Chronic constipation: Lessons from animal studies

Best Practice & Research Clinical Gastroenterology 25 (2011) 59–71

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Best Practice & Research Clinical Gastroenterology

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Chronic constipation: Lessons from animal studies Natalia Zarate, MD, PhD, Post CCT in Gastroenterology a, Nick J. Spencer, Senior Lecturer b, * a

Centre for Academic Surgery (GI Physiology Unit), Barts and The London Queen Mary’s School of Medicine and Dentistry, London, UK Discipline of Human Physiology, Flinders Medical Science and Technology Cluster, GPO Box 2100, Flinders University, Adelaide, South Australia 5001, Australia b

Keywords: Colon Constipation Colonic migrating motor complex Peristalsis

Chronic constipation is a highly debilitating condition, affecting a significant proportion of the community. The burden to the health care system and impact on individual patients quality of life is immense. Unfortunately, the aetiology underlying chronic constipation is poorly understood and animal models are being used increasingly to investigate possible intrinsic neurogenic and myogenic mechanisms leading to relevant colonic sensori-motor dysfunction. Recently, major advances have been made in our understanding of the mechanisms that underlie propagating contractions along the large intestine, such as peristalsis and colonic migrating motor complexes in laboratory animals, particularly in guinea-pigs and mice. The first recordings of cyclical propagating contractions along the isolated whole human colon have now also been made. This review will highlight some of these advances and how impairments to these motility patterns may contribute to delayed colonic transit, known to exist in a proportion of patients with chronic constipation. Ó 2011 Elsevier Ltd. All rights reserved.

Introduction In mammals, the large bowel performs various interrelated functions: absorption of water and electrolytes, fermentation of colonic contents by bacterial flora, storage of feces and their transport and evacuation. To achieve all these functions, a variety of complex patterns of colonic motility are required. There is now strong circumstantial evidence that dysfunctional colonic motor activity can lead to symptoms such as diarrhoea or constipation, abdominal pain and bloating. Unfortunately, in humans, * Corresponding author. Tel.: þ61 8 8204 4241; fax: þ61 8 8204 5768. E-mail address: nicholas.spencer@flinders.edu.au (N.J. Spencer). 1521-6918/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bpg.2010.12.003

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there is limited knowledge about the stimuli that initiate and modulate each pattern of contractility and whether neural and/or myogenic mechanisms control their generation and frequency of occurrence. This lack of understanding is due in part to the remarkably complex electromechanical activity in the human colon, the lack of suitable techniques to study the organ, its inaccessibility and the need for prolonged investigations. It is for this reason that laboratory animals are increasingly being used as models to unravel possible intrinsic mechanisms underlying colonic pathophysiology. It is important to acknowledge, that there is, at present, no animal model whose anatomy and function fully resembles that of the human colon, so it is important to exercise caution when extrapolating major conclusions that are derived from animal studies. Definition of chronic constipation Chronic constipation is a debilitating condition affecting both physical and emotional functioning. It is one of the most frequent complaints of the lower GI tract. Its prevalence is higher in females [1,2] and increases progressively with age [1–3] in both genders. Treatment is frequently unsatisfactory as laxatives provide temporal relief and require chronic use [4]. Because of high prevalence, the health care burden of constipation is substantial, with an estimated $1.7 billion (direct and indirect) costs that can be attributed to management of constipation, in the US alone [5]. Multiple definitions of constipation have been used in the literature, frequently not reflecting the patient perception. The American College of Gastroenterology Chronic Constipation Task Force has defined chronic constipation as ‘..unsatisfactory defecation that results from infrequent stools, difficult stool passage, or both.’ [6]. More recently, the Rome criteria defined chronic functional constipation on the basis of the presence of two or more of a list of six symptoms related to the ability to defecate. In the absence of structural or metabolic aetiologies, patients with chronic constipation may be subclassified on the basis of physiological measures including speed of colonic transit and efficacy of evacuation. This reflects the view that functional constipation is caused by a disorder of colonic transit (slow transit constipation), impaired evacuation (rectal evacuatory disorder) or abnormal rectal perception (rectal hyposensation) [6–8]. Constipation is not restricted to adults, with high prevalence also being reported in children. In fact, 10–25% of consults in paediatric gastroenterology clinics have been attributed to an evaluation and/or treatment of constipation [9]. Unfortunately, there is an intrinsic difficultly in defining constipation in children, due to a reliance of interpretation of symptoms by parents. This is further confounded by several different definitions for childhood constipation by different groups. Most studies report a similar prevalence between boys and girls and interestingly, socio-economic factors have not been found to be associated with prevalence of childhood constipation. Different mechanisms can lead to constipation and information obtained from animal models has the limitation of generally addressing only one or a few aspects of this multidimensional condition. Nonetheless, they provide the attractive possibility of dissecting in isolation the mechanisms governing colonic motility and exploring the consequences of their dysfunction. Loss of enteric neurons and interstitial cells of Cajal in humans and animal models The elderly community is most affected by constipation and in fact, it is estimated that greater than 50% of elderly patients arriving at nursing homes already have some degree of constipation [10]. The increased prevalence of constipation with age can be attributed to multiple factors: such as reduced mobility, reduced fluid and fibre intake and the use of drugs that affect colonic motor function. In addition, evacuatory function usually decreases with age, particularly in women due to detrimental changes in the pelvic floor [11]. However, emerging data suggests that neuromuscular degeneration within the colon of elderly individuals is also a likely contributing factor. In support of this, a large proportion of elderly patients with chronic constipation have impaired colonic transit, as a result of impaired, or ineffective propulsion of colonic contents [12,13]. However, the precise mechanisms that underlie impaired colonic propulsion with age are poorly understood. What is clear, is that with increasing age there is a substantial loss of enteric neurons [14–17]. For example, in the human colon, it has been reported that there is a 37% reduction in the number of myenteric neurons in a 65 year old otherwise healthy individual, compared to a 20–35 year old [15].

