ontogenically determined pattern of enteric NO neural maturation that may render the distal gut of premature piglets susceptible to injury. METHODS: NO synthase (cNOS and iNOS) activities were measured in the developing piglet gut wall and compared with gut from an intraluminal model of necrotizing enterocolitis (NEC) at different times. RESULTS: In premature animals, iNOS activity was significantly higher 3 hours after NEC induction compared with similarly treated 3-day-old piglets. INOS levels continued to rise 6 hours after NEC induction in prematures. Premature animals (labor induced by prostaglandins) failed to show such a rise in iNOS. In 3 day olds, iNOS levels increased significantly 16 hours after injury compared with the 3-hour group. CONCLUSIONS: iNOS production increases in premature piglets with NEC compared with full-term NEC animals and continues to rise in the presence of intestinal damage regardless of developmental status. Maternal administration of prostaglandins attenuates this rise in iNOS activity. Elevated NO production in premature gut may contribute to increased susceptibility to damage in NEC.

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.

GABA(A) receptor subunit messenger RNA expression in the enteric nervous system of the rat: implications for functional diversity of enteric GABA(A) receptors.

GABAergic neurons occur in the myenteric plexus and submucosa and their innervations of the gut, where GABA stimulates motor neurons, and non-neural cells via "central type" GABA(A) receptors. These receptors occur on half of the neurons in the rat intestine. The GABA(A) receptor is a ligand-gated chloride channel constructed from different subunit families (alpha, beta, gamma, delta, epsilon). In rat these exist as subtypes, alpha1-6, beta1-3, gamma1-3 and delta, defining the clinically relevant pharmacological features of GABA(A) receptors. However, the identity, distribution, and abundance of enteric GABA(A) receptor subunits are unknown. To identify and map the regional expression of GABA(A) receptor subunit messenger RNAs in the enteric nervous system, we assayed enteric RNA from the ileum of Sprague-Dawley rats by reverse transcription-polymerase chain reaction for alpha1-6, beta 1-3, gamma1-3, and delta subunit messenger RNAs. Subunit messenger RNA localization, was probed by in situ hybridization. Reverse transcription-polymerase chain reaction analysis of RNA from myenteric and submucosal nerve layers revealed the expression alpha1, alpha3, beta2, beta3, gamma1 and gamma3 subunit messenger RNAs. Little alpha4 and alpha6 and no alpha2, beta1, gamma2 or delta subunit messenger RNA were detected. In situ hybridization revealed that transcripts for alpha1, alpha3, alpha5 and beta2 subunits occur in both myenteric and submucous ganglia. However, beta3 messenger RNA was found only in myenteric plexus. The gamma1 subunit messenger RNA was also restricted to the cells in the myenteric plexus while gamma3 was found in cells of both nerve layers. In this study of the subunit messenger RNA expression profile of GABA(A) receptors within the enteric nerve layers we show an abundant, diverse and widespread distribution that is unique in comparison to the CNS. The distinctive and heterogeneous distribution of enteric GABA(A) subunits may be important in the integration of neural control of gut function.

Rat gastroduodenal motility in vivo: involvement of NO and ATP in spontaneous motor activity.

Our studies of fasted anesthetized rats have shown that all spontaneous relaxations of the antrum are nitric oxide (NO) dependent. Duodenal motility is patterned into propagating "grouped" motor activity interposed with "intergroup" periods of nonpropagating motor activity; in the duodenum, only intergroup relaxations are NO dependent. We examined the involvement of NO and ATP in spontaneous motor activities of the gastroduodenum in vivo: contractions and relaxations were recorded and analyzed simultaneously from the antrum (S1) and proximal duodenum (D1) of anesthetized Sprague-Dawley rats (n = 10/group), using extraluminal foil strain gauges. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral and intergroup relaxations, whereas grouped relaxations were enhanced (P < 0.05). These effects were reversed with L-arginine (300 mg/kg iv). L-NAME also increased (P < 0.05) the amplitude of duodenal contractions. ATP (8 mg. kg-1. min-1 iv) stimulated relaxations at S1 and D1 that were blocked by the P2-purinoceptor antagonist suramin (60 mg/kg iv). This treatment did not affect spontaneous antral relaxations; however, duodenal grouped relaxations were attenuated. Desensitization to the P2x-purinoceptor agonist alpha,beta-methylene ATP (300 micrograms/kg iv) gave results similar to suramin. In contrast, the P2y-purinoceptor agonist 2-methylthio-ATP (2-MeS-ATP; 360 micrograms/kg iv) evoked duodenal relaxations that were attenuated by L-NAME, and desensitization to 2-MeS-ATP attenuated intergroup relaxations. Spontaneous relaxations of the rat antrum and duodenal intergroup relaxations are NO dependent. Both gut regions relax in response to systemically administered ATP; this response is sensitive to suramin. Grouped duodenal relaxations display functional sensitivity to suramin and P2x- purinoceptor desensitization, indicative of the involvement of ATP and P2x purinoceptors. P2y purinoceptors must also be present; however, these occur on elements releasing NO. Although NO does not mediate grouped relaxations or duodenal contractions, the sensitivity of these responses to L-NAME indicates that the pathway(s) controlling these responses is modulated by NO.

