Colonic Nerves Research Articles

Activated mast cells in proximity to colonic nerves correlate with abdominal pain in irritable bowel syndrome

Background & Aims : The mechanisms underlying abdominal pain perception in irritable bowel syndrome (IBS) are poorly understood. Intestinal mast cell infiltration may perturb nerve function leading to symptom perception. We assessed colonic mast cell infiltration, mediator release, and spatial interactions with mucosal innervation and their correlation with abdominal pain in IBS patients. Methods : IBS patients were diagnosed according to Rome II criteria and abdominal pain quantified according to a validated questionnaire. Colonic mucosal mast cells were identified immunohistochemically and quantified with a computer-assisted counting method. Mast cell tryptase and histamine release were analyzed immunoenzymatically. Intestinal nerve to mast cell distance was assessed with electron microscopy. Results : Thirty-four out of 44 IBS patients (77%) showed an increased area of mucosa occupied by mast cells as compared with controls (9.2% ± 2.5% vs. 3.3 ± 0.8%, respectively; P < 0.001). There was a 150% increase in the number of degranulating mast cells (4.76 ± 3.18/field vs. 2.42 ± 2.26/field, respectively; P = 0.026). Mucosal content of tryptase was increased in IBS and mast cells spontaneously released more tryptase (3.22 ± 3.48 pmol/min/mg vs. 0.87 ± 0.65 pmol/min/mg, respectively; P = 0.015) and histamine (339.7 ± 59.0 ng/g vs. 169.3 ± 130.6 ng/g, respectively; P = 0.015). Mast cells located within 5 μm of nerve fibers were 7.14 ± 3.87/field vs. 2.27 ± 1.63/field in IBS vs. controls ( P < 0.001). Only mast cells in close proximity to nerves were significantly correlated with severity and frequency of abdominal pain/discomfort ( P < 0.001 and P = 0.003, respectively). Conclusions : Colonic mast cell infiltration and mediator release in proximity to mucosal innervation may contribute to abdominal pain perception in IBS patients.

Differential effect of nitric oxide synthase inhibition on sigmoid colon longitudinal and circular muscle responses to nicotine and nerve stimulation in vitro.

Nicotine has been shown to release nitric oxide from nerves in human sigmoid colon. This effect has been used to investigate the innervation and functional relationship of the longitudinal and circular muscle layers. Strips of longitudinal and circular muscle were obtained from 19 patients with colorectal cancer. The strips from ten patients were subjected to electrical field stimulation (EFS) in vitro using stimulus parameters for selective stimulation of nerves. The effect of nicotine 1-10 micromol/l on EFS responses was then measured in the presence and absence of a nitric oxide synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) 200 micromol/l. The effect of nicotine on spontaneous activity was investigated in the muscle strips from the other nine patients. Both longitudinal and circular strips responded to EFS with contraction. The time to achieve a peak contractile response (time to peak; TTP) was significantly longer (P<0.001) in circular strips. L-NAME reduced the mean(s.e.m.) TTP in circular muscle from 23.3(2.0) to 17.2(1.5) s (P=0.007) and altered its pattern of response to resemble that of longitudinal muscle. Nicotine 10 micromol/l reduced the contraction to EFS in circular (P<0.001) but not in longitudinal (P=0.347) muscle. The nicotine-induced reduction in circular muscle contraction was blocked by L-NAME 200 micromol/l (P=0.005). These findings suggest that nitric oxide release on neural stimulation is greater in circular than in longitudinal muscle.

Short-Chain Fatty Acids Decrease the Frequency of Spontaneous Contractions of Longitudinal Muscle via Enteric Nerves in Rat Distal Colon

Short-chain fatty acids (SCFAs) produced by the bacterial fermentation of carbohydrates in the cecum and proximal colon are reported to modify colonic motility as a luminal factor. Besides the physical stimuli in the distal colon, SCFAs in the intestinal lumen also seem to affect colonic motility under physiological concentrations. This study therefore used fasted rats to investigate the effect of SCFAs on the spontaneous contractions of longitudinal muscle (LM) in rat distal colon, including mucosa in vitro. The frequency of spontaneous contractions of LM strips from the distal colon was 9.4 +/- 0.5 contractions/20 min. The exogenous addition of >5 mM SCFAs decreased the frequency of spontaneous contractions of the LM to 6.1 +/- 0.8 contractions/20 min. Among SCFAs, only acetate elicited this inhibitory response. TTX and the combination of hexamethonium and granisetron abolished SCFA-induced inhibitory response, suggesting that this inhibitory response is mediated via the ENS, including nicotinic and 5-HT(3) receptors. In conclusion, it is suggested that SCFAs in rat distal colon decrease the frequency of spontaneous contractions of the LM and that SCFAs may contribute to colonic motility, including the peristaltic reflex, by regulating the frequency of spontaneous contractions of the LM through the enteric nervous system (ENS).

