Enteric nervous system abnormalities are present in human necrotizing enterocolitis: potential neurotransplantation therapy
© Zhou et al.; licensee BioMed Central Ltd. 2013
Received: 3 August 2013
Accepted: 11 November 2013
Published: 25 December 2013
Intestinal dysmotility following human necrotizing enterocolitis suggests that the enteric nervous system is injured during the disease. We examined human intestinal specimens to characterize the enteric nervous system injury that occurs in necrotizing enterocolitis, and then used an animal model of experimental necrotizing enterocolitis to determine whether transplantation of neural stem cells can protect the enteric nervous system from injury.
Human intestinal specimens resected from patients with necrotizing enterocolitis (n = 18), from control patients with bowel atresia (n = 8), and from necrotizing enterocolitis and control patients undergoing stoma closure several months later (n = 14 and n = 6 respectively) were subjected to histologic examination, immunohistochemistry, and real-time reverse-transcription polymerase chain reaction to examine the myenteric plexus structure and neurotransmitter expression. In addition, experimental necrotizing enterocolitis was induced in newborn rat pups and neurotransplantation was performed by administration of fluorescently labeled neural stem cells, with subsequent visualization of transplanted cells and determination of intestinal integrity and intestinal motility.
There was significant enteric nervous system damage with increased enteric nervous system apoptosis, and decreased neuronal nitric oxide synthase expression in myenteric ganglia from human intestine resected for necrotizing enterocolitis compared with control intestine. Structural and functional abnormalities persisted months later at the time of stoma closure. Similar abnormalities were identified in rat pups exposed to experimental necrotizing enterocolitis. Pups receiving neural stem cell transplantation had improved enteric nervous system and intestinal integrity, differentiation of transplanted neural stem cells into functional neurons, significantly improved intestinal transit, and significantly decreased mortality compared with control pups.
Significant injury to the enteric nervous system occurs in both human and experimental necrotizing enterocolitis. Neural stem cell transplantation may represent a novel future therapy for patients with necrotizing enterocolitis.
Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in neonates and the leading cause of death in premature babies . Current overall mortality rates are ~30%, with higher rates for very low birth weight babies. This high morbidity and mortality represent a significant medical problem and a costly burden to society.
Although the pathogenesis of NEC is not completely understood, prematurity is considered the most important risk factor for the development of the disease. In addition, exclusive formula feeding is associated with an increased incidence of NEC, while promotion of breast milk feeding may decrease the incidence of NEC [2–4]. The enteric nervous system (ENS), located in the wall of the intestine, is the largest and most complex division of the peripheral nervous system. The newborn ENS is not fully developed at birth and undergoes neuroplasticity during early postnatal stages . The content of neurotrophic factors and cytokines in human breast milk contribute to the postnatal development of the ENS . An immature ENS has been observed in premature infants, represented as decreased organized intestinal motility. It is thought that this intrinsic ENS immaturity may contribute to the development of NEC . Intestinal motility is mainly regulated by neurotransmitters produced by myenteric neurons. Neuronal nitric oxide synthase (nNOS)-producing neurons and choline acetyl transferase (chAT)-producing neurons are involved in the regulation of intestinal motility, and nNOS/chAT misbalance has been reported in certain intestinal motility disorders . Loss of enteric neurons in the submucosa has been demonstrated in patients with NEC, and some reports have shown NEC-induced myenteric plexus alterations [9, 10]. In addition, post-NEC complications such as intestinal dysmotility, stricture, and recurrent abdominal distention have been widely reported [11, 12]. The intestinal dysfunction that is present after successful medical or surgical management of NEC suggests that the compromised ENS is not fully recovered from the acute intestinal insult.
Similar to the central nervous system, neurons in the ENS have restricted potential for regeneration after injury. Limited neurogenesis may lead to a failure to reverse the neuronal cell loss that occurs during NEC, leading to compromised intestinal function long after recovery from the acute disease. Transplantation of intestinal enteric neurons to restore the neuronal cell loss associated with NEC may represent a future therapeutic option.
