Muscle hypertrophy and neuroplasticity in the small bowel in short bowel syndrome.

Short bowel syndrome (SBS) is a severe, life-threatening condition and one of the leading causes of intestinal failure in children. Here we were interested in changes in muscle layers and especially in the myenteric plexus of the enteric nervous system (ENS) of the small bowel in the context of intestinal adaptation. Twelve rats underwent a massive resection of the small intestine to induce SBS. Sham laparotomy without small bowel transection was performed in 10 rats. Two weeks after surgery, the remaining jejunum and ileum were harvested and studied. Samples of human small bowel were obtained from patients who underwent resection of small bowel segments due to a medical indication. Morphological changes in the muscle layers and the expression of nestin, a marker for neuronal plasticity, were studied. Following SBS, muscle tissue increases significantly in both parts of the small bowel, i.e., jejunum and ileum. The leading pathophysiological mechanism of these changes is hypertrophy. Additionally, we observed an increased nestin expression in the myenteric plexus in the remaining bowel with SBS. Our human data also showed that in patients with SBS, the proportion of stem cells in the myenteric plexus had risen by more than twofold. Our findings suggest that the ENS is tightly connected to changes in intestinal muscle layers and is critically involved in the process of intestinal adaptation to SBS.

The Surgical Treatment of Toxic Megacolon in Hirschsprung Disease.

OBJECTIVES: Enterocolitis remains the most significant cause of morbidity and mortality in Hirschsprung disease (HD). It could progress into toxic megacolon (TM)-acute dilatation of the colon as accompanying toxic complication of Hirschsprung enterocolitis. It is a devastating complication, especially in infants with so far undiagnosed HD. METHODS: A retrospective analysis of medical records of 4 infants with TM was performed. The diagnosis TM was determined on the basis of clinical information (abdominal pain or tenderness, abdominal distension, diarrhea, bloody diarrhea, and constipation), plain x-rays of the abdomen (segmental or total colonic dilation), and the presence of such criteria (fever, high heart rate, increased white blood cell count, C reactive protein, anemia, dehydration, electrolyte disturbances, hypotension). Surgical management and outcome was evaluated by retrospective chart review. RESULTS: The median duration of symptoms characteristic for TM was 3 days. Toxic megacolon was seen as the first manifestation of previously unknown HD in 3 patients; in 1 newborn, the contrast radiograph was suggestive of HD. In all patients, conservative treatment was failed. Three patients were treated with surgical decompression and ileostomy only. In all these cases, severe complications occurred, consequently 2 of them died. In 1 patient, a resection of the transverse dilated colon additionally was performed. This patient had no complications in postoperative period and survived. CONCLUSIONS: Because of the high mortality in patients with TM that were treated medically or with colonic decompression, a resection of massively distended part of the colon should be performed.

Quality of Life of Children with Short Bowel Syndrome from Patients' and Parents' Points of View.

Despite limited research, existing studies using generic quality of life (QOL) tools indicate decreased physical health and compromised emotional functioning in children with IF. This study investigates QOL in children with short bowel syndrome (SBS) and its determinants. The study included 57 pediatric patients with SBS treated at Mannheim's University Hospital between 1998 and 2014. To evaluate QOL, the KINDL questionnaire was used. Three age-specific questionnaire variants were employed, and parental proxy reports were collected. Most patients underwent intestinal lengthening procedures, with varying primary diagnoses. A comparison with healthy children from the patient's perspective revealed no difference but from the parent's perspective showed lower QOL in SBS patients, especially regarding physical and mental well-being. QOL varied with age, with 7-10-year-olds reporting the lowest scores. Several factors, including independence from parenteral nutrition and the presence of a complete colon, positively influenced QOL. The independence of parenteral nutrition and the presence of a complete colon positively influenced QOL. The Bianchi technique for intestinal lengthening has also shown promise but needs further research. The observation sample in this study is too small to generalize about the whole population of SBS patients. However, this study shows that many health and treatment factors affect QOL, and a large multicenter study is necessary. Our findings underline the importance of appropriate psychological support for children with SBS and their families.

Role of Inflammation and the NF-κB Signaling Pathway in Hirschsprung's Disease.

Hirschsprung's disease (HSCR, incidence 1/5000 live births) is caused by the failure of neural crest-derived precursors to migrate, survive, proliferate, or differentiate during the embryonic development of the Enteric Nervous System (ENS), which could be disrupted by many factors, including inflammatory processes. The NF-κB family controls several biological processes, including inflammation, neurogenesis, and cell migration. With the aim of studying the potential role of NF-κB in HSCR, we have analyzed the expression of the NF-κB main subunits and other NF-κB-related genes by RT-qPCR in HSCR tissue samples (sub-divided into ganglionic and aganglionic segments). We found decreased gene expression of the NF-κB main subunit RELA but also of NFKBIA, TNFA, TFGBR2, and ERBB3 in the pathologic distal aganglionic segments compared to the proximal ganglionic segments. Moreover, we could also confirm the lower protein expression of RelA/p65 in the aganglionic distal segments by immunofluorescence staining. Further, we show that the expression of RelA/p65 protein in the proximal segments concurs with lymphocyte infiltration in the bowel tissue, indicating a pro-inflammatory activation of p65 in the proximal ganglionic HSCR tissue in the patients analyzed. All in all, our findings suggest that the modulation of NF-κB signaling in the neuro-enteric system does obviously contribute to the pathological effects of HSCR.

Intra-arterial delivery of neurospheres into isolated perfused porcine colons: a proof of concept.