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Laboratory animals, particularly rodents, have emerged as ideal models for studies on the effects of ageing on gastrointestinal function because, similar to humans, they readily loose a large population of myenteric neurons with age [16]. They also show impaired colonic transit and constipation [18]. For example, in the intestine of aged guinea-pigs, there is a reduction of approximately half the number of myenteric neurons in the small intestine, with no reduction in the neuronal cell size [19]. Also, in rats, there is a clear reduction in the number of myenteric neurons, which has been directly associated with a reduction in output of fecal pellets with age [18]. Interestingly, in rats, the mass of feces produced during a 24 hr period was also significantly decreased with age, even though food intake was similar [18]. These detrimental changes occurred despite normal gastric emptying rates and normal rates of propulsion in the small intestine. This implies that similar to humans, colonic propulsion may be selectively impaired with age, due to a significant loss of myenteric neurons. Interstitial cells of Cajal (ICC) are now well accepted to be the intestinal pacemaker cells for myogenic slow waves and phasic contractions of the gut wal [20]. ICC have been studied in human colon samples obtained from patients with STC and in patients with megacolon. In patients with either of these conditions, a significant reduction in the number of ICCs was detected [8,21]. Interestingly, this reduction in ICC included a reduction in a specific class of ICC located at the submucosal border, which has been proposed as pacemaker cells within the human colon [22]. Gomez-Pinilla et al (2010) have showed the number of ICC to decline significantly with age, at approximately a rate of 10% per 10 years. These changes were described in various regions of the large bowel, in different ICC populations and in both men and women. It has also been reported that patients with megacolon have a marked reduction in c-kit immunoreactive cells in the dilated colon compared to the non-distended region [23]. This is particularly noteworthy because in animal models of transient bowel obstruction [24], a marked reduction in ICC density occurs in the dilated segment, proximal to the obstructed region and with resolution of the dilated segment, ICC numbers and pacemaker activity is restored. Interestingly, in children with idiopathic intractable constipation, an abnormal density of ICC has been reported, but manometric recordings from these patients failed to reveal major differences in motility. This lead to the important conclusion that manometric recordings alone are unlikely to be successful in revealing abnormalities in underlying neuromuscular apparatus [25]. The functional significance of a loss or reduction of ICC to colonic propulsion is still unclear, and this is one of the major unanswered questions for the future. In the human small bowel, it has been reported that a full term infant with intestinal pseudo-obstruction completely lacked an enteric nervous system and lacked ICC at the level of the deep muscular plexus, but still developed an intact ICC network at the level of the myenteric plexus [26]. In vitro recordings from the intestine of this infant revealed that not only were electrical slow waves still recorded from the smooth muscle, but so to were propagating contractions from this segment. This is consistent with results from animal studies, which have shown that in GDNF knockout mice, which also lack an enteric nervous system, ICC at the level of the myenteric plexus still develop and generate electrical slow waves in the absence of enteric nerves [27]. What is less clear is the mechanism that facilitates the propagation of contractions along the intestine without an enteric nervous system, as slow waves generally only propagate over short segments of intestine. Different patterns of contractility in the large intestine The human colon generates a diverse repertoire of motor patterns which includes: isolated phasic contractions, propagated contractions and periodic contractile activity, known as colonic motor complexes (CMCs) and rectal motor complexes (RMCs), see Ref. [28] for review. Unlike the colon of the majority of mammalian species, cyclical motor activity does not appear to be the primary motor pattern exhibited by the human colon, which is quiescent for substantial periods of time. In the canine colon CMCs consist of rthymic bursts of contractions lasting from 5 to 15 min at regular intervals that migrate orad or caudad over at least half the length of the colon and are thought to contribute significantly to transit [29]. RMCs and CMCs in the human colon are also defined as regular bursts of phasic pressure waves lasting >three minutes, with a contraction frequency of 2–6 cpm, between which are quiescent periods of >two minutes [30,31]. But, these contractions do not necessarily migrate and they predominate in the sigmoid and rectum, especially in the nocturnal period when they

