Current Gastroenterology Reports

, 16:363

Interstitial Cells of Cajal: Update on Basic and Clinical Science

Authors

    • Farncombe Family Digestive Health Research InstituteMcMaster University
  • Ji-Hong Chen
    • Department of Gastroenterology & HepatologyRenmin Hospital of Wuhan University
Neuromuscular Disorders of the Gastrointestinal Tract (S Rao, Section Editor)

DOI: 10.1007/s11894-013-0363-z

Cite this article as:
Huizinga, J.D. & Chen, J. Curr Gastroenterol Rep (2014) 16: 363. doi:10.1007/s11894-013-0363-z
Part of the following topical collections:
  1. Topical Collection on Neuromuscular Disorders of the Gastrointestinal Tract

Abstract

The basic science and clinical interest in the networks of interstitial cells of Cajal (ICC) keep growing, and here, research from 2010 to mid-2013 is highlighted. High-resolution gastrointestinal manometry and spatiotemporal mapping are bringing exciting new insights into motor patterns, their function and their myogenic and neurogenic origins, as well as the role of ICC. Critically important knowledge is emerging on the partaking of PDGFRα+ cells in ICC pacemaker networks. Evidence is emerging that ICC and PDGFRα+ cells have unique direct roles in muscle innervation. Chronic constipation is associated with loss and injury to ICC, which is stimulating extensive research into maintenance and repair of ICC after injury. In gastroparesis, high-resolution electrical and mechanical studies are beginning to elucidate the pathophysiological role of ICC and the pacemaker system in this condition. Receptors and ion channels that play a role in ICC function are being discovered and characterized, which paves the way for pharmacological interventions in gut motility disorders through ICC.

Keywords

Interstitial cells of Cajal (ICC)PDGFRα+Enteric nervous system (ENS)Chronic constipationGastroparesisInflammationColon motilityPacemaker cellsNitric oxideGuanylate cyclaseGastroparesisChronic constipationGut transitGastrointestinal transitIon channelsReceptors

Abbreviations

ICC

interstitial cells of Cajal

ICC-MP

ICC associated with the myenteric plexus (also called ICC-MY and ICC-AP)

ICC-DMP

ICC associated with the deep muscular plexus (small intestine)

ICC-SMP

ICC associated with the submuscular plexus (colon)

ICC-IM

Intramuscular ICC

c-Kit

tyrosine-protein kinase Kit or CD117

Ano1

Anoctamin-1

ENS

Enteric nervous system

PDGFRα+ cells

Platelet-derived growth factor receptor-alpha positive cells (specialized fibroblast-like cells)

LDC

Long Distance Contraction (colon)

RPMC

Rhythmic Propulsive Motor Complex (colon)

HAPC

High Amplitude Propulsive Contraction (an RPMC identified in human colon with amplitude > 100 mm Hg)

5HT

5-hydroxy tryptamine

Introduction

Gastrointestinal (GI) motility is critical for life, and therefore, the gut has developed multiple ways of performing mixing and propulsion of gut content, almost always using combinations of myogenic and neurogenic control systems. This is one reason why it is difficult to pinpoint a single cause for many neuromuscular disorders, and this is why deletion of a certain cell type or protein by disease or genetic manipulation in animal models does not always lead to severe malfunction and does not always pinpoint the role of the cell or protein in the normal functioning of the bowel. Two recent examples are the deletion of the enzyme that synthesizes serotonin in enterochromaffin cells (mediating nutrient- and distention-induced motor activities) [1] and the deletion of guanylate cyclase from gut smooth muscle cells (mediating nitric oxide [NO] induced relaxation) (Fig. 1) [3]. Both were expected to result in profound motility changes that did not materialize. In the first case, abolishing serotonin synthesis in enterochromaffin cells did not alter total GI transit, propulsion of rhodamine dextran down the small intestine, or bead expulsion in the colon. This does not mean necessarily that 5-hydroxy tryptamine (5HT) in enterochromaffin cells does not play a role in GI motility, but it does show that alternative mechanisms can fully accommodate transit of content. In the latter case, it appeared that innervation of interstitial cells of Cajal (ICC), also via stimulation of guanylate cyclase, could fulfill nitrergic innervation of the musculature [2••].
https://static-content.springer.com/image/art%3A10.1007%2Fs11894-013-0363-z/MediaObjects/11894_2013_363_Fig1_HTML.gif
Fig. 1

