P. F. Schmalz

Effect of Age on the Enteric Nervous System of the Human Colon

Abstract  The effect of age on the anatomy and function of the human colon is incompletely understood. The prevalence of disorders in adults such as constipation increase with age but it is unclear if this is due to confounding factors or age‐related structural defects. The aim of this study was to determine number and subtypes of enteric neurons and neuronal volumes in the human colon of different ages. Normal colon (descending and sigmoid) from 16 patients (nine male) was studied; ages 33–99. Antibodies to HuC/D, choline acetyltransferase (ChAT), neuronal nitric oxide synthase (nNOS), and protein gene product 9.5 were used. Effect of age was determined by testing for linear trends using regression analysis. In the myenteric plexus, number of Hu‐positive neurons declined with age (slope = −1.3 neurons/mm/10 years, P = 0.03). The number of ChAT‐positive neurons also declined with age (slope = −1.1 neurons/mm/10 years of age, P = 0.02). The number of nNOS‐positive neurons did not decline with age. As a result, the ratio of nNOS to Hu increased (slope = 0.03 per 10 years of age, P = 0.01). In the submucosal plexus, the number of neurons did not decline with age (slope = −0.3 neurons/mm/10 years, P = 0.09). Volume of nerve fibres in the circular muscle and volume of neuronal structures in the myenteric plexus did not change with age. In conclusion, the number of neurons in the human colon declines with age with sparing of nNOS‐positive neurons. This change was not accompanied by changes in total volume of neuronal structures suggesting compensatory changes in the remaining neurons.

Heme oxygenase 2 is present in interstitial cell networks of the mouse small intestine

BACKGROUND & AIMS The interstitial cell (IC) network may be of fundamental importance in regulating gastrointestinal motility. Intestinal smooth muscle cells are depolarized in the absence of ICs, and there are no spontaneous slow waves. The messenger molecules between IC network and smooth muscle are unknown. Exogenous administration of CO relaxes the opossum internal anal sphincter and the guinea pig ileum, and it modulates potassium current and membrane potential of circular smooth muscle cells of the human jejunum. The aim of this study was to determine whether heme oxygenase (HO)-1 and HO-2, enzymes that catalyze the production of CO, are present in the IC network of the mouse small intestine. METHODS Antibodies specific for c-Kit, HO-1, and HO-2 were used for immunohistochemistry. Confocal images were obtained and were volume rendered, and the images were converted into three-dimensional images. RESULTS HO-2-like but not HO-1-like immunoreactivity was found in IC networks associated with the myenteric plexus and the deep muscular plexus. CONCLUSIONS HO-2 but not HO-1 is present in the IC cell network of the mouse small intestine. The enzymatic activity of HO-2 will result in the endogenous production of CO in IC networks of the mouse small intestine.

Vasoactive intestinal polypeptide: a putative transmitter in the canine gastric muscularis mucosa.

The nature of the inhibitory transmitter in the canine gastric muscularis mucosae was studied in vitro using superfusion techniques. The inhibitory effect of nerve stimulation (10 V, 200 mus, 10 Hz) was not altered by adrenergic, cholinergic or serotonergic antagonists. Adenosine triphosphate had no effect on spontaneous mechanical activity. Nucleotide pyrophosphatase and apamin had no effect on the response to nerve stimulation. Alpha‐chymotrypsin abolished the inhibitory effect of nerve stimulation. Radioimmunoassay of the muscle indicated the presence of gastrin/cholecystokinin‐substance P‐ and vasoactive intestinal polypeptide (VIP)‐like immunoreactivity. Of the three peptides present, only VIP produced a concentration‐dependent relaxation. A substance with VIP‐like immunoreactivity was released during nerve‐induced relaxation of the muscle, and its release was blocked by tetrodotoxin and calcium‐depleted solution. The inhibitory effect of nerve stimulation was abolished by VIP antiserum. These data strongly support the hypothesis that VIP or a closely related peptide is an inhibitory neurotransmitter in the canine gastric muscularis mucosae.

The inhibitory effects of vasoactive intestinal polypeptide on the mechanical and electrical activity of canine antral smooth muscle.

1. The inhibitory effects of vasoactive intestinal polypeptide (VIP) on the electrical and mechanical activity of canine antral smooth muscle were investigated. 2. In concentrations ranging from 5 X 10(‐9) to 1 X 10(‐7) M, VIP decreased the force of spontaneous contractions but had no measurable effect on spontaneous action potential complexes. 3. VIP had no effect on the increase in the amplitude and duration of the plateau potential and on the amplitude of contraction caused by a maximally effective concentration of acetylcholine. 4. VIP caused a significant decrease in the force of contraction caused by ED50 and threshold concentrations of acetylcholine. However, VIP had no measurable effect on the increase in the size of the action potential plateau caused by either concentration of acetylcholine. 5. VIP antagonized the increase in the amplitude of the plateau potential and the force of contraction induced by pentagastrin. It had no consistent effect on the pentagastrin‐induced increase in frequency. 6. The data indicate that VIP acts as an inhibitor in this tissue in two distinct ways. It uncouples electromechanical coupling during spontaneous and acetylcholine‐induced electrical and mechanical activity, and antagonizes pentagastrin‐induced increases in electrical and mechanical activities.