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can last up to an hour [30,31] (Fig. 1). Mechanisms responsible for RMCs and CMCs in the human colon are currently unknown, but taking into account they do not typically propagate over large distances may be unlikely to contribute to progression of colonic transit and it has been postulated that they could act as a ‘braking mechanism’ to flow of colonic contents. The electrical activity in the canine colon is similar to the human, whose circular muscle layer exhibits slow wave activity also generated in the submucosa area by the ICC-SM [22]. Frequency of contractions within the CMCs in the human large bowel coincides with the maximum frequency of the ICC-SM generated slow wave (N. Zarate, personal observations). However, the role of ICC in this pattern of contractility in the human colon has not been determined. In the human colon, individual propagating pressure sequences (PS), rather than bursts of cyclical contractions, appear to be important for propulsion of stool [32,33] and for defecation itself. On the basis of their amplitude, PS are further classified as high amplitude propagating sequences (HAPS), or low amplitude propagating sequences (LAPS). HAPS are frequently associated with defecation, are rarely retrograde and the majority start in the right colon [34,35]. Interestingly, low amplitude PS in human colon were as likely to be associated with colonic movements as higher amplitude PS (Dinning et al, 2008). Surprisingly, there is still little knowledge about the mechanisms that underlie the generation of propagating contractions along the human colon. This is unfortunate because studies have shown differences in the characteristics of propagated contractions in slow transit constipation, mainly a significantly reduction in the frequency of HAPS and an absence of HAPS in 30% of patients studied [12]. More recent studies have also reported a reduction in the amplitude and extent of propagation of propagating pressure waves and an increase in the frequency of retrograde PS [36]. In children with slow transit constipation (STC), similar deficits in colonic motility have been reported as those identified in adults. Recent studies in children have identified a significant impairment in antegrade propagating motor activity and failure to respond to normal physiological stimuli [37]. Although high amplitude propagating sequences were found to be of normal frequency in children with STC, retrograde propagating sequences were significantly less frequent [37]. Propagated sequences in the human colon, particularly low amplitude sequences, resemble the colonic migrating motor complexes (CMMCs) described in small mammals, i.e rat and mouse. In an attempt to better understand the mechanisms underlying propagating contractile activity along the colon, many investigators have turned to laboratory animals such as mouse, rat and guinea-pig, whose enteric neurochemistry and neural pathways are remarkably similar to those reported in humans [38,39]. The mouse, in particular, has proved an ideal model to understand the basic control mechanisms underlying colonic propulsion. This is probably because owing to their small size, the entire

Fig. 1. Periodic motor activity of the canine and human colon. Regular bursts of phasic pressure waves lasting three minutes, with a contraction frequency of two/minute, between which are quiescent periods can be seen in both canine and human colon recordings. Canine complexes occur with a similar frequency as complexes in the human colon.

