Discrete localization of various fatty-acid-binding proteins in various cell populations of mouse retina

The analysis of myenteric neurons is becoming increasingly important for the assessment of enteric nervous system injury and degeneration occurring in motor disorders of the gut. Limited information is presently available on the quantitative estimation of myenteric neurons and glial cells in paraffin-embedded colonic sections; additional data would be useful for diagnostic purposes. In this morphometric study, we performed immunohistochemistry to count myenteric neurons and glial cells in paraffin sections of human colon. Serial cross sections of formalin-fixed paraffin-embedded full-thickness normal human left colon (n = 10, age-range: 50–72 years) were examined. HuC/D and S100β antigens were found to be the best markers for the detection of neurons and glial cells, respectively. Significant correlations were noted between the numbers of neurons/glial cells and the respective myenteric ganglion areas. These findings suggest that HuC/D-S100β-immunostained paraffin cross sections of human colon can be regarded as valuable tools for the quantitative estimation of myenteric neurons and glial cells. Based on the present method, only a limited number of paraffin sections are needed for reliable quantitative assessments of myenteric ganglion cells, thus allowing fast and simple approaches in the settings of the histopathological diagnosis of colonic motility disorders and retrospective evaluations of pathological archival tissue specimens. R. De Giorgio is the recipient of grants from the Fondazione Del Monte di Bologna e Ravenna and from the Fondazione Cassa di Risparmio, Bologna, Italy. The authors declare no conflicting interests.     

Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin‐embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.     

Ultrastructure of interstitial cells of Cajal in myenteric plexus of human colon

The role of the interstitial cells of Cajal (ICC) associated with the myenteric plexus (ICC-MP) as regulators of the motility of the colonic external muscle remains unclear. Ultrastructural studies of myenteric interstitial cells are lacking in human colon. We therefore characterized the distinctive ultrastructure of these cells in the myenteric region of the colon by transmission electron microscopy of the region between the main muscle layers in all parts of the colon in unaffected areas of resected specimens from nine adult human patients. ICC-MP were similar in various colonic regions and had myoid features such as scattered caveolae, prominent intermediate filaments, and cytoplasmic dense bodies. We found characteristic dense membrane-associated bands with a patchy basal lamina, invaginating cellular protrusions (peg and socket junctions) between ICC and between ICC and muscle cells, and close contacts (<100 nm) between ICC and nerves. No gap junctions were observed. Fibroblast-like cells (FLC) were abundant showing well-developed secretory organelles, including coated vesicles, but lacked prominent intermediate filaments and caveolae. FLC had a patchy basal lamina, and peg and socket junctions were observed between them. Macrophage-like cells frequently occurred in close apposition with FLC and, more seldomly, with ICC-MP. The ultrastructure of ICC and FLC in the myenteric region of the human colon thus differs characteristically, but significant overlaps in the ultrastructure between ICC and FLC might complicate any interpretation in pathological ultrastructural studies of the human colonic muscle layer. An erratum to this article can be found at     

An integrated assessment of histopathological changes of the enteric neuromuscular compartment in experimental colitis

Bowel inflammatory fibrosis has been largely investigated, but an integrated assessment of remodelling in inflamed colon is lacking. This study evaluated tissue and cellular changes occurring in colonic wall upon induction of colitis, with a focus on neuromuscular compartment. Colitis was elicited in rats by 2,4‐dinitrobenzenesulfonic acid (DNBS). After 6 and 21 days, the following parameters were assessed on paraffin sections from colonic samples: tissue injury and inflammatory infiltration by histology; collagen and elastic fibres by histochemistry; HuC/D, glial fibrillar acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), nestin, substance P (SP), von Willebrand factor, c‐Kit and transmembrane 16A/Anoctamin1 (TMEM16A/ANO1) by immunohistochemistry. TMEM16A/ANO1 was also examined in isolated colonic smooth muscle cells (ICSMCs). On day 6, inflammatory alterations and fibrosis were present in DNBS‐treated rats; colonic wall thickening and fibrotic remodelling were evident on day 21. Colitis was associated with both an increase in collagen fibres and a decrease in elastic fibres. Moreover, the neuromuscular compartment of inflamed colon displayed a significant decrease in neuron density and increase in GFAP/PCNA‐positive glia of myenteric ganglia, enhanced expression of neural SP, blood vessel remodelling, reduced c‐Kit‐ and TMEM16A/ANO1‐positive interstitial cells of Cajal (ICCs), as well as an increase in TMEM16A/ANO1 expression in muscle tissues and ICSMCs. The present findings provide an integrated view of the inflammatory and fibrotic processes occurring in the colonic neuromuscular compartment of rats with DNBS‐induced colitis. These morphological alterations may represent a suitable basis for understanding early pathophysiological events related to bowel inflammatory fibrosis.     