Rectal intraganglionic laminar endings are transduction sites of extrinsic mechanoreceptors in the guinea pig rectum

Background & aims : Vagal afferent mechanoreceptors in the upper gut have recently been identified morphologically as intraganglionic laminar endings (IGLEs), but little is known about the structure of mechanoreceptive endings elsewhere in the gastrointestinal tract. We have morphologically characterized the nerve endings of specialized mechanoreceptors in the rectum. Methods : Extracellular recordings from guinea pig rectal and colonic nerves were made, in vitro, in combination with rapid anterograde transport of biotinamide, to reveal the morphology of recorded fibers. Controlled distentions were used to activate mechanoreceptive afferent units, and von Frey hairs were used to identify their transduction sites. Results : Rectal mechanoreceptors were present in high density, had low thresholds, and adapted slowly to maintained distention. Each afferent unit had multiple small (<200-μm diameter) transduction sites (“hot spots”) at which they could be activated locally by application of a light von Frey hair (0.08–7 mN). Anterograde dye filling revealed characteristic rectal intraganglionic laminar endings (rIGLEs) in myenteric ganglia, significantly associated with hot spots, comparable to the IGLEs of vagal tension receptors, but smaller and less complex. Afferent fibers with these morphologic and physiologic features could not be recorded from colonic nerves innervating the large bowel proximal to the rectum. Conclusions : The rectum receives a dense afferent innervation by a distinct population of low-threshold, slowly adapting mechanoreceptors with specialized intraganglionic laminar endings (rIGLEs), which are not found more proximally in the colon.

Circumferential, not longitudinal, colonic stretch increases synaptic input to mouse prevertebral ganglion neurons.

The relationship between longitudinal and circular muscle tension in the mouse colon and mechanosensory excitatory synaptic input to neurons in the superior mesenteric ganglion (SMG) was investigated in vitro. Electrical activity was recorded intracellularly from SMG neurons, and muscle tension was simultaneously monitored in the longitudinal, circumferential, or both axes. Colonic intraluminal pressure and volume changes were also monitored simultaneously with muscle tension changes. The results showed that the frequency of fast excitatory postsynaptic potentials (fEPSPs) in SMG neurons increased when colonic muscle tension decreased, when the colon relaxed and refilled with fluid after contraction, and during receptive relaxation preceding spontaneous colonic contractions. In contrast, fEPSP frequency decreased when colonic muscle tension increased during spontaneous colonic contraction and emptying. Manual stretch of the colon wall to 10-15% beyond resting length in the circumferential axis of flat sheet preparations increased fEPSP frequency in SMG neurons, but stretch in the longitudinal axis to 15% beyond resting length in the same preparations did not. There was no increase in synaptic input when tubular colon segments were stretched in their long axes up to 20% beyond their resting length. The circumferential stretch-sensitive increase in the frequency of synaptic input to SMG neurons persisted when the colonic muscles were relaxed pharmacologically by nifedipine (2 microM) or nicardipine (3 microM). These results suggest that colonic mechanosensory afferent nerves projecting to the SMG function as length or stretch detectors in parallel to the circular muscle layer.

Involvement of histamine H3 receptors in scratching behaviour in mast cell-deficient mice.

