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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.
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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.
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Síndrome do Intestino Curto , Criança , Ratos , Humanos , Animais , Síndrome do Intestino Curto/etiologia , Síndrome do Intestino Curto/metabolismo , Nestina , Ratos Sprague-Dawley , Íleo/metabolismo , Íleo/cirurgia , Modelos Animais de Doenças , Plasticidade NeuronalRESUMO
BACKGROUND: Demographic change will lead to a serious shortage of doctors in the inpatient and outpatient sectors in the next years. To ensure that medical care is provided in the future, various models of medical assistant professions have been created in the past, both at state and federal level. Due to the diversity of these training courses, advanced training courses and study paths, it is becoming increasingly difficult to keep track of fields of application, focal points of activities, etc. METHOD: An analysis of the currently existing models and professions of physician assistants was carried out. The models considered are VERAH, AgnESzwei, EVA, MoNi, MoPra, OTA/ATA, CTA, ANP and PA. RESULTS: VERAH, AgnESzwei, EVA, MoNi and MoPra carry out their activities in primary care. OTA/ATA, CTA, ANP and PA, on the other hand, are active in clinical inpatient care (PA also in outpatient care). All curricula show significant differences in entrance requirements, length of time and content. VERAH and AgnESzwei, EVA, MoNi and MoPra have the lowest entrance requirements and the shortest advanced training. VERAH and AgnESzwei focus on case management, while OTA/ATA or CTA training and ANP as well as PA studies focus on practical clinical use and the transfer of medical knowledge. While CTAs and OTAs/ATAs can work in the surgery field only and ANP mainly in the nursing field, PA can work both in the clinical and outpatient field. CONCLUSIONS: Due to the already existing doctors shortage, which will increase in the future, medical care can only be guaranteed by interprofessional team building. To this end, the professional associations and the associations of Statutory Health-Insurance physicians are called to develop appropriate models and remuneration.
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Assistentes Médicos , Currículo , Alemanha , Humanos , Programas Nacionais de Saúde , Atenção Primária à SaúdeRESUMO
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.
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Enterocolite/cirurgia , Doença de Hirschsprung/cirurgia , Megacolo Tóxico/cirurgia , Enterocolite/diagnóstico por imagem , Evolução Fatal , Doença de Hirschsprung/diagnóstico por imagem , Humanos , Lactente , Recém-Nascido , Masculino , Megacolo Tóxico/diagnóstico por imagem , Megacolo Tóxico/etiologia , Estudos RetrospectivosRESUMO
BACKGROUND: The coexistence of congenital diaphragmatic hernia (CDH) with esophageal atresia (EA) has only been reported occasionally in literature. Series of patients from a single institution with comparison of different postnatal therapeutic approaches have not been reported. We describe our management in this unique cohort of patients and discuss the procedures that can lead to successful outcomes in this association of congenital anomalies. METHODS: The surgical approaches and outcome of six neonates with CDH associated with EA and distal tracheo-esophageal fistula (TEF) are discussed. RESULTS: Five newborns were treated surgically, while one patient with trisomy 18 only received palliative treatment. In four patients TEF was ligated during laparotomy for CDH repair. Secondary surgery was performed for correction of EA via thoracotomy after 4-6 weeks (primary anastomosis in two patients, Foker's-technique in one patient, one patient deceased prior to secondary surgery). All three surviving patients required fundoplication due to severe gastro-esophageal reflux during the first year of life. Two patients also required dilatation for anastomotic stricture. In one preterm infant correction of both malformations was accomplished during one surgical intervention. The herniated organs were eventrated and temporarily placed into a silastic bag to allow a mediastinal shift to the left. Thus a continuous ventilation of the right lung with minimal compression and sufficient oxygenation was possible during esophageal repair via a right-sided thoracotomy and extrapleural approach. No further surgery was required so far. CONCLUSIONS: Definitive surgical correction in newborns with CDH and EA was so far accomplished with multiple surgical interventions. Ligation of TEF via an abdominal approach with repair of CDH followed by delayed repair of EA is prone to stenosis and gastro-esophageal reflux due to loss of esophageal length. With a new combination of established surgical methods a single-staged correction of both malformations is possible. This new approach might help to preserve sufficient length of esophagus to accomplish primary anastomosis without tension and therefore avoid long-term morbidity and repetitive surgeries.