Butyrate enemas enhance both cholinergic and nitrergic phenotype of myenteric neurons and neuromuscular transmission in newborn rat colon

Postnatal changes in the enteric nervous system (ENS) are involved in the establishment of colonic motility. In adult rats, butyrate induced neuroplastic changes in the ENS, leading to enhanced colonic motility. Whether butyrate can induce similar changes during the postnatal period remains unknown. Enemas (Na-butyrate) were performed daily in rat pups between postnatal day (PND) 7 and PND 17. Effects of butyrate were evaluated on morphological and histological parameters in the distal colon at PND 21. The neurochemical phenotype of colonic submucosal and myenteric neurons was analyzed using antibodies against Hu, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS). Colonic motility and neuromuscular transmission was assessed in vivo and ex vivo. Butyrate (2.5 mM) enemas had no impact on pup growth and histological parameters compared with control. Butyrate did not modify the number of Hu-immunoreactive (IR) neurons per ganglia. A significant increase in the proportion (per Hu-IR neurons) of nNOS-IR myenteric and submucosal neurons and ChAT-IR myenteric neurons was observed in the distal colon after butyrate enemas compared with control. In addition, butyrate induced a significant increase in both nitrergic and cholinergic components of the neuromuscular transmission compared with control. Finally, butyrate increased distal colonic transit time compared with control. We concluded that butyrate enemas induced neuroplastic changes in myenteric and submucosal neurons, leading to changes in gastrointestinal functions. Our results support exploration of butyrate as potential therapy for motility disorders in preterm infants with delayed maturation of the ENS.     

Development of interstitial cells of Cajal in the human digestive tract as the result of reciprocal induction of mesenchymal and neural crest cells

Neural crest cells (NCC) can migrate into different parts of the body and express their strong inductive potential. In addition, they are multipotent and are able to differentiate into various cell types with diverse functions. In the primitive gut, NCC induce differentiation of muscular structures and interstitial cells of Cajal (ICC), and they themselves differentiate into the elements of the enteric nervous system (ENS), neurons and glial cells. ICC develop by way of mesenchymal cell differentiation in the outer parts of the primitive gut wall around the myenteric plexus (MP) ganglia, with the exception of colon, where they appear simultaneously also at the submucosal border of the circular muscular layer around the submucosal plexus (SMP) ganglia. However, in a complex process of reciprocal induction of NCC and local mesenchyma, c‐kit positive precursors are the first to differentiate, representing probably the common precursors of ICC and smooth muscle cells (SMC). C‐kit positive precursors could represent a key impact factor regarding the final differentiation of NCC into neurons and glial cells with neurons subsequently excreting stem cell factor (SCF) and other signalling molecules. Under the impact of SCF, a portion of c‐kit positive precursors lying immediately around the ganglia differentiate into ICC, while the rest differentiate into SMC.     

Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.     

Interleukin-1beta activates specific populations of enteric neurons and enteric glia in the guinea pig ileum and colon