Although the roles of histamine H3 receptors have been studied in several tissues such as the brain, lung, spleen, colon and peripheral sensory nerve endings, the involvement of H3 receptors in skin responses particularly in relation to scratching behaviour are not well documented. This work was performed to study the effects of histamine H3 antagonists on scratching behaviour in mast cell-deficient mice. Histamine H3 antagonists iodophenpropit and clobenpropit, histamine and substance P were injected intradermally into the rostral part of the back of mast cell-deficient (WBB6F1 W/Wv) and wild-type (WBB6F1+/+) mice and scratching behaviour was measured for 60 min. The effects of H1 antagonists on scratching behaviour induced by H3 antagonists were also investigated. Intradermal injection of iodophenpropit and clobenpropit at doses of 10 and 100 nmol per site caused significant increases in scratching behaviour in both mast cell-deficient and wild-type mice. Histamine also caused a dose-related increase in the incidence of scratching behaviour, and a significant effect was observed at a dose of 100 nmol per site in both mast cell-deficient and wild-type mice. Substance P was also effective in causing scratching behaviour in both mast cell-deficient and wild-type mice. However, histamine H1 antagonists diphenhydramine and chlorphenamine failed to inhibit H3 antagonist-induced scratching behaviour in both types of mice. Our results indicated that intradermal injection of H3 antagonists induces scratching behaviour and that chemical mediators other than histamine seem to be involved in the response.

Morphological characteristics and immunohistochemical detection of nicotinic acetylcholine receptors on intestinofugal afferent neurones in guinea-pig colon.

Intestinofugal afferent neurones (IFANs) provide excitatory synaptic input to abdominal prevertebral ganglion neurones. Input is greatly reduced during blockade of nicotinic acetylcholine receptors (nAChRs) in the wall of the colon, suggesting two projection pathways: a direct pathway without synaptic interruption and an indirect pathway interrupted by at least one nicotinic cholinergic synapse. This study aimed to characterize the morphology of IFANs and examine the distribution of nAChRs on them. We identified IFANs in guinea-pig colon by retrograde labelling with fluorescent tracer DiI placed either on the lumbar colonic nerves in vitro or inferior mesenteric ganglion in vivo. Confocal laser scanning microscopy and computerized image-processing software were used for 3D image reconstruction. Approximately 70% of identified IFANs had Dogiel type I-like morphology, the remainder were Dogiel type II-like. In vivo labelled IFANs were injected with Lucifer Yellow and immunostained for nAChRs using monoclonal antibody MAb35. Approximately 3% of total plasma membrane surface of IFANs with Dogiel type I morphology had MAb35-IR. In contrast, <1% of membrane surface of IFANs with Dogiel type II morphology had MAb35-IR. The finding that IFANs displayed immunostaining for nAChRs suggests the presence of putative nicotinic synapses.

β2- and β4-Subunit Composition of Nicotinic Acetylcholine Receptors in the Guinea Pig Inferior Mesenteric Ganglion: Immunohistochemical and Electrophysiological Studies

1 Bogomolets Institute of Physiology, National Academy of Sciences of Ukraine, Kyiv, Ukraine. Correspondence should be addressed to L. P. Voitenko (e-mail: lvoit@serv.biph. kiev.ua). Affinity-purified rabbit antibodies (Ab) against extracellular fragments 193-204 of nicotinic acetylcholine receptor (nAChR) β2 and β4 subunits were used in the experiments with immunoperoxidase labeling of guinea pig inferior mesenteric ganglion (IMG) neurons and intracellular recording of excitatory postsynaptic potentials (EPSP) from these units. Since the epitopes of these Ab are close to an acetylcholine-binding site located at the interface of α and β subunits, we expected that the Ab can block acetylcholine-mediated EPSP responses of IMG neurons, thus enabling us to determine the involvement of β2and β4-containing nAChR in synaptic transmission through intermesenteric pathways. Microscopic examination revealed the presence of two cell types in IMG preparations: principal ganglionic neurons (PGN), 48-60 μm in diameter, and small cells of unknown origin, 6-18 μm in diameter. The β4-specifi c Ab labeled 59.8 ± 7.5% of PGN, while the β2-specifi c Ab labeled less than 1% of these cells, the β4-positive label being much more intensive than the β2-positive one. The β4-specifi c labeling was similar to that revealed with the α3-specifi c Ab. It should be noted that the percentage of α5-immunoreactive IMG neurons differed insignifi cantly from that of α3-labeled PGN. The somata of small cells were labeled with the β4-specifi c Ab too, and a group of β2-positive small cells was also found. Both β4and β2-positive neurons were localized near the origins of the main nerve trunks branching from the IMG (mesenteric, colonic, and hypogastric nerves). In electrophysiological experiments, the β4-specific Ab decreased the EPSP amplitudes by 25.5 ± 1.8% in all 10 PGN tested. In contrast, the β2-specific Ab did not suppress the synaptic responses under study. Our findings showed that α3-, α5-, and β4-containing nAChR are present in the guinea pig IMG neurons and are involved in synaptic transmission through the intermesenteric pathways. Probably, the α3β4 nAChR subtype is more widespread that is consistent with the generally recognized nature of ganglionic nAChR.

