RESUMO
BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.
Assuntos
Colo , Sistema Nervoso Entérico , Doença de Hirschsprung , Imageamento Tridimensional , Microscopia Confocal , Humanos , Doença de Hirschsprung/patologia , Doença de Hirschsprung/diagnóstico por imagem , Doença de Hirschsprung/cirurgia , Colo/inervação , Colo/patologia , Colo/diagnóstico por imagem , Criança , Lactente , Sistema Nervoso Entérico/patologia , Sistema Nervoso Entérico/diagnóstico por imagem , Pré-Escolar , Adolescente , Adulto , Recém-Nascido , Pessoa de Meia-Idade , Feminino , Masculino , Adulto Jovem , Plexo Mientérico/patologia , Plexo Mientérico/diagnóstico por imagem , Íleo/diagnóstico por imagem , Íleo/inervação , Íleo/patologia , Fatores EtáriosRESUMO
BACKGROUND & AIMS: The presence of myenteric plexitis in the proximal resection margins is a predictive factor of early postoperative recurrence in Crohn's disease. To decipher the mechanisms leading to their formation, T-cell interactions with enteric neural cells were studied in vitro and in vivo. METHODS: T cells close to myenteric neural cells were retrospectively quantified in ileocolonic resections from 9 control subjects with cancer and 20 patients with Crohn's disease. The mechanisms involved in T-cell adhesion were then investigated in co-cultures of T lymphocytes with enteric glial cells (glia). Finally, the implication of adhesion molecules in the development of plexitis and colitis was studied in vitro but also in vivo in Winnie mice. RESULTS: The mean number of T cells close to glia, but not neurons, was significantly higher in the myenteric ganglia of relapsing patients with Crohn's disease (2.42 ± 0.5) as compared with controls (0.36 ± 0.08, P = .0007). Co-culture experiments showed that exposure to proinflammatory cytokines enhanced T-cell adhesion to glia and increased intercellular adhesion molecule-1 (ICAM-1) expression in glia. We next demonstrated that T-cell adhesion to glia was inhibited by an anti-ICAM-1 antibody. Finally, using the Winnie mouse model of colitis, we showed that the blockage of ICAM-1/lymphocyte function-associated antigen-1 (LFA-1) with lifitegrast reduced colitis severity and decreased T-cell infiltration in the myenteric plexus. CONCLUSIONS: Our present work argues for a role of glia-T-cell interaction in the development of myenteric plexitis through the adhesion molecules ICAM-1/LFA-1 and suggests that deciphering the functional consequences of glia-T-cell interaction is important to understand the mechanisms implicated in the development and recurrence of Crohn's disease.
Assuntos
Adesão Celular , Técnicas de Cocultura , Doença de Crohn , Molécula 1 de Adesão Intercelular , Plexo Mientérico , Neuroglia , Linfócitos T , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Doença de Crohn/patologia , Doença de Crohn/imunologia , Doença de Crohn/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Plexo Mientérico/patologia , Plexo Mientérico/metabolismo , Plexo Mientérico/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Neuroglia/imunologia , Estudos Retrospectivos , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
AIMS: Our main goals were to investigate the effects of L-glutathione (1%) treatment in Walker-256 tumor-bearing rats by analyzing immunoreactive neurons (IR), responsive to the nNOS enzyme and 3-Nitrotyrosine, in their jejunum myenteric plexus. Moreover, the oxidative state and inflammatory process in these animals were investigated. METHODS: Four experimental groups were utilized: control (C), control treated with L-glutathione (CGT), Walker-256 tumor-bearing rats (TW), and Walker-256 tumor-bearing rats treated with L-glutathione (TWGT). After 14 days of tumor inoculation, the jejunum was collected for immunohistochemical techniques and assessment of oxidative status. Plasma was collected to evaluate oxidative status and measure cytokines. RESULTS: The TW group exhibited a decrease of reduced glutathione in their jejunum, which was prevented in the L-glutathione treated TWGT group. TW animals presented pronounced oxidative stress by increasing levels of lipoperoxidation in their jejunum and malondialdehyde in their plasma; however, the L-glutathione treatment in TWGT group was not able to avoid it. The total antioxidant capacity was altered in groups TW and TWGT, yet the last one had a better index in their plasma. The IL-10, and TNF-α levels increased in TWGT animals. The nNOS-IR neuron density decreased in the jejunum myenteric plexus of the TW group, which was avoided in the TWGT group. The nNOS +3-Nitrotyrosine neurons quantification did not show significative alterations. CONCLUSION: The treatment with L-glutathione (1%) imposed an important defense to some parameters of oxidative stress induced by TW-256, leading to neuroprotection to the loss in the nNOS-IR neuron density.