In the current study we have documented ENS abnormalities in patients with NEC. Furthermore, we have administered neural stem cells (NSCs) to rat pups with experimental NEC, documented engraftment of the transplanted NSCs in injured intestine, and confirmed improved ENS integrity and intestinal motility post transplantation.
Materials and methods
Human intestinal tissue collection
Approval for the use of human intestinal tissues was obtained from the Institutional Review Board of Nationwide Children’s Hospital, Columbus, OH, USA (Protocol #06-00267). This protocol met the guidelines for waiver of informed consent. Intestinal tissue specimens were obtained from 18 neonates with acute NEC (gestational age range 24 to 37 weeks, mean gestational age 32.1 ± 2.7 weeks, age range 4 to 21 days; mean age 10.7 ± 5.5 days) and eight control patients (gestational age range 28 to 36 weeks, mean gestational age 35.9 ± 1.4 weeks, age range 1 to 6 days; mean age 1.7 ± 1.4 days) with small bowel atresia. All patients chosen for examination underwent initial bowel resection with ostomy creation followed by subsequent stoma closure 2 to 3 months later. Intestinal samples were obtained from the antimesenteric border of the intestines for consistency, given that the ENS distribution in the intestine is variable . Intestinal samples were subjected to RNA extraction, and to hematoxylin and eosin staining and immunohistochemistry.
Neural stem cell culture
Enteric NSCs were generated using a modification of a previously described method . Time mated c57BL/6-Tg (pan-EGFP) mice (Jackson Laboratory, Bar Harbor, MA, USA) were sacrificed and intestines from 12.5-day post‒coitum embryos were dissected into Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12 (DMEM/F12; Invitrogen, Carlsbad, CA, USA). Intestines were dissociated in 50 μg/ml dispase and 50 μg/ml collagenase (Worthington Biochemical, Freehold, NJ, USA) for 60 minutes at 37°C. Intestines were triturated and filtered through 40 μm cell strainers to obtain single cell suspensions. Cells were cultured in 35 mm Petri dishes in NSC culture medium consisting of DMEM/F12 containing 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen), supplemented with 2 mmol/l l-glutamine (Invitrogen), 7.5% (v/v) chick embryo extract (Gemini Bio-products, West Sacramento, CA, USA), 1% (v/v) N2 medium supplement (Sigma‒Aldrich, St Louis, MO, USA), 20 ng/ml mouse basic fibroblast growth factor, and 20 ng/ml mouse epidermal growth factor (Sigma‒Aldrich). NSCs grew as free-floating cellular aggregates known as neurosphere-like bodies.
Animal model of experimental necrotizing enterocolitis
All animal procedures were approved by the Institutional Animal Care and Use Committee of the Research Institute at Nationwide Children’s Hospital (Protocol #04203AR). Experimental NEC was induced as we have described previously [15, 16]. Rat pups were delivered on day 21 of gestation by Cesarean section from timed pregnant rats (Harlan Sprague–Dawley, Indianapolis, IN, USA). Newborn rat pups in the experimental NEC group were maintained in an incubator at 37°C and gavage fed with hypertonic formula containing 15 g Similac 60/40 (Ross Pediatrics, Columbus, OH, USA) in 75 ml Esbilac (Pet-Ag, New Hampshire, IL, USA), a diet that provided 836.8 kJ/kg daily. Pups were exposed to hypoxia with 100% nitrogen for 1 minute followed by hypothermia at 4°C for 10 minutes twice daily beginning 60 minutes after birth for 3 days, with intragastric feeding of lipopolysaccharide (2 mg/kg; Sigma‒Aldrich) 8 hours after birth.