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colon can be removed and studied in vitro, without damage to the intrinsic pathways responsible for CMMC generation or propagation [40–42] (Fig. 2). Recent advances in our understanding of the mechanisms underlying colonic migrating motor complexes It has been shown that CMMCs in mice are important for the propulsion of colonic contents [43]. What has remained elusive, however, is the location of the intrinsic pacemaker that determines the cyclical generation and frequency of CMMCs [41,44]. Although, it has been known for many years that the CMMC pacemaker must lie within the colon itself, since removal of the colon from mammals does not prevent CMMCs [42,45–47], this left the challenging task of identifying the pacemaker cells responsible for CMMC and determining what mechanisms entrain CMMC propagation along the colon. Recent breakthroughs have been made in this area. Originally, it was thought that cyclical generation of CMMCs required release of 5-HT from the mucosa, since antagonists of 5-HT receptors abolish CMMCs [48] and removal of the mucosa was initially thought to abolish CMMCs [49]. However, when these studies were repeated more carefully and thoroughly, and by actually recording 5-HT release in real time from the mucosa, it was shown that removal of the mucosa abolished 5-HT release, but did not abolish the generation or propagation of spontaneous CMMCs [50]. These results have now been reproduced by other laboratories. Furthermore, when antagonists of 5-HT3 receptors were applied to preparations of colon that were devoid of mucosa, they were found to have the same inhibitory effect in slowing the CMMC pacemaker [50]. This latter result was particularly important because it showed that 5-HT3 receptor antagonists must inhibit the CMMC pacemaker frequency via an action

Fig. 2. Schematic of the preparation of human descending colon used for mechanical recordings of circular muscle contractile activity in vitro. B, photomicrograph showing the human descending colon bathed and mounted in an organ bath in warm (36 C) oxygenated Krebs solution. Mechanical recordings were made simultaneously from the oral, mid and anal ends of the isolated descending colon. C, shows spontaneous propagating contractions along the descending colon preparation occurring about every 5– 7 min. D, shows an in vitro mechanical recording from an intact whole colon removed from a patient with chronic constipation. Recordings were made from the ascending, transverse and descending colon simultaneously. Cyclical propagating contractions occur at a similar frequency as the descending colon in a non-constipated colons (c.f. panel C).

