Chandar Singaram

Differences in the reducing power along the rat GI tract: Lower antioxidant capacity of the colon

The ability of the gastrointestinal (GI) tract, as well as other tissues, to cope with reactive oxygen species (ROS) efflux in pathological events is determined partly by epithelial antioxidant levels. These levels are comprized of tissue antioxidant enzymes and low molecular weight antioxidants (LMWA). While glutathione levels and the activity of enzymatic antioxidants along the GI tract have been studied, the contribution of the overall LMWA to the total antioxidant capacity has not yet been determined. In this study the overall antioxidant activity in the mucosa/submucosa and muscularis/serosa of various sections along the small intestine and colon of the rat was evaluated by determining the reducing power, which reflects the total antioxidant activity derived from LMWA, using cyclic voltammetry. The activity of the antioxidant enzymes superoxide dismutase (SOD) and catalase was also measured. The reducing power (total antioxidant activity) was higher in the mucosa/submucosa of the small intestine as compared to the mucosa/submucosa of the colon. Similarly, catalase and SOD activity in the mucosa/submucosa of the small intestine was significantly higher than in the mucosa/submucosa of the colon. Differences were also observed in the reducing power and SOD activity in the muscularis/serosa of the rat small intestine as compared to the colon. The low antioxidant capacity in the colon may facilitate reactive oxygen species (ROS)-mediated injury and lead to inflammatory diseases such as ulcerative colitis, specifically in the colon.

Peptidergic and nitrinergic denervation in congenital esophageal stenosis

Congenital esophageal stenosis (CES) is a rare disorder with narrowed esophageal lumen that presents as dysphagia from childhood and that is often associated with tracheobronchial remnants or webs. The pathogenesis of CES is unknown. The aim of this study was to examine the histological and immunohistochemical features of CES. Esophagi from 2 young adults with CES and 3 controls with no motility disorders underwent routine H&E staining, trichrome staining for collagen, and detailed immunocytochemical studies for general neuronal markers (protein gene product 9.5, neuron-specific enolase, and S-100) and neurotransmitters (vasoactive intestinal polypeptide, substance P, and galanin) and nitric oxide synthase by beta-nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase and a specific NO synthase antibody. Quantitative experiments compared the numbers of myenteric neurons and amounts of fibers at the circular muscle. CES esophagi showed infiltration of neutrophils in the myenteric plane, without any increase in collagen. NADPH-diaphorase histochemistry showed a significant reduction of myenteric nitrinergic neurons (7 +/- 3.4 vs. 2.7 +/- 1.8 neurons per high-power field) and fibers at the circular muscle. Other peptidergic neurons studied were not significantly reduced in CES. The specific total lack of NO inhibitory innervation may be an important mechanism in the pathogenesis of stenosis and aperistalsis of the esophagus in this disorder.

Electrophysiological and pharmacological responses of chronically denervated lower esophageal sphincter of the opossum

BACKGROUND & AIMS Achalasia is characterized by loss of myenteric neurons and incomplete relaxation of the lower esophageal sphincter (LES). The aim of this study was to develop an achalasia model in the opossum using the surfactant benzyldimethyltetradecylammonium chloride (BAC). This study further characterizes the achalasia model. METHODS BAC or saline was injected circumferentially into the LES of 14 adult opossums. Eight months after injection, manometry, isolated muscle bath studies, electrical field stimulation, and histochemical analysis were performed. RESULTS Manometrically, the LES of BAC-treated opossums showed higher pressures (38.7 +/- 12 mm Hg vs. 17 +/- 3.0 mm Hg) and reduced esophageal body contraction amplitudes (4.2 +/- 3 mm Hg vs. 27.4 +/- 12 mm Hg). Isolated muscle strips challenged with carbachol and sodium nitroprusside contracted and relaxed similarly to controls. Electrical field stimulation failed to induce relaxation in BAC-treated tissue but did induce contraction. Contractile responses were markedly reduced by tetrodotoxin and atropine in BAC-treated animals and controls. An altered nitric oxide system was shown by the lack of response to L-arginine and N omega-nitro-L-arginine. Histology showed loss of myenteric neurons and increased cholinergic nerve bundles. CONCLUSIONS Loss of NO inhibitory myenteric neurons markedly reduces the relaxation of the LES, and histology and pharmacological responses suggest a proliferation of cholinergic nerves into the LES contributing to the static elevated pressures of the amyenteric LES.

Ca2+ channel blockade by verapamil inhibits GMCs and diarrhea during small intestinal inflammation

The aim of this study was to investigate whether the blockade of L-type Ca2+ channels with verapamil suppresses giant migrating contractions (GMCs) and therefore diarrhea during small intestinal inflammation. Small intestinal inflammation was induced by infection with the nematode Trichinella spiralis. T. spiralisinfection alone significantly increased the frequency of GMCs and decreased the frequency of phase III activity in the small intestine for 9 days. The increased frequency of GMCs was associated with diarrhea. Immunohistochemical staining with specific antibodies indicated that the number of neutrophils and mast cells increased significantly in the jejunal lamina propria during T. spiralis infection. Only the neutrophils increased significantly in the muscularis externa of the jejunum. Myeloperoxidase (MPO) activity increased significantly in the jejunal and ileal lamina propria. Daily verapamil administration during T. spiralis infection significantly reduced the frequency of GMCs and diarrhea but had no further significant effect on the already reduced frequency of phase III activity. Verapamil administration, however, did not reduce MPO activity or immunocyte infiltration in the jejunum or ileum. We conclude that blockade of L-type Ca2+ channels selectively reduces the frequency of GMCs and therefore diarrhea during small intestinal inflammation. The decreased frequency of GMCs is not secondary to a reduction in the inflammatory response.