Fos expression was used to assess whether the proinflammatory cytokine interleukin-1beta (IL-1beta) activated specific, chemically coded neuronal populations in isolated preparations of guinea pig ileum and colon. Whether the effects of IL-1beta were mediated through a prostaglandin pathway and whether IL-1beta induced the expression of cyclooxygenase (COX)-2 was also examined. Single- and double-labeling immunohistochemistry was used after treatment of isolated tissues with IL-1beta (0.1-10 ng/ml). IL-1beta induced Fos expression in enteric neurons and also in enteric glia in the ileum and colon. For enteric neurons, activation was concentration-dependent and sensitive to indomethacin, in both the myenteric and submucosal plexuses in both regions of the gut. The maximum proportion of activated neurons differed between the ileal (approximately 15%) and colonic (approximately 42%) myenteric and ileal (approximately 60%) and colonic (approximately 75%) submucosal plexuses. The majority of neurons activated in the myenteric plexus of the ileum expressed nitric oxide synthase (NOS) or enkephalin immunoreactivity. In the colon, activated myenteric neurons expressed NOS. In the submucosal plexus of both regions of the gut, the majority of activated neurons were vasoactive intestinal polypeptide (VIP) immunoreactive. After treatment with IL-1beta, COX-2 immunoreactivity was detected in the wall of the gut in both neurons and nonneuronal cells. In conclusion, we have found that the proinflammatory cytokine IL-1beta specifically activates certain neurochemically defined neural pathways and that these changes may lead to disturbances in motility observed in the inflamed bowel.     

Spatial relationship between telocytes,interstitial cells of Cajal and the enteric nervous system in the human ileum and colon

Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high-resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC-telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function.     

Crohn’s disease of the colon: ultrastructural changes in submuscular interstitial cells of Cajal

Interstitial cells of Cajal (ICC) at the submuscular border of the human colon (ICC-SMP) are the proposed pacemaker cells of the musculature. In patients with Crohn’s disease (CD) of the colon, ICC-SMP showed characteristic cytological changes from controls. The changes comprised secondary lysosomes in connection with lipid droplets and cytoplasmic vacuoles or multiple empty, confluent and often outbulging vacuoles merging with cisterns of granular endoplasmic reticulum and clusters of glycogen granules. These changes were most pronounced in patients with macroscopical mucosal inflammation but were also demonstrable in uninvolved colonic segments. Relationships of ICC to other cells were undisturbed. The changes were selective to ICC-SMP, as glial cells, muscle cells and fibroblast-like cells at the submuscular border showed no cytological alterations compared with controls. Varicosities of the submuscular plexus were often empty and dilated. Fibroblast-like cells selectively encased macrophages and mast cells. The cytological changes in ICC-SMP in CD are thus similar to changes seen in ulcerative colitis and may be of pathophysiological significance with regard to the motility and sensory disturbances seen in patients with CD.     

Development of the enteric nervous system,smooth muscle and interstitial cells of Cajal in the human gastrointestinal tract

The generation of functional neuromuscular activity within the pre-natal gastrointestinal tract requires the coordinated development of enteric neurons and glial cells, concentric layers of smooth muscle and interstitial cells of Cajal (ICC). We investigated the genesis of these different cell types in human embryonic and fetal gut material ranging from weeks 4–14. Neural crest cells (NCC), labelled with antibodies against the neurotrophin receptor p75^NTR, entered the foregut at week 4, and migrated rostrocaudally to reach the terminal hindgut by week 7. Initially, these cells were loosely distributed throughout the gut mesenchyme but later coalesced to form ganglia along a rostrocaudal gradient of maturation; the myenteric plexus developed primarily in the foregut, then in the midgut, and finally in the hindgut. The submucosal plexus formed approximately 2–3 weeks after the myenteric plexus, arising from cells that migrated centripetally through the circular muscle layer from the myenteric region. Smooth muscle differentiation, as evidenced by the expression of -smooth muscle actin, followed NCC colonization of the gut within a few weeks. Gut smooth muscle also matured in a rostrocaudal direction, with a large band of -smooth muscle actin being present in the oesophagus at week 8 and in the hindgut by week 11. Circular muscle developed prior to longitudinal muscle in the intestine and colon. ICC emerged from the developing gut mesenchyme at week 9 to surround and closely appose the myenteric ganglia by week 11. By week 14, the intestine was invested with neural cells, longitudinal, circular and muscularis mucosae muscle layers, and an ICC network, giving the fetal gut a mature appearance.A.S.W. is funded by a PhD studentship awarded to A.J.B. by the Child Health Research Appeal Trust.     

