Xuan-Yu Wang

Cholinergic and nitrergic innervation of ICC-DMP and ICC-IM in the human small intestine.

With functional evidence emerging that interstitial cells of Cajal (ICC) play a role in smooth muscle innervation, detailed knowledge is needed about the structural aspects of enteric innervation of the human gut. Conventional electronmicroscopy (EM), immunohistochemistry and immuno‐EM were performed on the musculature of the distal human ileum focusing on ICC associated with the deep muscular plexus (ICC‐DMP) and intramuscular ICC (ICC‐IM). ICC‐DMP could be identified by EM but not by c‐Kit immunohistochemistry. Immuno‐EM revealed that ICC‐DMP were innervated by both cholinergic and nitrergic nerves, and were the only cells to possess specialized synapse‐like junctions with nerve varicosities and gap junction contacts with smooth muscle cells. c‐Kit positive ICC near the deep muscular plexus were not ICC‐DMP, but ICC‐IM located in septa. ICC‐IM were innervated by both cholinergic and nitrergic nerves but without specialized contacts. Varicosities of both nerve types were also found scattered throughout the musculature without specialized contact with any ICC. No ICC showed immunoreactivity for neuronal nitric oxide synthase. As ICC‐DMP form synapse‐like junctions with cholinergic and nitrergic nerves and gap junction contacts with muscle cells, it is hypothfesized that ICC‐DMP hold a specialized function related to innervation of smooth muscle of the human intestine.

Pathology of interstitial cells of Cajal in relation to inflammation revealed by ultrastructure but not immunohistochemistry.

The role of interstitial cells of Cajal associated with Auerbachs plexus (ICC-AP) in the pathophysiology of inflammation-induced abnormalities in gut motor activity is poorly understood. Therefore we applied a well-described model of inflammation (infection by Trichinella spiralis) to the mouse small intestine where the structure and function of ICC-AP are best known. Electron microscopic evaluation revealed that 1 to 3 days after infection, selective and patchy damage to the ICC processes occurred, thereby disrupting contacts between these ICC and smooth muscle cells as well as ICC and nerves, which was associated with disordered electrical activity and abnormal peristalsis. Ten to 15 days after infection, damage to ICC-AP was maximal and now involving the cell body and major processes. Marked synthetic activity and regrowth of their processes occurred from day 3 onward and recovery was completed at day 40 after infection. No changes to the network of ICC-AP were seen with c-Kit immunohistochemistry. From day 1 after infection, macrophages infiltrated the AP area, making close contact including peg-and-socket-like junctions with smooth muscle cells and ICC-AP but up to day 6 after infection without any sign of phagocytosis. By day 6 after infection, lymphocytes entered the musculature forming close contacts with ICC-AP. This was not associated with damage to ICC-AP but with proliferation of rough endoplasmic reticulum. From day 23 onward, immune cells withdrew from the musculature except macrophages, resulting in a markedly increased population of macrophages in the AP area at day 60 after infection.

Two Independent Networks of Interstitial Cells of Cajal Work Cooperatively with the Enteric Nervous System to Create Colonic Motor Patterns

Normal motility of the colon is critical for quality of life and efforts to normalize abnormal colon function have had limited success. A better understanding of control systems of colonic motility is therefore essential. We report here a hypothesis with supporting experimental data to explain the origin of rhythmic propulsive colonic motor activity induced by general distention. The theory holds that both networks of interstitial cells of Cajal (ICC), those associated with the submuscular plexus (ICC–SMP) and those associated with the myenteric plexus (ICC–MP), orchestrate propagating contractions as pacemaker cells in concert with the enteric nervous system (ENS). ICC–SMP generate an omnipresent slow wave activity that causes propagating but non-propulsive contractions (“rhythmic propagating ripples”) enhancing absorption. The ICC–MP generate stimulus-dependent cyclic depolarizations propagating anally and directing propulsive activity (“rhythmic propulsive motor complexes”). The ENS is not essential for both rhythmic motor patterns since distention and pharmacological means can produce the motor patterns after blocking neural activity, but it supplies the primary stimulus in vivo. Supporting data come from studies on segments of the rat colon, simultaneously measuring motility through spatiotemporal mapping of video recordings, intraluminal pressure, and outflow measurements.