Integrative control of rectoanal reflex in guinea pigs through lumbar colonic nerves.

The aim of the present study was to analyze the neuromodulation of rectoanal reflex activity by lumbar sympathetic nerves in guinea pigs. The mechanical activities of the rectum were recorded with a balloon connected to a pressure transducer, and those of the internal anal sphincter (IAS) were recorded with a custom-made strain gauge force transducer. Gradual and sustained rectal distension evoked the rectoanal reflex, causing cholinergic contractions of the rectum and synchronous nitrergic relaxations of the IAS. Section of the lumbar colonic nerves enhanced both rectal contractions and IAS relaxations. Section of the 13th thoracic cord abolished both rectal contractions and IAS relaxations, but section of the lumbar colonic nerves restored them. Lumbar sympathectomy and pithing sacral cords greatly diminished these rectal contractions and IAS relaxations, but the intrinsic reflex component remained. NG-nitro-L-arginine methyl ester enhanced the intrinsic reflex-mediated contraction of the rectum and abolished reflex-mediated relaxation of the IAS and converted into cholinergic contractions. The present results indicate that the extrinsic lumbar inhibitory outflow causes marked inhibition of the rectoanal reflex via the lumbar colonic nerves.

Effects of extrinsic autonomic inputs on expression of c-Fos immunoreactivity in myenteric neurons of the guinea pig distal colon

c-Fos protein is a nuclear protein coded by c-fos proto-oncogene subsequent to synaptic activation of the neurons. We used immunohistochemical methods to visualize the expression of c-Fos protein in myenteric neurons of the guinea pig distal colon and examined the effects of the extrinsic autonomic inputs on the enteric circuits. No c-Fos immunoreactivity was observed in the colonic segments fixed immediately after removal from the animal body. A number of c-Fos-immunoreactive nuclei of myenteric neurons, however, appeared in all preparations that were incubated in Krebs solution in vitro ( n=10). Application of tetrodotoxin (0.2 μM) abolished the expression of c-Fos-immunoreactivity ( n=6), but hexamethonium (100 μM) failed to decrease the number of c-Fos-positive neurons despite a complete suppression of spontaneous peristaltic movements ( n=5). Neither the electrical stimulation ( n=8) nor the severing of the pelvic nerves ( n=5) changed the number of c-Fos-positive neurons. Application of clonidine, an α 2-agonist, (0.1 μM) abolished the expression of c-Fos protein in all preparations ( n=5), while denervation of the sympathetic fibers in the lumbar colonic and hypogastric nerves in vivo increased the number of c-Fos-positive neurons ( n=5). The results indicate that the enteric circuit in the distal part of the gastrointestinal tract is under tonic inhibition by the sympathetic nervous system from the lumbar spinal cord. c-Fos immunoreactivity expressed in the colonic preparations in vivo might be the results of enhanced activation of non-nicotinic receptors after removal of the sympathetic inhibition.

Colonic Mechanoreceptor Input to Neurons in Guinea Pig Caudal Mesenteric Ganglion

The background activity of pacemaker-like neurons (PLN) of the guinea-pig caudal mesenteric ganglion (CMG) and their reflex responses to colonic distention were studied on combined isolated preparations including the CMG and a colon segment connected with the lumbar colonic nerves. The results allow us to suggest that the “spontaneous” and reflex activity of PLN is of a peripheral origin. Synaptic transmission in the peripheral colonofugal nerve pathways is mediated by acetylcholine and substance P (SP). The SP-induced excitation of PLN probably involves activation of the tachykinin NK3 receptors.

Iatrogenic painful neuropathic complications of surgery in cancer.

It is estimated that at least one out of four patients with cancer complains of pain originating from nerve injury. Nerve injury may result from direct invasion/compression by tumour, or by remote effect of the cancer such as paraneoplastic polyneuropathy. In many cases, the nerve injury is caused by medical therapy, or surgical interventions. Pain generated by drugs or medical acts is called iatrogenic. A common iatrogenic neuralgia is chemotherapy induced painful polyneuropathy. This neuropathy typically affects mostly the small myelinated and unmyelinated nerve fibres. Surgical and anaesthesiological interventions also frequently cause direct nerve stretch or section. Some interventions, particularly those requiring extended resection, have a higher incidence of painful sequelae. Limb and colon amputation, nerve dissection, mastectomy and thoracotomy are the most common interventions for cancer known to cause nerve injury. As pain clinicians, we focus attention on the painful consequences of surgical interventions because there is evidence that a more accurate surgical approach and possibly a prophylactic prevention of the neuralgia may reduce the painful sequelae of nerve injury.