Animal model of recovery from necrotizing enterocolitis
Animals that survived the exposure to experimental NEC described above were recovered by discontinuing exposure to hypoxia/hypothermia on day 3 of life, and then switching pups to normal formula feeding for an additional 4 days to mimic recovery from NEC. On day 3, an intraperitoneal (i.p.) injection of NSCs was performed as described by Martucciello and colleagues . Experimental pups received a single i.p. injection of NSC (50,000 cells in 50 μl DMEM/F12) into the right lower quadrant, while control animals received the same amount of medium carrier intraperitoneally. Normal control pups, designated the breast milk group, were breast fed using surrogate mothers and were not exposed to stress.
Immunohistochemistry and confocal microscopy
Tissue sections (4 μm thickness) from 4% paraformaldehyde-fixed paraffin-embedded human or rat intestine were subjected to immunohistochemistry with specific primary antibodies: mouse anti-human neuronal protein HuC/D (10 μg/ml), rabbit anti-nNOS (2 μg/ml), chicken anti-green fluorescence protein (GFP; 1 μg/ml) (all from Invitrogen); chicken anti-glial fibrillary acid protein (GFAP; 1:1,000), rabbit anti-protein gene product 9.5 (1:500), rabbit anti-peripherin (1:100) (all from Millipore, Billerica, MA, USA); and rabbit anti-cleaved caspase 3 (1:500; Cell Signaling, Danvers, MA, USA). After being washed in phosphate-buffered saline, sections were incubated with fluorophore-conjugated goat anti-mouse IgG (Alexa 647; Molecular Probes, Eugene, OR, USA), anti-rabbit IgG (Alexa 488; Molecular Probes) or anti-chicken Igγ (Cy3; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) at room temperature. Sections were counterstained with 4′,6-diamidino-2-phenylindole. For immunocytochemistry of NSCs, free-floating neurosphere-like bodies were seeded in poly-d-lysine/laminin-coated eight-well chamber slides (BD Bioscience, Bedford, MA, USA) and cultured for an additional 12 hours. To detect nestin expression, neurospheres were incubated with mouse monoclonal antibody Rat-401 (1:100; Developmental Studies Hybridoma Bank of the University of Iowa, Iowa City, IA, USA) overnight at 4°C and then recognized by fluorophore-conjugated goat anti-mouse IgG (Cy3; Jackson ImmunoResearch Laboratories). For NSC differentiation studies, neurospheres were grown in differentiation medium (DMEM/F12 + 10% fetal bovine serum) for 4 days. Differentiated cells were recognized by monoclonal rabbit anti-neuronal class III β-tubulin (Tuj1, 1 μg/ml; Covence, Princeton, NJ, USA) and polyclonal chicken anti-GFAP (1:1,000) respectively. Fluorescence images were captured by confocal microscopy (LSM 710; Carl Zeiss, Thornwood, NY, USA). Images were analyzed using image analysis software (Image Pro Plus; Media Cybernetics, Silver Springs, MD, USA).
Morphology of myenteric plexuses
Cross-sections from human intestinal specimens were used to identify enteric ganglia in myenteric plexuses located between the intestinal longitudinal and circular muscular layers. Intestinal tissue strips were dissected from the anti-mesenteric border of the intestines, and 10 serial sections with a thickness of 4 μm each were examined in order to reduce the possibility of biased observations. Human myenteric plexuses were identified by groups of HuC/D immunoreactive neuronal somata surrounded by glial cells. Total myenteric ganglion numbers in each section were recorded. Neurons were recognized by HuC/D-immunoreactivity, which was displayed as an artificial gray color, and neurons were quantified as the mean neuronal number per ganglion. nNOS-expressing functional neurons immunoreactive for nNOS were also counted, and the ratio of nNOS-expressing neurons to total HuC/D immunoreactive neurons per ganglia was calculated. The appearance of glial cells in the myenteric plexus, as displayed by GFAP immunoreactivity, showed very small nuclei with elongated and interconnected processes in the myenteric plexus, making it very difficult to count the glial cells directly. Since myenteric ganglia mainly consist of neurons and supportive glial cells, total glial cell numbers in each ganglion were extrapolated by subtracting the number of neurons from the total cell numbers as recognized with 4′,6-diamidino-2-phenylindole staining. Lastly, the myenteric neuronal cell area (HuC/D+-stained area) and the myenteric ganglion area were measured in blinded samples using Image Pro Plus software (Media Cybernetics), and the ratio of the areas was calculated .