Role of enteric glia in intestinal physiology: effects of the gliotoxin fluorocitrate on motor and secretory function

The role of enteric glia in gastrointestinal physiology remains largely unexplored. We examined the actions of the gliotoxin fluorocitrate (FC) on intestinal motility, secretion, and inflammation after assessing its efficacy and specificity in vitro. FC (100 microM) caused a significant decrease in the phosphorylation of the glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diaz-4-yl)amino]-2-deoxyglucose in enteric glial cultures and a reduction in glial uptake of the fluorescent dipeptide Ala-Lys-7-amino-4-methylcoumarin-3-acetic acid in both the ileum and colon. Dipeptide uptake by resident murine macrophages or guinea pig myenteric neurons was unaffected by FC. Incubation of isolated guinea pig ileal segments with FC caused a specific and significant increase in glial expression of the phosphorylated form of ERK-1/2. Disruption of enteric glial function with FC in mice reduced small intestinal motility in vitro, including a significant decrease in basal tone and the amplitude of contractility in response to electrical field stimulation. Mice treated with 10 or 20 micromol/kg FC twice daily for 7 days demonstrated a concentration-dependent decrease in small intestinal transit. In contrast, no changes in colonic transit or ion transport in vitro were observed. There were no changes in glial or neuronal morphology, any signs of inflammation in the FC-treated mice, or any change in the number of myenteric nitric oxide synthase-expressing neurons. We conclude that FC treatment causes enteric glial dysfunction, without causing intestinal inflammation. Our data suggest that enteric glia are involved in the modulation of enteric neural circuits underlying the regulation of intestinal motility.     

5-Hydroxytryptophan activates colonic myenteric neurons and propulsive motor function through 5-HT4 receptors in conscious mice

Serotonin [5-hydroxytryptamine (5-HT)] acts as a modulator of colonic motility and secretion. We characterized the action of the 5-HT precursor 5-hydroxytryptophan (5-HTP) on colonic myenteric neurons and propulsive motor activity in conscious mice. Fos immunoreactivity (IR), used as a marker of neuronal activation, was monitored in longitudinal muscle/myenteric plexus whole mount preparations of the distal colon 90 min after an intraperitoneal injection of 5-HTP. Double staining of Fos IR with peripheral choline acetyltransferase (pChAT) IR or NADPH-diaphorase activity was performed. The injection of 5-HTP (0.5, 1, 5, or 10 mg/kg ip) increased fecal pellet output and fluid content in a dose-related manner, with a peak response observed within the first 15 min postinjection. 5-HTP (0.5-10 mg/kg) dose dependently increased Fos expression in myenteric neurons, with a maximal response of 9.9 +/- 1.0 cells/ganglion [P < 0.05 vs. vehicle-treated mice (2.3 +/- 0.6 cells/ganglion)]. There was a positive correlation between Fos expression and fecal output. Of Fos-positive ganglionic cells, 40 +/- 4% were also pChAT positive and 21 +/- 5% were NADPH-diaphorase positive in response to 5-HTP, respectively. 5-HTP-induced defecation and Fos expression were completely prevented by pretreatment with the selective 5-HT4 antagonist RS-39604. These results show that 5-HTP injected peripherally increases Fos expression in different populations of cholinergic and nitrergic myenteric neurons in the distal colon and stimulates propulsive colonic motor function through 5-HT4 receptors in conscious mice. These findings suggest an important role of activation of colonic myenteric neurons in the 5-HT4 receptor-mediated colonic propulsive motor response.     

Subtractive hybridization unravels a role for the ion cotransporter NKCC1 in the murine intestinal pacemaker

In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. Our aim was to identify ICC-specific genes and their function in the mouse jejunum. Suppression subtractive hybridization using two independent ICC-deficient mouse models identified 56 genes putatively downregulated in the muscularis propria compared with wild-type littermates. Differential expression was confirmed by real-time quantitative PCR for the tyrosine kinase receptor KIT, the established marker for ICC, and for the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Immunoreactivity for NKCC1 was detected in myenteric ICC but not in the ICC population located at the deep muscular plexus. NKCC1 was also expressed in enteric neurons and mucosal crypts. Bumetanide, an NKCC1 inhibitor, reversibly affected the shape, amplitude, and frequency of the slow waves. Similar alterations were observed in NKCC1 knockout mice. These data support the hypothesis that NKCC1 expressed in myenteric ICC is involved in the mechanism of slow waves in the murine jejunum.     