Are interstitial cells of Cajal (ICC) a conduit for nitrergic nerves to innervate smooth muscle? Yes, likely. Here, we see that when nitric oxide (NO) is generated in the musculature by an NO donor (DEA-NO) in the fundus of a mouse from which guanylate cyclase (the NO intracellular receptor) is knocked out in both smooth muscle cells and ICC (SM/ICC-GCKO), the musculature cannot relax (c). The graphs show relaxation of a precontracted fundus muscle strip subjected to a NO donor. When guanylate cyclase is knocked out in all smooth muscle cells (a), there is still significantly capability for relaxation, likely mediated by ICC. When guanylate cyclase is knocked out in ICC only (b), there is near normal relaxation via the direct smooth muscle pathway. Data are from [2••], shown with permission

Several networks of ICC are key players in the control of GI motility. They generate electrical pacemaker activity (rhythmic slow oscillations of membrane potential) that provides the musculature with the mechanism to produce propulsive rhythmic contractile activity [4], and they appear to be conduits for muscle innervation [2••]. They may also be critical for transmission of sensory information [5, 6•, 7]. Thus, extensive research is underway to further elucidate their role in normal physiology and discover their contribution to the pathophysiology of neuromuscular disorders of the GI tract [8] (Table 1). This review highlights new work from the last 3 years, but ICC research has been so prolific that not all the important works can be cited in the limited space available; hence, the review is restricted to certain areas of interest. Research on ICC outside the gut [29] has also been productive, but this review does not discuss such studies.
Table 1

Recent studies on relationships between interstitial cells of Cajal (ICC) injury and gastrointestinal (GI) disease

Disease

Injury to ICC

GI Dysfunction

Relationship Between ICC Injury and GI Dysfunction Proposed or Shown / Notes

Reference

Ulcerative colitis

Decrease in c-Kita in full thickness archival biopsies

  

[9]

Crohn’s disease

ICC-MP in terminal ileum show ultrastructural injury

 

Communication with mast cells appears to provide recovery

[10]

ICC-MP showed no degeneration or cytological changes

  

[11]

ICC-DMP shows ultrastructural injury

 

Injury selective to ICC

[12]

Slow transit constipation

Reduction in Ano1 immuno-reactivity

Constipation

Not specifically studied

[13]

Appendicitis

Loss of c-Kita, ultrastructural injury but no loss of cells

Inflammation

Injury related to degree of inflammation, full recovery when inflammation resolved

[14]

Idiopathetic and diabetic gastroparesis

Ultrastructural injury to ICC, differences between types

  

[15]

Loss of c-Kita

Delayed gastric emptying

Inverse correlation between ICC count and 4-h gastric retention in DG but not IG

[16••]

Loss of c-Kita

  

[17]

Loss of c-Kita

 

High resolution mapping with 256 electrodes shows slow wave irregularities

[18•]

Diabetic gastropathy

Ultrastructural injury to ICC, abnormal slow waves in vitro

  

[19]

Chagas disease

Loss of c-Kita

Megacolon

C-kit positivity related to extent of dilation

[20]

Loss of c-Kita

With and without megacolon

C-Kit was significantly more reduced in patients with megacolon

[21]

Intestinal atresia

Loss of c-Kita

atresia

Loss of c-Kit in proximal but not distal part

[22]

Loss of c-Kita

atresia

No significant difference of loss in proximal and distal part

[23]

Streptozotocin-induced diabetes in rats

Reduction in c-kit protein levels

Delayed gastric emptying

Curcumin (reducing oxidative stress) improves gastric emptying and c-kit protein levels

[24]

Reduction in c-Kita in gastric antrum

 

Impaired up-regulation of HO-1 expression associated with ICC injury

[25]

More than 50 % loss of c-Kita in small intestine

 

Slow wave mapping with 121 electrodes showed no changes

[26]

Diabetic enteropathy in mice

No change in ICC in small intestine and colon (immuno and electron microscopy)

