Keywords

27.1 Introduction

Hirschsprung’s disease (HSCR) is the most common congenital gut motility disorder and is characterized by the absence of ganglion cells and the presence of hypertrophic nerve trunks affecting the distal bowel (aganglionosis). In the majority of patients (~80%) the extent of aganglionosis is limited to the rectosigmoid region, while a longer segment extending proximally from the internal anal sphincter (IAS) is involved in the remaining ~20% [1]. The gold-standard treatment for this condition is the pull-through (PT) operation. While several variations on this procedure exist, the underlying principle of the PT operation is the transanal resection of the tonically contracted aganglionic segment of bowel with anastomosis of the normally innervated colon to a point just inside the dentate line, preserving the IAS [2].

Despite undergoing a technically well-performed PT operation, many patients experience persistent bowel symptoms at long-term follow-up, including constipation, soiling, and recurrent Hirschsprung-associated enterocolitis (HAEC) [3, 4]. While a demonstrable mechanical cause can be shown in many of these patients, a proportion is experiencing ganglionic bowel dysfunction as the underlying cause. This chapter explores the phenomenon of ganglionic bowel dysfunction in HSCR, including potential mechanisms by which it occurs.

27.2 Causes of Persistent Bowel Symptoms Following Pull-Through Surgery for Hirschsprung’s Disease

The range of operations performed to correct HSCR and the varied means of monitoring postoperative outcome (parental questionnaire/validated scoring systems/subjective assessment) make comparison of the outcomes obtained in each study difficult, with the results of each being judged on the merits of the study. For ease, persistent symptoms following PT surgery can be categorized as obstructive symptoms (constipation, recurrent HAEC) and incontinence symptoms [5].

Assessing the prevalence of constipation after PT surgery depends on how it is defined. Studies with statistically meaningful numbers of patients have reported constipation ranging from 9.8% to 55% [3, 6,7,8,9,10,11,12]. Postoperative HAEC occurs in 2–35% of patients who have undergone a PT operation [13, 14].

Fecal incontinence and associated soiling is one of the key contributors to the quality of life reported by patients who undergo PT surgery for HSCR. Normal fecal continence is a function of rectal reservoir function, anal canal sensation, and normal anal sphincter function [3]. While loss of rectal reservoir is an inherent side effect of the PT procedure, loss of anal canal sensation may occur due to the commencement of endorectal dissection too close to the dentate line. Interference with the sphincter complex may occur owing to excessive stretching during total transanal endorectal PT or retrorectal dissection during a Duhamel PT [15].

As with constipation, the incidence of post-PT incontinence and soiling may be considerably higher when independently assessed than has been previously thought [3]. Soiling has been reported in various follow-up studies in 12–56% of patients, a small proportion of whom are patients with true incontinence as opposed to overflow soiling [6, 8, 9, 11, 12, 16].

Patients with persistent bowel symptoms may fall into one or more of the following categories of patients: (1) those with mechanical obstruction, due to a stricture or an obstructive cuff of tunica muscularis following a Soave PT procedure; (2) those with recurrent, retained, or acquired aganglionosis; (3) patients with intestinal dysmotility; (4) patients with internal anal achalasia; (5) functional megacolon due to stool holding behaviors [4]. While those with a mechanical obstruction or a retained segment of aganglionic bowel have a demonstrable surgically treatable cause, as is also somewhat the case with internal anal sphincter achalasia, getting control over ongoing bowel symptoms in those with dysmotility or an acquired megacolon can be challenging.

27.3 Investigation of Persistent Bowel Symptoms After the Pull-Through Operation for Hirschsprung’s Disease

All patients presenting with persistent bowel symptoms following a pull-through operation should have a detailed clinical assessment, specifically trying to elucidate information about abdominal distension, explosive stools, consistency of stool, vomiting, weight gain, use of any medications that affect gastrointestinal motility, and details about the PT operation and any postoperative complications. A physical examination will also yield information about nutritional status, abdominal distension, and the gross appearance of the anal canal, and whether there are any palpable strictures. A contrast enema is a valuable initial investigation in these individuals as it provides information regarding bowel caliber and motility [4, 5].

