Keywords
Hirschsprung's disease - Hirschsprung-associated enterocolitis - Pathophysiology -
translational research
Introduction
Hirschsprung Disease
Hirschsprung disease (HSCR), first described by Harald Hirschsprung,[1] is defined as a congenital disease that is characterized by the absence of intrinsic
ganglion cells in the submucosal and myenteric plexuses of the hindgut/distal colon.[2] The incidence of HSCR has been reported to be approximately 1/5,000 live births.[3] Clinical symptoms of HSCR in newborns include functional obstruction characterized
by delayed passage of meconium within the first 24 to 48 hours of life, abdominal
distension, vomiting, and constipation.[4]
There has been continued development in our understanding of the pathogenesis of the
disease, and the development of a variety of surgical options. Medical research into
HSCR has steadily grown over the last three decades ([Fig. 1]), with continued interest in translational models which can help to identify the
underlying pathogenesis and explore novel treatment. The current standard treatment
for HSCR is surgical pull-through operation resecting the aganglionic bowel, and the
normal ganglionic intestine is then anastomosed to the rectum or anus. However, the
procedure requires part of the intestine to be removed leading to various post-operative
complications. Fistula or stenosis of the anastomosis may appear as short-term complications,
and long-term outcomes can include fecal incontinence or recurrent constipation.[5]
[6]
[7]
[8] Furthermore, 20 to 38% of HSCR patients may develop enterocolitis despite technically
successful surgery, a condition referred as Hirschsprung-associated enterocolitis
(HAEC). These associated complications can have long-term impacts on the quality of
life of patients and necessitate a deeper understanding of both the disease's pathogenesis
and its associated complications.
Fig. 1 Peer-reviewed publications for Hirschsprung disease (blue) and Hirschsprung-associated enterocolitis (green) on PubMed over the past 30 years. Search query of: “Hirschsprung” [Title/Abstract]
OR “Hirschsprung's” [Title/Abstract] OR “HSCR” [Title/Abstract] for the Hirschsprung's
disease manuscripts. Search query of: “Hirschsprung associated enterocolitis” [Title/Abstract]
OR “HAEC” [Title/Abstract] for the Hirschsprung-associated enterocolitis manuscripts.
Hirschsprung-Associated Enterocolitis
HAEC is the most common and serious complication of HSCR, with the potential to develop
into generalized sepsis, potentially leading to a high mortality rate. HAEC classically
occurs in HSCR patients with abdominal distention, fever, and foul-smelling stools.
It accounts for most of the morbidity and mortality of children with HSCR.[9]
[10] Although the surgical correction of HSCR is mostly successful, challenges remain
as HAEC can still develop.[11]
[12] Additionally, the incidence of HAEC appears to be unchanged between the pre-operative
and post-operative periods in both animal models and humans.[13]
[14]
[15]
The pathogenesis of HAEC appears complex and is not completely understood yet.[10] It usually presents with both gastrointestinal (GI) and generalized symptoms ranging
from fever and diarrhea to bloodstained stools and septic shock, while in chronic
cases, it affects growth and development.[16]
[17] Currently, treatments for HAEC are relatively nonspecific and can consist of a combination
of antibiotics, rectal irrigations, and bowel rest.[18]
[19] These measurements are often directed toward treating the acute symptoms rather
than targeting the factors that are thought to contribute to the disease. Recent research
into the pathogenesis of the disease with animal models, and a better understanding
of the underlying etiology may substantially improve the quality of life of the patients
who developed HAEC.
Etiology of HAEC
Investigations focused on histopathologic changes in HAEC-affected bowel have suggested
that the distal obstruction in HSCR is likely the leading cause of HAEC.[20]
[21] However, clinical observation of continued susceptibility to HAEC after pull-through
procedure contradicts this conclusion.[13]
[15] The etiology of HAEC is poorly understood and controversial. The many proposed potential
pathological processes of HAEC can be classified into three main abnormalities of
the intestinal homeostasis, namely ones of (1) intestinal barrier dysfunction, (2)
abnormal innate immune response, and (3) abnormal microbiota.