Assuntos
Neoplasias , Neurônios Nitrérgicos , Ratos , Animais , Jejuno , Ratos Wistar , Neuroproteção , Estresse Oxidativo , Glutationa/metabolismo , Plexo Mientérico/patologia , Neoplasias/metabolismo , Neoplasias/patologiaRESUMO
BACKGROUND: In this prospective cohort study, we evaluated features of "adult-onset megacolon with focal hypoganglionosis." METHODS: We assessed the radiologic, endoscopic, and histopathologic phenotyping and treatment outcomes of 29 patients between 2017 and 2020. Data from community controls, consisting of 19,948 adults undergoing health screenings, were analyzed to identify risk factors. Experts reviewed clinical features and pathological specimens according to the London Classification for gastrointestinal neuromuscular pathology. KEY RESULTS: The median age of the patients with adult-onset megacolon with focal hypoganglionosis at symptom onset was 59 years (range, 32.0-74.9 years), with mean symptom onset only 1 year before diagnosis. All patients had focal stenotic regions with proximal bowel dilatation (mean diameter, 78.8 mm; 95% confidence interval [CI], 72-86). The comparison with community controls showed no obvious risk factors. Ten patients underwent surgery, and all exhibited significant hypoganglionosis: 5.4 myenteric ganglion cells/cm (interquartile range [IQR], 3.7-16.4) in the stenotic regions compared to 278 cells/cm (IQR, 190-338) in the proximal and 95 cells/cm (IQR, 45-213) in the distal colon. Hypoganglionosis was associated with CD3+ T cells along the myenteric plexus. Colectomy was associated with significant symptom improvement compared to medical treatment [change in the Global Bowel Satisfaction score, -5.4 points (surgery) vs. -0.3 points (medical treatment); p < 0.001]. CONCLUSIONS AND INFERENCES: Adult-onset megacolon with focal hypoganglionosis has distinct features characterized by hypoganglionosis due to inflammation. Bowel resection appears to benefit these patients.
Assuntos
Megacolo , Humanos , Adulto , Pessoa de Meia-Idade , Idoso , Estudos Prospectivos , Megacolo/patologia , Colo/patologia , Plexo Mientérico/patologia , ColectomiaRESUMO
Eosinophilic myenteric ganglionitis (EMG) is a rare pathologic finding within the Auerbach myenteric plexus characterized by eosinophilic infiltration on light microscopy. The plexus's ultimate obliteration results in chronic intestinal pseudo-obstruction (CIPO). EMG is almost exclusively seen in the pediatric population. The diagnosis of EMG is made through full-thickness rectal biopsy and EMG is not detectable through routine screening measures such as imaging or colonoscopy. The current treatment modality for this disorder is not standardized, and has often been treated with systemic steroids given its eosinophilic involvement. This case presents a 73-year-old male with chronic constipation presenting with new obstipation in the setting of recent orthopedic intervention requiring outpatient opioids. Admission radiographs were consistent with sigmoid volvulus. Following endoscopic detorsion, exploratory laparotomy revealed diffuse colonic dilation and distal ischemia requiring a Hartmann's procedure. Surgical pathology revealed EMG, increasing the complexity of subsequent surgical decision-making after his urgent operation.