Rat gastroduodenal motility in vivo: interaction of GABA and VIP in control of spontaneous relaxations.

Spontaneous relaxations occurring within motor activity in the rat gastroduodenum in vivo can be distinguished by their dependence on either nitric oxide (NO) or ATP. We examined the interaction of gamma-aminobutyric acid (GABA) and vasoactive intestinal peptide (VIP) within pathways controlling this activity in the antrum (S) and duodenum (D) of anesthetized Sprague-Dawley rats, using miniaturized extraluminal foil strain gauges oriented perpendicular to (S1, D1) or in the axis of (S2) the circular smooth muscle. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral relaxations and, in the duodenum, nonpropagating "intergroup" relaxations. The GABAA receptor antagonist bicuculline (350 micrograms/kg sc) had similar effects. The GABAA agonist 3-amino-1-propanesulfonic acid stimulated L-NAME-sensitive relaxations at S1 and D1. Propagating "grouped" responses were unchanged. VIP (6 micrograms/kg iv) always induced a relaxation of the duodenum, which was attenuated by bicuculline and L-NAME. VIP caused simultaneous responses at S1 and S2; however, the antrum displayed either contraction or relaxation in response to VIP. All antral relaxations in response to VIP were attenuated (P < 0. 05) by L-NAME; however, only VIP-induced relaxations at S1 were sensitive to bicuculline. VIP-induced contractions were also unaffected. GABAA receptors mediate the pathway(s) controlling NO-related spontaneous relaxations of the antrum and duodenal circular muscle. All VIP-induced relaxations are mediated by NO. Spontaneous relaxations of the rat gastroduodenum include responses that involve a GABAAergic NO-related pathway, which is targeted by VIP. In addition, VIP can target NO relaxations of the antrum via other pathways.

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.

Dysplastic nitrergic neurons in the rectum of a patient with rectal ectasia.

We present a histochemical study of resected colon from a 13-year-old boy diagnosed with rectal ectasia. Laminar preparations and sectioned tissue of rectum were assayed histochemically for nitric oxide (NO) synthase activity by reducing nitro blue tetrazolium salt in the presence of the cofactor NADPH. Tissue preparations displayed intensely labelled neurons and fibers throughout the gut wall. Laminar preparations of Meissner's plexus showed a hyperplasia of ganglia and NO-related neurons throughout the length of the resected rectum compared with normal bowel. Sectioned tissue of the Auerbach's plexus demonstrated a normal number of ganglia and NO-related neurons. As well, the ectatic bowel showed a proliferation of nerve fibers in keeping with the degree of circular smooth muscle hypertrophy. This proliferation may represent a reactive phenomenon secondary to the functional obstruction. The NO histochemical technique may form the basis of further investigations in defining the cause of this functional obstruction.

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 Henle's 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.

The effects of gamma-radiation on intestinal motor activity and faecal pellet expulsion in the guinea pig.

The effects of whole-body gamma-radiation (10 Gy) on intestinal motor activity was examined in the small and large intestine of the guinea pig 18 hr post irradiation. Neurally mediated relaxations of isolated gut bath preparations were generally unaffected. However, the contractile responses to direct smooth muscle stimulation with the cholinergic muscarinic agonist carbachol or ganglionic stimulation of intrinsic cholinergic motor neurones were significantly increased in the duodenum and colon but not the jejunum. This increased sensitivity to cholinergic stimulation was reflected in an increased contractility and a shift in the concentration-response curves for carbachol. The specificity of radiation actions for cholinergic mediated contractions was further supported by the observation that histamine-evoked contractions were unaffected. In a second series of experiments we examined the effects of gamma-radiation on the rate of pellet expulsion from freshly excised colons. Both colons from irradiated animals and nonirradiated colons exposed to carbachol showed significantly faster rates of pellet expulsion, indicative of increased propulsive motility. Pretreatment of animals with 0.5 mg/kg sc of the 5HT3 receptor antagonist Granisetron prevented the effect of radiation and reduced the pellet expulsion rate to below normal. These results indicate that gastrointestinal motility disturbances seen in organ-bath preparations of the intestine from rats exposed to whole-body gamma-radiation may be related to an increased sensitivity of the cholinergic muscarinic system.

