Motility Disorders
Amy E. Noffsinger
Normal gastrointestinal motility depends on intact neuromuscular function including both intrinsic and extrinsic innervation, interstitial cells of Cajal (ICCs), and the activity of smooth and striated muscles. Extrinsic control of peristalsis includes the sympathetic (thoracolumbar) and parasympathetic (vagal) innervation in the ganglionated plexuses. Intrinsic control includes the enteric nervous system (ENS), muscle cells, and ICC, with the latter serving as both pacemaker cells and as intermediaries of enteric innervation (1,2). The ENS is estimated to contain approximately 80 to 100 million neurons, and it has been referred to as the “second brain” because of its ability to function in the absence of neural input from the central nervous system (CNS) (3). Analysis of animal models (mostly transgenic and knockout animals) has been critical to our understanding of the genes required for ENS development (Table 10.1) and has also been critical to furthering our understanding of developmental or acquired diseases that result in crippling motility disorders in children and adults (Table 10.2) (4,5,6,7).
▪ DEVELOPMENT OF THE ENTERIC NERVOUS SYSTEM
The neural crest is a transient, migratory population of multipotent cells that emerges from the dorsal aspect of the neural tube during early development and gives rise to a remarkable variety of differentiated cell types. These neural crest cells (NCCs) have the capacity for long-range migration, and the journey they take across the dynamic landscape of the developing embryo exposes them to myriad signals from diverse surrounding microenvironments, which vary by developmental stage and site. The paths of the migrating NCCs are directed by molecular guidance clues mediated by the binding of external ligands with receptors of the NCC surface. The differentiation, morphology, and patterning of NCC derivatives and tissues with which they interact are also influenced by local interactions (8).
The ENS develops from the coordinated migration, expansion, and differentiation of NCCs from three axial levels of the neural crest (vagal, rostral-truncal, and lumbosacral) (9). NCCs enter the foregut and hindgut to become enteric neural crest-derived cells (ENCCs) (3). ENCCs migrate in a rostral-to-caudal direction, colonizing the gut and generating the majority of the ENS, giving rise to neurons, ganglia, and glia. The ENCCs advance as multicellular strands along the entire length of the developing gut. The process begins with the production of a sufficient pool of pre-enteric progenitor NCCs that emigrate from the neural tube to the foregut. A subpopulation of ENCCs crosses from the midgut to the hindgut via the mesentery during a short developmental period in which those regions are transiently juxtaposed. This transmesenteric migration contributes considerably to the colonization of the distal intestine by ENCCs, and interference with this shortcut delays hindgut innervation. The process requires glial cell line-derived neurotrophic factor (GDNF) signaling provided by hindgut mesenchyme and vascularderived signals allowing the ENCCs to sense environmental cues and adapt to changes in their migratory fields. Impaired transmesenteric migration of ENCCs may contribute to the pathogenesis of some forms of Hirschsprung disease (HD) (see below) (10). Sacral NCCs also contribute to a subset of enteric ganglia, neurons, and glia in the hindgut, colonizing the colon in a caudal-to-rostral wave. NCCs migrate to the gut at a stage when they are morphologically indistinguishable from surrounding mesenchymal cells.
Vagal nerve trunks can be found in the upper esophagus by the 5th fetal week. A number of factors serve as guidance clues for the axons from the cell bodies in the nodose ganglia and dorsal motor nucleus to find their enteric targets (11). Nerve trunks extend on the outer surface of the gut wall and neuroblasts are found along the gastric cardia by the 6th week, the cephalic limb of the midgut by the 7th week, and the entire gut up to the distal half of the colon and rectum by the 8th week. By the 12th week, they have migrated
as far as the rectum. The myenteric plexus forms just outside the circular muscle coat. Then, the longitudinal muscle coat develops. Neuroblasts migrate from the myenteric plexus to form the submucosal plexus; this is completed during the third and fourth fetal months (12). Following NCC migration, the cells proliferate and the neurons mature, developing processes and synapses that allow for communication within ganglia, between ganglia, and between the ganglia and smooth muscle cells (Fig. 10.1). This process is completed in the cranial bowel segments first and finishes more distally. Interruption of this process at different stages produces various myenteric plexus abnormalities ranging from agenesis to incomplete maturation. Interruption of the orderly cranial-caudal migration of NCCs explains the variable lengths of aganglionic segments in HD. Incomplete caudal-rostral migration may explain the zonal form of HD (13).
as far as the rectum. The myenteric plexus forms just outside the circular muscle coat. Then, the longitudinal muscle coat develops. Neuroblasts migrate from the myenteric plexus to form the submucosal plexus; this is completed during the third and fourth fetal months (12). Following NCC migration, the cells proliferate and the neurons mature, developing processes and synapses that allow for communication within ganglia, between ganglia, and between the ganglia and smooth muscle cells (Fig. 10.1). This process is completed in the cranial bowel segments first and finishes more distally. Interruption of this process at different stages produces various myenteric plexus abnormalities ranging from agenesis to incomplete maturation. Interruption of the orderly cranial-caudal migration of NCCs explains the variable lengths of aganglionic segments in HD. Incomplete caudal-rostral migration may explain the zonal form of HD (13).
TABLE 10.1 INTESTINAL PHENOTYPE IN DISRUPTED GENES NECESSARY FOR ENS DEVELOPMENT | ||||||||||||||||||||||||||||
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TABLE 10.2 PATHOLOGY OF ENTERIC NEUROMUSCULAR DISEASE | ||||||||||||||||||||||||||
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Components of the extracellular matrix play a major role in specifying where NCCs migrate. Precursors arriving in the gut are multipotential, and the enteric microenvironment to which they migrate dictates their phenotypic differentiation. Enteric neuronal migration and differentiation involves complex interactions of lineage-determined elements, including transcription factors, tyrosine kinase receptors and their ligands, the extracellular matrix, and specific adhesion molecules (Table 10.3) (3,4,14,15). Among the more important are the tyrosine kinase receptors (Trk-A, Trk-B, and Trk-C), ligands including the neurotrophin family of growth factors
that promote neuronal differentiation, growth, and survival (16,17). Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and other molecules, decrease neuronal death evoked by a number of agents ranging from mechanical, chemical, or ischemic injury. The nerves in HD fail to express the Trk-C tyrosine receptor and its ligand neurotrophin, supporting the notion that neurotrophic factors critical for cellular survival and differentiation may play a role in the pathogenesis of HD (18).
that promote neuronal differentiation, growth, and survival (16,17). Neurotrophins, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and other molecules, decrease neuronal death evoked by a number of agents ranging from mechanical, chemical, or ischemic injury. The nerves in HD fail to express the Trk-C tyrosine receptor and its ligand neurotrophin, supporting the notion that neurotrophic factors critical for cellular survival and differentiation may play a role in the pathogenesis of HD (18).
 FIG. 10.1 Fetal plexus stained with antibody to neurofilament protein. A: A 13-week fetus with only rare cells immunoreactive for neurofilament protein. Note the lack of dendritic processes. B: A 15-week fetus showing further development of the neuroblastic cells and the beginning of the appearance of dendritic processes. C: A 17-week fetus with better developed processes and the beginning appearance of submucosal plexuses with fibers having neural extensions. |
Glial cell line-derived nerve growth factor (GDNF) and neurturin (NTN) are important in NCCS migration into and along the small and large intestines and the esophagus, a process that is RET dependent (19). To activate RET, the neurotrophins, including GDNF, form a tripartite complex with RET and a member of four extracellular GPI-linked alpha receptors (GFR&agr; 1-4). Endothelin-3, the ligand for endothelin receptor B (EDNRB), inhibits the chemoattraction of NCC to GDNF (20). GDNF is mitogenic, neurotrophic, and chemoattractive to NCCs in the embryonic colon. GDNF attracts ENCCs to the gut and prevents them from straying away once they get there (21). It is thought that in RET and GDNF deficiency, the ENS cell precursors undergo apoptotic cell death prior to or during their invasion of the foregut mesenchyme (22). A deficiency of NTN or its corresponding receptor complex GFR&agr; also results in abnormalities of the ENS, which are less severe, such as reduced myenteric plexus innervation and abnormal gut motility. GDNF and NTN’s ability to promote the survival, proliferation, and differentiation of multipotent ENS progenitors is stage specific. Later in development, other neurotrophic factors support enteric development.
TABLE 10.3 KEY MOLECULES THAT AFFECT ENS DEVELOPMENT AND STRUCTURE | ||||||||||||||||||||||||||||||||||
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During ENS development, ENCCS self-renew and begin expressing markers of both neural and glial lineages as they populate the intestine. Also as part of their normal development and migration, some of the NCCs undergo apoptosis. Failure for this to occur can result in hyperganglionosis (23).
▪ NORMAL NEUROMUSCULAR STRUCTURE
The normal neuromusculature of the gut consists of its innervation (various types of neurons), ganglia, glia, ICC, neuromuscular junctions, and smooth muscle cells. There are also striated muscles in the esophagus and anus. Abnormalities in any of these may result in abnormal motility of the affected site. Often, more than one structure may be abnormal.
Innervation
The ENS is the most complex portion of the peripheral nervous system. It consists of nearly one hundred million autonomously functioning neurons distributed in the myenteric and submucosal plexuses. Myenteric nerve trunks consist
of both extrinsic (sympathetic and parasympathetic) and intrinsic neurons. The ENS contains three ganglionated plexuses in the gut wall: the myenteric (Auerbach plexus located between the longitudinal and circular muscle layers of the muscularis propria) and the submucosal (Meissner) plexus found in the submucosa). A third lesser known plexus, which derives from extrinsic nerves, lies in the inner quarter of the circular muscle coat adjacent to the submucosa and is rich in ICCs (24). These plexuses extend uninterrupted from the esophagus to the anus, innervating the mucosa, muscle layers, and blood vessels. The submucosal plexus mainly controls epithelial secretion whereas the myenteric plexus controls gut motility. The plexuses are synaptically connected in reflex circuits that are modulated by the CNS. They contribute to gastrointestinal neural control of at least four physiologic effector systems: the visceral smooth muscle responsible for motility and sphincteric functions, the mucosa responsible for ion transport including gastric acid secretion and intestinal fluid and electrolyte homeostasis, the immune cells responsible for mucosal immunity (including mast cell and perhaps macrophages), and mucosal blood flow. Functionally important mast cell mediators with neural actions in the human ENS are histamine, proteases, cytokines, chemokines, and serotonin receptors. Mast cells synthesize, store, and release NGF and are in direct contact with nerve fibers suggesting that they are essential for nerve growth and repair. Mediator release from mast cells is modulated by neuropeptides released from enteric and visceral afferent nerves (25). Macrophages are also often associated with intrinsic and extrinsic innervation. Sometimes they are in tight registration with dendrites and axons of myenteric neurons and varicosities along the length of the sympathetic axons (26).
of both extrinsic (sympathetic and parasympathetic) and intrinsic neurons. The ENS contains three ganglionated plexuses in the gut wall: the myenteric (Auerbach plexus located between the longitudinal and circular muscle layers of the muscularis propria) and the submucosal (Meissner) plexus found in the submucosa). A third lesser known plexus, which derives from extrinsic nerves, lies in the inner quarter of the circular muscle coat adjacent to the submucosa and is rich in ICCs (24). These plexuses extend uninterrupted from the esophagus to the anus, innervating the mucosa, muscle layers, and blood vessels. The submucosal plexus mainly controls epithelial secretion whereas the myenteric plexus controls gut motility. The plexuses are synaptically connected in reflex circuits that are modulated by the CNS. They contribute to gastrointestinal neural control of at least four physiologic effector systems: the visceral smooth muscle responsible for motility and sphincteric functions, the mucosa responsible for ion transport including gastric acid secretion and intestinal fluid and electrolyte homeostasis, the immune cells responsible for mucosal immunity (including mast cell and perhaps macrophages), and mucosal blood flow. Functionally important mast cell mediators with neural actions in the human ENS are histamine, proteases, cytokines, chemokines, and serotonin receptors. Mast cells synthesize, store, and release NGF and are in direct contact with nerve fibers suggesting that they are essential for nerve growth and repair. Mediator release from mast cells is modulated by neuropeptides released from enteric and visceral afferent nerves (25). Macrophages are also often associated with intrinsic and extrinsic innervation. Sometimes they are in tight registration with dendrites and axons of myenteric neurons and varicosities along the length of the sympathetic axons (26).