Ultrastructural evidence for communication between intramuscular vagal mechanoreceptors and interstitial cells of Cajal in the rat fundus

Abstract  To assess whether afferent vagal intramuscular arrays (IMAs), putative gastrointestinal mechanoreceptors, form contacts with interstitial cells of Cajal of the intramuscular type (ICC‐IM) and to describe any such contacts, electron microscopic analyses were performed on the external muscle layers of the fundus containing dextran‐labelled diaminobenzidin (DAB)‐stained IMAs. Special staining and embedding techniques were developed to preserve ultrastructural features. Within the muscle layers, IMA varicosities were observed in nerve bundles traversing major septa without contact with ICC‐IM, contacting unlabelled neurites and glial cells. IMA varicosities were encountered in minor septa in contact with ICC‐IM which were not necessarily in close contact with muscle cells. In addition, IMA varicosities were observed within muscle bundles in close contact with ICC‐IM which were in gap junction contact with muscle cells. IMAs formed varicosities containing predominantly small agranular vesicles, occasionally large granular vesicles and prejunctional thickenings in apposition to ICC‐IM processes, indicating communication between ICC and IMA via synapse‐like contacts. Taken together, these different morphological features are consistent with a hypothesized mechanoreceptor role for IMA‐ICC complexes. Intraganglionic laminar ending varicosities contacted neuronal somata and dendrites in the myenteric plexus of the fundus, but no contacts with ICC associated with Auerbach’s plexus were encountered.

Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease

Abstract  Crohns disease associated dysmotility has been attributed to fibrosis and damage to enteric nerves but injury to interstitial cells of Cajal (ICC) could also be involved. We assessed ICC in specimens obtained from patients with Crohns disease and determined the relation between ICC and the inflammatory infiltrate, particularly mast cells (MC) using quantitative immunohistochemistry and electron microscopy. Ultrastructural injury to ICC was patchy in all ICC subtypes but ICC‐Auerbachs plexus (AP) showed damage more frequently, i.e. swelling of mitochondria, decreased electron density, autophagosomes and partial depletion of the cytoplasm. Light microscopy confirmed a significant decrease in c‐kit immunoreactivity for ICC‐AP and an increased number of MC in the muscularis externa. Electron microscopy showed MC exhibiting piecemeal degranulation and making frequent and selective membrane‐to‐membrane contact with all types of injured ICC which suggests chronic release of granule content to affect ICC. Extent of ICC injury was not associated with duration of the disease. In conclusion, ultrastructural injury and loss of ICC‐AP is evident in Crohns disease. Epidemiological and morphological data suggest that ICC have the capacity to regenerate in spite of the chronic insult. The muscularis hosts a marked number of MC that exhibit piecemeal degranulation associated with ICC and may facilitate ICC maintenance.

Changes in Interstitial Cells of Cajal at the Deep Muscular Plexus Are Associated with Loss of Distention-Induced Burst-Type Muscle Activity in Mice Infected by Trichinella spiralis

The physiology and pathophysiology of the network of interstitial cells of Cajal associated with the deep muscular plexus (ICC-DMP) of the small intestine are still poorly understood. The objectives of the present study were to evaluate the effects of inflammation associated with Trichinella spiralis infection on the ICC-DMP and to correlate loss of function with structural changes in these cells and associated structures. We used immunohistochemistry, electron microscopy, and assessment of distention-inducing electrophysiological parameters in vitro. Damage to ICC-DMP was associated with a loss of distention-induced patterns of electrical activity normally associated with distention-induced peristalsis. Consistently, the timing of recovery of ICC-DMP paralleled the timing of recovery of the distention-induced activity. Nerve varicosities associated with ICC-DMP including cholinergic nerves, assessed by immunoelectron microscopy and whole mount double labeling, paralleled injury to ICC-DMP thus contributing to impaired excitatory innervation of smooth muscle cells. Major additional changes included a remodeling of the inner circular muscle layer, which may affect long-term sensitivity to distention after infection. In conclusion, transient injury to ICC-DMP in response to T. spiralis infection is severe and associated with a complete lack of distention-induced burst-type muscle activity.