Real-time reverse-transcription polymerase chain reaction
Longitudinal muscle–myenteric plexus (LMMP) strips consisting of the myenteric plexus and the innervated longitudinal muscles were dissected from fresh human intestines. Total RNA was reverse-transcribed with random hexamers using a first-strand cDNA synthesis kit (Invitrogen-Gibco). Real-time reverse-transcription polymerase chain reaction (RT-PCR) was carried out using a SYBR Green RT-PCR kit (Applied Biosystems, Branchburge, NJ, USA) and an ABI Prism 770 Sequence Detection System (Applied Biosystems). Human nNOS and chAT were amplified using the following primers: nNOS sense, 5′-GGCCCATATTAATCCCTCGT-3′ and nNOS anti-sense, 5′-ACATGAGGGCTCTGCTCACT-3′; chAT sense, 5′-CCACTCCATTCCCACTGACT-3′ and chAT anti-sense, 5′-GAGACGGCGGAAATTAATGA-3′. Amplification of the housekeeper gene (glyceraldehyde 3-phosphate dehydrogenase) cDNA was used as an internal control for quantification. Quantification was performed using Relative Quantification Software, version 1.01 (Applied Biosystems).
LMMP strips of intestines from rat pups were isolated 4 days after NSC transplantation. Tissue lysates were subjected to SDS-PAGE and proteins were transferred to nitrocellulose membranes and subjected to immunoblot analysis. Peripherin, GFAP, and nNOS were detected using primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Blots were developed with the ECL Plus system (Amersham Biosciences, Piscataway, NJ, USA) using Hyperfilm (Amersham Biosciences) for exposure. The intensity of immunoreactive bands on western blots was quantified using the Scion Image Program (Scion Corporation, Frederick, MD, USA) and expressed as the mean ± standard error of the mean.
Intestinal motility assay
Four days after NSC transplantation, rat pups were made nil per os for 4 hours followed by administration of 0.2 ml methylene blue-labeled 10% dextrose solution (Sigma-Aldrich) delivered by duodenal gavage. Animals were euthanized 5 minutes after introduction of the dye. Intestinal transit was measured from the pylorus to the most distal point of migration of the blue dye. The total length of the small intestine was measured and the ratio of dye migration distance/total intestinal length was calculated and transit times were expressed as a percentage of the total intestinal length .
All data are presented as the mean ± standard error of the mean. Statistical analyses were performed using the Student t test or one-way analysis of variance (SigmaPlot 11.0; Systat Software, Inc. San Jose, CA, USA). Absolute P values are shown, with P <0.05.
Enteric nervous system is injured in human necrotizing enterocolitis
Since nNOS and chAT are important regulators of intestinal motility, we next investigated nNOS and chAT mRNA expression in our intestinal samples. Since nNOS-expressing neurons are abundant in the myenteric plexus compared with the submucosal plexus , we intentionally removed the circular muscular layer and its tightly attached submucosal plexus in order to prepare LMMP strips for RT-PCR analysis of the myenteric plexus alone in these experiments. Real-time RT-PCR confirmed that acute NEC was associated with significantly decreased expression of both nNOS and chAT (Figure 2E, F). Whereas nNOS expression remained low, chAT expression increased at the time of stoma closure. The disturbed expression of nNOS and chAT in the intestine during and after NEC suggests that ENS functions are disrupted even during the recovery phase.