The structure of the myenteric plexus is not uniform throughout the gut. In the esophagus, the myenteric plexus is sparse with few ganglia. In the lower 5 cm, the esophagus has thick bundles of nerve fibers that cross the gastroesophageal junction and radiate to the periphery of the stomach through several branches. The plexus in the stomach is uniform with intermediate and intrafascicular ganglia. A thick nerve bundle encircles the pylorus and gives off branches on either side to the antrum and the duodenum. In the duodenum and proximal jejunum, the plexus is regular, but in the mid small intestine, the longitudinal interganglionic fascicles are more prominent than the circumferential fascicles. Distally, this pattern reverses. A thick nerve bundle encircles the ileocecal junction. In the cecum and proximal colon, the plexus is sparse with large intermediate and intrafascicular ganglia. In the midcolon to the rectum, the plexus is dense with parafascicular and intrafascicular ganglia (27).
The plexuses contain neurons, ganglion cells, neuronal processes, and a neuropil formed by neuronal processes (some of extrinsic origin and others issued by intrinsic neurons) and glial processes. Since neural fibers from many different origins exist within the neuropil, the neuronal organization is extremely complex with controls coming from both the intramural and extramural ganglia. Functionally, neurons of the ENS fall into five types: (a) motor neurons, efferent neurons, or effector neurons acting to control smooth muscle tone in the wall of the gut; (b) vasomotor neurons, which control vascular muscle tone; (c) secretory neurons, other effector neurons regulating exocrine and endocrine secretion; (d) sensory neurons, which carry sensory information to the CNS; and (e) interneurons that provide communication between different neurons and the gut wall (Fig. 10.2). All the neuronal types intermingle in the myenteric and submucosal ganglia and are integrated in a continuous overlapping network along the gut. Neurons of the ENS may also be classified by their transmitters. Utilizing this classification, there are at least five types of neurons: (a) cholinergic (acetylcholine containing), (b) adrenergic (norepinephrine containing), (c) GABA-ergic (&ggr;-aminobutyric acid containing), (d) peptidergic (peptide containing), and (e) nitrergic, which are nonadrenergic noncholinergic (NANC) peptidergic nerves. Nitrergic nerves constitute 25% to 40% of the total myenteric neuronal population (28).
 FIG. 10.2 Diagram showing the interactions of sensory neurons and motor neurons with interneurons, epithelial cells, blood vessels, and portions of the muscularis mucosae. |
Normal gut function requires a balance between the release of excitatory neurotransmitters and inhibitory transmitters. The peristaltic reflex is a response to mechanical or chemical stimulation of the gut wall that produces a contraction above and a relaxation below the point of stimulation. As gastrointestinal contents move along the gut, mucosal enterochromaffin cells are stimulated by the luminal content and release 5-hydroxytryptamine (5-HT), which acts on 5-HT3 and 5-HT4 receptors on the mucosal terminals of intrinsic primary afferent neurons (IPANs) whose cell bodies reside in submucosal and myenteric ganglia (29). In the myenteric plexus, IPANs synapse with other IPANs to form a feed-forward network; they synapse directly with motor neurons and with orally and anally projecting interneurons (30). IPANs release acetylcholine (Ach) and substance P as excitatory neurotransmitters. IPANs are not the only neurons
capable of detecting mechanical stimulation of the gut wall. There are also multifunctional neurons that can act as mechanosensors as well as interneurons. These neurons respond to gut wall stretching or local mechanical stimuli. IPANs and multifunctional neurons connect with interneurons, which synapse with other interneurons to form long pathway circuits that mediate propulsive activity. Interneurons synapse with excitatory motor neurons in orally directed pathways and inhibitory motor neurons in anally directed pathways. Orally directed interneurons use Ach as their primary neurotransmitter, whereas interneurons in descending pathways use Ach and adenosine triphosphate (ATP) as their primary excitatory neurotransmitters. Other excitatory neurotransmitters include tachykinins such as substance P and neurokinin A (31). Inhibitory neurons release nitric oxide (NO), VIP, and ATP or &bgr;-nicotinamide adenine dinucleotide (32). Neural NO is produced by neural nitric oxide synthase (nNOS) (33). NO is also produced by the smooth muscle cells (34). VIP and NO are cotransmitters in the NANC nerve-mediated smooth muscle relaxation and part of VIP actions may be mediated by NO (Fig. 10.3) (35). The contraction on the oral side and relaxation on the anal side of a stimulus produce the pressure gradient needed to propel stool along the length of the gut.
capable of detecting mechanical stimulation of the gut wall. There are also multifunctional neurons that can act as mechanosensors as well as interneurons. These neurons respond to gut wall stretching or local mechanical stimuli. IPANs and multifunctional neurons connect with interneurons, which synapse with other interneurons to form long pathway circuits that mediate propulsive activity. Interneurons synapse with excitatory motor neurons in orally directed pathways and inhibitory motor neurons in anally directed pathways. Orally directed interneurons use Ach as their primary neurotransmitter, whereas interneurons in descending pathways use Ach and adenosine triphosphate (ATP) as their primary excitatory neurotransmitters. Other excitatory neurotransmitters include tachykinins such as substance P and neurokinin A (31). Inhibitory neurons release nitric oxide (NO), VIP, and ATP or &bgr;-nicotinamide adenine dinucleotide (32). Neural NO is produced by neural nitric oxide synthase (nNOS) (33). NO is also produced by the smooth muscle cells (34). VIP and NO are cotransmitters in the NANC nerve-mediated smooth muscle relaxation and part of VIP actions may be mediated by NO (Fig. 10.3) (35). The contraction on the oral side and relaxation on the anal side of a stimulus produce the pressure gradient needed to propel stool along the length of the gut.
The innervation of the musculature is particularly dense at the level of the sphincters. The anal sphincter has the densest adrenergic innervation found in the GI tract. Most of its fibers originate from the superior mesenteric ganglion, but some derive from ganglionic neurons of the sacral sympathetic chain. Adrenergic fibers are also plentiful in the sphincter of Oddi. Nerve fibers containing VIP are numerous in the musculature of the gastroesophageal junction, the pylorus, and the sphincter of Oddi. The pyloric sphincter has the highest nNOS levels (34). The internal anal sphincter also contains high levels nNOS (34). Nitrergic neurotransmission may be abnormal in a variety of diseases, and it may be disrupted by deficient neural migration, differentiation, growth, neurodegeneration, inflammatory damage, or defective regulation of the enzyme nNOS (36).
 FIG. 10.3 Nitric oxide (NO) mediates vascular relaxation. NO is produced in endothelial cells by the action of calcium-dependent nitric oxide synthase (NOS). Mediators such as acetylcholine and bradykinin stimulate its production. NO acts on smooth muscle cells through a process involving conversion of GTP to cyclic GMP, affecting relaxation. NO released into the interstitium may produce damaging free radicals after interacting with superoxide molecules. |
The normal mature colon contains approximately 7 ganglion cells/mm of myenteric plexus, the jejunum 3.6/mm, and the ileum 4.3/mm in 3 &mgr;m sections (37). Ganglion cells lie approximately 1 mm apart; they may occur in clusters of one to five cells in normal adults (38). At birth, normal enteric ganglia contain both mature and immature neurons. Normal neonates often have plentiful, prominent ganglion cells, but they appear small when they are immature (12). Premature infants have more immature neurons than do term infants. Mature neurons are larger than immature neurons and they have a distinct cell membrane, a vesicular nucleus, and a large amount of basophilic cytoplasm. Immature neurons are small cells with dark nuclei, clumped chromatin, and scant cytoplasm (Fig. 10.4). A variety of stains can be used to highlight the various components of the ENS (Table 10.4).
Glia
Enteric glial cells (EGCs) represent an extensive but relatively poorly described cell population in the GI tract. Glial cells are the predominant cell type in the ENS and the dense synaptic neuropil within enteric ganglia is solely composed of neurons and enteric glia. Major populations of glia exist in both the myenteric and submucosal plexuses (39). They are also found outside the ENS in the circular muscle (40). EGCs share anatomical, structural, and electrophysiological properties with astrocytes (41). They express high levels of glial fibrillary acidic protein (GFAP) and S100.
 FIG. 10.4 Fetal gut stained with hematoxylin-eosin. Note the presence of immature neuroblastlike cells without clearly identifiable ganglia (arrows). |
TABLE 10.4 MARKERS USEFUL IN EVALUATING INTESTINAL MOTILITY DISORDERS | ||||||||||||||||||||||||||||||||||||||||||||||||||
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Different populations of EGCs probably represent unique classes of glial cells serving different functions. As noted, enteric glia are involved in almost every gut function including motility, mucosal secretion, and host defenses. Subepithelial glial cells seem to have a trophic and supporting relationship with intestinal epithelial cells. Glia in the enteric ganglia are activated by synaptic stimulation suggesting an active role in synaptic transmission. They also have a neuroprotective role controlling neuronal maintenance, survival, and function (42).
Interstitial Cells of Cajal
C kit-positive precursors of ICCs first appear in the stomach at week 7 forming a layer in the outer gastric wall around myenteric plexus elements. They develop from a mesenchymal precursor cell that is also a precursor for smooth muscle cells (43). Between weeks 9 and 11, some differentiate into mature ICCs. ICCs in the myenteric plexus develop first, followed by those within the muscle and within connective tissue septa enveloping muscle bundles. All of the various types of ICC have developed by the 4th gestational month (44).
C kit, insulin, and insulinlike growth factor are important for ICC development and maintenance. C kit is a tyrosine kinase transmembrane receptor whose main ligand is stem cell factor (SCF). The interaction of SCF and kit is fundamental for ICC development, survival, and maintenance (45). C kit knockout mice show gut dilation and absent peristalsis, evidence of the critical role that these cells play in regulating gut motility.
Although ICCs form only 5% of the cells in the musculature of the gut, they play a critical role in regulating smooth muscle function and GI motility in cooperation with the ENS (46,47). ICCs are also electrically coupled to the smooth muscle cells (48) and form a network within and between muscle bundles. They generate the electrical slowwave pacemaker signal to smooth muscle, set smooth muscle membrane potentials, mediate nitrergic and cholinergic neurotransmission, and are mechanoreceptors (49,50).