An examination of the anal canal under anesthesia is performed to check the integrity of the anal canal, whether the dentate line is intact in its full circumference, and depending on the type of PT operation performed, whether there is an obstructing Soave cuff, or a dilated rectal pouch. Retained aganglionic or transition zone bowel is evaluated by means of a full-thickness rectal biopsy. In the absence of a mechanical obstruction or a damaged anal canal/disrupted dentate line, motility studies including anal manometry and a radio-opaque marker study may provide information about localized or diffuse dysmotility and colonic hyperactivity or atony. Management options depending on the nature of the abnormality found will range from anal dilatations or a redo PT operation, to medical treatment with loperamide, dietary modification, bowel irrigations, or intra-sphincteric botulinum toxin therapy [4, 5].

27.4 The Extent of the Transition Zone in Hirschsprung’s Disease

The transition zone (TZ) represents the segment of colon in HSCR at the interface between the tonically contracted aganglionic colon and the “normal” colon, which contains ganglion cells and non-hypertrophied nerve trunks. Characteristically, TZ colon is neuroanatomically abnormal despite ganglion cells being present [17]. Localizing the transition zone prior to curative PT surgery for HSCR is important in operative planning, especially if considering a total transanal approach over a combined transabdominal-transanal approach.

By far the most widely used means of achieving this is the preoperative contrast enema [2]. Radiologically, the TZ typically appears as a site where large caliber dilated colon abruptly funnels into a narrow caliber colon [18, 19]. The presence of irregular contractions, spasm, and mucosal irregularities may also be used as secondary signs indicative of TZ colon [19].

However, concordance between the radiological TZ on contrast enema and the histological TZ varies widely. In neonates and in those with total colonic aganglionosis the colon can appear normal in caliber [20]. Frongia et al. reported 94.4% concordance of contrast enema with the histologically assessed proximal level of aganglionosis in those with RSHD, although it was only 50% accurate in those with LSHD [21]. Muller et al. levelled the TZ according to the corresponding vertebral level and found that, when those with no visible radiological transition zone are excluded, the levels of the radiological and histological TZ were only concordant in 51.9% of children with rectal or rectosigmoid aganglionosis [22]. A striking finding in a study of similar objective by Jamieson et al. was that the radiological TZ was consistently more distal than the histological TZ in those in whom there was discordance (62.5% of cases) [19].

Failure to recognize TZ colon can lead to a retained aganglionic segment following PT surgery, with the potential need for a redo-PT [23]. One retrospective series reported a TZ PT rate of 15% (13 patients), with incorrect interpretation of the intraoperative biopsy being the cause in 7 patients and partial circumference aganglionosis with a ganglionic intraoperative biopsy being the cause in 5 patients [17]. Lawal et al. reported a series of 93 patients referred with persistent obstructive symptoms following PT surgery for HSCR. As part of their workup, a rectal biopsy was performed. Twenty-five patients (26.9%) were found to have retained aganglionic or transition zone bowel, 11 of whom had rectosigmoid disease [23].

There are no agreed criteria defining what constitutes the histological TZ in HSCR. In general, features such as hypoganglionosis, the presence of hypertrophic nerve trunks, and partial circumference aganglionosis are widely quoted characteristics of transition zone colon [17]. Partial circumference abnormalities of innervation pattern have been well described in HSCR. White and Langer (2000) prospectively examined the circumferential distribution of ganglion cells in transition zone colon in 12 patients with HSCR and found a “leading edge” of ganglion cells extending for up to 2.4 cm in the myenteric plexus and 2.1 cm in the submucosal plexus [24]. Das et al. described a series of 20 patients with long-segment aganglionosis, in whom they evaluated the quadrantic innervation of the colon after pull-through surgery. They noted incomplete ganglionation of the full circumference in the transition zone in 8 patients [18].