Generation of neurospheres from fetal intestine
Enteric nervous system is injured in experimental necrotizing enterocolitis
Neural stem cell transplantation preserves enteric nervous system integrity in experimental necrotizing enterocolitis
Neural stem cell transplantation protects the intestines, promotes motility, and increases survival in experimental necrotizing enterocolitis
Although the pathophysiology of NEC remains elusive, immaturity of the intestinal tract has been implicated in its development. The ENS is comprised of neurons and supportive glial cells present in two major plexuses, the myenteric plexus and the submucosal plexus, that control the physiologic functions of the gastrointestinal tract . A significant reduction in glial cells, associated with a gradual deterioration of intestinal neurons, has been identified in intestine resected for NEC [9, 10]. In this study, we have confirmed that NEC is associated with neuronal and glial cell loss in the ENS. Importantly, the numbers of enteric glial cells remained low in the intestines from NEC patients even months after the acute episode when the patients underwent stoma closure. This suggests that intrinsic immaturity of the ENS, or dysfunctional glial cells, may predispose premature babies to NEC. The decrease in glial cells with NEC is surprising given that ischemia or inflammation is a powerful stimulus to glial cell proliferation or GFAP expression, such as that which occurs with 2,4,6-trinitrobenzene sulfonic acid-mediated inflammation of the gastrointestinal tract , or in inflammatory diseases of the gut such as ulcerative colitis . Acute failure of enteric glia could be an upstream event in the cascade that precipitates the ischemic insult resulting in NEC. A previous study showed that ablation of enteric glial cells leads to fulminant and fatal intestinal inflammation, which is very consistent with the histopathologic changes seen in NEC . While the mechanisms of neuronal cell loss have not been studied in patients with NEC, deprivation of neurotropic factors such as nerve growth factor and glial-derived neurotrophic factor that are normally provided by supportive glial cells may lead to activation of apoptotic pathways. In the current study, we demonstrated the presence of neuronal apoptosis via activation of a caspase-3-dependent pathway. Strong activated caspase-3 immunoreactivity was evident in the myenteric and submucosal ganglia of the intestines from NEC patients, with similar findings observed in the ENS of animals subjected to experimental NEC. Recent reports showed that GFAP-positive glia exert an anti-apoptotic effect on intestinal epithelial cells and preserve mucosal integrity by limiting epithelial damage and promoting epithelial reconstitution during inflammation [28, 29]. Dysfunctional glia in the immature ENS of premature newborns, or lack of growth factors in formula feeding, may contribute to the impairment of the epithelial lining that leads to acute NEC.
Loss of specific neuronal subpopulations in the myenteric plexus has been reported during aging or disease, or in animals with specific genetic defects, indicating that neuronal subpopulations respond differently to injury or growth factor deprivation [30–32]. We found that patients with NEC have loosely packed myenteric neurons, with small neurons remaining in the myenteric ganglia. It is possible that the larger myenteric neurons were affected more dramatically, leading to loss of these cells. Preferential loss of large ganglion cells has also been reported by others [33, 34], and large motor neurons appeared to be more vulnerable to some diseases or to be the first group to die during injury [35, 36]. Further study of the vulnerability of some specific neuronal subpopulations such as motor neurons during NEC is required.
Among the subpopulations of neurons in the ENS, nNOS-immunoreactive neurons are more abundant in the myenteric plexus compared with the submucosal plexus . The constitutive expression of nNOS in the gastrointestinal tract maintains the regulated production of low levels of nitric oxide, which acts as a predominant inhibitory neurotransmitter. Nitric oxide, mainly produced by enteric neurons and glial cells, causes relaxation of the intestinal smooth muscle in response to nerve stimulation, which is important in gut motility [37, 38]. Nitric oxide signaling also regulates proliferation and differentiation of neural precursor cells [39, 40]. Although changes in nNOS with NEC have not been reported, there is one report of decreased nNOS expression in a rat model of lipopolysaccharide-induced intestinal injury . This is the first report to demonstrate that clinical NEC is associated with significantly decreased expression of nNOS not only during the acute event, but also months later. This decreased nNOS expression may explain the long-term intestinal dysmotility seen in NEC patients even after recovery from the acute event.