Increased fecal output, increased intestinal transit

Change in enteric nerves likely cause of dysmotility

[27]

Diabetic gastroparesis in mice

Loss of c-Kita

Delayed gastric emptying

Gastric emptying and ICC restored by upregulation of heme oxygenase I (reducing oxidative stress)

[28]

ICC-MP, ICC associated with the myenteric plexus; ICC-DMP, ICC associated with the deep muscular plexus

ac-Kit (tyrosine-protein kinase Kit or CD117) = counting c-Kit immuno-positive cells or assessing percentage of area of c-Kit positivity using anti c-Kit antibodies. In such studies, mast cell immuno-positivity is usually excluded

Structure–Function Relationships of ICC

ICC are pacemaker cells for many rhythmic motor activities throughout the gut. The first modern paper to promote the concept of ICC as pacemaker cells was written in Italian but is now translated into English [30], accompanied by an introduction highlighting the significance of structural investigations for ultimate understanding of organ function [31]. Morphological investigations in humans still dominate our search for the role of ICC in gut dysfunction. ICC have been assessed in many biopsies or surgical material related to neuromuscular disorders by immunohistochemistry and electronmicroscopy (Fig. 2). Most frequently, ICC density is measured using some type of quantification of c-Kit immunohistochemistry [9, 14, 17, 33, 34], but anoctamin-1 (Ano1) immunohistochemistry is also employed [13] and will become more prevalent. There are not yet universally adopted standards of sampling and quantification; hence, each study has to be evaluated on its own merits. Although ICC quantification is a first important step and ICC loss has been associated with loss of function [16••], it has to be acknowledged that we do not know how much loss of ICC organs can tolerate before reduction or loss of function develops [35]. Also, loss of c-Kit immuno-staining of ICC might be caused by loss of the c-Kit receptor and might not accurately assess loss of cells [14]. In a model of diabetes, loss of more than 50 % of ICC did not appear to affect any parameter of slow wave activity, suggesting that the network of pacemaker cells can withstand a marked reduction in ICC density [26]. Furthermore, ICC form a coupled network with PDGFRα+ cells, are coupled to smooth muscle cells, and are heavily innervated; hence, dysfunction of ICC can be related to loss of coupling to other cells or injury to other cells to which they are electrically coupled. Although a likely role for ICC in the pathophysiology of motility disorders based on structural abnormalities is clearly established, the functional confirmation of dysfunction due to ICC injury is still to be discovered in most cases. The search for evidence for a role of injury to ICC in motor dysfunction has to take into account the age of animals used in models of disease, since the number and volume of ICC networks in the normal human stomach and colon decline with age [36]. How much this affects function is not established at this moment.
https://static-content.springer.com/image/art%3A10.1007%2Fs11894-013-0363-z/MediaObjects/11894_2013_363_Fig2_HTML.gif
Fig. 2

Is there structural evidence supporting a role for interstitial cells of Cajal (ICC) in enteric innervation to smooth muscle cells? Yes. For example, the ICC associated with the deep muscular plexus (ICC-DMP) in the human small intestine shown here are connected by synapse-like junctions to enteric nerve varicosities and by gap junctions to smooth muscle cells. In disease, one of the first signs of ICC injury involves the breakdown of connections with enteric nerves. An ICC-DMP (white*) intercalated between an individual nerve varicosity (N, enlarged in the left bottom square) and an outer circular smooth muscle cell (OCM, enlarged in the right top square). The ICC–nerve junction is 25 nm wide and shows increased prejunctional membrane density at the varicosity side (inset white arrow). Gap junctions occurred between ICC-DMP and the outer circular muscle cells (inset arrow). ICM, inner circular muscle; small arrows, caveoli; m, mitochondria; small arrowheads, basal lamina. In disease, the cell organelles and membrane structure can be altered, cells can be lost, and connections with inflammatory cells can be studied. Reproduced from [32]

Control of Colonic Motor Patterns

We have come a long way from inferring gut motor function from agonist-induced contractions on muscle strips or from measuring a “motility index” of in vivo pressure recordings that grouped all motor activities together into a number that said nothing about the type of contractions or motor patterns. In particular, spatiotemporal mapping techniques of video recordings of motor activities in vivo or in whole organs in vitro have given us unprecedented insight into details of motor patterns, far exceeding knowledge gained from classical manometry or in vitro muscle strip studies. Spatiotemporal mapping and high-resolution manometry will dominate searches for physiological and pharmacological control of motility for some time to come.