C kit (CD117) is the traditional marker for ICCs, but they may also express CD34, CD44, vimentin, platelet-derived growth factor receptor, Wilms tumor gene protein WT-1, and calretinin (51). However, it should be noted that CD34 and vimentin are also expressed by fibroblasts. There may be two different types of ICCs, the traditional one that is labeled by c kit and a second type that is labeled by PDGFR&agr; (52) perhaps explaining the presence of PDGFR&agr;-positive gastrointestinal stromal tumors (see Chapter 19). It is known that not all ICCs stain with antibodies to c kit (49).
ICCs are found from the esophagus to the anal sphincter, but the distribution in their types differs in different areas. They are most frequent in the esophageal side of the lower esophageal sphincter (LES) but not on the gastric side (53). In the esophagus, gastric cardia, and fundus, they are present in the muscularis propria but not in the myenteric plexus or submucosa. ICCs are more numerous in the corpus and antrum than in the fundus. They occur within the smooth muscle and at the submucosal border where they appear as bipolar cells. They do not concentrate around the myenteric plexus as they do in the intestines. They are more prominent in the small intestine than in the colon, but their arrangements are similar (54). The jejunum contains 12 to 20 (mean 16.2)/mm length of intermyenteric plexus (55). They are most prominent around and between the myenteric plexus ganglia and are organized bundles of up to five cells with overlapping processes (24). These bundles extend into the adjacent portions of the circular and longitudinal muscles and the interlamellar septa (56). The cells are multipolar and form a reticular network. Submucosal ICCs are present in the small and large intestines.
ICCs are affected in various gastrointestinal conditions, some associated with inflammation (Table 10.5) (48,57,58,59). These diseases can cause a decrease in ICC number and/or disrupt ICC structure or function ultimately affecting GI motility. It is currently unknown, however, how much loss of ICCs organs can tolerate. In addition, the number and
volume of ICC networks in the stomach and colon declines with age (60). ICCs are able to recover and repopulate following removal of an injurious process (48). ICCs also have the ability to redifferentiate into other cell types following injury. These redifferentiated cells retain their c kit immunoreactivity (48) although they resemble smooth muscle or fibroblasts (61).
volume of ICC networks in the stomach and colon declines with age (60). ICCs are able to recover and repopulate following removal of an injurious process (48). ICCs also have the ability to redifferentiate into other cell types following injury. These redifferentiated cells retain their c kit immunoreactivity (48) although they resemble smooth muscle or fibroblasts (61).
TABLE 10.5 DISORDERS INVOLVING ABNORMALITIES IN INTERSTITIAL CELLS OF CAJAL | ||||||||||||||||||||
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Muscles
Intestinal smooth muscle layers include the muscularis mucosae and the muscularis propria. The latter has an inner circular and an outer longitudinal layer that extends from the upper esophagus to the anal canal (Fig. 10.5). The only exception to this occurs in the stomach, where three muscle layers are present. The inner circular layer is organized in lamellae separated by connective tissue septa. The septa are in continuity with the connective tissue space between the inner and outer layers of the muscularis propria in the area of the myenteric plexus. In the cecum and in parts of the colon, the longitudinal muscle is attenuated except in the regions where it forms thick cords, that is, the taeniae coli. At the junctions between adjacent organs, the muscular coat rearranges to form sphincters including the pharyngoesophageal, esophagogastric, pyloric, ileocecal, and anal sphincters. The function of these sphincters is based on physiologic and pharmacologic characteristics of the musculature and on their innervation.
Circular muscle from the esophagus to the internal anal sphincter behaves as an electrical syncytium resulting from nexuses between the plasma membranes of contiguous muscle fibers. These nexuses function as intracellular pathways for excitation conduction between adjacent cells. Even in the absence of neural influences, these syncytial properties allow three-dimensional spread of excitation (39).
 FIG. 10.5 Hematoxylin-eosin-stained section demonstrating the circular and longitudinal smooth muscle layers of the muscularis propria.This layer is present throughout the gastrointestinal tract. |
The fibers of the muscularis mucosae and muscularis propria express smooth muscle actin, desmin, and smoothelin (54). The latter is a key component of the contractile apparatus of fully differentiated contractile smooth muscle cells. Smoothelins are actin-binding proteins that are expressed abundantly in visceral (smoothelin-A) and vascular (smoothelin-B) smooth muscle. They are essential for functional contractility of intestinal motility. Absence of the protein leads to a clinical picture resembling intestinal pseudo-obstruction (62). Of note, the inner layer of the muscularis propria of the normal ileum and occasionally the normal colon often fails to express smooth muscle actin. The reason for this lack of expression is unknown and is of no clinical consequence.
The cells of the muscle layers contain numerous receptors, allowing them to respond to neural signals, as well as other stimulatory and inhibitory signals during the digestive process. Contraction of the circular layer constricts the lumen; contraction of the longitudinal layer shortens the digestive tube. When the bowel becomes obstructed or the intestinal lumen distends on a persistent basis, the muscle increases in volume through both hypertrophy and hyperplasia. Smooth muscle hyperplasia also follows myenteric ablation.
▪ GENERAL FEATURES OF MOTILITY DISORDERS
Motility disturbances constitute a complex array of clinical and pathological disorders that are histopathologically classified according to the “London Classification” (63) (Table 10.6). This classification includes disorders associated with histologic abnormalities in nerves (enteric visceral neuropathies) and smooth muscle cells (enteric myopathies) or alterations in ICC (enteric mesenchymopathies) or from a combination of alterations in several cell types (64). Enteric
neuropathies can be subdivided into two major entities: degenerative neuropathies without any inflammatory component and inflammatory neuropathies referred to as myenteric ganglionitis. Intestinal neuropathies appear to be more common than intestinal myopathies. Defects in ICCs are more common than previously recognized.
neuropathies can be subdivided into two major entities: degenerative neuropathies without any inflammatory component and inflammatory neuropathies referred to as myenteric ganglionitis. Intestinal neuropathies appear to be more common than intestinal myopathies. Defects in ICCs are more common than previously recognized.
TABLE 10.6 LONDON CLASSIFICATION OF GASTROINTESTINAL NEUROMUSCULAR PATHOLOGY | |
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Most patients with congenital myenteric plexus abnormalities fall into one of six categories: (a) aganglionosis, (b) hypoganglionosis, (c) hyperganglionosis, (d) ganglionic immaturity, (e) alterations in the ICC, and (f) poorly classified abnormalities. Pathogenesis of ENS developmental disorders results from genetic defects, failed neural crest migration or differentiation, anoxia, or inflammation. Developmental neural diseases occur alone or coexist with systemic disorders such as congenital abnormalities or neurofibromatosis.
Dysganglionoses are a heterogeneous group of ENS anomalies including HD, intestinal neuronal dysplasia (IND), internal anal sphincter neurogenic achalasia, and hypoganglionoses. Unfortunately, the pathologic features are not always readily classifiable. Furthermore, individual patients may manifest more than one morphological and/or molecular abnormality including aganglionosis, hypoganglionosis, neuronal nuclear inclusions, apoptosis, neural degeneration, inflammation, IND, neuronal hyperplasia, ganglioneuromas, or ICC alterations (64). There are enteric neuropathies that result in gut dysmotility in the absence of systemic or easily identified neuromuscular disorders. Examples include autonomic neuropathies, parkinsonism, and multiple sclerosis. A combination of molecular derangements may contribute to the degenerative process that leads to enteric neuronal loss. These include disorders of intracellular Ca2+ signaling, mitochondrial dysfunction, oxidative stress, and alterations in neurotransmitter or signal transduction pathways (64).
Motility disorders occur at any age and may be primary or may complicate systemic diseases. Primary motility diseases more typically affect children than adults. They may remain limited to the gut, as in HD, or they may be part of a generalized peripheral autonomic neuropathy, as in familial visceral neuropathies. Primary motility disorders may be familial or sporadic. Familial disorders are inherited as both autosomal recessive and autosomal dominant diseases. Secondary conditions such as drug injury, infections, connective tissue disorders (scleroderma-associated myopathy), metabolic disorders (diabetes mellitus, hypothyroidism), paraneoplastic or postinfectious syndromes, or amyloidosis more frequently affect adults.
The clinical and/or pathological findings of gastrointestinal motility disorders may be subtle or dramatic. Affected patients may present with dysphagia, nausea, vomiting, diffuse esophageal spasm, gastroparesis, intestinal pseudoobstruction, diarrhea, constipation, or intestinal diverticulosis. Symptoms differ depending on whether the upper or lower GI tract is involved. Nausea, vomiting, and intolerance to food intake are the predominant symptoms associated with upper GI involvement. Alterations in bowel habits dominate lower tract involvement. Abdominal pain with food intake is characteristic of both upper and lower tract disease. The inability for normal food intake and failure to maintain a normal body weight, chronic pain, and repeated surgeries result in a greatly impaired quality of life and significant mortality (65). Some patients become very malnourished with extreme weight loss.
Extraintestinal manifestations depend on the nature of the underlying disease; some help define specific syndromes. Features suggesting autonomic dysfunction include postural dizziness, difficulties in visual accommodation to bright light, and sweating abnormalities. Recurrent urinary tract infections and difficulty emptying the urinary bladder suggest a general visceral neuromyopathic disorder. Bedridden patients, such as those with dementia, stroke, and spinal cord injuries, are particularly prone to developing megacolon and chronic pseudo-obstruction. Patients on pain medications and antidepressants or tranquilizers may also develop chronic constipation.
Motility disorders are clinically diagnosed using specific physiological measurements of gastrointestinal motor function, including scintigraphy, gastroduodenojejunal manometry, and surface electrogastrography. The clinician will also often seek the pathologist’s assistance to rule out the presence of infiltrative lesions, such as amyloidosis, or connective tissue diseases or to document the presence of neuromuscular abnormalities. Assessment of motility is done once a mechanical obstruction has been excluded. A transit profile of the stomach, small bowel, and colon can be performed. If the cause of a transit disturbance is unclear, manometry using a multilumen tube with sensors in the distal stomach and proximal small intestine can differentiate a neuropathic process (normal amplitude contractions but abnormal patterns of contractility) from a myopathic process (low-amplitude contractions in the affected segments) (66,67). Colonic manometry and tone measurement facilitate identification of colonic inertia by the poor response to feeding or to medications such as bisacodyl (66). In patients with motility disorders, one also needs to identify the complications of the disorder including bacterial overgrowth, enterocolitis, dehydration, and malnutrition.
Even though the clinical or gross findings of motility disorders may be dramatic, the histological features are often inconspicuous, and they may also overlap with nonspecific neural and/or muscular histological abnormalities accompanying other conditions. Additionally, some patients with a clinically evident motility disorder may have histologic abnormalities that have not been well described or placed into specific syndromes. In other patients, there may be alterations in neurotransmitters that may or may not associate with any morphological abnormalities. These most commonly involve alterations in the nitrergic neural system or changes in vasoactive intestinal polypeptide (VIP) or substance P (SP) containing nerves. Changes may also be present in the muscle cells or ICCs. Thus, patients may have a neuropathy, a myopathy, or what has been termed a mesenchymopathy (defects in ICCs). These may exist alone, but some patients may have abnormalities in more than one cell type.