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independent of 5-HT release from enterochromaffin (EC) cells in the mucosa. These findings have significant therapeutic ramifications for the control of CMMC frequency, and potentially colonic transit, because we can now direct therapies to target the intrinsic neural circuitry in the myenteric plexus, which is known to directly initiate CMMC generation and propagation. In humans, a selective highaffinity 5HT4 receptor agonist is known to stimulate high amplitude propagated contractions [51], enhancing colonic propulsion [52] and evidence suggests this drug is efficacious for the treatment of chronic constipation [53]. In vitro recordings of rat colon have revealed two major types of spontaneous contractile activity: high frequency low amplitude (HFLA) contractions and giant, low frequency high amplitude (LFHA) contractions [54,55]. Giant contractions migrate regularly over variable lengths of the rat colon at a frequency of w40/hour and are called giant migrating contractions (GMC). The use of this term has contributed to confusion in the literature, as it is also used to define the predefecatory propagated high amplitude contractions seen in the dog colon. Phasic contractions have been proposed to contribute to mixing of colonic contents and GMC to the propulsion of colonic contents. Control mechanisms of GMC require the cooperation of neurogenic and myogenic systems. The tonic release of nitric oxide (NO) by enteric neurons inhibits spontaneous GMC and pharmacological suppression of nitrergic neurons activity significantly increases their amplitude and frequency in vitro [54,56] and in vivo [57,58]. The importance of endogenous neural release of NO in the control of propagating CMMCs and their underlying myoelectrical correlates was first shown in isolated mouse colon where inhibitors of NO synthesis increased the frequency of CMMCs [45] and their underlying myoelectric correlates [59]. Interestingly, the human colon is also under the control of nitrergic innervation, as blockade of NO synthesis has a similar stimulatory effect on the frequency of proximal colonic PS [60]. Dissection studies performed in the rat colon by [55] provided evidence that the phasic contractile and electrical activity depended on the presence of the ICC-SMP network, whilst LFHA contractions persisted after removal of the submucosa. They likely depended on the myenteric plexus region [55,61] as they could not be recorded after removal of the longitudinal muscle layer which also removes the myenteric region. The myogenic origin of LFHA and HFLA contractility is supported by their TTXinsensitive nature and their sensitivity to L-type Ca antagonists [55]. Furthermore, studies performed in Ws/Ws mutant rats, have identified a marked reduction in the density of ICC networks [61] and shown an absence of the regular patterns of HFLA and LFHA contractions. At the present time, the contribution of the ICC network in the generation of propagated sequences in the human colon is unknown. Propagating contractions along the isolated intact whole human colon – is there a difference in chronic constipation? In vivo recordings from human rectum and colon have identified rectal motor complexes [62] and colonic motor complexes [62,63], respectively. The mean interval between rectal motor complexes has been reported to occur with a mean interval of w92 min, where each contraction burst typically lasts between 10 and 20 min [64]. Other studies seem to report a similar interval between colonic motor complexes and rectal motor complexes in vivo, see Ref. [28], for review. These characteristics are of particular interest, since recently we have characterised the motor activity along the isolated whole colon of human patients with chronic constipation and compared these motility patterns with those recorded from patients without constipation. In isolated full-length segments of human colon, we have recorded propagating contractions that occur with a mean interval of 4.2  0.7 min and half duration of 52.3  8.1 sec (Fig. 2). These contractions can propagate over significant lengths of human colon (ascending to descending colon) in healthy control subjects; and in the colons removed from patients with chronic constipation (Fig. 2). When focal transmural electrical nerve stimuli were applied to localised regions of colon, it has been possible to evoke a premature propagating contraction, which appear indistinguishable from rhythmically occurring propagating contractions (see Fig. 3). These preliminary studies reveal, for the first time, the existence of cyclical contractions in isolated, intact, whole human colon, which can propagate over significant distances that must occur independently of any spinal neural inputs, or endogenous hormonal influences. Furthermore, they can be evoked by brief trains of enteric nerve stimuli, similar to electrically-evoked CMMCs in the isolated colon of rodents [46] (Fig. 4).

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Fig. 3. Mechanical activity recorded from the isolated intact whole human colon. Spontaneous propagating contractions occur approximately every five minutes. A train of transmural electrical nerve stimuli (70 V, 0.5 ms, three seconds) were applied to the descending colon which evoked an orally propagating contraction which reached the ascending colon (see arrow). The right hand panel shows the oral propagation of the electrically-evoked propagating contraction.

Inhibition of colonic transit in response to colonic elongation – ‘The Occult reflex’ Recent studies in isolated guinea-pig distal colon have shed new light onto one of the potential mechanisms that may underlie impaired colonic transit, leading to chronic constipation [65]. This novel mechanism we termed ‘the Occult reflex’. An interesting aspect of the ‘Occult reflex’ was that its activation was dynamic and triggered by acute longitudinal distension (activated in seconds to minutes following stretch) and was not reliant upon any pathological insult, or a slow decay in the number of enteric neurons with age. The Occult reflex was first identified when we found that elongation in the length of the colon had profound inhibitory effects on the underlying neural circuitry responsible for colonic propulsion [65]. Initially, we showed that circumferential stretch applied to isolated segments of guinea-pig colon potently activated a repetitively firing peristaltic reflex, consisting of synchronised firing of ascending and descending interneurons [66]. When longitudinal stretch was applied to these same preparations (in which circumferential stretch was still present), it was found that as the length of the colon increased, so to did the degree of inhibition of the peristaltic reflex in the circular muscle [65]. In fact, the extent of the inhibitory action of the ‘Occult reflex’ was graded according to the extent of longitudinal stretch [65]. How might the ‘Occult reflex’ be activated in vivo? When an impacted distal colon was removed from euthanised guinea-pigs that contained multiple fecal pellets, its length measured approximately 160% longer than the length of an empty colon. Conversely, it was noted that as each pellet was expelled from an impacted colon, there was a gradual shortening in colonic length, which was associated with an increase in emptying velocity of subsequent pellets. Initially, pellet evacuation velocity from an impacted colon was slow (0.3 mm/s)