In the case of intestinal barrier dysfunction, viral and/or bacterial infectious pathogens
may lead to the development of HAEC but the causative organisms remain elusive with
conflicting data identified.[22]
[23]
[24] Studies have also demonstrated that there are decreased turnover and alterations
in various subsets of mucins which play a fundamental role in the luminal barrier
integrity of patients with HSCR.[25] Although there exists variation in the literature among what mucin receptors are
suspected of being involved,[26]
[27] interestingly in most cases these findings seem to extend beyond the aganglionic
region of the bowel.
Researchers have shown that there is a defect of adaptive immunity in HSCR. Cheng
et al[28] found that splenic lymphopenia and a small-sized spleen existed in Ednrb knockout mice, which is characterized by a 5 to 20-fold reduction in the amount of
CD19+ mature B cells, CD4+ T cells, and CD8+ T cells. Other studies have also shown that in severe HAEC, thymic involution, splenic
lymphopenia, and suppression of B-cell production were also present.[29] These results mimic the clinical manifestations seen in neonates who develop sepsis
and highlight the importance of early management. Secretory IgA is also important
for intestinal immunity, Moore et al identified an increase in IgM and IgG populations
without a concomitant IgA increase in the aganglionic and transitional segments of
the intestine in HSCR patients.[30] Of note, the reduction of B-cell-produced secretory IgA have been detected in Ednrb knockout mouse model and have shown that this may be due to a combination of both
an intrinsic B-cell defect in antibody production and an extrinsic defect in IgA transport
in HAEC.[31] In addition, the abnormalities of glial cells may lead to HAEC.[32]
[33]
The HAEC recurrence rate can have quite a varying range with as high as 50% recurrence
in some cases.[34] This may be attributable to sustained histopathological alterations in the intestinal
mucosa or the immunodeficiencies/dysfunction of intestinal immunity mentioned above.
Interestingly, the assessment of stool specimens from a patient using amplified ribosomal
DNA restriction analysis has suggested that the recurrence may be associated with
a specific distribution of intestinal flora, which is influenced by antibiotic use.[22] Using high throughput sequencing, Li et al showed that HSCR patients without HAEC
had relatively distinct and more stable microbiota relative to their HAEC patient
counterparts.[35] The microbiotas in HSCR patients were characterized by a higher prevalence of Bacteroidetes,
while HAEC patients had a higher prevalence of Proteobacteria. Neuvonen et al also
found a significantly increased abundance of Proteobacteria in HAEC patients and a
decrease in enterobacteria and Bacilli.[36] In fact, in early 2020, Tang et al were able to identify a group of 21 microbiome
signature operational taxonomic units that can predict postoperative HAEC with an
85% accuracy.[37] These studies among others suggest the potential of modulating the gut microbiome
as a potential way to prevent the development or progression of HAEC in HSCR patients.
Animal model of HSCR
Animal models of HSCR play a critical role in our understanding of the important anatomical
features, pathogenesis, and underlying molecular mechanisms of the disease. They are
also vital in allowing us to model disease progression changes with novel treatments
and in finding strategies to assess the effectiveness of treatment. Over the last
few decades, several models of HSCR have been developed. These models have been used
in numerous studies on a wide range of animals like mice, rats, rabbits, and pigs
and have helped to effectively model human aganglionosis that is seen in HSCR. Additionally,
the animal models have also allowed us to appropriately assess disease progression
and find associated markers that have since opened the door to a wide spectrum of
potential targets to reduce negative outcomes in patients. Animal models can generally
be broken down into three main types of models, namely, chemical or teratogen-induced
models, and genetic knockout models ([Table 1]).