Histologic examination using conventional H&E stains is usually augmented with the use of special stains (Table 10.4) or, less commonly, ultrastructural examination. Some histologic changes are so subtle that they require precise neuronal counting to document their presence. However, this is fraught with problems, since the number of nerves and ganglia varies with age, location, other disease processes, and section thickness.
Treatment of motility disorders ranges from dietary changes or pharmacological treatment to surgery or intestinal or stem cell transplantation. Prokinetic drugs such as cisapride, metoclopramide, linaclotide, and octreotide benefit some patients. Patients with acute colonic pseudo-obstruction may benefit from neostigmine treatment (68). Bowel decompression through gastrostomy, jejunostomy, or colonic stent placement may help some patients.
Candidates for transplantation include those receiving total parenteral nutrition (TPN) with frequent episodes of sepsis, limited intravenous access to nutritional support, or impending liver failure. However, small bowel transplantation is challenging and there is currently an increasing interest in transplantation of embryonic or neuroepithelial stem cells as a potential source of cell replacement therapy in ENS disorders (41,69). These cells can be generated from a number of tissue sources including the gut itself (70).
▪ HIRSCHSPRUNG DISEASE (SYNONYMS: AGANGLIONIC MEGACOLON, CONGENITAL MEGACOLON, AGANGLIONOSIS)
Etiology and Pathophysiology of the Disease
HD is a congenital disorder of the ENS characterized by intestinal megacolon, aganglionosis, neural hyperplasia, and the absence of enteric neurons along variable lengths of the intestines. It is thought to be the result of abnormal migration, proliferation, differentiation, survival, and/or apoptosis of ENCCs during embryogenesis. It is a heterogeneous genetic disorder with autosomal dominant, autosomal recessive, and polygenic forms of inheritance. Because molecular mechanisms required for ENS precursor migration, proliferation, and differentiation are complex, many distinct mutations are associated with HD. As a result, HD displays a highly variable clinical phenotype with variation in recurrence risk by gender, familiality, length of the aganglionic segment of bowel, and associated phenotypes.
HD is a polygenetic disorder characterized by mutations in a wide array of genes that control tyrosine kinase function and the neurotrophins that play crucial roles in neuronal differentiation and maturation. There are greater than 10 genes that have been identified as contributors to the HD phenotype; affected genes are listed in Table 10.7 (71,72,73,74,75,76,77,78,79,80). Although extensive research has identified many key factors in the pathogenesis of HD, a large number of cases remain genetically undefined. Therefore, additional unidentified genes or modifiers likely contribute to the etiology and pathogenesis of HD.
RET Alterations
The RET proto-oncogene is the major gene associated with HD, with up to 50% of familial and 3% to 35% of sporadic cases demonstrating an associated mutation in RET (81,82,83). The RET gene is located on chromosome 10q11.2 and encodes a receptor tyrosine kinase. RET is expressed in neurons of the ENS and it plays a key role in the glial cell line-derived neurotrophic factor (GDNF)/GDNF coreceptor a (GFRa1) signaling pathway in development of the ENS (84,85). Over 100 mutations in RET have been identified, including large-scale deletions, microdeletions, insertions, missense, nonsense, and splicing mutations (86,87,88,89). These mutations occur throughout the gene, without any mutational hot spots (87). Loss-of-function mutations in RET are associated with HD; gain-of-function RET mutations occur in association with familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2 (MEN2). Certain
“Janus” mutations in RET result in both MEN2A and HD phenotypes (90).
“Janus” mutations in RET result in both MEN2A and HD phenotypes (90).
TABLE 10.7 SELECTED MUTATIONS PRESENT IN HIRSCHSPRUNG DISEASE | |||||||||||||||||||||||||||||||||||||||||||||
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The majority of HD-associated RET mutations result in either decreased levels of the RET protein or loss of its function (91). There are multiple RET kinase domain sequence variants in HD with multiple functional effects. A minority of HD variants abrogate ret kinase function; the remaining are phosphorylated and transducer intracellular signals. These sequence variants also impact maturation, stability, and degradation of RET proteins all of which lead to impaired RET protein activity (92). They fall loosely into two groups: frameshift or missense mutations that disrupt the structure of the intracellular tyrosine kinase domains or missense mutations in exon 2, 3, 5, or 6 of the extracellular domain (93). Patients with mutations of the intracellular domain have either short segment- or long segment-type HD, whereas those with mutations in the extracellular domain all have long segment-type HD.
In addition to the presence of specific RET mutations that may lead to the development of HD, there are also RET intragenic polymorphisms that lead to various clinical phenotypes (94). Noncoding mutations in RET can increase susceptibility to other HD-associated mutations (95,96,97). These noncoding mutations may alter regulatory elements or cellular functions such as transcription or translation or may be associated with linked susceptible loci (98). While many of these noncoding mutations have a low penetrance (95), they nevertheless may act synergistically with other mutations to affect the disease phenotype (98). Nongenetic factors such as vitamin A can influence the penetrance of the aganglionosis. Retinoic acid prevents efficient precursor migration (99).
Endothelin Pathway
Endothelin signaling is necessary for normal ENCC migration and likely plays a role in maintenance of an ENCC permissive environment. The endothelin 3 (EDN3) gene is located on chromosome 13q22. The EDN3 gene product is a secreted protein expressed by gut mesenchymal cells (100). EDN3 binds to a G-protein-coupled receptor, EDNRB, on migrating ENCCs. The EDN3-EDNRB signaling pathway is involved in regulation of normal ENCC migration and maintains them in an undifferentiated, proliferative state (101,102).
Approximately 5% of HD patients have heterozygous mutations in EDN3-EDNRB pathway-associated genes (103,104). EDN3 and EDNRB mutations are associated with both syndromic (Waardenburg-Shah) and nonsyndromic forms of HD (97,104). Patients with Waardenburg-Shah syndrome have colonic aganglionosis, pigmentation abnormalities, and sensorineural deafness (83). The autosomal recessive form of the syndrome is associated with EDN3 or EDNRB mutations, and an autosomal dominant form is associated with mutations in SOX10 (97,104).
Other Genetic Associations
Ten to fifteen percent of patients with HD have associated congenital anomalies or other diseases (Table 10.8). Ten percent of patients have Down syndrome; 5% have other serious neurologic abnormalities (105). Several genes may modify the severity of the HD phenotype in patients with or without coexisting IND. These may lie near 21q22 (106) and may account for the high prevalence of HD among patients with trisomy 21 (106). RET promoter and intron 1 variations have been shown to be associated with HD and the intronic single nucleotide polymorphism 2 (SNP2[re2435357]) is associated with Down syndrome-associated HD (107).
Nongenetic Factors
Alterations in the intrinsic gastrointestinal innervation may also contribute to the clinical and pathologic features of HD.
VIP and NO, components of the NANC system that relax smooth muscle and form part of the inhibitory component of the peristaltic reflex, are absent (108,109,110). However, extrinsic parasympathetic, cholinergic, and sympathetic adrenergic innervation persist. As a result, the distal aganglionic bowel is under constant, unopposed extramural stimulation so that it becomes narrowed, spastic, and unable to support peristalsis.
VIP and NO, components of the NANC system that relax smooth muscle and form part of the inhibitory component of the peristaltic reflex, are absent (108,109,110). However, extrinsic parasympathetic, cholinergic, and sympathetic adrenergic innervation persist. As a result, the distal aganglionic bowel is under constant, unopposed extramural stimulation so that it becomes narrowed, spastic, and unable to support peristalsis.
TABLE 10.8 OTHER ABNORMALITIES AFFECTING PATIENTS WITH HIRSCHSPRUNG DISEASE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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The pathogenesis of HD-associated enterocolitis, which affects some patients, is not well understood. It is likely to result from toxemia due to bacterial stasis in the dilated colonic lumen. Risk factors for enterocolitis include a delayed diagnosis of HD, long-segment disease, family history for HD, female gender, and trisomy 21 (111). There may also be an increased risk of enterocolitis in patients who have hypoganglionosis as part of their disease and who undergo surgery for their HD (112).
Clinical Features
HD affects 1 in every 5,000 to 30,000 live births; 80% of patients are male (113,114). Approximately 4% to 6% of cases are familial (113), especially when the megacolon extends to the cecum. Five percent of patients have an affected sibling (115). Nine percent of patients have total colonic aganglionosis (113). Less severe short-segment disease (about 80% of cases) is more common than long-segment disease and displays a more pronounced gender bias.
HD is the most commont form of congenital intestinal obstruction, often presenting with delayed passage of meconium in the first 24 to 48 hours of life. Up to 80% of cases are symptomatic and are, therefore, diagnosed during the first year of life; 10% first present in adulthood. Typically, the lack of propulsive movements and inhibitory reflexes in an intestinal segment leads to abdominal distension, vomiting, severe constipation, and marked dilation of the proximal ganglionic segment. Infants with obstruction, but without megacolon, should be suspected of having HD involving the entire colon. Reduced food intake and malabsorption result in failure to thrive. As the affected patient’s nutritional status deteriorates, infections may worsen the underlying motility problem. Some patients develop mucosal prolapse at the junction of the ganglionic and aganglionic bowel due to differential luminal pressures in these bowel segments. Mucosal prolapse is more prominent in older patients and correlates with disease duration. HD patients may also present in the neonatal period with perforation due to a coexisting necrotizing enterocolitis (NEC). The enterocolitis has an ischemic basis that may be based in part on an early and sustained disruption of the colonic microbiome (116). It is characterized by mucosal ulceration, colonic bleeding, perforation, sepsis, and toxemia. Patients may also have upper GI symptoms with abnormal transit in the small bowel and altered esophageal and gastric motility (117).
Pathological Findings
Several forms of HD are recognized.
Classical form: The aganglionic segment begins in the distal colorectum and extends proximally for variable distances into the adjoining proximally dilated bowel (Fig. 10.6).
Short-segment form: The aganglionic segment involves several centimeters of the rectum and rectosigmoid. The aganglionic segment may be as short as 3 cm, so that this variant may be missed if the biopsy is taken too high above the pectinate line.
Long-segment HD. The aganglionic segment extends beyond the sigmoid and involves a variable length of colon but does not extend beyond the cecum. This form affects less than 10% of patients (118).
Total colonic aganglionosis. The entire colon is involved along with variable lengths of ileum, jejunum, and even the stomach. It almost always presents in the first weeks of life. In rare cases, there can be skip areas that contain ganglion cells. In such cases, careful mapping of the bowel will help determine the ganglionated and nonganglionated
areas (119). Some cases are histologically similar to shortsegment and long-segment disease, whereas in others, the bowel is aganglionic, but there is little or no neural hyperplasia. The latter finding can lead to a false-negative diagnosis.