Only a small number of studies have examined the total extent of the histological TZ in HSCR. A small series by Swaminathan and Kapur measured the gradient change in ganglion cell density in the myenteric plexus moving from aganglionic to ganglionic colon, and estimated the extent of the TZ to be 1–4 cm in length [25]. In a well-designed study examining the resected specimens of 15 individuals with short-segment HSCR (aganglionic segments 1–10 cm in length), Kapur and Kennedy examined several parameters in attempting to delineate the TZ: partial circumference (>1/8) aganglionosis; hypoganglionosis; hypertrophic submucosal nerves; Glut1-positive submucosal innervation; submucosal hyperganglionosis; and the presence of so-called ectopic ganglia in the lamina propria, tunica muscularis, or serosa. They found that the TZ extent was ~5 cm, while the extent of partial circumference aganglionosis is approximately 1–3 cm [26].

A more recent study from the same institution examines the features of the TZ in an expanded cohort of patients, 20% of whom had long-segment disease. In addition to the histological features described earlier, gangliosclerosis (the presence of dense fibrosis usually seen around the myenteric plexus) and eosinophilic periganglioneuritis were also included as criteria in describing the TZ. This study found that although major histological changes such as myenteric hypoganglionosis extended for ~5 cm proximal to the aganglionic segment, subtler histological changes such as eosinophilic periganglioneuritis could extend for up to 15 cm [27].

The use of intraoperative frozen section evaluation of seromuscular colonic biopsies looking for normal ganglionic distribution is a mandatory component of the PT operation for HSCR. Thick sections should be used (14–16 μm) to avoid an erroneous determination of sample aganglionosis [20]. Once a biopsy showing a normal ganglionic pattern is taken, most authors recommend citing the proximal resection margin at a point several centimeters proximal to this biopsy. In his description of the laparoscopic-assisted PT operation in 2002, Georgeson recommended a 10–15 cm margin of ganglionic colon be resected prior to completing the anastomosis [28]. Schäppi et al. recommend that the proximal resection margin be at least 2–3 cm of colon proximal to the first biopsy showing normal ganglion density [20]. Prior to completion of the colo-anal anastomosis it is also highly recommended that a full circumference donut of the proximal resection margin is evaluated by frozen section to allow detection of abnormal distribution of the ganglion cells.

27.5 Abnormalities of Excitatory and Inhibitory Innervation in the Ganglionic Colon in Hirschsprung’s Disease

HSCR is one of the most common causes of neonatal intestinal obstruction. Functionally, it is characterized by the presence of a spastic contracted aganglionic segment of bowel causing functional obstruction, which manifests as failure to pass meconium, abdominal distension, bilious vomiting, and enterocolitis [2, 14, 29]. The underlying mechanism giving rise to this contracted segment is thought to concern an imbalance of excitatory and inhibitory neurotransmitters in the aganglionic colon [2].

Enteric smooth muscle cell contractility is modulated by opposing actions of inhibitory and excitatory neurotransmitters and neuropeptides released by the Enteric Nervous System (ENS). The chief excitatory neurotransmitter is acetylcholine (ACh), which acts through nicotinic and muscarinic acetylcholine receptors [30]. Key neuropeptides involved in eliciting an excitatory post-junctional response include substance P (SP), neurokinin A and neurokinin B, which exert their actions through neurokinin receptors NK1, NK2, and NK3, respectively [30]. A range of non-adrenergic non-cholinergic substances act as inhibitory neurotransmitters. Neuronal nitric oxide synthase (nNOS) catalyzes the conversion of L-arginine to nitric oxide in nerve cells. Its activity co-localizes with that of NADPH-diaphorase in nitrergic neurons [31]. Nitric oxide is one of the most important neurotransmitters involved in the relaxation of smooth muscle. It acts by inducing slow hyperpolarization in smooth muscle [30]. Vasoactive intestinal peptide (VIP) causes smooth muscle relaxation by inducing hyperpolarization through a mechanism that is, as of yet, poorly understood. Purinergic neurotransmission largely governs induction of the fast inhibitory junction potential in the smooth muscle syncytium through actions of ATP and β-NAD on purinergic receptors [30].

