ENTERIC NERVOUS SYSTEM AND ENTERIC NEUROPATHIES
Enteric neuropathies comprise a vast and disparate array of congenital and acquired disorders of the enteric nervous system (ENS) (1–8). These disorders range from a complete absence of enteric nerves in part of the bowel (Hirschsprung disease, HSCR) to more subtle abnormalities of ENS structure or function (grouped within intestinal pseudoobstruction). This range of defects reflects the enormity of the ENS in terms of neuronal numbers and diversity, akin to the central nervous system (9–11). Truly normal functioning of the ENS relies not only on its own structural integrity and interaction with other key elements of the neuromusculature (ie, smooth muscle and interstitial cells of Cajal) but also on a large number of modulating influences. Apart from the central nervous system, these influences include the immune and endocrine systems. As a result, in addition to gastrointestinal motility, the ENS exerts control over immune and therefore inflammatory processes, endocrine and exocrine secretions, and the intrinsic microcirculation of the gut. Not only can these be primarily or secondarily disordered in intestinal neuropathies but each represents potential targets for therapy.
CURRENT THERAPIES AND OUTCOME
At the present time, therapeutic strategies for enteric neuropathies per se remain limited to surgery and the provision of artificial nutrition. Unquestionably, these interventions have transformed the lives of sufferers, most of whom would otherwise not have survived beyond the neonatal period.
The role of surgery in intestinal neuropathies has extended far beyond the simple resection of abnormal dysmotile bowel. It has provided a powerful tool for diagnosis and the timely decompression of functionally obstructed bowel aimed to reduce downstream resistance to flow of luminal contents and further limit bowel decompensation (12).
Despite surgical intervention to optimise tailored feeding strategies, any enteral feeding that is established is often inadequate to sustain life, and this is where parenteral nutrition has really revolutionalised management and overall survival of children suffering from severe intestinal neuropathies (12–14). Mortality rates, however, remain on the order of 8% to 20%, and mostly relate to iatrogenic complications of central venous catheter–related sepsis and parenteral nutrition–related liver failure (12–17).
The poor outcome of enteric neuropathies is perhaps best illustrated by Hirschsprung disease, in which despite substantial surgical expertise and relatively rare use of parenteral nutrition, the postoperative morbidity data are compelling. A long-term follow-up study of 48 patients with total colonic aganglionosis by Tsuji et al (18) showed that 94% survived. Of the survivors, faecal incontinence was present in 82% of patients at 5 years, 57% at 10 years, and 33% at 15 years follow-up. On anthropometric follow-up, 63% of patients with total colonic aganglionosis were failing to thrive at 15 years. Ludman et al (19) showed that long-term incontinence is common in patients with HSCR regardless of the extent of aganglionosis.
Such data highlights the need to develop new therapies for enteric neuropathies. In the last decade, the tremendous advances in molecular biology and genetics have significantly enhanced our understanding of ENS development and function. They have facilitated not only our appreciation of the pathogenesis of enteric neuropathies but also, coupled with equivalent progress in the fields of pharmacology and stem-cell biology, the identification of novel tools and targets for therapies (20). Such therapies would either manipulate the functional output of the ENS that is present or replace/replenish components of an inadequate ENS. This article focuses particularly on our work with ENS stem cells as potential therapeutic tools for enteric neuropathies
POTENTIAL FUTURE THERAPIES
Manipulation of the ENS
In many children with intestinal neuropathies, although ENS function is clearly disordered, its structural integrity appears to be intact. The modulation of the ENS in such disorders is an area of considerable interest. Potential therapies include the use of pharmacological agents that address the following:
- Imbalances between excitatory and inhibitory influences within the ENS. A number of enteric neurotransmitters, receptors, and neuroendocrine factors appears to have key roles in regulating ENS function (21). Serotonin (5-hydroxytryptamine, 5-HT) is an important enteric neuroendocrine transmitter with both sensory and motor functions (22). A number of 5-HT receptors are expressed by neuronal cells in the gastrointestinal tract and appear to function in either excitatory (5-HT1P, 5-HT3, and 5-HT4) or inhibitory (5-HT1A) roles (21–23). The use of specific 5-HT receptor agonists and antagonists has been shown to alter gastrointestinal motility experimentally and these have been used in adults, predominantly with irritable bowel syndrome (21). Little, if any, data exist for their use in children at the present time.
- Inflammatory neuropathies of the enteric nervous system. It is increasingly recognised that there is often an ongoing and significant inflammatory process occurring within the ENS in both acquired and congenital enteric neuropathies. So-called ganglionitis has been widely reported in the adult and paediatric literature, along with reported improvement in gastrointestinal motility with the use of immunomodulating agents (24–28). The use of such therapies in enteric neuropathies is inevitable, given the explosion in the use of novel immunotherapy for gut-associated inflammation.
- Key cell signalling pathways involved in ENS development. The unravelling of such mechanisms has allowed the identification of putative factors that may be useful to promote optimal ENS structure and function. These include a number of mediators in cell signalling pathways (2,7). This is destined to be a main growth area and is discussed below.
Another technique that shows some promise is electrical pacing of the gastrointestinal tract. Gastric, small-intestinal, and colonic pacing have been used to either recapitulate normal electrical activity or override abnormal activity in an effort to stimulate more normal motility (29–31). This technology is still largely experimental, invasive, and not practicable. Much more data are needed before it can be applied to the paediatric population.