Table 1
Animal models of Hirschsprung disease
|
Type of model
|
Model
|
Chemical used or target gene
|
Underlying mechanism
|
|
Chemical-induced models
|
Benzalkonium chloride (BAC) rat model
|
Benzalkonium chloride (BAC), a cationic surfactant
|
BAC ablates the myenteric neurons and glia of the intestine leading to the development
of aganglionosis.[38]
[39]
[40]
[41]
|
|
Retinoic acid
|
Several potential targets, two common ones are retinol-binding protein 4 and retinaldehyde
dehydrogenase 2
|
Retinoic acid is required for lamellipodia formation and enteric neural crest cell
migration.[43]
|
|
Ibuprofen
|
Ibuprofen, a nonselective cyclooxygenase inhibitor
|
Ibuprofen reduces enteric neural crest cell migration, lamellipodia formation, and
levels of motility genes like Rac1.[45]
|
|
Mycophenolate
|
Mycophenolate inhibits inosine-5′-monophosphate dehydrogenase
|
Prevents de novo guanine nucleotide synthesis which leads to defects in multiple neural
crest derivatives and intestinal aganglionosis.[46]
[47]
|
|
Genetic models
|
Ret gene
|
Ret gene knockdown or GDNF deletion/mutation or Ntn knockout
|
Ret gene encodes a receptor tyrosine kinase for GDNF, Ntn, Atm, and Psp.[48] Knockdown models or GDNF knockout produces aganglionosis and plays an important
role of Ret gene and have been shown in human HSCR in up to 50% of familial cases.[51]
[52]
[53]
[54]
|
|
Endothelin gene
|
Ednrb or Edn3 mutation
|
Endothelin receptor b and Edn3 are required for proper ENCC migration and expression.[62]
[63] Mutations have been seen in human HSCR patients and Ednrb knockout is a commonly used animal model of HSCR.[66]
[67]
|
|
Other models (Sox 10, Phox2b, Pax3, Shh, and Ihh)
|
Various mutation models that affect ENCCs
|
Mutation in various genes that affect ENCC development as they leave the neural tube,
migrate to the intestine, or allow for ENCC differentiation can lead to aganglionosis
that mimics HSCR.[68]
[69]
[70]
[71]
|
Abbreviations: ENCC, enteric neural crest cell; GDNF, glial cell line-derived neurotrophic
factor; HSCR, Hirschsprung disease.
Chemical-Induced Models of HSCR
The benzalkonium chloride (BAC) rat model of HSCR was first described in 1978 and
has become a well-established model since then to investigate the underlying mechanism
of HSCR as it is highly similar in its progression to human HSCR.[38]
[39]
[40] BAC is a cationic surface-acting agent that attaches to cell membranes and through
selective neuronal ablation leads to irreversible depolarization, cell damage, and
death.[41] In the intestinal region where BAC is used, the myenteric neurons and glia are almost
completely ablated leading to the development of aganglionosis which mimics human
HSCR.[41] The submucosal neurons, however, are not affected by BAC treatment. This model has
the advantage of having had many years of use and is relatively inexpensive with good
long-term survival.
Another chemical model involves depleting retinoid signaling or vitamin A. Downregulation
of retinol-binding protein 4 and retinaldehyde dehydrogenase 2 have both shown that
the retinoic acid pathway, in which they are involved, plays a critical role in colonic
aganglionosis confirming the importance of retinoic acid (active form of vitamin A)
signaling in ganglionic development.[42] The underlying mechanism is believed to be the diminished retinoic acid activity
which leads to impaired lamellipodia formation and reduces enteric neural crest cell
(ENCC) migration in response to glial cell line-derived neurotrophic factor (GDNF).[43] These findings suggest that vitamin A deficiency may act in combination with potential
genetic factors to increase disease penetrance and expression.[43]
[44]
Other chemical models have also been explored and shown to reduce ENCC migration.