Zonal colonic aganglionosis (synonym: skip-segment HD): A short bowel segment is involved in this form of HD (Fig. 10.7). Ganglion cells are present, both proximal and distal to the aganglionic segment. This form of the disease is thought to be a consequence of a failure by the transmesenteric migrating ENCCs to remerge with the ENCCs that take the longer route along the gut wall leaving a gap in the enteric innervation (see earlier section on ENS development) (10). It may also have an ischemic basis in some patients. The majority of patients with this form of the disease are males (75%), and the majority of cases (92%) occur in patients with total colonic aganglionosis with the remaining cases representing rectosigmoid HD. Of the total aganglionosis cases in one study, 41% had the skip area in the transverse colon, 27% in the ascending colon, 9% in the cecum, and 23% had multiple skip areas (120). Normal distal innervation or a skip area containing some ganglia is present within an area of aganglionosis (121). Such cases are thought to occur as a result of mesenteric or extramural migration of ENCCs into an aganglionic segment of bowel (120). If biopsies are performed on the unaffected segments, the diagnosis will be missed.
HD with coexisting intestinal neuronal dysplasia (IND). In HD, IND lies within, or just proximal to, the aganglionic transitional zone. Patients have also been described with aganglionosis involving the entire colon and terminal ileum and coexisting jejunal and gastric IND. The IND accounts for residual symptoms in HD patients following pull-through operations (122).
 FIG. 10.6 Gross specimen of a patient with Hirschsprung’ disease. Note the contracted distal bowel, which tapers distally. The proximal bowel is dilated. |
 FIG. 10.7 Zonal aganglionosis. The distal bowel lies to the right of the photograph and the proximal bowel to the left. The aganglionic segment is the contracted segment in the middle. The proximal bowel appears dilated and featureless, with loss of the mucosal markings. |
The widely dilated, fluid-filled, hypertrophic colon empties into a funnel-shaped transitional zone extending to the anus (Fig. 10.6). Plain abdominal films may show air-fluid levels. The transition zone can be appreciated both on double-contrast exams and on CT scans (123). The correlation between the level of the radiographic transition zone on contrast enema and the length of the aganglionosis remains low while identifying the transitional zone on CT scans appears to provide a better correlation with the histology (123). The anal canal and rectum are small and empty, and the anal sphincter is tight. In adults, an abrupt, smooth rectal transition zone with proximal colonic dilation, in the setting of an appropriate clinical history, suggests the diagnosis.
A diagnosis of HD is usually made on a suction rectal biopsy containing both the mucosa and submucosa since the aganglionosis coincides closely in both the submucosal and myenteric plexuses. Biopsies are usually taken 2 cm from the pectinate line and at about a 5-cm distance. In very small neonates, the biopsy is taken just above the pectinate line and as high as can be taken safely without risking perforation. Two biopsies are preferred to increase the chances of an adequate biopsy, to overcome the possibility of a hypoganglionic segment, and to provide guidance as to the length of the aganglionic segment. Full-thickness rectal biopsy is reserved for patients in whom the diagnosis cannot be made with a more superficial biopsy.
Typical features of HD include aganglionosis (Fig. 10.8) and increased numbers of hypertrophic, nonmyelinated, submucosal and myenteric plexus cholinergic nerves (part of the extrinsic parasympathetic innervation) (Fig. 10.9).
Hypertrophic nerve trunks are observed more commonly in the rectosigmoid region than more rostrally (124). While neural hyperplasia is characteristic of HD, it should be noted that neural hyperplasia may be present in other disorders (Table 10.9). Ganglion cells are absent from both plexuses in the distal narrowed bowel and are decreased in number in the first few centimeters of the transitional zone. An increase in the number of ganglion cells occurs as one progresses proximally into the funnel-shaped transitional zone and into the normally innervated bowel. The transitional zone usually occurs over a short distance with ganglia appearing almost simultaneously in both the myenteric and submucosal plexuses. Some patients have longer transitional zones than do others; prominent nerve trunks may be present for several centimeters. The transitional zone may contain abnormally shaped ganglia. Some transition zones show features of IND (see below).
Hypertrophic nerve trunks are observed more commonly in the rectosigmoid region than more rostrally (124). While neural hyperplasia is characteristic of HD, it should be noted that neural hyperplasia may be present in other disorders (Table 10.9). Ganglion cells are absent from both plexuses in the distal narrowed bowel and are decreased in number in the first few centimeters of the transitional zone. An increase in the number of ganglion cells occurs as one progresses proximally into the funnel-shaped transitional zone and into the normally innervated bowel. The transitional zone usually occurs over a short distance with ganglia appearing almost simultaneously in both the myenteric and submucosal plexuses. Some patients have longer transitional zones than do others; prominent nerve trunks may be present for several centimeters. The transitional zone may contain abnormally shaped ganglia. Some transition zones show features of IND (see below).
 FIG. 10.8 A: Section of normal colon stained with anti-neuron-specific enolase (NSE) to highlight the submucosal ganglion. B: Serial section stained with anti-S100. The ganglion cells are negative. C: Hirschsprung’ disease stained with anti-NSE. Ganglion cells are absent, and there is nerve fiber proliferation. This is seen better at higher-power magnification. D: Serial section stained with anti-S100. Nerve fibers are positive. |
In premature infants, it may be difficult to recognize the immature ganglion cells due to their small size and
inconspicuous nuclei (Fig. 10.3). They may form rosettelike structures arranged around a central neuropil-type matrix producing a horseshoelike structure. The immature ganglia may mimic macrophages, smooth muscle cells, endothelial cells, and Schwann cells. Immature ganglion cells are usually present in clusters called neural units, which underlie the muscularis mucosae. These are easier to see than individual ganglion cells, and consist of a central pale neurofibrillary region with a surrounding mixture of small cells including immature ganglion cells and supporting Schwann cells (125). Immature ganglion cells have smaller and darker nuclei with a lower nuclear to cytoplasmic ratio compared to ganglion cells in older children and adults (126). They also have inconspicuous nucleoli and eccentric pear-shaped gray-blue cytoplasm without the stippled Nissl substance seen in mature ganglion cells. The characteristic features may not be present in each cell at each level (126).
inconspicuous nuclei (Fig. 10.3). They may form rosettelike structures arranged around a central neuropil-type matrix producing a horseshoelike structure. The immature ganglia may mimic macrophages, smooth muscle cells, endothelial cells, and Schwann cells. Immature ganglion cells are usually present in clusters called neural units, which underlie the muscularis mucosae. These are easier to see than individual ganglion cells, and consist of a central pale neurofibrillary region with a surrounding mixture of small cells including immature ganglion cells and supporting Schwann cells (125). Immature ganglion cells have smaller and darker nuclei with a lower nuclear to cytoplasmic ratio compared to ganglion cells in older children and adults (126). They also have inconspicuous nucleoli and eccentric pear-shaped gray-blue cytoplasm without the stippled Nissl substance seen in mature ganglion cells. The characteristic features may not be present in each cell at each level (126).
 FIG. 10.9 Hypertrophic nerve in Hirschsprung’ disease stained with an antibody to S100. |
TABLE 10.9 CONDITIONS ASSOCIATED WITH HYPERPLASIA OF THE MYENTERIC PLEXUS | |||||||
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Identification of even sparse numbers of ganglion cells is sufficient to exclude HD (126). The ganglia may be highlighted with special stains. However, it should be kept in mind that immature ganglia may fail to stain with ganglionic markers. Patients may also have a relative loss of ICC (59), although this finding is not present in all studies (127). Increased numbers of mast cells, often in direct contact with the hypertrophic nerves, are present, especially in the aganglionic segment. These produce NGF, cytokines, and prostaglandins, which stimulate neural growth and are thought to play a role in neural repair. The number of mast cells in the transitional segments is significantly less than in ganglionated segments and, interestingly, is increased in all bowel layers in aganglionic areas, particularly in the mucosa and submucosa (128,129). While hypertrophic nerves may be seen, patients with long-segment HD, including those with total aganglionosis, may completely lack nerve fibers in the circular layer (124). Furthermore, patients with both Down syndrome and HD have a marked reduction in neurite meshworks in the circular layer (124).
Frequently, the bowel wall proximal to the aganglionic segment is biopsied at the time of surgery, to ensure that the proximal resection margin is normal. Submucosal nerve trunks greater than 40 &mgr;m in diameter or abnormal-appearing ganglia strongly correlate with abnormal innervation and aganglionosis (Fig. 10.10) (130). However, the presence of hypertrophic nerve fibers is insufficient to make a diagnosis of HD. The degree of neural hypertrophy varies and increases with age. Hypertrophic nerves may not be present in ultrashort or long-segment HD. If hypertrophic nerve trunks or abnormal ganglion cells are present in frozen sections, the surgeon should extend the resection proximally and monitor it with additional frozen sections to identify a region that contains completely normal neural structures in order to prevent recurrent disease. Once resection specimens are received in the laboratory, the extent of the aganglionosis should be determined and the status of the proximal margin should be ascertained, if this was not done intraoperatively. Patients who develop postoperative symptoms may have either a retained portion of the transitional zone with neuronal dysplasia or an aganglionic segment or they may have developed an acquired disorder secondary to postoperative ischemia or infection.
If enterocolitis develops, the histology may include crypt dilation with mucin depletion, cryptitis, crypt abscesses, mucosal ulcers, transmural necrosis, and perforation. Enterocolitis affects both ganglionic and aganglionic intestinal segments and resembles other forms of enterocolitis. Pneumatosis intestinalis may be present. One occasionally sees abnormal submucosal blood vessels in HD. The abnormal arteries are most conspicuous in the transitional zone where they appear thickened and may show bizarre microscopic changes. Adventitial fibromuscular dysplasia, as evidenced by increased collagen around the internal elastic lamina, marked hypertrophy of the media, and obliterative endarteritis, also develops (131). The vascular changes predispose to ischemic injury.
 FIG. 10.10 Hirschsprung disease. A: Full-thickness section through the wall demonstrates the usual layers. B: High-power magnification of the myenteric plexus shows abnormal small ganglion cell in the transitional area. C: A higher-power magnification of another abnormal-appearing ganglion cell. D: Abnormal ganglion compared to a normal ganglion (E). |
Special Techniques for Evaluating HD specimens
Some individuals base the diagnosis of HD solely on examination of serial sections of rectal biopsy specimens, whereas others use acetylcholinesterase (ACE) staining as the primary diagnostic tool. Others use a combination of the two. The basis of the ACE test is that normal nerves do not express the enzyme, but it is increased in HD. Acetylcholinesterase (ACE) enzymatic staining (Fig. 10.11) demonstrates an increased network of hyperplastic, coarse, thickened, irregular, cholinergic nerve fibers within the muscularis mucosae and lower mucosa. The lamina propria fibers travel in a plane parallel to the mucosal surface. Numerous submucosal small nerve fibers and smaller and larger nerve trunks may also be present. This pattern is evident even in the most distal biopsies, including those from the mucocutaneous junction. Increased ACE-positive nerve fibers are consistently present
in short- and long-segment HD, but they may be absent in total colonic aganglionosis. ACE-staining patterns are also less dramatic in neonates than in older individuals as the intensity of the staining appears to increase with age. Weak staining in neonates may lead to false-negative diagnoses. The results of ACE staining are not always straightforward and the results are not always uniform leading to both falsepositive and false-negative results.
in short- and long-segment HD, but they may be absent in total colonic aganglionosis. ACE-staining patterns are also less dramatic in neonates than in older individuals as the intensity of the staining appears to increase with age. Weak staining in neonates may lead to false-negative diagnoses. The results of ACE staining are not always straightforward and the results are not always uniform leading to both falsepositive and false-negative results.