Current therapy for intestinal dysmotility is limited mainly to palliation, and new treatments for this debilitating condition are clearly needed. A promising intervention involves NSC transplantation. In efforts to develop stem cells therapies for ENS disorders, several sources of NSCs have been explored, including NSCs derived from totipotent embryonic stem cells, NSCs derived from the fetal or adult central nervous system, NSCs derived from fetal or adult intestine (enteric NSCs) and NSCs derived from non-neuronal sources (mesenchymal stem cells, amniotic fluid-derived stem cells) . Enteric NSCs are isolated from the intestine and form neurospheres that contain nestin-positive NSCs capable of differentiating into neurons and glial cells when growth factors are withdrawn. Enteric NSCs, by virtue of their intestinal origin, may be more suitable for therapeutic purposes since they may respond better to intestinal-specific environmental cues upon transplantation [42, 43]. Several reports have already described functional success of NSC transplantation for other diseases. NSCs transplanted either by direct injection into the intestinal wall or by i.p. administration led to the engraftment of NSCs in the myenteric plexus, and the generation of neurons in ganglionated small bowel and colon as well as aganglionic rectum . Whereas direct intramural injection resulted in clumps of cells, i.p. injection resulted in diffuse engraftment throughout the gastrointestinal tract, representing a clinically viable route of administration, especially if generalized areas of intestine need to be treated. We have previously shown that i.p. administration of mesenchymal stem cells is an effective delivery route of stem cells in experimental NEC, as it avoids passive entrapment of transplanted cells in the pulmonary capillary bed . Based on this, we used i.p. administration of NSCs in the current study, and documented engrafted cells in the submucosal and myenteric plexuses after transplantation. However, i.p. injection of NSCs to breast-fed normal rat pups did not result in engraftment of cells in the intestines (data not shown), suggesting that the cytokines and chemokines secreted by the injured intestine in experimental NEC are crucial in directing the mobilization, migration and homing of stem cells to the injured ENS. Others have shown that enteric NSCs isolated from embryonic intestine were committed to differentiate into neurons and glial cells . Although the precise local factors responsible for transplanted NSC differentiation have not been identified, our results suggest that the gut environment provides a predominantly neurogenic drive for NSCs, with differentiated neurons but not glial cells identified in the recipient intestine post transplantation.
For future clinical neurotransplantation in human patients with NEC, collection of NSCs from fetal gut or from postnatal myenteric plexuses is not clinically practical. In a parallel study, we found that NSCs can be selectively induced from amniotic fluid-derived multipotent stem cells. Based on this, amniotic fluid could be collected at the time of delivery, amniotic fluid-derived stem cells cultured, and the cells induced into NSCs in the laboratory. The abundant availability of amniotic fluid meets the requirement for large numbers of stem cells for transplantation. NSC transplantation may represent a novel future potential therapeutic strategy for those premature newborns at highest risk of developing NEC, or those with early stages of the disease.
In summary, our results confirm significant ENS damage during clinical and experimental NEC. Engraftment of enteric NSCs led to significantly increased nNOS expression in the ENS, to preserved intestinal integrity with elongation of villous length, to significantly improved intestinal motility, and to significantly increased survival after experimental NEC. These results support a future potential clinical role for enteric NSC transplantation in patients with NEC.
Choline acetyl transferase
Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F12
Enteric nervous system
Glial fibrillary acid protein
Green fluorescent protein
Longitudinal muscle–myenteric plexus
Neuronal nitric oxide synthase
Neural stem cell
Reverse-transcription polymerase chain reaction.
The authors thank pediatric surgeons from the Department of Pediatric Surgery of Nationwide Children’s Hospital for providing human intestinal tissue specimens. This work was supported by the National Institutes of Health (R01 GM61193 and R01 DK074611).
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