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Anormalidades Múltiplas/cirurgia , Atresia Esofágica/cirurgia , Esofagoplastia/métodos , Hérnias Diafragmáticas Congênitas/cirurgia , Herniorrafia/métodos , Fístula Traqueoesofágica/cirurgia , Feminino , Seguimentos , Refluxo Gastroesofágico/cirurgia , Humanos , Lactente , Recém-Nascido , Masculino , Fístula Traqueoesofágica/congênito , Resultado do TratamentoRESUMO
AIM: To characterize the influence of location, species and treatment upon RNA degradation in tissue samples from the gastrointestinal tract. METHODS: The intestinal samples were stored in different medium for different times under varying conditions: different species (human and rat), varying temperature (storage on crushed ice or room temperature), time point of dissection of the submucous-mucous layer from the smooth muscle (before or after storage), different rinsing methods (rinsing with Medium, PBS, RNALater or without rinsing at all) and different regions of the gut (proximal and distal small intestine, caecum, colon and rectum). The total RNA from different parts of the gut (rat: proximal and distal small intestine, caecum, colon and rectum, human: colon and rectum) and individual gut layers (muscle and submucosal/mucosal) was extracted. The quality of the RNA was assessed by micro capillary electrophoresis. The RNA quality was expressed by the RNA integrity number which is calculated from the relative height and area of the 18 S and 28 S RNA peaks. From rat distal small intestine qPCR was performed for neuronal and glial markers. RESULTS: RNA obtained from smooth muscle tissue is much longer stable than those from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, on ice it is stable at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. RNA obtained from the submucosal/mucosal layer always showed a much worse amplification rate than RNA from muscle tissue. In general RNA harvested from rat tissue, either smooth muscle layer or submucosal/mucosal layer is much longer stable than RNA from human gut tissue, and RNA obtained from smooth muscle tissue shows an increased stability compared to RNA from submucosal/mucosal tissue. At RT muscle RNA degrades after one day, while the stability on ice lasts at least three days. Cleaning and separation of gut layers before storage and use of RNALater, maintains the stability of muscle RNA at RT for much longer periods. Different parts of the gut show varying degradation periods. The RNA from muscle and submucosal/mucosal tissue of the proximal small intestine degrades much faster than the RNA of distal small intestine, caecum or colon with rectum. RNA obtained from the submucosal/mucosal layer always showed a much more reduced amplification rate than RNA from muscle tissue [ß-Tubulin III for muscle quantification cycle (Cp): 22.07 ± 0.25, for ß-Tubulin III submucosal/mucosal Cp: 27.42 ± 0.19]. CONCLUSION: Degradation of intestinal mRNA depends on preparation and storage conditions of the tissue. Cooling, rinsing and separating of intestinal tissue reduce the degradation of mRNA.
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Mucosa Intestinal/química , Intestinos/química , Músculo Liso/química , Estabilidade de RNA , RNA Mensageiro/análise , Manejo de Espécimes/métodos , Animais , Animais Recém-Nascidos , Pré-Escolar , Dissecação , Eletroforese Capilar , Humanos , Lactente , Mucosa Intestinal/anatomia & histologia , Intestinos/anatomia & histologia , Músculo Liso/anatomia & histologia , Neuroglia/química , Neurônios/química , RNA Ribossômico 18S/análise , RNA Ribossômico 28S/análise , Ratos Sprague-Dawley , Especificidade da Espécie , Temperatura , Fatores de TempoRESUMO
Stem cell therapies seem to be an appropriate tool for the treatment of a variety of diseases, especially when a substantial cell loss leads to a severe clinical impact. This is the case in most neuronal cell losses. Unfortunately, adequate neural stem cell sources are hard to find and current alternatives, such as induced programmed stem cells, still have incalculable risks. Evidence of neurogenesis in the adult human enteric nervous system brought up a new perspective. In humans the appendix harbors enteric neuronal tissue and is an ideal location where the presence of neural stem cells is combined with a minimal invasive accessibility. In this study appendices from adults and children were investigated concerning their neural stem cell potential. From each appendix tissue samples were collected, and processed for immunohistochemistry or enteric neural progenitor cell generation. Free-floating enteric neurospheres (EnNS's) could be generated after 6 days in vitro. EnNS's were either used for transplantation into rat brain slices or differentiation experiments. Both transplanted spheres and control cultures developed an intricate network with glia, neurons and interconnecting fibers, as seen in primary enteric cultures before. Neuronal, glial and neural stem cell markers could be identified both in vitro and in vivo by immunostaining. The study underlines the potential of the enteric nervous system as an autologous neural stem cell source. Using the appendix as a potential target opens up a new perspective that might lead to a relatively unproblematic harvest of neural stem cells.