 FIG. 10.11 Acetylcholinesterase staining reactions. The positive reaction is shown by the blackish brown color. A: Normal bowel characterized by the presence of thin, wispy, acetylcholinesterasepositive nerve fibers within the muscularis mucosa. None are present in the overlying lamina propria of the mucosa. B: Patient with Hirschsprung’ disease demonstrating thick, irregular fibers both within the muscularis mucosae and extending up into the lamina propria and around the glands. (Pictures courtesy of Dr. Kevin Bove, Cincinnati Children’s Hospital.) |
The common procedure for the ACE staining is as follows: two mucosal suction biopsies are obtained 3 to 4 cm above the pectinate line. There is a lack of consensus on the distance from the pectinate line. Some recommend lower biopsies at 1 to 2 cm to detect ultrashort HD. However, this area contains a physiological hypoganglionic area. Specimens are generally considered to be inadequate if they contain squamous epithelium or skeletal muscle as the biopsies may come from this physiological zone. One specimen is snap-frozen immediately, and the other is placed in formalin for paraffin embedding. The optimal size of the biopsy is 3 to 3.5 mm in diameter with a minimum of 1 mm of submucosa (some recommend the examination of as many as 100 levels of the biopsy) (132). Several paraffin sections are used to complement the evaluation of acetylcholinesterase activity. Delayed freezing after specimen delivery may cause false-negative results due to enzyme degradation. The frozen sample is cut in a plane perpendicular to the mucosal surface. Since fast snap-frozen tissue is not always available for the analysis, a reliable marker than can be used in formalin-fixed paraffinembedded material is needed. The special stains listed in Table 10.4 help delineate specific structures (133,134,135).
Calretinin staining has recently been shown to be a simple and reliable tool for diagnosing HD (136). It compares favorably with results of ACE staining and it can be performed on formalin-fixed paraffin-embedded samples. In the majority of cases with aganglionosis, bowel calretinin staining is negative. Patients with normal ganglionated segments show calretinin-positive nerves in all the bowel wall layers. In general, the staining is more appreciable in the lamina propria, muscularis mucosae, and submucosa than in deeper layers of the bowel wall. The nerves show a granular, nonhomogeneous staining pattern and outline their very fine structure focally forming a fine fibrillary network in the lamina propria. Ganglion cells also stain with the antibody. Calretinin staining may be helpful in highlighting the ganglia and the nerves, but calretinin-positive nerve fibers may extend into the aganglionated portion of the bowel (137).
Treatment and Prognosis
Surgery is invariably necessary to treat symptomatic HD and HD-associated enterocolitis. Incomplete resection of the transitional zone between histologically normal intestine and aganglionic bowel in HD is a putative cause of postoperative dysmotility. The proximal margin of the transitional zone is often difficult to delineate and the length of the transitional zone is difficult to determine. At present, a conservative approach based on frozen section examination of the entire proximal margin of the resection specimens to exclude obvious circumferential aganglionosis (contiguous gap between ganglia of more than one eighth of the circumference), hypoganglionosis (continuous string of myenteric ganglia composed of 1 or 2 ganglion cells without surrounding neuropil), or hypertrophic nerves (>2 nerves with widths ≥40 &mgr;m/hpf) seems prudent (138).
Persistent constipation is the most important long-term postsurgical problem in patients with HD. Inadequate resection, anastomotic strictures, coexisting IND, or achalasia of the internal anal sphincter may also cause these sequelae. Today, there is active interest in the use of transplanted neural crest-derived stem cells to treat the disease (139,140).
▪ INTERNAL ANAL SPHINCTER ACHALASIA
Internal anal sphincter achalasia is a disorder characterized by the failure of the internal anal sphincter to relax in the presence of ganglion cells in rectal suction biopsies and normal ACE staining. This disorder used to be referred to as the
ultrashort form of HD, but it is now recognized as a separate and distinct entity. It results from abnormal innervation of the internal anal sphincter. Normal relaxation of the internal anal sphincter occurs secondary to activation of the intramural NANC nerves (141) via NO, the transmitter in NANC signaling (142). In anal sphincter achalasia, there is loss of NANC function in the anal sphincter due to abnormalities in NOS and NADPH-diaphorase. There is also a reduction in ICCs (143). The diagnosis is established by anorectal manometry, which shows the absence of the rectosphincteric reflex. Patients are treated by internal sphincter myotomy or botulinum toxin injections.
ultrashort form of HD, but it is now recognized as a separate and distinct entity. It results from abnormal innervation of the internal anal sphincter. Normal relaxation of the internal anal sphincter occurs secondary to activation of the intramural NANC nerves (141) via NO, the transmitter in NANC signaling (142). In anal sphincter achalasia, there is loss of NANC function in the anal sphincter due to abnormalities in NOS and NADPH-diaphorase. There is also a reduction in ICCs (143). The diagnosis is established by anorectal manometry, which shows the absence of the rectosphincteric reflex. Patients are treated by internal sphincter myotomy or botulinum toxin injections.
▪ HYPOGANGLIONOSIS (PSEUDOHIRSCHSPRUNG DISEASE)
Three forms of hypoganglionosis appear to exist: immaturity of the ganglia, congenital hypoganglionosis, and acquired hypoganglionosis. Hypoganglionosis may complicate either HD or IND or may exist in the absence of these diseases in a localized or disseminated form. In cases of disseminated hypoganglionosis, abnormal involvement of the entire colon requires total colectomy, whereas hypoganglionosis complicating adult HD usually only requires partial colectomy or anorectal surgery (112). Hypoganglionosis is characterized by sparse and small myenteric ganglia and low or absent ACE activity in the lamina propria and sparse myenteric plexus and muscular ICCs (144). Patients with isolated hypoganglionosis show decreased numbers of nerve cells, decreased plexus area, and increased distance between ganglia. In congenital hypoganglionosis, the number and the size of the ganglion cells are small at birth. The size of the ganglia tends to increase over time but not their number. As a result, the dysmotility does not improve with age.
Acquired hypoganglionosis is a late-onset disorder characterized by degeneration of the ganglion cells and gliosis. It may be the result of circulatory disturbances, intramural inflammation, and infections. After resection of the affected segment, the prognosis is usually good (145). Adult hypoganglionosis is considered to be a form of dysganglionosis. Hypoganglionosis, which can clinically mimic HD, is characterized by scarce ganglion cells and a reduced number of parasympathetic nerves in the intestinal wall. Symptoms include refractory constipation that can lead to complications such as fecalomas with erosion and ulceration. These can cause bleeding or perforation, partial or complete intestinal obstruction, or vascular and respiratory compromise due to massive colonic distension. Unlike adult HD, adult hypoganglionosis is not mainly confined to the rectosigmoid (146). Furthermore, the hypoganglionic segments may be multiple throughout the length of the intestine especially if the hypoganglionosis coexists with HD. Patients with hyperganglionosis also show transition zones. According to some, hypoganglionosis is more accurately diagnosed with full-thickness biopsy staining with nicotinamide adenine dinucleotide phosphate-diaphorase staining than by ACE staining (147).
The cause of hypoganglionosis is likely to be multifactorial and different in different patients. In some cases, an inborn hypoplasia of the parasympathetic myenteric plexus may be the cause (148).
▪ INTESTINAL NEURONAL DYSPLASIA
IND was introduced as an entity by Meier-Ruge in 1971 who described two subtypes: type A and type B (149). Type A is characterized by decreased or immature gastrointestinal sympathetic innervation, and type B is characterized by increased numbers of ganglion cells, a dysplastic submucosal plexus, and defective neuronal nerve fiber differentiation (150). IND type A is also known as hypoganglionosis. IND type B is also known as hyperganglionosis. The entity known as oligoneuronal disease, which is sometimes called the hypogenetic type of dysganglionosis (148), may also be a form of IND type A.
In 1991, when the classification of congenital malformations of colorectal innervation was published by a study group of pathology, other ganglionic and neural malformations were noted (151). In 1992, Meier-Ruge introduced a number of previously undescribed dysganglionoses, which he labeled nonclassifiable (NCD). Patients with IND may have other associated intestinal malformations such as colonic atresia (152). In HD, the aganglionic bowel is characterized by hypertrophic nerve trunks and increased numbers of adrenergic and cholinergic fibers, whereas IND type B is characterized by dysplasia of parasympathetic nerves, hyperganglionosis, and giant ganglia. There is a close association of the hypertrophic nerves in both disorders with mast cells (153). Patients with IND may have associated congenital anomalies.
Intestinal Neuronal Dysplasia Type A
IND type A is very rare, and the symptoms caused by its associated hypoganglionosis resemble those seen in HD. The disorder is characterized by immaturity or hypoplasia of the extrinsic sympathetic nerves supplying the gut (64). Hypoganglionosis occurs in three forms: (a) an isolated form occurring as a segmental or even disseminated disease, (b) hypoganglionosis of variable length adjacent to an aganglionic segment in HD, and (c) hypoganglionosis in combination with IND type B of a proximal segment. Hypoganglionosis may result from a developmental hypoplasia of the myenteric plexus, possibly due to the absence or abnormal expression of neurotrophic factors.
In newborns, there may be delayed meconium discharge or neonatal constipation in affected infants. Small children generally have rare bowel evacuations that respond to enemas. The colon becomes dilated and contains fecalomas. With increasing age, these fecal masses can be palpated through the abdominal wall. Distension causes intermittent colicky pain, often relieved by massive flatulence. Some children experience overflow discharge of sometimes bloody stool. The diagnosis of hypoganglionosis is usually difficult
to establish. X-ray studies, determination of transit times, and anorectal manometry are unreliable indicators of the disease.
to establish. X-ray studies, determination of transit times, and anorectal manometry are unreliable indicators of the disease.
Patients with IND type A have reduced numbers of myenteric ganglia and myenteric plexus neurons and no or low colonic mucosal and muscularis mucosae ACE levels, with secondary hypertrophy of the muscularis mucosae and the circular muscle of the muscularis propria. Absent or small submucosal and myenteric ganglia containing only one or two ganglia and immature neuroblastlike cells extend throughout the affected parts of the gut. Some patients have irreversible neuronal degeneration. Accurate diagnosis of hypoganglionosis is facilitated by examination of the myenteric plexus by NADPH-diaphorase staining in fullthickness sections (117). Patients with IND type A may also have reduced numbers of ICC, perhaps contributing to the dysmotility (144).
There is no consensus of how few ganglion cells there should be to make a diagnosis of hypoganglionosis. Meier-Ruge suggests that a 10-fold decrease in the number of ganglion cells per centimeter of bowel as compared to normal bowel is diagnostic (150). The distance between the ganglia in hypoganglionosis is nearly double that of the normal bowel. The treatment of hypoganglionosis (IND type A) is resection of the affected bowel and a pull-through operation.
IND Type B
In contrast to IND type A, the incidence of IND type B varies from 0.3% to 40% of rectal suction biopsies (150). The mean age at diagnosis is 1.5 years. It occurs as an isolated disorder or it complicates other disorders (Table 10.10) (64,154). The etiopathogenesis of IND type B remains obscure. It is generally accepted that it is caused by a delay in ENS maturation, although its association with chronic intestinal obstruction and HD indicates that it may be a secondary response to bowel obstruction or inflammation in the fetal or postnatal period (155).