Apoptotic cell death of human interstitial cells of Cajal

Abstract  Interstitial cells of Cajal (ICC) are specialized mesenchyme‐derived cells that regulate contractility and excitability of many smooth muscles with loss of ICC seen in a variety of gut motility disorders. Maintenance of ICC numbers is tightly regulated, with several factors known to regulate proliferation. In contrast, the fate of ICC is not established. The aim of this study was to investigate whether apoptosis plays a role in the regulation of ICC numbers in the normal colon. ICC were identified by immunolabelling for the c‐Kit receptor tyrosine kinase and by electron microscopy. Apoptosis was detected in colon tissue by immunolabelling for activated caspase‐3, terminal dUTP nucleotide end labelling and by ultrastructural changes in the cells. Apoptotic ICC were identified and counted in double‐labelled tissue sections. They were identified in all layers of the colonic muscle. In the muscularis propria 1.5 ± 0.2% of ICC were positive for activated caspase‐3 and in the circular muscle layer 2.1 ± 0.9% of ICC were positive for TUNEL. Apoptotic ICC were identified by electron microscopy. Apoptotic cell death is a continuing process in ICC. The level of apoptosis in ICC in healthy colon indicates that these cells must be continually regenerated to maintain intact networks.

Distribution of interstitial cells of Cajal and nitrergic neurons in normal and diabetic human appendix.

Abstract  The objective of this study was to determine the distribution of enteric nerves and interstitial cells of Cajal (ICC) in the normal human appendix and in type 1 diabetes. Appendixes were collected from patients with type 1 diabetes and from non‐diabetic controls. Volumes of nerves and ICC were determined using 3‐D reconstruction and neuronal nitric oxide synthase (nNOS) expressing neurons were counted. Enteric ganglia were found in the myenteric plexus region and within the longitudinal muscle. ICC were found throughout the muscle layers. In diabetes, c‐Kit positive ICC volumes were significantly reduced as were nNOS expressing neurons. In conclusion, we describe the distribution of ICC and enteric nerves in health and in diabetes. The data also suggest that the human appendix, a readily available source of human tissue, may be useful model for the study of motility disorders.

Bicarbonate Secretion by the Rat Colon: Effect of Intraluminal Chloride and Acetazolamide

Summary To examine the conditions under which bicarbonate is secreted by the colon, rats were perfused by a recirculation technique using solutions containing sodium and chloride, one or other of these ions, or neither. All solutions contained bicarbonate in concentrations greater than those in portal venous blood. In 12 animals, bicarbonate was secreted when chloride was a constitutent of the luminal contents, but bicarbonate was absorbed when chloride was excluded from perfusing solutions. Acetazolamide reduced the secretion of bicarbonate and the absorption of sodium, chloride, and water when sodium chloride solutions were used, but the drug had no effect on water and electrolyte transport when a chloride-free solution was used. These results suggest that carbonic anhydrase has a role in the exchange of bicarbonate and chloride across the mucosa of the colon and, further, they imply anion exchange may be related to the mechanism of sodium and water transport in the colon.

The three-dimensional structure of myenteric neurons in the guinea-pig ileum.

Myenteric neurons of the guinea-pig ileum were intracellularly filled with the fluorescent dye Lucifer Yellow, optically sectioned with a confocal microscope and volume reconstructed to recreate 3-D images of the cells. The resulting images provide information not evident from regular microscopy. The somata varied in cross-section from flat-oval to nearly circular, and their surface membranes were marked by invaginations and protrusions significantly increasing the surface area of the somatic membrane. The neurons could be divided into four morphological classes: Dogiel type I, Dogiel type II, filamentous, and intermediate. There was no clear correlation between cell class and the shape of the soma in cross-section. The dendritic processes of all the neurons studied extended in an orad-caudad or circumferential direction of the bowel wall. When the filled neurons were viewed edge-on, the spatial arrangement of the processes was confined to a plane that had a thickness less than the thickness of the parent soma. The broad, short dendrites of Dogiel type I neurons were oval or nearly circular in cross-section. Directly measured quantitative data were obtained for the volume and surface area of the somata and visible processes. The structural details reported herein are likely to have important implications regarding the functional properties of individual enteric ganglion neurons and circuits of enteric ganglion neurons.

Central innervation of neurones in the inferior mesenteric ganglion and of the large intestine of the cat

1. Segmental, lumbar sympathetic outflow to neurones in the cat inferior mesenteric ganglion and to the large intestine were studied. Synaptic responses of neurones in the inferior mesenteric ganglion were recorded intracellularly, in vitro, during electrical stimulation of preganglionic fibres in the lumbar white rami. Synaptic responses consisted of excitatory post‐synaptic potentials and/or action potentials.

Light, electron, and confocal microscopic study of the mouse superior mesenteric ganglion

The superior mesenteric ganglion (S.m.g.), a sympathetic prevertebral ganglion, is an integrating center for gastrointestinal reflexes. Many details of its structure are still lacking. In the present study, mouse S.m.g. neurons were studied by light, electron, and confocal microscopy. Neurons had an average of 5–6 primary dendrites. Total dendritic length averaged 963 μm. Confocal microscopy and three‐dimensional reconstructed images revealed cell body surface features, precise location where axons and dendrites emerged from it, cell body size, and extent of dendritic projection in three axes. Cell body diameter and dendritic projections were less in the dorsoventral than in the rostrocaudal or mediolateral axes. Cell body surface area and volume averaged 4,271 μm2 and 4,908 μm3, respectively. Dendritic surface areas and volumes were 5–6 times larger. Two main neuron types (projecting caudally or rostrally) were distinguished. The former were found throughout the S.m.g., whereas the latter were found only in the cephalad region, comprising about 40% of neurons found there. Rostrally projecting neurons had fewer primary dendrites, fewer total dendritic branches, and shorter total dendritic length than caudally projecting neurons. There were regional differences in percentage of neurons responding to electrical stimulation of left or right hypogastric, lumbar colonic, or left splanchnic nerves but not in nerve fibers connecting the S.m.g. and celiac ganglion. A greater percentage of caudally than rostrally projecting cephalad neurons responded to stimulation of any nerve trunk. These results indicate that the mouse S.m.g. contains at least two distinct types of neurons that differ in their morphology and their source of preganglionic synaptic input.