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Fig. 4. Spontaneous colonic migrating motor complexes (CMMCs) recorded from isolated whole mouse colon. CMMCs occur spontaneously approximately every three minutes and are abolished by tetrodotoxin, confirming their neurogenic origin. Spontaneous CMMCs in isolated mouse colon show strong similarities to cyclical propagating contractions along isolated human colon, see Fig. 2.

compared to the final velocity of the residual fecal pellets that propagated along the empty colon (1.1 mm/s). Overall, it was found that a direct correlation existed between the degree of colonic elongation and transit velocity [67]. It is suspected that the Occult reflex provides a mechanism to allow the colon to increase in length to accommodate more pellets, whilst providing inhibition of the propulsive motor activity underlying emptying. Oral and anal intrinsic reflexes activated by colonic elongation Recent intracellular electrophysiological recordings from the guinea-pig distal colon were made to identify which class(es) of sensory neuron may underlie the activation of the ‘Occult reflex’. There are two major classes of sensory neuron that lie within the enteric nervous system, known as afterhyperpolarizing (AH) neurons and S-neurons [68,69]. Activation of these sensory neurons by stimuli has been clearly shown not to require any inputs from the spinal cord, peripheral nervous system or central nervous system [68,70,71]. A population of myenteric S-neurons (interneurons) were found to likely underlie the activation of the Occult reflex [65], which respond to only graded amounts of circumferential stretch, while others appear to respond only to longitudinal stretch, by triggering activation of specific neural circuits [67], It was previously established that in response to circumferential stretch of the colon, the activated peristaltic reflex circuitry consisted of repetitively firing oral excitatory junction potentials (EJPs) that were temporally synchronised in time with anal inhibitory junction potentials (IJPs) in both smooth muscle layers [66]. Similar to the Occult reflex, the sensory neurons underlying the initiation of this activity were generated by mechanosensory S-interneurons, whilst surprisingly, AH sensory neurons were found to be electrically silent [68]. Mechanosensory interneurons were found to have stretch-sensitive neuronal processes that innervated the circular muscle [68]. Further experimentation revealed that the inhibitory Occult reflex activated by colonic elongation is more complex than initially thought [65], since both descending inhibitory (Occult) and ascending excitatory intrinsic nerve pathways are activated by longitudinal stretch (elongation) [67]. One possibility is that the descending inhibitory elongation reflex, the Occult reflex, is mediated by nitric oxide synthase positive descending interneurons [72]. Whereas, the ascending excitatory (potentiating) reflex activated by colonic elongation is likely to be one of the 3 classes of ascending cholinergic interneurons. This requires further detailed investigation.

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What might be the physiological role of the Occult reflex in the body? The physiological role of the Occult reflex is likely to be associated with accommodation and storage, rather than directly linked to the propulsion of contents, but this is largely speculation. Although the Occult reflex per se has not been demonstrated in human colon, an elongated colon has been reported in human patients with constipation. It is of interest that patients with slow transit constipation have been associated with a reduction in amplitude and frequency of high amplitude propulsive sequences this might lead to a slower movement of contents along the large bowel [73,74]. In summary, the Occult reflex is activated by elongation of the distal colon, such as occurs when the colon is impacted and contains multiple fecal pellets. This elongation of the colon activates intrinsic neural circuitry leading to a slowing of transit velocity. Sophisticated neural pathways appear to be activated during elongation, which activates both descending inhibitory (Occult) and ascending potentiating reflex circuitry. This means that colonic transit and velocity of fecal pellet propulsion would be subject to a balance between the two reflexes activated by elongation [67]. Colorectal aganglionosis and the implications for chronic constipation In the past decade, there has been a resurgence of interest in the developmental and molecular deficits underlying the onset of rectal aganglionosis associated with Hirschsprung’s disease. Despite

Fig. 5. Piebald mice heterozygous for the endothelin B receptor gene (Ednrb) have a short aganglionic segment and develop impaired CMMCs in the terminal colorectum (see:Ref. [76]). A, small white spotting indicates incomplete expression of Ednb gene. B, the colon of these offspring never develops megacolon, but shows impaired CMMCs (see C) in the terminal aganglionic segment. Tetrodotoxin abolished residual CMMCs in the proximal ganglionic region. D, shows the aganglionic region in an Ednb heterozyogous mouse. These heterozygous offspring which were originally thought to have normal colonic motility and an absence of any aganglionosis.