The genetic basis and congenital origin of IND type B were established based on a study of affected monozygotic twins and on reports of families in which several members had biopsy-proven IND type B across multiple generations. However, no specific genetic alteration has been identified to date (156,157). Since HD and IND type B often coexist, a common molecular pathway in the genesis of both pathologies may also exist. Mutations in the most relevant genes implicated in HD, such as RET and GDNF, have generally not been found in IND type B in several series (158,159). However, a certain combination of single nucleotide polymorphisms in the RET proto-oncogene have been found in some patients. In addition, patients with MEN2B and concomitant IND type B have RET mutations. In some cases, IND and neurofibromatosis are familial and associate with a tandem duplication in the NFI gene and a reciprocal translocation (t15;16)(q26.3;q12.1) (160).
IND type B clinically both mimics and complicates HD, as discussed in an earlier section. Patients present with nausea/vomiting, diarrhea, constipation, intestinal dilation, neonatal enterocolitis, intestinal obstruction, intussusception, and volvulus. Symptoms develop insidiously, with progressive development of severe constipation that sometimes results in overflow incontinence (150). Some patients eventually spontaneously develop normal colonic motility (150). However, a significant number of patients develop severe intra-abdominal complications during the perinatal period, including NEC, meconium ileus, or bowel perforations. Such complications are especially common in premature neonates. Complications of the disease can lead to severe colitis and even death in adults (161).
TABLE 10.10 CONDITIONS THAT MAY BE ASSOCIATED WITH INTESTINAL NEURONAL DYSPLASIA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Pathological Findings
Neuronal dysplasia can be diffuse, involving both the small and large intestine, or it may remain confined to a single intestinal segment. Extensive disease may involve the stomach and esophagus. The bowel grossly appears either normal or variably dilated.
Controversy exists over the diagnostic criteria of IND type B. In one study, a group of three pathologists agreed on the diagnosis in only 14% of children without aganglionosis
(162). The diagnostic criteria of IND type B have included prominent hyperplasia of the parasympathetic myenteric and submucosal plexuses characterized by increased numbers of neurons and ganglia (Fig. 10.12); giant submucosal ganglia containing 7 to 15 ganglion cell hypertrophic nerve bundles containing increased numbers of thickened, beaded, and disorganized axons (Fig. 10.12); increased ACE activity in mucosal, submucosal, and arterial adventitial nerves (150); a proliferation of fine nerve fibers in the lamina propria and circular muscle; and the presence of isolated ganglion cells in the submucosa, muscularis mucosae, or lower mucosa (Fig. 10.13) (64,148,150,162,163). The myenteric ganglia may be large and almost continuous with numerous readily identifiable neurons. The finding of giant ganglia, while almost always seen in IND type B, is not specific for it and this feature may be present in the proximal colon in patients with HD and in some patients with hypoganglionosis. The diagnosis of IND type B is further complicated by the fact that the density of ganglion cells in the myenteric plexus decreases significantly with age during the first 3 to 4 years of life and estimates of nerve cell density are influenced by section thickness (164). Many neurons contain bizarre nuclei and poorly defined cytoplasm. Hypertrophic nerve bundles contain an increased number of thickened, beaded, and disorganized axons. Inflammatory changes, as might be seen in visceral myopathies or neuropathies, are absent. Prominent hypertrophy of the circular and longitudinal muscle layers also occurs.
(162). The diagnostic criteria of IND type B have included prominent hyperplasia of the parasympathetic myenteric and submucosal plexuses characterized by increased numbers of neurons and ganglia (Fig. 10.12); giant submucosal ganglia containing 7 to 15 ganglion cell hypertrophic nerve bundles containing increased numbers of thickened, beaded, and disorganized axons (Fig. 10.12); increased ACE activity in mucosal, submucosal, and arterial adventitial nerves (150); a proliferation of fine nerve fibers in the lamina propria and circular muscle; and the presence of isolated ganglion cells in the submucosa, muscularis mucosae, or lower mucosa (Fig. 10.13) (64,148,150,162,163). The myenteric ganglia may be large and almost continuous with numerous readily identifiable neurons. The finding of giant ganglia, while almost always seen in IND type B, is not specific for it and this feature may be present in the proximal colon in patients with HD and in some patients with hypoganglionosis. The diagnosis of IND type B is further complicated by the fact that the density of ganglion cells in the myenteric plexus decreases significantly with age during the first 3 to 4 years of life and estimates of nerve cell density are influenced by section thickness (164). Many neurons contain bizarre nuclei and poorly defined cytoplasm. Hypertrophic nerve bundles contain an increased number of thickened, beaded, and disorganized axons. Inflammatory changes, as might be seen in visceral myopathies or neuropathies, are absent. Prominent hypertrophy of the circular and longitudinal muscle layers also occurs.
 FIG. 10.12 A 4-month-old infant with IND type B. The hypertrophic nerves are accentuated by an S100 immunostain. |
Only about 5% of the ganglia in IND type B are giant ganglia (164) and finding one giant ganglion is insufficient for establishing the diagnosis (165). Occasional giant ganglia can be found in normal individuals without constipation or in patients with diverticular disease (166,167). It has been suggested that a diagnosis of IND type B only be made if in the submucosa or 30 serial sections 15% to 20% of the ganglia are giant ganglia with more than 8 nerve cells (165). The presence of giant ganglia may be age independent, whereas hyperplasia of the submucosal plexus and increases in ACE activity in the nerve fibers of the lamina propria appear to be age-dependent findings that disappear with maturation of the ENS. Therefore, neural hyperplasia is significantly more common in neonates less than 4 weeks of age than in older individuals (168).
Patients with IND may also exhibit ICC hyperplasia (169) or a reduced number of ICCs (170). When ICC hyperplasia is marked, it can be visible grossly as a thick, white, fibrous band between the inner circular and outer longitudinal muscle layers throughout the full length of the resected bowel. This is most commonly seen in children with NF1. Microscopically, this bandlike layer consists of haphazardly arranged spindled to oval-shaped cells. The nuclei are long and oval in shape with slightly tapered ends and possess hyperchromatic or clumped chromatin and occasional small nucleoli. The cells have a moderate amount of eosinophilic cytoplasm; mitotic figures are rare. The muscle layers are partially replaced by these hyperplastic spindle cells, and focally, the full thickness of the inner muscular layer can be involved. Residual myenteric plexus can be identified in the midst of the hyperplastic cells. The hyperplastic cells are c kit positive.
Patients with IND often have large numbers of mast cells in the bowel wall compared to the normal colon. Mast cells produce NGFs that support the development and functional maintenance of the sympathetic and cholinergic neurons, and they may be important in the neuronal hyperplasia seen in this condition (129). We have also seen endocrine cell hyperplasia associated with hyperganglionosis in the neonate.
Individuals with IND type B also often have secondary changes in the muscularis propria. There may be areas of significant muscle atrophy in one or another layer of the muscularis propria. Alternatively, there may be hyperplasia of either the circular or longitudinal layer of the muscularis propria or hyperplasia and atrophy may both coexist. These changes may be focal or diffuse in nature and they may be present in the circular layer in some parts of the gut and in the longitudinal layer in others. These secondary changes undoubtedly reflect abnormal innervation of the muscle layers and the neuromuscular junction.
Overall, it would be desirable to have better quantitative diagnostic criteria of IND to distinguish normal variants from pathologic conditions, particularly in very young children. Moore et al. introduced a morphologic scoring system based on the finding of hyperganglionosis, giant ganglia, neuronal maturity,
heterotopic neuronal cells, and ACE activity in the lamina propria, muscularis mucosae, or adventitia of submucosal blood vessels. Hyperganglionosis and increased ACE activity of nerve fibers in the lamina propria had major importance in this scoring system (107). The best diagnostic indicator of IND in adults may be the detection of 6 to 10 giant ganglia with greater than 7 nerve cells in 15 biopsy sections (171).
heterotopic neuronal cells, and ACE activity in the lamina propria, muscularis mucosae, or adventitia of submucosal blood vessels. Hyperganglionosis and increased ACE activity of nerve fibers in the lamina propria had major importance in this scoring system (107). The best diagnostic indicator of IND in adults may be the detection of 6 to 10 giant ganglia with greater than 7 nerve cells in 15 biopsy sections (171).
 FIG. 10.13 Area of hyperganglionosis in the same specimen illustrated in Figure 10.12. A: Low magnification showing a large collection of ganglion cells lying in the hypertrophic muscle and in the lower portion of the mucosa. B: Higher magnification showing the large numbers of ganglion cells. |
The majority of patients outgrow their disease as the ENS matures. Patients with persistent symptoms are managed medically with prokinetic agents, colonic irrigations, and cathartics. If bowel symptoms persist after 6 months of conservative treatment, surgery is often considered. Resection and pull-through operations may be indicated in extensive IND.
Some patients with a history of intestinal inflammatory conditions (ulcerative colitis and ischemia) may develop secondary IND type B with features that are virtually identical to the pediatric form of the disease. These cases are thought to represent an acquired form of the disease (172).
▪ NONCLASSIFIABLE DYSGANGLIONOSES
Nonclassified dysganglionoses were divided into types 1 to 4 by Meier-Ruge in 1992 (173). Among them, almost a third were characterized as mild dysganglionosis (type1) showing only marginal malformation of the submucosal plexus. Another third showed hypogenesis of the submucosal plexus (type 2). The diagnosis of hypogenesis of the submucosal plexus (type 2) has a substantial clinical impact and may be seen with or without aganglionosis (174). Surgery was often required to treat the lesion, which can extend as far proximally as the ileum. Heterotopic ganglion cells within the mucosa characterize type 3 dysganglionosis and heterotopic nerves of the muscularis propria in both the circular and the longitudinal layers characterize type 4. Type 4 is rare, but highly pathologic, generally requiring resection of the affected segment of the colon (173,174).
▪ HYPERGANGLIONOSIS AND GANGLIONEUROMATOSIS
Patients with hyperganglionosis and diffuse ganglioneuromatosis (Fig. 10.14) almost always have MEN2B and a mutation in the RET gene. Patients with MEN2B exhibit a diffuse ganglioneuromatous proliferation in the GI tract. This may lead to dysmotility, constipation, and chronic pseudo-obstruction. Diarrhea develops when associated with enterocolitis. These symptoms may occur in infancy as MEN2B is a dominantly inherited disorder, which in 95% of cases is the result of M918T missense mutation in the RET proto-oncogene that encodes a tyrosine kinase receptor. This is expressed particularly in neural crest-derived cells including enteric ganglia (175). The remaining 5% of patients have a mutation at codon 883 (176). The mutation alters RET substrate specificity in a ligand-independent fashion (a gain-of-function mutation) (177) that increases susceptibility to endocrine tumors (medullary thyroid carcinoma, adrenal pheochromocytoma, and parathyroid tumors).