The colon has always been perceived as the organ with the most complex, inconsistent motor activities. Recent studies have shown detailed motor patterns of the rat colon [37•, 38•], rabbit colon [38•, 39, 40, 41], mouse colon [42•], and human colon [43•, 44]. Detailed characteristics of various motor patterns are becoming known, and their relationships to the two muscle layers, to sensory and motor neurons, to ICC, and to nutrients and microbiota are slowly becoming clearer. In all studies, the dramatic rhythmicity of motor patterns is striking. Our studies in the rat colon revealed various rhythmic activities that are known to be or proposed to be linked to ICC pacemakers. In most studies, a relatively low amplitude but high frequency (10–14/min) propulsive activity is recognized, which is often referred to as ripples. We found it to be an almost constant feature of the proximal rat colon, and often it propagated retrograde, especially immediately following strong propulsive antegrade contractions. There is little doubt that ripples are associated with the omnipresent slow wave activity in the colon generated by ICC associated with the deep muscular plexus (ICC-DMP), but, interestingly, ripples are not the dominant (propulsive) motor activity. Movie 1 show the ripples associated with the dominant propulsive motor activity, which we termed long-distance contraction (LDC), since in a fluid-filled colon, it travels always from the proximal to the distal colon and keeps the colon contracted for many seconds, clearing all liquid content out of the colon (Fig. 3) [45•]. A different propulsive motor pattern was named rhythmic propulsive motor complexes (RPMCs) [37•] (Movie 2), which is dominant in the distal colon. RPMCs are contractions that are usually much shorter in the length of propagation, have a slower velocity of propagation, can occur retrograde, but can also be effective in clearing the colon. The LDCs and RPMCs have a rhythm usually between 0.5 and 4/min in the isolated whole rat colon, and they are proposed to be associated with the ICC associated with the myenteric plexus (ICC-MP), in concert with the enteric nervous system (ENS) [45•]. That is, the rhythm of propulsive activities is governed by slow wave activity from the ICC-MP, and the ICC-MP network throughout the colon is proposed to be involved in the propagation of the propulsive activities. The laboratories of Jimenez and Takaki provided the evidence for colonic ICC-MP being pacemaker cells [46, 47], and we propose that the LDCs have proximal ICC-MP as pacemaker, whereas the RPMCs probably have their rhythm determined by ICC-MP of the mid-colon [45•, 48]. ICC-MP do not generate omnipresent pacemaker activity; it has to be induced. Distention (even in the presence of nerve blockade), excitatory stimuli such as muscarinic agonists, or enteric nerves can produce the stimulus. The propulsive motor patterns can also develop in the absence of neural activity, but in vivo and in whole segments under physiological conditions, it is the ENS that provides the dominant stimulus. It is likely that different neural programs exist for the generation of the two propulsive motor patterns.
https://static-content.springer.com/image/art%3A10.1007%2Fs11894-013-0363-z/MediaObjects/11894_2013_363_Fig3_HTML.gif
Fig. 3

Can we deduce interstitial cells of Cajal (ICC) function from spatiotemporal maps? Not easily. It is not unambiguous to deduce ICC function from spatiotemporal maps. Here, we see motor patterns (image representations of motor activity based on the changes of the colon diameter over time) created from video recordings of the whole rat colon. a In a quiescent colon, fluid-infusion induced a long distance contraction (LDC), which was followed without further stimulus by rhythmic LDCs. Ripples followed the LDCs in the proximal colon. The white band preceding the first LDC is the distention caused by fluid infusion. The white areas preceding spontaneous LDCs are relaxations preceding the contraction. b Interrupted LDCs showed a transient relaxation at about one third down the colon length. The interruption (relaxation) is seen as a white spot on the black contraction. c A tandem contraction (an LDC and a rhythmic propulsive motor complex [RPMC] occurring in tandem) is shown followed by a spontaneous LDC. d Five rhythmic LDCs are shown together with RPMCs in the mid to distal colon. Ripples were seen in the proximal colon. Can we deduce ICC function? The ripple frequency and propagation characteristics are very likely determined by the ICC associated with the submuscular plexus . The frequency of the stronger propulsive contractions is likely determined by ICC associated with the myenteric plexus (see the text), but this is under heavy neuronal control; hence, abnormalities in propulsive activity are not easily linked exclusively to ICC. Modified from [45•] with permission