While data concerning the distribution of excitatory and inhibitory enteric motor neurons in the ganglionic bowel in humans with HSCR are sparse, two studies using a murine model of HSCR have reported abnormal patterns of expression. The earlier of the two studies described a deficiency of nitrergic and peptidergic neurons proximal to the aganglionic segment in lethal spotted mice [32]. More recently, Zaitoun et al. described an inverse relationship between neuronal density and expression of nNOS and choline acetyltransferase, a key enzyme in ACh synthesis, in endothelin B-receptor (EDNRB)-deficient mice, with nNOS expression being reduced in aganglionic bowel and relatively elevated in ganglionated colon, while the converse was true of ChAT [33]. A similar relationship has been reported in a small cohort of children with HSCR. When compared to healthy colon, the imbalance in expression of these two neurotransmitters was not merely due to relative deficiency but rather pathological overexpression of ChAT and nNOS in aganglionic and ganglionic bowel, respectively. Such abnormality may account for the segmental or generalized atony seen in the colons of some patients following a PT operation. This same study found that VIP and Substance P were expressed at normal levels in the ganglionic colon in HSCR [34].

27.6 5-Hydroxytryptamine (Serotonin) and Hirschsprung-Associated Enterocolitis

Hirschsprung-associated enterocolitis (HAEC) is the most serious complication of HSCR, and is the leading cause of disease-related mortality. It occurs in 17–50% of patients with HSCR and may occur before or after a pull-through operation [14, 35]. Although a scoring system for HAEC exists, there is no agreed definition of this condition. It is typically described as an inflammatory disease of the colon leading to a spectrum of symptoms ranging from abdominal distension and loose stools to life-threatening toxic megacolon [2, 36]. The etiology and pathogenesis of HAEC are still incompletely understood. It has been proposed that intestinal barrier dysfunction, abnormal innate immunity, and the presence of a disturbed microbiome are all potential contributors to its etiology [14]. However, given that the primary abnormality in HSCR is the absence of enteric ganglia in the distal colon, it follows that the enteric nervous system may have a role in the pathogenesis.

5-Hydroxytryptamine (5-HT), commonly known as serotonin, is a major neuroendocrine signaling molecule. While the gut is the single largest reservoir of 5-HT, the wide range of its functions therein have only been elucidated relatively recently. Most enteric serotonin is stored in the mucosa in the enterochromaffin (EC) cells. Approximately 1–5% of enteric serotonin is stored in the serotonergic enteric nerves, where it acts as a neurotransmitter [37, 38]. The rate limiting step in the synthesis of 5-HT is the conversion of L-tryptophan to 5-hydroxytryptophan by tryptophan hydroxylase (TPH). The conversion of 5-hydroxytryptophan to 5-HT then occurs rapidly through the actions of L-amino acid decarboxylase [39]. However, the synthesis pathway of enteric 5-HT differs depending on whether 5-HT is being synthesized in EC cells, where the TPH1 isozyme of TPH predominates, or in serotonergic neurons, where TPH2 predominates [37, 39].

5-HT has many roles, including activation of intrinsic reflexes such as peristalsis, vasodilation, and secretion. When released from EC cells, mucosal 5-HT has been demonstrated to promote inflammation – an activity that is counterbalanced by its re-uptake via the serotonin transporter (SERT) [37, 40]. Abnormal mucosal 5-HT activity has been demonstrated in inflammatory and functional bowel disorders such as ulcerative colitis and irritable bowel syndrome [41]. Conversely, neuronal 5-HT is anti-inflammatory and neuroprotective, an activity that has obvious importance in the setting of inflammation and enterocolitis, as neuronal damage can frequently result [40]. It has previously been reported that populations of mucosal enterochromaffin cells are deficient in the ganglionic bowel of children with HSCR who have previously had HAEC. It is unclear if this is a facilitator or an effect of HAEC [42].