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Sistema Nervoso Entérico/citologia , Trato Gastrointestinal/citologia , Células-Tronco Neurais/citologia , Adolescente , Adulto , Idoso , Animais , Técnicas de Cultura de Células/métodos , Criança , Pré-Escolar , Humanos , Lactente , Pessoa de Meia-Idade , Ratos , Adulto JovemRESUMO
The largest part of the peripheral nervous system is the enteric nervous system (ENS). It consists of an intricate network of several enteric neuronal subclasses with distinct phenotypes and functions within the gut wall. The generation of these enteric phenotypes is dependent upon appropriate neurotrophic support during development. Glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor-2 (FGF2) play an important role in the differentiation and function of the ENS. A lack of GDNF or its receptor (Ret) causes intestinal aganglionosis in mice, while fibroblast growth factor receptor signaling antagonist is identified as regulating proteins in the GDNF/Ret signaling in the developing ENS. Primary myenteric plexus cultures and wholemount preparations of wild type (WT) and FGF2-knockout mice were used to analyze distinct enteric subpopulations. Fractal dimension (D) as a measure of self-similarity is an excellent tool to analyze complex geometric shape and was applied to classify the subclasses of enteric neurons concerning their individual morphology. As a consequence of a detailed analysis of subpopulation variations, wholemount preparations were stained for the calcium binding proteins calbindin and calretinin. The fractal analysis showed a reliable consistence of subgroups with different fractal dimensions (D) in each culture investigated. Seven different neuronal subtypes could be differentiated according to a rising D. Within the same D, the neurite length revealed significant differences between wild type and FGF2-knockout cultures, while the subclass distribution was also altered. Depending on the morphological characteristics, the reduced subgroup was supposed to be a secretomotor neuronal type, which could be confirmed by calbindin and calretinin staining of the wholemount preparations. These revealed a reduction up to 40 % of calbindin-positive neurons in the FGF2-knockout mouse. We therefore consider FGF2 playing a more important role in the fine-tuning of the ENS during development as previously assumed.
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Sistema Nervoso Entérico/metabolismo , Fator 2 de Crescimento de Fibroblastos/deficiência , Neurônios/metabolismo , Animais , Calbindina 2 , Calbindinas , Morte Celular , Células Cultivadas , Sistema Nervoso Entérico/crescimento & desenvolvimento , Sistema Nervoso Entérico/patologia , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Fractais , Genótipo , Interpretação de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/metabolismo , Neuritos/patologia , Neurônios/classificação , Neurônios/patologia , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteína G de Ligação ao Cálcio S100/metabolismoRESUMO
INTRODUCTION: The transplantation of neural crest derived stem cells (NCSC) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. Influences of HSCR-smooth-muscle-protein extracts upon isolated myenteric plexus cells, dissociated dorsal root ganglia and NCSC were studied in vitro to investigate the quality of this microenvironment effects. METHODS: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) and transforming-growth-factor-ß-1 (TGFß-1) concentration were measured in the homogenates from the individual segment using ELISA. Myenteric plexus and dissociated dorsal root ganglia (DRG) cultures, as well as NCSCs were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival, and branching was evaluated. RESULTS AND CONCLUSIONS: The amount of the factors varied considerably between the individual segments and also from patient to patient. Four major expression patterns could be detected. While all extracts tested lead to a significant increase in neurite outgrowth compared to the control, extracts from proximal segments tended to have more prominent effects. In one experiment, extracts from all individual segments of a single patient were tested. Neurite outgrowth, neuronal survival, and branching pattern varied from segment to segment, but all HSCR-muscle-protein extracts increased neuronal survival and network formation. Smooth muscle protein from aganglionic bowel supports the survival and outgrowth of myenteric neurons and NCSCs and is so an appropriate target for neural stem cell treatment.