Expression of Ca(2+)-activated K(+) channels, SK3, in the interstitial cells of Cajal in the gastrointestinal tract

A role for small-conductance Ca²⁺-activatedK⁺ (SK) channels on spontaneous motility of thegastrointestinal tract has been suggested. Although four subtypes of SKchannels were identified in mammalian tissues, the subtypes of SKchannel expressed in the gastrointestinal tract are still unknown. Inthis study, we investigated the expression and localization of SKchannels in the gastrointestinal tract. RT-PCR analysis showsexpression of SK3 and SK4 mRNA, but not SK1 or SK2 mRNA, in the ratintestine. SK3 immunoreactivity was detected in the myenteric plexusand muscular layers of the stomach, ileum, and colon.SK3-immunoreactive cells were stained with antibody forc-kit, a marker for the interstitial cells of Cajal (ICC), but not with that for glial fibrillary acidic protein in the ileum andstomach. Immunoelectron microscopic analysis indicates that SK3channels are localized on processes of ICC that are located close tothe myenteric plexus between the longitudinal and circular musclelayers and within the muscular layers. Because ICC have been identifiedas pacemaker cells and are known to play a major role in generating theregular motility of the gastrointestinal tract, these results suggestthat SK3 channels, which are expressed specifically in ICC, play animportant role in generating a rhythmic pacemaker current in thegastrointestinal tract.

     

Appearance of interstitial cells of Cajal in the human midgut

Several subtypes of the interstitial cells of Cajal (ICC) form networks that play a role in gastrointestinal motor control. ICC express c-kit and depend on signaling via Kit receptors for development and phenotype maintenance. At 7-8 weeks of development, c-kit-immunoreactive (c-kit-IR) cells are present in the human oesophagus, stomach and proximal duodenum wall. In the remaining small and large bowel, c-kit-IR cells appear later. The object of the present study is to determine the timing of the appearance of c-kit-IR ICC in the parts of the digestive tube originating from the midgut (distal duodenum, jejunum, ileum and proximal colon). Specimens were obtained from eight human embryos and 11 fetuses at 7-12 weeks of gestational age. The specimens were exposed to anti-c-kit antibodies to investigate ICC differentiation. The differentiation of enteric neurons and smooth muscle cells was immunohistochemically examined by using anti-PGP9,5 and anti-desmin antibodies, respectively. In the distal duodenum, jejunum and ileum, c-kit-IR cells emerged at week 9 at the level of the myenteric plexus in the form of a thin row of cells encircling the inception of the ganglia. These cells were multipolar or spindle-shaped with two long processes and corresponded to the ICC of the myenteric plexus. In the proximal colon, c-kit-IR cells emerged at week 9-10 in the form of two parallel belts of cells extending at the submucosal plexus and the myenteric plexus levels. We conclude that ICC develop following two different patterns in the human midgut.     

Possible involvement of muscularis resident macrophages in impairment of interstitial cells of Cajal and myenteric nerve systems in rat models of TNBS-induced colitis

Resident macrophages are distributed in the network of interstitial cells of Cajal (ICC) and the myenteric nerve within the myenteric plexus. We evaluated changes in chemoattractant protein mRNA expression in macrophages and neutrophils, the ICC, nerve and macrophages in the myenteric plexus of model rats with TNBS-induced colitis. Chemoattractant proteins, MCP-1, GRO, MIP-2 and CINC-2α were upregulated in the colonic muscle layer after inflammation. Leukocyte infiltration and MPO activity were increased in the muscle layer. Electron microscopy indicated an irregular contour of the myenteric ganglia into which numerous macrophages had penetrated. Macrophages were also distributed near the ICC in the inflamed myenteric plexus. Immunohistochemistry showed that the ICC network and myenteric nerve system had disappeared from the inflamed region, whereas the number of resident macrophages was increased. TTX-insensitive, possibly ICC-mediated, rhythmic contractions of circular smooth muscle strips and enteric neuron-mediated TTX-sensitive peristalsis in the whole proximal colon tissue were significantly inhibited in the inflamed colon, indicating that the ICC-myenteric nerve system was dysfunctional in the inflamed muscle layer. Their accumulation around the myenteric nerve plexus and the ICC network suggests that macrophages play an important role in inducing intestinal dysmotility in gut inflammation.