Cell replacement in aganglionic intestines is a promising, yet merely experimental tool for the therapy of congenital dysganglionosis of the enteric nervous system like Hirschsprung disease. While the injection of single cells or neurospheres to a defined and very restricted location is trivial, the translation to the clinical application, where large aganglionic or hypoganglionic areas need to be colonized (hundreds of square centimetres), afford a homogeneous distribution of multiple neurospheres all over the affected tissue areas. Reaching the entire aganglionic area in vivo is critical for the restoration of peristaltic function. The latter mainly depends on an intact nervous system that extends throughout the organ. Intra-arterial injection is a common method in cell therapy and may be the key to delivering cells or neurospheres into the capillary bed of the colon with area-wide distribution. We describe an experimental method for monitoring the distribution of a defined number of neurospheres into porcine recta ex vivo, immediately after intra-arterial injection. We designed this method to localize grafting sites of single neurospheres in precise biopsies which can further be examined in explant cultures. The isolated perfused porcine rectum allowed us to continuously monitor the perfusion pressure. A blockage of too many capillaries would lead to an ischaemic situation and an increase of perfusion pressure. Since we could demonstrate that the area-wide delivery of neurospheres did not alter the overall vascular resistance, we showed that the delivery does not significantly impair the local circulation.

Associations of Mucosal Nerve Fiber Innervation Density with Hirschsprung-Associated Enterocolitis: A Retrospective Three-Center Cohort Study.

OBJECTIVE: Hirschsprung's disease (HSCR) is a congenital intestinal neurodevelopmental disorder characterized by the absence of enteric ganglion cells in the distal colon. Although Hirschsprung-associated enterocolitis (HAEC) is the most frequent life-threatening complication in HSCR, to date reliable biomarkers predicting the likelihood of HAEC are yet to be established. We established a three-center retrospective study including 104 HSCR patients surgically treated between 1998 and 2019. MATERIALS AND METHODS: Patient-derived cryopreserved or paraffin-preserved colonic tissue at surgery was analyzed via ßIII-tubulin immunohistochemistry. We subsequently determined extrinsic mucosal nerve fiber density in resected rectosigmoid specimens and classified HSCR patients accordingly into nerve fiber-high or fiber-low groups. We compared the distribution of clinical parameters obtained from medical records between the fiber-high (n = 36) and fiber-low (n = 68) patient groups. We assessed the association between fiber phenotype and enterocolitis using univariate and multivariate logistic regression adjusted for age at operation. RESULTS: Enterocolitis was more prevalent in patients with sparse mucosal nerve fiber innervation (fiber-low phenotype, 87%) compared with the fiber-high phenotype (13%; p = 0.002). In addition, patients developing enterocolitis had a younger age at surgery (3 vs. 7 months; p = 0.016). In the univariate analysis, the odds for enterocolitis development in the fiber-low phenotype was 5.26 (95% confidence interval [CI], 1.67-16.59; p = 0.005) and 4.01 (95% CI, 1.22-13.17; p = 0.022) when adjusted for age. CONCLUSION: Here, we showed that HSCR patients with a low mucosal nerve fiber innervation grade in the distal aganglionic colon have a higher risk of developing HAEC. Consequently, histopathologic analysis of the nerve fiber innervation grade could serve as a novel sensitive prognostic marker associated with the development of enterocolitis in HSCR patients.

Impact of cryopreservation on viability, gene expression and function of enteric nervous system derived neurospheres.

Introduction: Impairment of both the central and peripheral nervous system is a major cause of mortality and disability. It varies from an affection of the brain to various types of enteric dysganglionosis. Congenital enteric dysganglionosis is characterized by the local absence of intrinsic innervation due to deficits in either migration, proliferation or differentiation of neural stem cells. Despite surgery, children's quality of life is reduced. Neural stem cell transplantation seems a promising therapeutic approach, requiring huge amounts of cells and multiple approaches to fully colonize the diseased areas completely. A combination of successful expansion and storage of neural stem cells is needed until a sufficient amount of cells is generated. This must be combined with suitable cell transplantation strategies, that cover all the area affected. Cryopreservation provides the possibility to store cells for long time, unfortunately with side effects, i.e., upon vitality. Methods: In this study we investigate the impact of different freezing and thawing protocols (M1-M4) upon enteric neural stem cell survival, protein and gene expression, and cell function. Results: Freezing enteric nervous system derived neurospheres (ENSdN) following slow-freezing protocols (M1-3) resulted in higher survival rates than flash-freezing (M4). RNA expression profiles were least affected by freezing protocols M1/2, whereas the protein expression of ENSdN remained unchanged after treatment with protocol M1 only. Cells treated with the most promising freezing protocol (M1, slow freezing in fetal calf serum plus 10% DMSO) were subsequently investigated using single-cell calcium imaging. Freezing of ENSdN did not alter the increase in intracellular calcium in response to a specific set of stimuli. Single cells could be assigned to functional subgroups according to response patterns and a significant shift towards cells responding to nicotine was observed after freezing. Discussion: The results demonstrate that cryopreservation of ENSdN is possible with reduced viability, only slight changes in protein/gene expression patterns and without an impact on the neuronal function of different enteric nervous system cell subtypes, with the exception of a subtle upregulation of cells expressing nicotinergic acetylcholine receptors. In summary, cryopreservation presents a good method to store sufficient amounts of enteric neural stem cells without neuronal impairment, in order to enable subsequent transplantation of cells into compromised tissues.