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Microambiente Celular , Trato Gastrointestinal/patologia , Doença de Hirschsprung/patologia , Plexo Mientérico/crescimento & desenvolvimento , Plexo Mientérico/patologia , Agregação Celular , Diferenciação Celular , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Gânglios Espinais/metabolismo , Gânglios Espinais/patologia , Regulação da Expressão Gênica , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Doença de Hirschsprung/genética , Doença de Hirschsprung/cirurgia , Humanos , Lactente , Recém-Nascido , Proteínas Musculares/metabolismo , Neuritos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Extratos de Tecidos , Fator de Crescimento Transformador beta/metabolismoRESUMO
BACKGROUND AND AIMS: The transplantation of neural crest derived stem cells (NCSC's) is a potent alternative for the treatment of Hirschsprung's disease (HSCR). Cells to be transplanted should find an appropriate microenvironment to survive and differentiate. To investigate the quality of this microenvironment, effects of HSCR-smooth-muscle-protein extracts upon NCSC's were studied in vitro. METHODS: Postnatal human gut from children undergoing colonic resection due to HSCR was divided in segments. Smooth muscle was dissected and homogenized. Glial-cell-line-derived-neurotrophic-factor (GDNF) concentration was measured in the homogenates from the individual segment using ELISA. NCSC's were exposed to protein extracts derived from ganglionic and aganglionic HSCR segments, and their effect upon neurite outgrowth, survival and branching was evaluated. RESULTS: The amount of the factors varied considerably between the proximal and distal segments, and also from patient to patient. While extracts from proximal segments tended to have more prominent effects, all HSCR-muscle-protein extracts increased neuronal survival and network formation. CONCLUSION: Muscle protein from aganglionic bowel supports the survival and outgrowth of NCSC's and is so an appropriate target for neural stem cell treatment.
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Colo/patologia , Doença de Hirschsprung/patologia , Proteínas Musculares/metabolismo , Músculo Liso/metabolismo , Células-Tronco/citologia , Diferenciação Celular , Pré-Escolar , Colectomia , Colo/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/cirurgia , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Proteínas Musculares/genética , RNA Mensageiro/genética , Transplante de Células-Tronco/métodos , Células-Tronco/metabolismoRESUMO
The human enteric nervous system (ENS) derives from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. During development of the NCC, a rearrangement of various cytoskeletal intermediate filaments such as nestin, peripherin, or alpha-internexin takes place. Although all are related to developing neurons, nestin is also used to identify neural stem cells. Until now, information about the prenatal development of the human ENS has been very restricted, especially concerning potential stem cells. In this study the expression of nestin, peripherin, and alpha-internexin, but also of neuronal markers such as protein gene product (PGP) 9.5 and tyrosine hydroxylase, were investigated in human fetal and postnatal gut. The tissue samples were rapidly removed and subsequently processed for immunohistochemistry or immunoblotting. Nestin could be detected in all samples investigated with the exception of the 9th and the 12th week of gestation (WOG). Although the neuronal marker PGP9.5 was coexpressed with nestin at the 14th WOG, this could no longer be observed at later time points. Alpha-internexin and peripherin expression also did not appear before the 14th WOG, where they were coexpressed with PGP9.5. This study reveals that the intermediate filament markers investigated are not suitable to detect early neural crest stem cells.
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Sistema Nervoso Entérico/metabolismo , Proteínas de Filamentos Intermediários/biossíntese , Mucosa Intestinal/metabolismo , Neurônios/metabolismo , Adolescente , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , Criança , Pré-Escolar , Sistema Nervoso Entérico/embriologia , Sistema Nervoso Entérico/crescimento & desenvolvimento , Idade Gestacional , Humanos , Immunoblotting , Imuno-Histoquímica , Lactente , Recém-Nascido , Intestinos/embriologia , Intestinos/crescimento & desenvolvimento , Glicoproteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , Nestina , Periferinas , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/biossíntese , Ubiquitina Tiolesterase/biossínteseRESUMO
BACKGROUND: The human enteric nervous system (ENS) descends from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. The development of this entity strongly depends on the supply of an appropriate support with trophic factors during organogenesis. The lack of important factors, such as glial cell line-derived neurotrophic factor, leads to severe disturbances in the ENS and, thus, to motility disorders in children. The isolation of neuronal precursor cells as well as their transplantation after expansion in vitro is therefore a hopeful new approach concerning all forms of dysganglionosis in children. METHODS: We therefore established a way to isolate and expand precursor cells from the developing and postnatal human ENS. Bowel samples were obtained from human fetuses and children (from the 9th week of gestation to 5 years postnatal). Myenteric plexus was isolated by enzymatical digestion and cultivated until spheroid aggregates, the so-called neurospheres, developed. These neurospheres could be differentiated and also be transplanted after dissociation into aganglionic bowel in vitro. RESULTS: Enteric neurospheres could be grown from different gestational ages, including postmortem material. Undifferentiated proliferating precursor cells were kept in culture for up to 72 days and could be differentiated in neurons and glial cells in vitro. CONCLUSION: The first results using isolated enteric neurospheres in aganglionic bowel are quite promising and are a basis to develop an appropriate cell therapy for all kinds of dysganglionosis, especially for cases where a surgical approach is not sufficient or not even possible.