ENaC subunits are components of the mechanosensor of colonic mechanosensory afferent nerves projecting to prevertebral ganglia

ENaC subunits are components of the mechanosensor of colonic mechanosensory afferent nerves projecting to prevertebral ganglia

ENaC subunits are components of the mechanosensor of colonic mechanosensory afferent nerves projecting to prevertebral ganglia

ENaC subunits are components of the mechanosensor of colonic mechanosensory afferent nerves projecting to prevertebral ganglia

Vanilloid receptor 1 immunoreactivity in inflamed human bowel

Vanilloid receptor 1 (VR1) is expressed by sensory neurons. Once activated, these neurons evoke the sensation of burning pain and release neuropeptides that induce neurogenic inflammation. We used immunoblotting and immunostaining to estimate the density of VR1 in colonic tissues of patients with inflammatory bowel disease and of controls. Our study results indicate that VR1 immunoreactivity is greatly increased in colonic nerve fibres of patients with active inflammatory bowel disease. Thus, the discovery of new drugs that can bind the VR1 receptor, or antagonise endogenous inflammatory substances that activate this receptor, could lead to new therapies for pain and dysmotility.

Myenteric plexus of obese diabetic mice (an animal model of human type 2 diabetes).

The myenteric plexus of the gastrointestinal tract was investigated in the obese diabetic mouse, an animal model of human type 2 diabetes. Sections were immunostained by the avidin-biotin complex method, using a general nerve marker, protein gene product 9.5 (PGP 9.5), as well as antibodies to several important neurotransmitters. Computerized image analysis was used for quantification. When diabetic mice were compared with controls, no difference was found in the density of PGP 9.5-immunoreactive (IR) nerve fibres in antrum, duodenum or colon. In antrum and duodenum, diabetic mice showed a decreased number of vasoactive intestinal peptide (VIP)-IR neurons in myenteric ganglia as well a decreased relative volume density in myenteric plexus (though not significantly in antrum, p=0.073). No difference was found regarding VIP-IR nerves in colon. The volume density of nitric oxide synthase (NOS)-IR nerve fibres was decreased in antrum and duodenum of diabetic mice, whereas no difference was found in colon. The density of galanin-IR nerve fibres was decreased in duodenum. Whereas neuropeptide Y (NPY)- and vesicular acetylcholine transporter (VAChT)-IR nerve fibres was increased in density in colon of diabetic mice, no difference was found in antrum and duodenum. Regarding substance P, there was no difference between diabetic and control mice in antrum, duodenum or colon. The present study shows that gut innervation is affected in this animal model of human type 2 diabetes. It is possible that the present findings may have some relevance for the gastrointestinal dysfunctions seen in patients with type 2 diabetes.

Rectal mucosal biopsy compared with laparoscopic seromuscular biopsy in the diagnosis of intestinal neuronal dysplasia in children with slow-transit constipation

Background/Purpose: Intestinal neuronal dysplasia (IND) as a cause for severe chronic constipation remains controversial. The aim of this study is to examine the correlation between a deficiency of substance P (SP) immunoreactive nerve fibers in the colon and enzyme histochemistry of rectal biopsies in children with slow-transit constipation. Methods: Fifty children with intractable constipation have been assessed by rectal biopsies examined with histochemical staining for lactate dehydrogenase, and 32 children among those 50 have been studied by laparoscopic seromuscular biopsy of the colon labelled with antibodies to SP using immunofluorescence methods. Results: Four children have evidence of IND. Fifteen children, including all 4 IND cases, showed a deficiency of SP immunoreactivity. There is a significant correlation between giant ganglia and SP deficiency (P <.01). Conclusion: This study is attempting to propose that a deficiency of SP immunoreactivity in colonic circular muscle nerves may be used as a histologic marker for slow-transit constipation and that IND may be a small subset of patients with SP deficiency. J Pediatr Surg 35:1724-1727. Copyright © 2000 by W.B. Saunders Company.