The key to many controversies related to myogenic versus neurogenic origins of motor patterns is that abolishment of a motor pattern by tetrodotoxin (TTX) does not show exclusive control by the ENS. First, a nonneural stimulus might create the same pattern under different conditions, and second, neurally dependent ICC pacemaker activities might underlie a motor pattern that would be inhibited by TTX. Most motor patterns are orchestrated by an intricate combination of neurogenic and myogenic control systems [38•, 45•].

In studies on muscle strips of the human colon, slow rhythmic contractions at about 1/min were observed [43•, 49], possibly related to cyclic motor complexes recorded in whole human colons in vitro at 0.25/min [50]. Spencer et al. proposed that the pacemaker is intrinsic to the ENS, although a polarized intrinsic neural reflex was not demonstrated. In the study by Carbone et al., the strong rhythmic contractions were shown not to be due to burst firing by motor neurons, but to rely on intrinsic properties of the muscular apparatus. The rhythm was speculated to occur through motor neurons exciting but not driving the pacemaker system, likely originating in ICC-MP [43•].

Although good studies on the human colon using traditional manometry are emerging, such as a recent 24-h colonic manometry study [51], such studies focus on the most marked colonic activities, such as the so-called high-amplitude (>100 mm Hg) propulsive contractions (HAPCs) that might occur only a few times in 24 h [51]. The study of Singh et al. [51] had as one of the objectives to differentiate myopathies from neuropathies. Myopathies were deduced from the presence of most motor activities, albeit of low amplitude, and neuropathies from the absence of responses to a meal or the absence of occurrence of HAPCs. Future studies using high-resolution manometry will better define motor patterns, and much more basic research will reveal the myogenic and neurogenic parts of the control mechanism of human colonic motility.

Interactions Between ENS and ICC

Innervation of ICC serves to modulate pacemaker activity, serves to facilitate the sensory function of ICC networks, and serves to mediate nitrergic innervation. ICC are heavily innervated; every ICC is innervated by multiple varicosities of enteric sensory and/or motor nerves via synapse-like structures [52] (Fig. 2). This is different from smooth muscle cells that are primarily innervated through varicosities in the extracellular space. Although nerve varicosities can be as close as 20 nm to smooth muscle cells, in general they are innervated by nerve varicosities that are further away from the cell membrane, thereby creating the possibility of innervation of multiple cells through single varicosities or a string of varicosities along an axon [53]. When excitatory nerves increase the pacemaker frequency of the stomach [54], the frequency of peristaltic contractions will increase (if the general muscle excitation surpasses the mechanical threshold). Enteric sensory AH neurons have been shown to communicate with ICC, likely affecting intestinal pacemaker activity [52].