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Sistema Nervoso Entérico/citologia , Enteropatias/cirurgia , Intestinos/inervação , Doenças do Sistema Nervoso/cirurgia , Transplante de Células-Tronco/métodos , Coleta de Tecidos e Órgãos/métodos , Células Cultivadas , Pré-Escolar , Feminino , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Intestinos/citologia , GravidezRESUMO
The enteric nervous system in vertebrates is the most complex part of the peripheral nervous system. Concerning chemical coding, ultrastructure and neuronal circuits, it is more similar to the central than to the peripheral nervous system. Its networks, the myenteric and submucous plexus are integrated in the gut wall. The enteric nervous system is a system of high plasticity, which not only changes during pre- and postnatal development, but also with disease or changing dietary habits. The Aim of this study was to elucidate changes in protein expression during the first two postnatal weeks in the rat myenteric plexus. Colonic and duodenal myenteric plexus from newborn (P1) and fourteen-day old (P14) Sprague-Dawley rats was isolated following a procedure that combines enzymatic digestion and mechanical agitation. The neuronal tissue was collected and processed for two-dimensional gel electrophoresis (2-DE). The obtained 2-D gels were stained with silver for image analysis or with colloidal Coomassie for subsequent protein identification. Gels from the various samples showed a high degree of consistence concerning protein-spots found in all preparations. Nevertheless, there was a number of proteins that were clearly detected in one sample but not, or only in significantly smaller amounts in the other. Several differentially expressed proteins in the postnatal myenteric plexus were identified with MALDI-TOF mass spectrometry. Especially stathmin, polyubiquitin and heterogeneous nuclear ribonucleoprotein seem to play an important role in pre- and postnatal development. 2-DE combined with mass spectrometry can help to identify pathological relevant proteins in the enteric nervous system, and so deliver a valuable tool for the early diagnosis of also central nervous system diseases by using biopsies from the gut.
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Plexo Mientérico/crescimento & desenvolvimento , Plexo Mientérico/metabolismo , Proteoma/análise , Animais , Animais Recém-Nascidos , Eletroforese em Gel Bidimensional , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
INTRODUCTION: After successful experimental autologic-allotopic ileum mucosa transplantation, we investigated the remodeling of the transplanted submucous and mucous plexus, which is essential for the motility of the created colon coat-ileum mucosa complex. METHOD: In 8 beagles we transplanted ileum mucosa in a demucosed vascularized transverse colon segment, which was reanastomosed with the small bowel immediately after transplantation. Four weeks later the animals were sacrificed and histology specimens taken from the anastomosis site of the colon coat-ileum mucosa complex, allowed comparison between transplanted and normal mucosa in the same section. After fixation in 4% formaldehyde and PBS the samples were embedded in paraffin and 7 micro m sections were prepared. The distribution of nerve fibers and submucous ganglia were examined in dewaxed sections, using antisera against protein gene product (PGP9.5), a general neuronal marker. RESULTS: The submucosal ganglia were prominent in all samples but they were smaller and the submucous nerve cells within the ganglia were less numerous compared to the controls. The innervation of the transplanted ileum mucosa was reduced as the number of nerve fibers in the mucosal villi was decreased. Besides these neuromorphologic changes the transplanted mucosa showed a slightly higher rate of shortened villi compared to normal ileum mucosa. CONCLUSIONS: After ileum mucosa transplantation the submucosal ganglia are smaller and less numerous. Furthermore there is a considerable loss of nerve fibers in the mucosal layer. Additionally a loss of microvilli in the transplanted ileum mucosa was found. Whether these findings represent a state of remodeling or a slow atrophy of the enteric nervous system in the transplanted areas is currently under investigation.
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Íleo/transplante , Mucosa Intestinal/transplante , Neurônios/patologia , Transplante de Órgãos/métodos , Animais , Cães , Gânglios Autônomos/patologia , Motilidade Gastrointestinal/fisiologia , Íleo/inervação , Íleo/patologia , Íleo/fisiologia , Mucosa Intestinal/inervação , Mucosa Intestinal/patologia , Mucosa Intestinal/fisiologia , Modelos AnimaisRESUMO
The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm(2) culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.