Serotonergic nerves have previously been shown to make extensive synapses with myenteric ICC – the pacemaker cells of the bowel – as well as nitrergic neurons. This indicates a functional role for serotonergic neurons in modulating nitrergic neurotransmission and pacemaker activity [43]. The intimate spatial arrangement of serotonergic nerves with nNOS-positive nerves is also important as it is thought that, through the intercession of descending inhibitory serotonergic neuronal activity, the release of nitric oxide from nNOS-positive neurons suppresses excitatory cholinergic activity and limits the rate at which high-amplitude peristaltic activity is propagated in mice [44]. Using TPH2 as a surrogate marker for neuronal 5-HT, it has been shown in children with HSCR that serotonergic nerves are reduced in density in the ganglionic colon of children who had experienced HAEC prior to their PT procedure, with the exception of children who had undergone a diverting/levelling colostomy in the neonatal period. The finding of reduced serotonergic neuron density in the aganglionic, transition zone, and ganglionic bowel in these children with HSCR complicated by HAEC is probably reflective of enterocolitis-mediated neuronal damage. The vicious circle of enterocolitis and loss of neuroprotective serotonergic neurons may thus occur [45].

27.7 The S.I.P Syncytium in Hirschsprung’s Disease

The movement of intestinal contents in the gut requires coordinated concentric contractions of the smooth muscle layers of the gut wall. The orderly excitatory stimulation of specific smooth muscle cells (SMC) to contract and inhibitory stimulation of others to relax to achieve these peristaltic contractions is conducted by the ENS [46, 47]. Central to the process of peristalsis is the propagation of slow wave electrical activity through the electrical syncytium formed by the SMC, ICC and Platelet-Derived Growth Factor Receptor-α-positive (PDGFRα+) cells, known as the SIP syncytium [48]. ICC have a key role as electrical pacemaker cells in the gut. They are coupled loosely to SMC by gap junction proteins. This loose coupling limits the likelihood of electrical waves dissipating across a large smooth muscle area [46]. The systole-diastole nature of electrical slow wave activity leads to ordered periods of smooth muscle contraction and relaxation, which are essential to propel intestinal contents along the alimentary tract for digestion.

ICC act as intermediaries between enteric motor neurons and SMC, modulating inhibitory and excitatory signals from the enteric nervous system [49]. Abnormalities of ICC distribution have been observed in a range of gastrointestinal disorders including inflammatory bowel disease, idiopathic slow-transit constipation, and necrotizing enterocolitis [50]. At microscopy, these cells can be identified by their immunopositivity for c-kit and anoctamin-1. It has long been recognized that ICC networks are markedly deficient in the aganglionic colon in HSCR. More recently, several studies have demonstrated that ICC networks are less dense in the ganglionic bowel in HSCR [51, 52].

PDGFRα+ cells are fibroblast-like cells that share morphological similarities with ICC but are c-kit negative. They are arranged in discrete networks in the myenteric plexus but also in the tunica muscularis where they form part of the SIP syncytium. They are thought to be involved in smooth muscle relaxation in the bowel and express the small conductance calcium-activated potassium (SK3) channel, which, when activated, facilitates purine-mediated smooth muscle relaxation. The density of both PDGFRα+ cells and SK3 channel expression have both been shown to be reduced in the aganglionic colon in HSCR and, in a proportion of patients, in the ganglionic colon [53, 54].

In addition to these findings indicating abnormalities in the density of cells needed for normal gut motility in the ganglionic colon in HSCR, abnormalities in the expression of functional cellular components in the SIP syncytium have also been described. Gap junctions are channels that couple cells electrically and chemically. They consist of two hemichannels composed of connexin proteins. Several connexins (Cx26, Cx36, Cx43) are involved in mediating the intercellular signaling responsible for colonic motility. Despite being expressed across all the cell types in the SIP syncytium, many of which are reduced in density in the ganglionic bowel of children with HSCR, only Cx26 has been found to be underexpressed in the SIP in HSCR ganglionic colon [55, 56]. Reduced expression of Kv7 channels, which play a role in the membrane excitability of ICCs, and Nav1.9 channels, which play a role in the repolarization cycle of the action potential in specific nerve populations in the colon, have also been described [57, 58].