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Fig. 6. Changes in the density of myenteric ganglia in the ganglionic region of mice heterozygous for the piebald (Ednrb) mutation. Left hand side of figure shows that genetic characteristics of piebald offspring can be visually based on degree of coat colour white spotting. Right hand side of figure shows the dramatic loss of myenteric ganglia in the proximal to mid colon.

this, there still appears to be a remarkable lack of understanding regarding colonic motility in both the ganglionic and aganglionic colorectum from mammals that develop Hirschsprung’s disease. This is rather surprising, since the major problems in Hirschsprung’s patients are associated with impaired colonic motility and improper propulsion of colonic content. Originally, it was thought that only the aganglionic segment was affected in patients with Hirschsprung’s disease and that following resection and removal of the aganglionic segment, the remaining ganglionated segment was fully functional and morphologically intact. However, emerging data now shows that the ganglionic region of colon in mammals with rectal aganglionosis also exhibits a significant loss of myenteric neurons. In addition to at least 8 different genetic mutations which are known to lead to varying lengths of aganglionosis, there are also a variety of modifying genes which can alter the length of aganglionosis [75]. In support of this, it was recently shown that heterozygote offspring of the piebald strain of mouse, which were thought to be normal and served as experimental controls, also show a significant reduction in Ednrb gene expression (reduced by 90%) and significant loss of myenteric neurons, with a short segment aganglionosis. Interestingly, these heterozygote offspring do not develop megacolon and they do live a normal murine life span, but have impaired propagation of CMMCs [76] (Fig. 5 & 6). This data is particularly relevant to humans, because many heterozygote carriers of a variety of Hirschsprung’s disease mutations may live with short or ultrashort segment rectal aganglionosis and experience periods of impaired colonic transit and/or constipation, without knowledge of their genetic pedigree or phenotype [77,78]. This recent knowledge may aid in future diagnosis, particular in young patients with idiopathic constipation who are born to parents that may potentially carry genes, or modifying genes that are known to lead to short segment rectal aganglionosis. Summary Recordings obtained from animal models have played a major role in allowing us to understand some of the complex motor patterns known to exist in the human colon. This is a necessary step to gain understanding of the abnormal circuits underlying colorectal motor disorders. Taking into account the substantial socio-economic and personal implications of chronic constipation, investigations into the mechanisms governing colonic sensori-motor activity should be a priority for the basic science and medical communities. We are at the point when information obtained from in vitro investigations of animal models and in vivo studies in human must be integrated to enhance understanding and create a comprehensive model of human colon propulsive activity in health and disease.

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Conflict of interest statement None. Acknowledgements We wish to acknowledge the financial support provided by the National Health and Medical Research Council (NH&MRC) of Australia to grant # 535033 & 535034. Practice points  Laxatives are commonly used for treatment of chronic constipation, but as soon as laxative use ceases, constipation often returns. There is a need for better understanding of the mechanisms underlying the cause.  Incidence of constipation increases with age, which coincides with a significant loss of enteric neurons and ICC. However, many cell types are lost with age. Whether loss of neurons and ICC is responsible for impaired transit and constipation has not been demonstrated.  Develop clinical approaches to determine whether a proportion of patients with idiopathic chronic constipation have undiagnosed short or ultrashort colorectal aganglionosis inherited from parents who are carriers of genes encoding Hirschsprung’s disease.

Research agenda  Further develop in vitro studies on isolated intact human colon to characterise the mechanisms underlying impaired colonic transit in patients with chronic constipation.  Identify the classes of intrinsic sensory neuron underlying the different components of the ‘Occult’ reflex.  Determine whether acute ablation of Kit-positive ICC in the colon of laboratory animals induces changes in colonic transit and whether these changes in transit can be restored following regeneration and recovery of ICC populations.  Understand the relationship between HAPCs and LAPCs recorded from human colon in vivo with the propagating contractions recorded from intact human colon in vitro.

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