A method for simultaneous recording and assessment of gut contractions and relaxations in vivo.

The normal gut exhibits both contractions and relaxation from a resting tonus. Pathological and pharmacological information may be gleaned from separate measurement of these activities. Methods for recording and analyzing gut contractions have been presented before. We present an extension to the methods, utilising simple foil strain gauges tested here in male Sprague-Dawley rats. Coupled with a computer-based data acquisition and analysis system, we could assess the relaxation and contraction waves recorded simultaneously and from different gut regions in vivo. In contrast with the antrum, where spontaneous motor activity consisted of low frequency relaxations and random contractions, spontaneous duodenal motility was patterned into periodic groups of intense activity interposed by periods of low amplitude, low frequency contractions and relaxations. This grouped activity was propagatory, reminiscent of migrating myoelectrical complexes. When challenged with the ulcerogen cysteamine-HCl (56 mg/100 g s.c.), only duodenal motor activity was affected. Moreover, this treatment had differential effects in the duodenum. Patterned motility was no longer distinguishable, and contraction frequency and amplitude were increased while relaxation amplitude was decreased. This method affords a particularly sensitive and more precise assessment of both contractile and relaxant motor activity in vivo, before and after drug treatment.

gamma-Aminobutyric acid stimulates electrolyte transport in the guinea pig ileum in vitro.

BACKGROUND & AIMS: An extensive enteric distribution of GABAergic neurons and nerve fibers has been identified in a number of species, including humans, but the role of gamma-aminobutyric acid (GABA) in mucosal physiology is unclear. The study was designed to determine if GABA stimulates electrolyte transport in an in vitro guinea pig ileum model. METHODS: The localization of GABAergic innervation in the submucosa and mucosa was determined using autoradiography. The effects of GABA and its analogues on electrolyte transport were measured in stripped guinea pig ileum mounted in Ussing chambers. Sensory afferent-evoked secretion after GABAA receptor blockade was also assessed. RESULTS: GABA and the GABAA receptor agonist 3-amino-1-propanesulfonic acid, but not the GABAB agonist baclofen, caused a bicuculline- and tetrodotoxin-sensitive, biphasic increase in short-circuit current. The response to 3-amino-1-propanesulfonic acid was partially reduced by atropine, implicating cholinergic secretomotor neurons, and by the histamine H1 antagonist pyrilamine, suggesting the involvement of a histamine-releasing cell. The GABAA receptor antagonist bicuculline and 3-amino-1-propanesulfonic acid-induced tachyphylaxis, but not the GABAA-associated chloride channel blocker picrotoxinin, caused a modest reduction in the secretory responses to capsaicin. CONCLUSIONS: Activation of submucosal GABAA receptors elicits a multifactorial secretory response but plays a minor role in capsaicin-sensitive, afferent-evoked secretion.

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).

Localization of GABAA receptor immunoreactivity in NO synthase positive myenteric neurones.

GABAA receptors were localized within laminar preparations of the rat distal colon myenteric plexus using a monoclonal antibody (mAb 62-3G1) to the affinity purified GABAA receptor/benzodiazepine receptor/Cl- channel complex. The immunofluorescence procedure showed that approximately half of the myenteric ganglion cells displayed extensive GABAA receptor labelling of their soma. This population was further characterised by treating some GABAA-receptor-labelled laminar preparations for the histochemical demonstration of nitric oxide (NO) synthase-related NADPH-dependent diaphorase activity. A subpopulation of the GABAA-receptor-immunoreactive cells (35%) were also found to display intense NO-synthase-related activity. These findings extend our understanding of the GABAA-receptor-related innervation of the rat gut wall herein referred to as 'A-GABAergic' and provides an anatomical basis for the pharmacologically-identified GABA-nitrergic pathway in the mammalian gut.

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.