Because ICC are directly innervated and because they have gap junction contact with smooth muscle cells, the idea that ICC are actual conduits for innervation of smooth muscle cells, consistent with Ramon y Cajal’s original hypothesis [55], has gained considerable attention. But are ICC “mediators of smooth muscle innervation”? This term is used when the hypothesis is discussed that a smooth muscle function is affected by innervation via ICC, where ICC are deemed to be a conduit without necessarily a specific function other than transmitting the signal from nerve to muscle. For example, using WWv mice that lack intramuscular ICC (ICC-IM), electrical stimulation of nitrergic nerves [56] did not cause significant muscle relaxation, and stimulation of cholinergic nerves [57] did not lead to excitatory junction potentials in smooth muscle leading to the conclusion that innervation did not occur via direct communication between nerves and smooth muscle but that ICC was an essential intermediary. Subsequent experiments showed that the Ws rat fundus, also lacking ICC-IM, readily relaxed to the same type of stimulation of nitrergic nerves [58] or contracted normally via cholinergic neurotransmission [59], indicating that direct innervation of smooth muscle was the major pathway. Other studies showed mixed results [60]. The discrepancies between these studies was solved elegantly by Friebe and his coworkers, who first showed that all nitrergic inhibition was mediated by cGMP generated by guanylate cyclase [3]. Then they showed that mice that lack guanylate cyclase specifically in smooth muscle cells had normal nitrergic innervation. It was suggested that relaxation might be mediated by ICC or PDFGRα+ cells or that NO might release VIP from enteric nerves [3]. A subsequent study showed that mice that lacked guanylate cyclase in both smooth muscle and ICC did not show any nitrergic innervation [2••] or, more correctly, that an NO donor (Fig. 1) or electrical field stimulation of enteric nerves did not relax smooth muscle cells. Hence, direct innervation to smooth muscle leads to normal relaxation, but, stunningly, when this pathway is blocked through deletion of the smooth muscle guanylate cyclase, the ICC pathway appears to be able to give complete and near normal smooth muscle relaxation. This occurs via NO-induced cGMP in ICC, which then transmits this signal to smooth muscle cells, by cGMP passing through gap junction and/or by hyperpolarizing the smooth muscle cells. There were differences noted in ICC or smooth-muscle-mediated relaxation. NO-induced relaxation was normal in muscle that was precontracted with 10 μM carbachol and also was normal in the presence of nifedipine, whereas the ICC-mediated pathway was absent under these conditions. This needs further investigations. The conclusion is that two or more parallel ways of neurally mediated nitrergic smooth muscle relaxation exist. It is possible that other cells, such as the PDGRF α+ cells, which contain guanylate cyclase cells, are also involved. Klein and coworkers used a c-Kit CreERT2 knock-in allele to target ICC [61•]. This achieved a 50 % reduction in ICC that resulted in loss of slow wave activity and severely reduced transit, confirming the critical role of ICC in normal motor function. In the small intestine, electrical field stimulation to induce neurally mediated inhibitory and excitatory junction potentials failed, but in the colon, completely normal fast and slow inhibitory junction potentials remained. This provides evidence for a role of ICC in neurotransmission, but it also shows that normal inhibitory innervation to the muscle is present in the colon of these mice. Therefore, their conclusion that “ICC . . . are essential for transmission of signals from enteric neurons to GI smooth muscle cells” may be a bit of an overinterpretation in light of their data on the colon and in light of other studies indicating parallel innervation.

There is little doubt that nitrergic innervation plays an important role in normal gut motility, yet when colonic motility was studied in nNOS knock-out mice, all normal motility features were present, although the frequency of propulsive activity and transit were moderately affected [42•]. Even in the study of Groneberg et al. [2••], transit in almost all mice with guanylate cyclase knocked out in both smooth muscle cells and ICC showed total gut transit times in the normal range. This again shows that alternative pathways are present for many gut functions.

What do these new insights into ICC nerve interactions contribute to our understanding of the pathophysiology of GI motor dysfunction? For example, in chronic constipation, it is well established that ICC are diminished. This means that innervation may be compromised, although to what extent is still difficult to assess—first, because of the existence of parallel pathways [2••] and because the direct muscle innervation appears to be more powerful [2••]. Moreover, we do not know whether the ICC pathway and the direct muscle pathway are completely overlapping. It is possible that certain neuronal programs preferentially use one pathway over the other [62]; this is an interesting topic for further investigations.