Intramuscular interstitial cells of Cajal associated with mast cells survive nitrergic nerves in achalasia

Abstract  Achalasia is dominated by injury to inhibitory nerves. As intramuscular interstitial cells of Cajal (ICC‐IM) are proposed to form functional units with nitrergic nerves, their fate in achalasia may be critically important. We studied the relationship between loss of nitrergic nerves and injury to ICC‐IM in patients with achalasia and determined associations between ICC‐IM and mast cells (MC), using quantitative immunohistochemistry and electron microscopy. Loss of neuronal nitric oxide synthase (nNOS) immunoreactivity was completed within 3 years of acquiring achalasia. Thereafter, progressive ultrastructural injury to remaining nerve structures was evident. Within the first 2 years, the number of ICC‐IM did not decline although ultrastructural injury was already present. Thereafter, loss of ICC‐IM occurred unrelated to duration of disease. Damage to ICC‐IM appeared unrelated to nerve injury. A significant MC infiltration was observed in the musculature; the number of MC was positively related to the persistent number of ICC‐IM. Mast cell formed close contacts with ICC‐IM and piecemeal‐degranulation occurred towards ICC‐IM. In conclusion, injury to ICC‐IM in achalasia is variable, but not related to duration of disease and injury to nitrergic nerves. MC are prominent and form close functional contact with ICC‐IM which may be responsible for their relatively long survival.

Interstitial cells of Cajal and neuromuscular transmission in the rat lower oesophageal sphincter.

Abstract  The distribution of interstitial cells of Cajal (ICC) and neurotransmission were investigated in lower oesophageal sphincter (LES) circular muscle strips from Sprague–Dawley (SD) rats, Ws/Ws mutant rats and their wild‐type (+/+) siblings. Intramuscular c‐Kit‐positive cells, confirmed to be ICC‐IM by electron microscopy, were observed throughout both muscle layers from SD and +/+ rats. In contrast, c‐Kit‐positive, ultrastructurally typical ICC‐IM were absent in Ws/Ws. LES strips from Ws/Ws rats showed increased spontaneous contractile activity. Strips from SD and +/+ rats, responded to electrical neuronal stimulation with a relaxation that was in part L‐NNA and in part apamin sensitive, followed by a contraction which was decreased by atropine. In Ws/Ws rats, similar to +/+ rats, neurally mediated relaxation was L‐NNA and apamin sensitive and the contraction was decreased by atropine. We conclude that in the rat LES, relaxation is mediated by NO and an apamin‐sensitive mediator, and contraction primarily by acetylcholine. Despite the absence of c‐Kit‐positive ICC, nerve–muscle interaction can be accomplished likely by diffusion of neurotransmitters to the smooth muscle cells. The lack of c‐Kit‐positive ICC is related to an increase in the basal tone and spontaneous contractile activity. The presence of fibroblast‐like ICC in Ws/Ws rats might represent immature ICC whose possible functions need further investigation.

On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP(3)R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP(3) and IP(3)-induced calcium release from the SR. Immunohistochemistry proved the expression of IP(3)R type I (IP(3)R-I), but not type II (IP(3)R-II) and type III (IP(3)R-III) in ICC-MP, indicating the involvement of the IP(3)R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP(3) synthesis and IP(3)-induced calcium release from the SR, via the IP(3)R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP(3)-activated SR calcium channel and IP(3) metabolism.

Role of interstitial cells of Cajal in the generation and modulation of motor activity induced by cholinergic neurotransmission in the stomach

Background  Interstitial cells of Cajal (ICC) are intimately linked to the enteric nervous system and a better understanding of the interactions between the two systems is going to advance our understanding of gut motor control. The objective of the present study was to investigate the role of ICC in the generation of gastric motor activity induced by cholinergic neurotransmission.