One such example is ibuprofen, a nonselective cyclooxygenase inhibitor, which has
been shown to reduce ENCC migration and lamellipodia as well as levels of motility
genes like Ras-related C3 botulinum toxin substrate 1 (Rac1).[45] Another example is mycophenolate, a potent, reversible, non-competitive inhibitor
of inosine-5′-monophosphate dehydrogenase, which is required for de novo guanine nucleotide
synthesis and has also been shown to reduce ENCC migration and lamellipodia.[46] Like other chemical models, this reduction in ENCC migration and lamellipodia leads
to intestinal aganglionosis. However, just a delay in neural crest cell migration
with the eventual development of a normal enteric nervous system (ENS) was founded
in both studies. Additionally, inhibition of inosine-5′-monophosphate dehydrogenase
2 (Impdh2) causes a defect in multiple neural crest derivatives which leads to intestinal
aganglionosis, craniofacial abnormalities, and malformations of major vessels.[47] The chemical-induced models seemed to represent the models of perturbed ENS development,
the genetic models have been established to overcome these hurdles.
Genetic Models of HSCR
HSCR has a complex genetic etiology with several genes and loci being potentially
associated with either isolated HSCR or syndromic forms. Implicated genes often play
a critical role in regulating proper ENCC migration and/or appropriate enteric nervous
system development. Disruptions in these genes lead to remarkably phenotypic expression
in animal models similar to human HSCR. Two of the most well-studied genes that have
produced useful HSCR animal models are the Ret gene and the endothelin (Edn) gene family.[48]
The Ret gene encodes a receptor tyrosinase kinase which has four ligands, GDNF, neurturin
(Ntn), artemin (Atm), and persephin (Psp),[48] with another receptor (glycosylphosphatidylinositol-linked receptor) to form a complex
that is important for molecular adhesion and neural crest migration.[49]
[50]
Ret gene mutations have been shown to play a vital role in human HSCR, accounting for
15 to 20% of patients with sporadic HSCR and up to 50% in familial cases.[51]
[52]
[53]
[54] A total/global knockout of the Ret gene in mice causes complete intestinal aganglionosis; however, it also leads to
kidney agenesis and death of the mouse pups at birth.[55]
[56]
[57]
[58] GDNF deletion has also been shown to produce a similar phenotype as it is a critical
Ret activator. GDNF/Ret signaling has been shown to play a critical role in the formation
of specific neuron subtypes and enteric nervous system development overall. Ntn has
also been shown to play a critical role in the maintenance of enteric neurons and
ganglia and in promotion of neuronal differentiation and the ENCC proliferation.[59]
[60] Ntn-deficient mice have reduced nerve fiber density with associated abnormalities
in bowel motility.[59]
[61]
Endothelins are peptides with receptors that normally are responsible for constricting
blood vessels and affecting blood pressure. The endothelins act via surface transmembrane
receptors, endothelin receptor A (Ednra), and endothelin receptor B (Ednrb).[48] Ednrb and Edn3 are both expressed in the enteric neurons and gut mesenchymal cells
of fetuses and are required for appropriate enteric neural migration and expression.[62]
[63] The Edn3–Ednrb pathway has been shown to play an important role in ENCC migration
and in maintaining enteric progenitors.[64]
[65] Heterozygous Ednrb mutations have been identified in non-syndromic HSCR patients.[66] Homozygous mutations of Ednrb and Edn3 genes by contrast have been reported in HSCR patients with the type-2 Waardenburg
syndrome. Interestingly, Ednrb mutations have been associated with short-segment HSCR, while Ret mutations appear to be associated mainly with long-segment aganglionosis.[66] Neural crest cell-specific deletion of Ednrb has also been shown to lead to reduced
neuronal density in the ENS of the colon and may account for the dysmotility seen
in HSCR.[67] The Ednrb knockout/knockdown models provide both mechanistic and translational understanding
of HSCR and its associated symptoms.
Other gene mutations have also been implicated in ENCC differentiation or migration.