ICC and Inflammation

An inflammatory process can cause significant damage to the neuromuscular apparatus, but the gut organs also have a dramatic capability to recover from the most serious injuries. ICC can be very sensitive to an inflammatory process, but the injury can be quite selective to subpopulations of ICC. In an animal model of inflammation using Nippostrongylus Brasiliensis, ICC-DMP were seen to be virtually wiped out at day 30 postinfection, whereas ICC-MP showed little structural and functional injury [63]. In mice infected by Trichinella Spiralis, ICC-DMP showed significant injury at day 10 postinfection but fully recovered at day 30 [64]. In the same model, ultrastructural studies showed that the ICC-MP had undergone significant damage but c-Kit immunohistochemistry did not reveal injury; recovery was almost complete at day 40 postinfection [65]. Human appendicitis showed a relationship between degree of inflammation, ultrastructural injury to ICC, and loss of the c-Kit receptor, although no marked loss of ICC was observed [14]. An interesting phenomenon shown in many studies, including this appendicitis study, is that one of the first injuries to ICC is the retraction of processes and subsequent loss of contact with nerves. Importantly, interval appendicitis (surgery performed weeks after inflammation has receded through antibiotic treatment) showed marked recovery of ICC injury [14]. In Crohn’s disease, ICC in the terminal ileum can be significantly diminished and injured, but there is no linear relationship between years of disease and diminished presence of ICC [10]. In fact, in long-standing Crohn’s disease, ICC networks can be quite normal, and it was hypothesized that mast cells might play a positive role in the maintenance of a healthy ICC population [10]. Also, in the colon of Crohn’s disease, although ICC-MP appeared less dense, they were ultrastructurally normal [11]; ICC associated with the submuscular plexus (ICC-SMP), however, showed ultrastructural signs of injury, whereas neighboring glial cells and fibroblast-like cells were undisturbed, with mast cells connecting to the fibroblast-like cells [12]. In ulcerative colitis, a recent immunohistochemical study found that all ICC types were reduced (loss of c-Kit) by more than 50 % in the left colon, together with significant neuronal damage [9]. Mast cells were markedly increased in the musculature. In a postinfectious IBS rat model using wild type and cytolethal distending toxin (CDT) negative strains of C. jejuni, it was demonstrated that Campylobacter CDT is an important factor in the development of chronic altered bowel patterns, rectal inflammation, and reduced ICC-DMP [66].

In conclusion, inflammation induces, most often, ultrastructural changes to ICC and, most often, loss of the c-Kit receptor, but it does not always lead to loss of ICC. Hence, reduction of c-Kit density is not equivalent to loss of ICC; on the other hand, normal c-kit immunohistochemistry might still be associated with ICC structural injury. Importantly, injury to ICC can be restricted to a subset of ICC, and, often, associated cells are undisturbed, except nerves that are usually affected. Gut tissue shows a dramatic capability of recovery to injury. Even with chronic inflammation, the fate of ICC is not doomed; it appears possible that partial recovery can occur, possibly mediated by mast cells. Of great interest are the factors that are responsible for regeneration, and many factors have been identified [67], including recently anoctamin-1 [68, 69]. Important issues for future studies are the injury to communication between ICC and neighboring cells—in particular, nerves, smooth muscle cells, PDGFRα+ cells, and immune cells—as well as the correlation between structural injury and loss of specific ICC functions. The study of impaired recovery of ICC or under- or overexpression of growth factors may contribute to the role of ICC in chronic neuromuscular disorders, and the study of epigenetics might reveal critical factors involved [70].

ICC and Pacemaker Dysfunction in Gastroparesis

Gastroparesis is a term used for patients with real or perceived gastric retention accompanied by nausea and/or vomiting and/or bloating. It is no doubt a heterogeneous disorder, but despite that, it appears that the most common intrinsic defect is being recognized in the ICC [71]. Recent studies using full-thickness biopsies have confirmed this, together with the finding of CD45 and CD68 immune cell infiltration in the musculature [17]. Interestingly, ICC counts inversely correlated with 4-h gastric retention in diabetic gastroparesis, but not in idiopathic gastroparesis. There was also a significant correlation between loss of ICC and enteric nerves in diabetic, but not in idiopathic, gastroparesis [16••]. No distinguishing features were found between ultrastructural injuries in both types of gastroparesis [15]. Injury was not observed in ICC-associated PDGFRα+ cells [72•]. There was a similar immune infiltrate in diabetic and idiopathic gastroparesis, but ICC injury was not correlated with this [16••]. Heme oxygenase-1 might be important in ICC maintenance during gastroparesis, as deduced from a study on streptozotocin-induced diabetes in rats [25], confirming an earlier study [28]. In the same model, curcumin, which has antioxidant and antiinflammatory properties, upregulated stem cell factor and c-Kit and was protective for ICC injury [24].