Control of peripheral sympathetic prevertebral ganglion neurones by colonic mechanosensory afferents

Sympathetic innervation of the gastrointestinal tract arises from abdominal prevertebral ganglia (PVG) comprising the coeliac, superior mesenteric, and inferior mesenteric ganglia. These ganglia are integrating centres between the central nervous...

Selective Demonstration of Mural Nerves in Ganglionic and Aganglionic Colon by Immunohistochemistry for Glucose Transporter-1 Prominent Extrinsic Nerve Pattern Staining in Hirschsprung Disease

c Objective.—Hypertrophic nerves have long been considered a histopathologic feature of the aganglionic segment in Hirschsprung disease, but they remain incompletely explained. The purpose of this study was to define the nature and diagnostic importance of hypertrophic nerves in Hirschsprung disease and to clarify their relation to nearby smaller nerve fibers. Methods.—We used an immunoperoxidase staining technique to compare the distribution of 2 nerve markers—erythrocyte-type glucose transporter (GLUT-1), a marker of perineurium, and nerve growth factor receptor, a marker of both nerve fibers and perineurium—in aganglionic tissue (12 resected specimens and 4 rectal biopsies) and control tissue (6 autopsy specimens and 17 rectal biopsies) of children. Results.—In control ganglionic tissue, the myenteric and submucosal areas contained only occasional GLUT-1‐positive nerves (usually less than 50 mm in diameter), but extramural extrinsic (serosal) nerves were invariably positive for GLUT-1. In aganglionic tissue, GLUT-1‐positive nerves in the myenteric and submucosal areas were frequent and included both large (50‐150 mm) and small (,50 mm) diameter nerves. Nerve growth factor receptor‐positive fibers were frequent in all layers of all tissue studied. In aganglionic bowel, a distinct perineurium could be identified in the largest nerves, but nerve growth factor receptor had poor discrimination for small perineuriumsheathed nerves. Conclusion.—Most nerves, of both large and small diameter, in the myenteric and submucosal plexus of aganglionic bowel are GLUT-1 positive. Serosal extrinsic nerves stain identically, supporting the interpretation that the mural nerves are of extrinsic origin. Mural GLUT-1‐positive nerves, when they are multiple and especially when they are greater than 50 mm in diameter (a figure which may be used as a threshold for hypertrophic nerves), are suggestive of Hirschsprung disease. (Arch Pathol Lab Med. 2000;124:1314‐1319)

Selective demonstration of mural nerves in ganglionic and aganglionic colon by immunohistochemistry for glucose transporter-1: prominent extrinsic nerve pattern staining in Hirschsprung disease.

Hypertrophic nerves have long been considered a histopathologic feature of the aganglionic segment in Hirschsprung disease, but they remain incompletely explained. The purpose of this study was to define the nature and diagnostic importance of hypertrophic nerves in Hirschsprung disease and to clarify their relation to nearby smaller nerve fibers. We used an immunoperoxidase staining technique to compare the distribution of 2 nerve markers-erythrocyte-type glucose transporter (GLUT-1), a marker of perineurium, and nerve growth factor receptor, a marker of both nerve fibers and perineurium-in aganglionic tissue (12 resected specimens and 4 rectal biopsies) and control tissue (6 autopsy specimens and 17 rectal biopsies) of children. In control ganglionic tissue, the myenteric and submucosal areas contained only occasional GLUT-1-positive nerves (usually less than 50 microm in diameter), but extramural extrinsic (serosal) nerves were invariably positive for GLUT-1. In aganglionic tissue, GLUT-1-positive nerves in the myenteric and submucosal areas were frequent and included both large (50-150 microm) and small (<50 microm) diameter nerves. Nerve growth factor receptor-positive fibers were frequent in all layers of all tissue studied. In aganglionic bowel, a distinct perineurium could be identified in the largest nerves, but nerve growth factor receptor had poor discrimination for small perineurium-sheathed nerves. Most nerves, of both large and small diameter, in the myenteric and submucosal plexus of aganglionic bowel are GLUT-1 positive. Serosal extrinsic nerves stain identically, supporting the interpretation that the mural nerves are of extrinsic origin. Mural GLUT-1-positive nerves, when they are multiple and especially when they are greater than 50 microm in diameter (a figure which may be used as a threshold for hypertrophic nerves), are suggestive of Hirschsprung disease.