Nitrinergic and peptidergic innervations and their inter-relationships in human colon

The distribution and colocalization of nitrinergic and peptidergic nerves were examined in six human colons. The tissues were fixed, cryosectioned, and standard immunohistochemistry was performed for several known neuropeptides. The same sections were stained for NADPH-diaphorase to denote nitric oxide synthase. NADPH-diaphorase-positive myenteric neurons were counted and colocalization noted for each peptide, as well as for peptide terminations. Galanin was the only neuropeptide that colocalized to a significant extent (23.0 +/- 7.21%) with NADPH-diaphorase-positive myenteric neurons. Many neuropeptide-containing nerve fibers had extensive terminations onto NADPH-diaphorase-positive neurons. Vasoactive intestinal peptide was the only neuropeptide that colocalized with NADPH-diaphorase to any extent in nerve fibers within circular muscle (59.5 +/- 9.3%). Fiber distribution in the longitudinal muscles showed a similar, but less dense pattern. These observations provide morphological evidence for the presence of nitric oxide, a candidate nonadrenergic noncholinergic neurotransmitter in the human colon.

Immunohistochemical evaluations of ultrashort-segment Hirschsprung's disease

PURPOSE: Unlike classic Hirschsprungs disease, short-segment and ultrashort-segment varieties are usually found to be latent and milder. Ultrashort-segment Hirschsprungs disease may present as intractable chronic constipation in children over one year of age, adolescents, and adults. Anorectal myectomy has been shown in many instances to provide effective long-term treatment for certain patients with ultrashort-segment Hirschsprungs disease. Histologically, the affected segment in Hirschsprungs disease has been shown to have increased cholinergic nerves, lack of nitric oxide synthase-containing neuronal elements, and show moderate to severe loss of myenteric neurons. METHODS: Here, we report three cases that showed clinical and manometric evidence of ultrashort-segment Hirschsprungs disease. Two of the three patients responded well to myectomy. RESULTS: Detailed histologic and immunohistochemical evaluation of the internal anal sphincter and a comparison with three normal controls revealed absence of nitric oxide synthase-containing neurons in both cases that responded well to surgery and continued presence of these neurons in the patient who did not respond. A review of the current literature on various treatment modalities is included. CONCLUSIONS: Anorectal myectomy provides long-term relief of this chronic problem in a subgroup of patients with ultrashort-segment Hirschsprungs disease who lack nitrinergic neurons at the internal anal sphincter.

Evaluation of early events in the creation of amyenteric opossum model of achalasia

Abstract Benzyldimethyltetradecylammonium chloride (BAC) has previously been used to create amyenteric rat jejunal models. Fifteen opossums (D. virginiana) were injected with 10–15 mL 4 mM BAC or saline in the distal oesophagus and along with controls underwent oesophagoscopy, manometry and barium oesophagrams. Atropine and sodium nitroprusside were studied in six of the BAC‐treated and five controls using oesophageal manometry. Histologically several neuronal markers, B‐NADPH‐diaphorase and acetylcholine esterase histochemical staining were used. NADPH‐diaphorase activity was assayed at the lower oesophageal sphincter (LOS) and 3 and 5 cm above LOS in both groups. Oesophagoscopy of the treated animals showed no mucosal inflammation, or strictures. Manometrically, LOS pressures were significantly higher in the BAC‐treated group (25.7 ± 8.6 mmHg) when compared to controls (8.7 ± 1.8 mmHg). The oesophageal contraction amplitudes were similar in both groups. While sodium nitroprusside (SNP) significantly reduced the LOS pressure, atropine did not alter the resting LOS pressure in the BAC‐treated animals. Histologically at the LOS the treated group showed: (i) absence of myenteric neurons, in contrast to prominent NADPH‐diaphorase and other neuron and peptide markers in the control and (ii) ***increàse in the number of nerve bundles that were not positive for AchE. No differences were seen in the oesophageal body between the groups. The NADPH‐diaphorase assay showed a significant decrease of activity in the BAC‐treated LOS, but no differences in the oesophageal body compared to controls. Several of these radiologic, manometric and histological observations resemble features of achalasia and the mechanism of the tonic pressure increase at this early time point appears to be due to a non‐cholinergic mechanism.

Reinnervation of villi of rat jejunum following severe mucosal damage

We studied the time course of the regeneration of the jejunal mucosa of the rat after it was damaged by exposure to the surfactant, benzalkonium chloride. We placed particular emphasis on assessing the morphology of the nerve fibers within the villi during and after regeneration. The application of benzalkonium chloride resulted in virtually complete loss of villi within the treated segment; however, the crypts were only partially damaged. The mucosa began to regenerate within 6 hr of the insult. The villus lengths and crypt depths returned to pretreatment values within two to four days. The mucosal innervation was assessed through immunohistochemistry for vasoactive intestinal peptide (VIP), neuropeptide Y (NPY), and neuron-specific enolase (NSE). At all stages of regeneration, VIP, NPY, and NSE immunopositive fibers within the lamina propria extended to the tips of the villi. The density of the immunopositive fibers in the lamina propria at four days after mucosal insult was similar to that in control tissues regardless of the neuronal marker visualized. We conclude that the nerve fibers innervating the small intestinal mucosa grow at a rate of approximately 100 μm/day and that the entire length of each villus contains nerve fibers throughout the regeneration process. The innervation of the regenerated mucosa appears identical to that of control mucosa.