Replacement/Replenishment of ENS
Intestinal transplantation represents a rather extreme therapy for dysfunction of only 1 component of the enteric neuromusculature. Although there have been reports of increasing success, all paediatric intestinal transplant programmes are limited by the scarcity of donor organs and relatively high mortality and morbidity rates (16,32–40). Intestinal transplantation is likely, however, to remain an important therapy, arguably most beneficial in chronic cases of disease with decompensated bowel, those unlikely to respond to replenishment of individual components, or those with other reasons (eg, lack of vascular access).
ENS Stem Cells
One group of tools to replenish the ENS is their original building blocks, the ENS stem cells. These arise from the neural crest and invade the developing gut during embryogenesis. They survive throughout colonisation of the gut, migrate along its entire length, proliferate extensively, and ultimately differentiate into the neurons and glia that constitute a functioning ENS (41–43).
Recent studies have demonstrated that multipotent cells that retain the ability to form the ENS when transplanted to uncolonised or aganglionic gut are present within the gastrointestinal tract during development and into early postnatal life (44–49). We recently identified 1 such source of ENS stem cells (50). In these investigations, gut from embryonic and postnatal mice of up to 2 weeks of age was dissociated and cultured in a medium designed to encourage growth of neural crest–precursor cells. After a number of days, neurospherelike bodies (NLBs) were identified in the cultures. In addition to differentiated neurons and glia, NLBs also contained proliferating progenitors that were capable of giving rise to subsequent colonies also containing enteric neurons and glia (50).
When pieces of NLBs were grafted into normal and aganglionic early embryonic mouse gut, progenitors were able to colonise the gut and differentiate into appropriate enteric phenotypes at the appropriate locations (50). More important, ENS stem cells were shown to be similarly isolated from the ganglionic portion of Hirschsprung-like gut from 1 of the established HSCR mouse models, the miRet51 mutation (50,51). In this mutation, only the Ret51 isoform is expressed in the animals, which have a HSCR phenotype with distal colonic aganglionosis and early presentation with subacute intestinal obstruction (52). The differentiation profile of such cells, however, was shown to be abnormal, perhaps in keeping with their inability to fully colonise the developing gastrointestinal tract (51).
These preliminary studies suggested that ENS stem cell transplantation, both allogeneic and autologous, is feasible but requires considerable refinement and optimisation. Colonisation, proliferation, and differentiation within the recipient gut were potential limiting factors, and it became clear that controlled manipulation of developmental properties remains a major challenge. Recent data, however, suggest this may be possible. Endothelin 3, for example, inhibits reversibly the commitment and differentiation of ENS-progenitor cells along the neurogenic and gliogenic lineages, suggesting a role for this factor in the maintenance of multilineage ENS progenitors (53). Glial cell line–derived neurotrophic factor acting in the presence or absence of endothelin 3 significantly increased the proliferation of ENS progenitors, as well as neurite outgrowth (50,54). Glial cell line–derived neurotrophic factor consistently has been implicated in the process of directed migration of neural crest–derived ENS progenitors to facilitate colonization of the developing gut during embryogenesis (55,56). Vassilis Pachnis' group recently has shown that ENS progenitor cells isolated from mice with the Ret51 mutation are susceptible to genetic manipulation. Restoration of the Ret9 isoform within the Ret51 EPCs resulted in rescue of the differentiation phenotype (Natarajan and Pachnis, personal communication, 2007). Such findings and studies have enormous implications for pretransplantation priming of ENS stem cells, as well as the creation of receptive environments within recipient aganglionic gut. The latter is likely to be key, given that the target aganglionic gut is postnatal, and thus post–ENS formation and the pathogenesis of failed ENS formation may indeed lie in defects within the mesenchymal environment (eg, endothelin-3 mutations in HSCR).
There has been rewarding progress in translational human work with ENS stem cells. Our preliminary data have shown that ENS progenitor cell–containing NLBs can be generated from human embryonic gut and from postnatal gut, including from the ganglionic segment of HSCR patients. Although such cells can be identified using available neural crest cell markers, specific isolation remains a problem. This may be circumvented by the use of ENS stem cell–containing NLBs as “therapeutic packages.” Preliminary work has shown that human NLB-derived cells are capable of migration within aganglionic recipient gut and formation of ganglionlike clusters within the presumptive submucosal and myenteric plexi.
These investigations suggest that transplantation of ENS stem cells could be a viable alternative treatment for HSCR. Such cells can be harvested from postnatal gut, their biological properties manipulated, and ultimately transplanted into aganglionic gut to replenish components of the ENS. Perhaps the goal of complete ENS replenishment is unrealistic and somewhat naïve. Studies of the ageing gut, in which a scant surviving ENS functions in the absence of any overt functional obstruction, suggest that partial ENS reconstitution may be sufficient to restore some balance between inhibitory and excitatory influences within the neuropathic gut (57,58). Targeted replenishment (eg, sphincteric) may be more readily achievable but would only be useful in more localised disease. This therapeutic strategy may be useful for a number of congenital enteric neuropathies or those secondary to destructive or degenerative processes, but provides particular hope for aganglionic gut disorders such as Hirschsprung disease, in which postoperative morbidity in certain subgroups remains unacceptably high.
To professionals in the field, children and adults with enteric neuropathies represent a significant challenge in management. Many do not escape the surgeon's knife nor a lifetime in and out of hospital. Significant strides have been made in teasing away at the processes that underlie the complex workings of the enteric nervous system, the so-called second brain. This has given us tremendous insight into pathogenesis and led to the identification of multiple putative therapeutic tools. Without doubt, in the next decade their use will herald a shift away from the largely supportive management of enteric neuropathies to definitive curative therapies.
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