High-resolution mapping of electrical activities using arrays of hundreds of extracellular electrodes has given us much insight into the two-dimensional organization of pacemaker activity [73]. This technique has now established that abnormalities in pacemaker activity accompany gastroparesis, such as changes in pacemaker frequency, but also the occurrence of ectopic pacemakers and conduction block [18•]. The pacemaker region in the stomach was shown to be associated with high-velocity and high-amplitude localized circumferential propagation of slow waves. Interestingly, rapid circumferential propagation also emerges during a range of gastric dysrhythmias, elevating extracellular amplitudes and organizing transverse wavefronts, likely disturbing motility. Bidirectional coupling between ICC-MP and circular ICC-IM networks was suggested to be associated with these electrical activity patterns [73]. Also, in vitro, irregular, or loss of slow wave activity was observed in a model of gastroparesis [19].

In a detailed study on intestinal dysmotility in the RIP-I ⁄ hIFNb transgenic mice after 3.5 months of type 1 diabetes induced with streptozotocin, in contrast to most other models of diabetes, no changes in ileal and colonic ICC were observed, only selective changes in neurotransmission that appeared to underlie increased transit in the small intestine [27].

Continuing Discovery of Ion Channels and Receptors on ICC as Potential Targets for ICC Modulation

Our knowledge about mechanisms by which neurotransmitters and other regulatory substances affect ICC function is steadily increasing. Ion channels in the ICC membrane are discovered or further elucidated. Receptors are identified, and their agonists and functional roles explored. This accumulating knowledge will lead to our understanding of normal ICC function and how this can be modulated. The ideal would be to be able to selectively affect ICC function as pacemakers or mediators of innervation. But a lot of work is still needed to understand the intracellular workings of the ICC. To that end, the rhythmic ongoing transient calcium oscillations in ICC may play a key role (Movie 3) [74], and it is not surprising that many regulatory substances can affect intracellular calcium. Both neurotensin [75] and prostaglandin F2α [76] increase these calcium oscillations, and subsequently, nonselective cation channels are activated, leading to increased ICC excitation. Also, 5HT enhances spontaneous calcium oscillation in ICC-MP of the mouse small intestine via 5HT3 receptors and might, therefore, affect ICC excitability [77]. Intracellular calcium can also mediate the regulation of excitability by CCK via the CCK-1 receptor, activating the HCN channel in the gastric antrum [7880] initiating rhythmic inward currents. Pacemaker currents can also be activated by sphingolipids via sphingosine 1-phosphate [81]. Both histamine via H1 receptors [82] and Substance P can excite ICC via membrane depolarization [83]. The maxi chloride channel in ICC is also activated by intracellular calcium [84]. The maxi chloride channel is one of the candidates for the pacemaker channel in the ICC-MP of the small intestine, together with the chloride channel Ano1 [85, 86]. The maxi chloride channel in ICC has complicated kinetics—in particular, when patch clamping of in situ ICC is attempted and single-channel analysis has been hampered by the difficulty of assessing channel opening and closing transitions. Phase portrait analysis has given us exciting new ways of understanding channel gating in ICC [87, 88].

Conclusions

Many studies have been performed in recent years that bring us closer to our understanding of the physiological role of ICC in controlling GI motility and their role in pathophysiology of motility disorders. This review has highlighted some of these studies dealing with several but not all aspects of gut motor function. Of critical importance are the studies that highlight how ICC collaborate with nerves and other cells in and outside the pacemaker networks to perform functions as pacemakers and mediators of innervation. Detailed knowledge is accumulating on receptors and ion channels in the ICC cell membrane and intracellularly that will bring us in the near future to pharmacological experiments to alter or restore ICC function to affect motor abnormalities.

Acknowledgements

The study was financially supported by Grant 81170249 from the National Natural Science Foundation of China (NSFC) and by Grant MOP12874 from the Canadian Institutes of Health Research (CIHR). As always, we appreciate the discussions with Dr. Xuan-Yu Wang.

Compliance with Ethics Guidelines

Conflict of Interest

Dr. Huizinga states that this work was possible due to ongoing research support from the Canadian Institutes of Health Research (CIHR). Dr. Chen declares that this work was possible due to ongoing research support from the National Natural Science Foundation of China (NSFC).

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by the authors.

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© Springer Science+Business Media New York 2014