These include, Sox10, a member of the SRY-related family of transcription factors
and expressed in vagal ENCCs as they leave the neural tube.[68] Paired-like homeobox2b, a transcription factor expressed in migrating ENCCs, enteric
neurons, and glial cells.[69]
Pax3, a member of the paired-box containing family of nuclear transcription factors that
is expressed in neural cell precursors giving rise to an enhancer in the Ret gene,[70] and Sonic hedgehog, and Indian hedgehog genes also potentially affect the survival
and differentiation of ENCCs.[71] Targeted deletion of these genes among others all are viable options and provide
avenues in understanding what is potentially leading to aganglionosis, its underlying
causes, and how to best target therapeutics to address it.
Animal Models of HAEC
Genetic Murine Models: Understanding the Pathogenesis of HAEC
HAEC is responsible for the most serious morbidity and mortality from HSCR. Animal
models that can mimic the inflammatory process can help provide insight into the etiology
of HAEC as well as provide an avenue to test potential treatment options. Three rodent
models have been used to study HSCR and its associated entercolitis[15] ([Table 2]). These include Ednrb null mice,[62]
[72] piebald-lethal mice,[73]
[74] and endothelin receptor B-deficient rats.[75]
[76]
[77] There are additional mutant rodent models that have been used to study HSCR in general
and these include Endothelin-3 ligand-deficient mice,[63] Hoxb5 dominant-negative conditional (Cre-Lox) transgenic mice,[78] erbB2/nestin-Cre conditional mutant mice,[79] Dom spontaneous mutant mice,[80] conditional β-1 integrin knockout mice,[81] trisomy 16 mice,[82]
[83] and mice deficient in the c-ret proto-oncogene,[84] and the fmc/fmc (familial megacecum and colon) rat[85] which all have phenotypes that mimic HSCR.
Table 2
Hirschsprung disease models that have studied the associated enterocolitis (HAEC)
|
Type of model
|
Model
|
Overview
|
|
Genetic
|
Piebald-lethal mice[73]
[74]
|
Natural mutation that leads to a recessive phenotype identical to Ednrb knockout mice that disrupts enteric neural crest development.
|
|
Ednrb knockout mice[62]
[72]
|
Ednrb mutation reliably models human HAEC, and knockout allows for targeted assessments.
|
|
Ednrb-deficient rats[75]
[76]
[77]
|
Can be naturally occurring null mutation in Ednrb gene as in case of spotting lethal (sl) rats. Similar functional use to the knockout
model but using larger animals (rats).
|
|
Chemical induced
|
Benzalkonium chloride piglet[88]
|
Chemical induction under general anesthesia of 5-day-old piglets leading to induced
partial aganglionosis and HAEC-like development.
|
|
Ex vivo
|
Intestinal organoids[89]
|
Cultured organoids from intestinal stem cells and pluripotent stem cells of animals
or humans can help mimic intestinal conditions and be utilized to understand the effect
of chemical, physical, and environmental conditions which can lead to aganglionosis
and HAEC development.
|
Abbreviation: HAEC, Hirschsprung-associated enterocolitis.
By utilizing some of these models among other newly developed ones, several interesting
findings have been identified with respect to HAEC etiology which may provide therapeutic
targets. Chen et al found that in the Ednb knockout mouse model the intestinal cells of Cajal lost their C-KIT expression in
the dilated portion of the colon resulting in damaged pacemaker function and intestinal
motility.[86] They found that proinflammatory macrophage activation may act as a phenotypic switch
to intestinal cells of Cajal and as a result represent a promising therapeutic target
for HAEC. Another recent study using a glial cell line-derived neurotrophic factor
receptor α-1 (GFRa1) hypomorphic mouse model demonstrated that a 70 to 80% reduction
in GFRa1 results in HSCR and associated HAEC in mice and that this process proceeds
from goblet cell dysplasia and mucin abnormalities to epithelial damage.[87] Won et al used endothelin receptor B-deficient rats as a model for long-segment
HSCR and examined the myogenic mechanism involved in the intestinal obstruction of
this model.[76] They found that there was both increased contractility of the smooth muscle and
thickness of the intestinal muscular wall contributing to the intestinal obstruction
in this model, offering a more functional finding. The diversity of models available
and the variety of approaches being taken have greatly expanded our understanding
of HAEC as a result and have helped to reduce its mortality over the last few decades.