 FIG. 10.14 Patient with pseudo-obstruction, IND, and ganglioneuromatosis. A: Gross appearance of the bowel demonstrating numerous polyps. B: Histological examination shows a cellular proliferation that obliterates the normal mucosal-submucosal junction. C: Section through the base of a polyp showing a ganglioneuroma. D: Higher-power magnification shows the neural tissue and ganglion cells. E: Submucosal ganglion demonstrating peripheral fibrosis and abnormal architecture. F: The architecture of the myenteric plexus is abnormal, and there are numerous inflammatory cells within the myenteric plexus. (Case courtesy of Dr. E. Foucar, Department of Pathology, Presbyterian Hospital, Albuquerque, NM.) |
▪ NEURONAL IMMATURITY
Neuronal immaturity also known as neuronal maturational arrest syndrome is characterized by the failure of neural elements to mature properly. The ganglia appear immature histologically, and the patients present with clinical features resembling HD and IND. There may be overlap with IND. The underlying cause of failed neuronal maturation is unknown. Possible pathogenetic mechanisms include (a) failure of normal numbers of NCCs to migrate into the gut, (b) inadequate neural proliferation in the gut, or (c) lack of growth or death of neuroblasts once they arrive in the gut due to the failure of the local microenvironment to support
normal neuronal development during fetal life. There may also be a lack of neurotrophins since the latter are known to be important in neuronal development, differentiation, maturation, survival, and maintenance. There may also be absence or delayed maturation of the ICC, contributing to pseudo-obstruction in affected neonates.
normal neuronal development during fetal life. There may also be a lack of neurotrophins since the latter are known to be important in neuronal development, differentiation, maturation, survival, and maintenance. There may also be absence or delayed maturation of the ICC, contributing to pseudo-obstruction in affected neonates.
 FIG. 10.15 Infant with pseudo-obstruction and neuronal immaturity. The submucosa contains ring-shaped ganglia. |
The histologic features differ depending on the stage at which myenteric plexus development ceased. Patients exhibit several major histologic abnormalities: (a) no myenteric plexus seen in either H&E or specially stained sections, (b) small numbers of neuronal structures (ganglia and nerve trunks), or (c) an apparently normal myenteric plexus seen on H&E-stained sections, but a neural deficiency is shown by immunohistochemical staining. The immature neurons lack neurofilaments. The ganglion cells may also line up at the periphery of the ganglia (ring-shaped ganglia) as occurs in premature infants (Figs. 10.15 and 10.16). There is no associated inflammation or neural degeneration. These findings contrast with those seen in patients with HD or IND type B in that there is no neural hyperplasia, but as noted, there may be overlap with IND.
 FIG. 10.16 Infant with pseudo-obstruction and neuronal immaturity. This picture of the myenteric plexus is from the same case that is shown in this figure. A number of immature ganglion cells line up at the periphery of the myenteric plexus. |
Neuronal immaturity often spontaneously improves with conservative therapy and the normal development of the child unless it occurs as part of IND.
▪ ABSENT ENTERIC NERVOUS SYSTEM
The absence of nerves and ganglia from the stomach to the colon characterizes the absent ENS. This very rare disorder presents as severe perinatal pseudo-obstruction. No nerves or ganglia are present in either the submucosal or myenteric plexus or in the muscularis propria. S100 and ACE staining confirm the absence of enteric neural structures. Sparse PGP 9.5-positive extrinsic nerve fibers are present in the submucosa. Prominent ICCs are present in the area between the circular and longitudinal muscle layers in a normal distribution in some parts of the gut. These cells do allow for some contractile activity of the gut (45). In other parts, they are absent or in various stages of destruction. The overall prognosis is poor (178).
▪ PEDIATRIC MOTILITY DISORDERS NOT FITTING SPECIFIC DIAGNOSTIC CRITERIA
A number of children present clinically with pseudo-obstruction syndromes in the first few weeks, months, or years of life that lack the classical features of either HD or IND (type A or B). These cases are challenging for clinicians to manage and they often look to the pathologist for help in establishing a diagnosis that will guide them into the appropriate therapy for the affected children. Pathological features are not readily associated with clinical findings and vice versa.
These cases are challenging for the pathologist since they often lack features that allow them to be easily categorized (Fig. 10.17). The use of special stains to highlight the neurons, ganglia, glia, or ICC may help identify some of the underlying abnormalities that are subtle and may not have been detectable on standard H&E-stained sections. A specific patient with a motility disorder may show one or more of the following abnormalities: aganglionosis, hypoganglionosis, hyperganglionosis, neural hyperplasia including ganglioneuromas, neural intranuclear inclusions, inflammatory changes, neural degeneration or apoptosis, or alterations in the ICC.
Given the confusion surrounding the diagnosis of pediatric motility disorders, the question arises as to how to best report the changes present. Our current practice is to diagnose HD, IND, or neural immaturity if classical features of these diseases are present. In cases in which the pathological findings do not fit a specific diagnostic entity, we tend to be descriptive, listing the major findings. Perhaps a standardized template approach to reporting these cases could lead to a better understanding of pediatric motility disorders in general. Once such approach is shown in Table 10.11.
▪ FAMILIAL VISCERAL NEUROPATHIES
Hereditary familial visceral neuropathies are a rare group of genetic diseases characterized by pseudo-obstruction (Fig. 10.18) and by myenteric plexus abnormalities with
variable inheritance patterns and characteristic clinical and extraintestinal manifestations (Table 10.12). The nerves often appear vacuolated (Fig. 10.19). Silver stains or immunostains highlight both the number and the shape of the myenteric plexus neurons and nerve fibers.
variable inheritance patterns and characteristic clinical and extraintestinal manifestations (Table 10.12). The nerves often appear vacuolated (Fig. 10.19). Silver stains or immunostains highlight both the number and the shape of the myenteric plexus neurons and nerve fibers.
 FIG. 10.17 Example of a neuropathy in a 1-month-old with pseudoobstruction that is difficult to classify into currently recognized disorders. Ganglia are present in both the submucosal (A) and myenteric plexus (B). They are neither increased nor decreased in number, but they appear small and shriveled. There is no neural hyperplasia. |
 FIG. 10.18 Gross appearance of the dilated colon in a patient with pseudo-obstruction at the time of autopsy. Note the markedly dilated colon, which bulges out through the abdominal incision. |
TABLE 10.11 POSSIBLE TEMPLATE FOR REPORTING PEDIATRIC MOTILITY DISORDERS THAT FAIL TO MEET THE DIAGNOSTIC CRITERIA OF HIRSCHSPRUNG DISEASE OR INTESTINAL NEURONAL DYSPLASIA | ||||||||||||||||||||||||||||||||||||
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Neuronal Intranuclear Inclusion Disease
This disorder may be present in multiple family members and appears to be transmitted from the paternal side in an autosomal dominant fashion (179). This disease presents
with symptoms of intestinal pseudo-obstruction, diffuse neurological abnormalities, evidence of mild autonomic insufficiency, and denervation hypersensitivity of the smooth muscles (180,181). The disease usually presents in the neonatal period with pseudo-obstruction in the absence of other neurological symptoms (182). Patients characteristically pass less than one stool per week despite the use of laxatives and enemas, and they exhibit abnormal esophageal, small intestinal, and colonic motility. Patients also develop gastroparesis, neurogenic bladder, and atrophy in other organs. The abnormalities are restricted to the myenteric plexus, which shows a significant reduction in the number of neurons, a third of which contain a round, eosinophilic intranuclear inclusion (180). The diagnosis is made on a simple rectal biopsy by finding discrete eosinophilic intranuclear inclusions in submucosal ganglion cells (179). Most remaining neurons are misshapen with reduced numbers of nerve fibers in the nerve tracts. Ultrastructurally, these intranuclear inclusions consist of a membrane-bound random array of straight or slightly curving filaments. They have a characteristic beaded pattern with a periodicity of 15 to 30 nm and measure 17 to 27 nm in diameter. It is unclear whether the inclusions are a primary abnormality or occur secondary to an underlying disease. Similar inclusions occur in the neurons of patients with CNS disorders with gastrointestinal involvement.
with symptoms of intestinal pseudo-obstruction, diffuse neurological abnormalities, evidence of mild autonomic insufficiency, and denervation hypersensitivity of the smooth muscles (180,181). The disease usually presents in the neonatal period with pseudo-obstruction in the absence of other neurological symptoms (182). Patients characteristically pass less than one stool per week despite the use of laxatives and enemas, and they exhibit abnormal esophageal, small intestinal, and colonic motility. Patients also develop gastroparesis, neurogenic bladder, and atrophy in other organs. The abnormalities are restricted to the myenteric plexus, which shows a significant reduction in the number of neurons, a third of which contain a round, eosinophilic intranuclear inclusion (180). The diagnosis is made on a simple rectal biopsy by finding discrete eosinophilic intranuclear inclusions in submucosal ganglion cells (179). Most remaining neurons are misshapen with reduced numbers of nerve fibers in the nerve tracts. Ultrastructurally, these intranuclear inclusions consist of a membrane-bound random array of straight or slightly curving filaments. They have a characteristic beaded pattern with a periodicity of 15 to 30 nm and measure 17 to 27 nm in diameter. It is unclear whether the inclusions are a primary abnormality or occur secondary to an underlying disease. Similar inclusions occur in the neurons of patients with CNS disorders with gastrointestinal involvement.
TABLE 10.12 FINDINGS IN VISCERAL NEUROPATHIES | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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 FIG. 10.19 Sporadic visceral neuropathy. Note the extensive vacuolization of the neural elements in the myenteric plexus with the cells simulating signet ring cells. |
Autosomal Recessive Disease with Mental Retardation and calcification of Basal Ganglia
Some mentally disabled individuals present with episodes of pseudo-obstruction and malabsorption. The intestinal smooth muscle layers appear normal or reduced in thickness. The variably sized neurons are decreased in the colon but normal in the esophagus, stomach, and small intestine and they appear degenerated with misshapen pyknotic nuclei. The brain shows extensive foci of calcification within the subcortical white matter and a striking reduction of neurons in the basal ganglia (181).
Autosomal Dominant Visceral Neuropathies
Some patients have intestinal pseudo-obstruction predominantly affecting the small intestine, without evidence of central, autonomic, or peripheral nervous system involvement. Special stains show decreased numbers of degenerated neurons and axons and many ganglia contain only one or two neurons with decreased argyrophilia. These appear swollen, distorted, and vacuolated and they lack inclusions. Nerve fibers appear hypertrophic with swellings or beading. Inflammation is absent, but muscular hyperplasia may be present (181).
▪ SPORADIC VISCERAL NEUROPATHIES
Sporadic visceral neuropathy includes at least two distinct morphologic diseases affecting the myenteric plexus of any part of the GI tract. The changes are not familial and do not affect extragastrointestinal structures. The disorders typically affect adults. These degenerative changes can be either noninflammatory or inflammatory. In primary inflammatory neuropathies, the infiltrate in the myenteric plexus is predominantly lymphocytic or more rarely eosinophilic (64,183).
Type I Sporadic Visceral Neuropathy
In type I sporadic visceral neuropathy, the number of myenteric plexus neurons is reduced. The neurons that remain appear swollen and irregular and have slightly concave shapes. Their cell boundaries are sharply defined and have a number of distinct tapering processes emanating from the nerve bodies. The remaining neuronal processes may appear thickened and haphazardly arranged. Gliosis replaces the plexus. These areas are devoid of neurons and only a few axons remain within the glial scar. The changes are difficult to appreciate without the use of silver or immunohistochemical stains. The disorder differs from neuronal intranuclear inclusion disease by the absence of intranuclear inclusions and clubbed dendrites, the presence of swollen neurons with degenerated cytoplasm, and the presence of gliosis (181).
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