27.8 Colonic Mucosal Barrier Integrity in Hirschsprung’s Disease

Hirschsprung-associated enterocolitis (HAEC) is the most common life-threatening surgical emergency that can affect children with HSCR. Its diagnosis is primarily based on clinical grounds, with abdominal distension, explosive loose stools, fever, and vomiting as some of the key features [13, 14]. A scoring system with 83% sensitivity and 98.7% specificity for diagnosing HAEC has been devised to aid in clinician diagnosis [59]. Its etiology is uncertain and is thought to involve disturbance of the intestinal microbiome, impaired mucosal barrier function, abnormal innate immune response and bacterial translocation, and it affects both the aganglionic and the ganglionic colon [14, 60].

The intestinal microbiome in HSCR has been demonstrated to be less diverse than in healthy individuals, with larger proportions of pathogenic organisms such as Enterobacter and Bacilli [61]. In children with HSCR, Protobacteria and Bacteroidetes are more prevalent in the colons of children with a history of HAEC when compared to those without HAEC. The population of fungal organisms in fecal specimens from children with HAEC has also been shown to be less diverse with an expansion of Candida species [62]. Patients who have previously been treated for HAEC have been shown to have a microbiome more similar to that of children with active enterocolitis than those with HSCR who have never had HAEC [63]. In both cases, it is unclear if these findings are underlying contributors to the development of HAEC or as a result of HAEC. Further prospective studies should be able to elucidate this.

Intestinal mucosal barrier dysfunction is likely be play a strong role in the etiology of HAEC. Numerous components required for normal barrier function have been found to be deficient in the aganglionic and the ganglionic colon of children with HSCR. The potassium channel, TREK-1, is a stretch-mediated channel that is involved in mucosal barrier integrity, as well as mediating the response of gastrointestinal smooth muscle to neural stimulation and mechanical stretch. Its expression is reduced in both aganglionic and ganglionic colon in HSCR compared to controls [64].

Goblet cells in the colon produce a mucus layer that forms a bactericidal and bacteriostatic barrier against bacterial invasion. Nakamura et al. found that populations of goblet cells, as well as a host of factors required for normal goblet cell differentiation and maturation are deficient in both ganglionic and aganglionic colon in HSCR [65]. An abnormal immune response may also partly contribute to the susceptibility to enterocolitis seen both before and after PT surgery. The pro-inflammatory cytokine IL-36γ is overexpressed in the colons of children with HSCR compared to healthy controls [66].

27.9 Conclusions and Future Directions

The proportion of children requiring medical treatment for ongoing problems with constipation, soiling, and recurrent enterocolitis despite having a technically adequate PT operation for HSCR is not trivial. Key to avoiding this problem is the avoidance of retained segments of aganglionic or transition zone colon. This can be best avoided by resecting an adequate margin above the most distal normal intraoperative colonic biopsy and, where possible, doing a frozen section of the proximal resection margin to ensure the absence of partial circumference aganglionosis.

Despite having normal neuroanatomical architecture, the ganglionic colon in HSCR appears to be abnormal in a number of ways. Specifically, there is an imbalance of neurotransmitters, with excessive nitrergic innervation and deficient cholinergic innervation. Important modulators of neurotransmission in the enteric nervous system, such as neuronal serotonin, ICC and PDGFRα+ cells all appear to be deficient in the ganglionic colon in at least some patients with HSCR. The intestinal microbiome is also disrupted in children with HSCR, with those who have experienced HAEC demonstrating the least diversity. It is unclear if this is a cause or an effect as it applies to susceptibility toward HAEC.

Future studies will aim to elucidate if many of the findings described above are primarily abnormalities of the colon or occur as a result of the distension associated with congenital megacolon seen in HSCR. Understanding the basis for these abnormalities is important because even if aganglionosis can be reversed in the future (e.g., stem cell-based therapies), it may not yield a satisfactory result for the patient regarding obstructive symptoms or the occurrence of enterocolitis.