Chemical-Induced Model: Toward Clinical Translation
In an attempt to create a model of HAEC closer to the clinical representation of the
human disease, some investigators have employed in large animal such as piglets, whose
GI tract shares ontogenetic similarities close to that of humans. Arnaud et al used
a piglet model of iatrogenic rectosigmoid hypoganglionosis to study the impact of
the enteric nervous system on gut barrier function and microbiota development.[88] This is an effective study in using a larger animal model to demonstrate the effects
of hypoganglionosis especially in replicating the gut barrier issues and proinflammatory
states seen in smaller animal models. Adequate animal models will provide the ability
to assess medical options and especially surgical interventions. However, the use
of larger animal models like this has some severe disadvantages that may make them
of limited use. For instance, it is arduous to maintain a large enough number of piglets
for adequate statistical significance. Additionally, unlike mice, knockout models
and genetic markers are far more difficult to create, as transgenic lines are often
unavailable or producing such lines may take a long time and be very costly.
Ex Vivo Model (Organoids): Emerging Future Directions
Intestinal organoids provide a potential translational bridge between our current
animal models and the human studies. It has been shown that recent advances in culturing
of intestinal and pluripotent stem cells have allowed us to develop an intestinal
organoid model.[89] This model allows for the development of an in vitro model that can utilize either
human or animal tissue to produce mini self-encapsulated systems that mimic GI tissue
and by proxy allow us to both understand what is happening at the intestinal wall
and test potential treatments. Furthermore, the transplantation of these intestinal
organoids by enema has been shown to be able to rescue damaged colonic epithelium
in mice with dextran sulfate sodium-induced colitis.[90] These models may be expanded to study other GI diseases including HSCR and provide
a promising avenue for future research. The combination of mouse/rat models, piglet
model, intestinal organoids, and associated clinical data from human studies/samples
has allowed for a multipronged approach to studying HSCR and HAEC while expanding
our understanding of the disease and providing a platform to test varying potential
treatment options ([Fig. 2]).
Fig. 2 Diverse models of Hirschsprung disease and its associated colitis allow for a greater
potential of identifying possible viable treatments. Human clinical data and tissue,
mouse models that mimic HAEC, a piglet model that reproduces the aganglionosis, and
intestinal organoid models in combination that allow for a greater mechanistic understanding
of the disease and more opportunity to try varying potential treatments.
Conclusion and Future Perspective
Conclusion and Future Perspective
HAEC remains a challenging clinical condition with many unresolved problems. The pathophysiology
of the disease and the mechanisms that lead to the intestinal inflammation and damage
are yet to be fully characterized. Additionally, the clinical management and therapeutic
options for patients are restricted, as demonstrated by the consistent mortality and
morbidity rates of the patients affected. The usage of animal models has advanced
our knowledge on HAEC and helped in providing better overall management for patients.
These models are now well characterized and will continue to be of tremendous use
in the development of future studies designed to better understand the disease and
to translate the findings into clinical interventions. With the continued use of murine
models of HAEC and the more recent use of larger animals and intestinal organoids
derived from human tissue, there is great potential for translational therapeutics.
However, currently, there is a lack of clinical trials available in the field, and
thus, the management strategies remain quite limited. Greater effort should be placed
on translating animal study findings into the clinical application and assessing the
potential safety and efficacy of such treatment options as they may hold great promise
for HAEC patients in the future.