Assuntos
Pseudo-Obstrução Intestinal , Volvo Intestinal , Doenças do Colo Sigmoide , Masculino , Humanos , Criança , Idoso , Volvo Intestinal/complicações , Volvo Intestinal/diagnóstico , Volvo Intestinal/cirurgia , Colo , Pseudo-Obstrução Intestinal/patologia , Pseudo-Obstrução Intestinal/cirurgia , Plexo Mientérico/patologia , Colonoscopia , Doenças do Colo Sigmoide/complicações , Doenças do Colo Sigmoide/diagnósticoRESUMO
CONTEXT.: Intestinal neuronal dysplasia type B (IND B) is a complex entity involving the enteric nervous system, clinically manifested with constipation in infancy. Diagnosis has been established by histopathologic analysis of rectal biopsies. However, the criteria for the diagnosis have been questioned and modified, hindering diagnostic practice. OBJECTIVE.: To analyze the applicability of PTEN immunohistochemistry in the diagnosis of IND B and to compare with control cases and cases of Hirschsprung disease (HD). DESIGN.: PTEN immunohistochemical expression was analyzed in colorectal samples from 29 cases of IND B and compared with 4 control cases and 6 cases of HD. The pattern of PTEN immunoexpression was analyzed in glial cells of the submucosal and myenteric nerve plexuses and in neural fibrils of the muscularis propria using a scoring system. RESULTS.: Marked reduction or absence of PTEN expression was observed in glial cells of the submucosal nerve plexuses in all cases of the IND B group and in the myenteric nerve plexuses in 28 of 29 cases (96.5%). Lack of PTEN expression was detected in neural fibrils within the muscularis propria in 21 of 29 cases (72%) of the IND B group. PTEN expression was positive in the same neural structures of the control and HD groups. CONCLUSIONS.: PTEN immunohistochemistry may be a valuable tool in the diagnostic evaluation of IND B. Lack of or reduction of PTEN expression in neural fibrils within the muscularis propria suggests that involvement of the neuromuscular junction may be a key event in the pathogenesis of the motility disturbance occurring in IND B.
Assuntos
Sistema Nervoso Entérico , Doença de Hirschsprung , Humanos , Imuno-Histoquímica , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Doença de Hirschsprung/complicações , Doença de Hirschsprung/metabolismo , Doença de Hirschsprung/patologia , Constipação Intestinal/patologia , PTEN Fosfo-HidrolaseRESUMO
BACKGROUND: Immaturity of ganglia (IG), an allied disorder of Hirschsprung disease (AD-HSCR), develops as neonatal ileus, but the dysmotility spontaneously resolves after several months. The diagnosis of IG using HE staining is often difficult. We herein report a new pathological finding of IG called the 'palisading-like pattern', which may be helpful for improving the diagnostic accuracy. METHODS: Cases of IG that were managed over the past 28 years were retrospectively reviewed. We investigated the clinical course and pathological findings for Hematoxylin-Eosin (HE) staining. The conventional diagnostic criteria for IG were (1) a normal or slightly increased number of ganglion cells and (2) ganglion cells with small nuclei. RESULTS: Among the 155 cases, 28 were diagnosed with IG, and 10 were retrospectively confirmed by HE staining. A palisading-like pattern was confirmed at the time of the initial ileostomy (median age, 2.5 days), and the palisading-like pattern had completely disappeared by the time of stoma closure (median age, 215 days) in all 10 cases. A palisading-like pattern is not present in other diseases. CONCLUSIONS: Even if immunostaining data are not available for a further analysis, the detection of a palisading-like pattern on HE staining makes an accurate diagnosis possible. LEVEL OF EVIDENCE: LEVEL IV.
Assuntos
Doença de Hirschsprung , Obstrução Intestinal , Pré-Escolar , Gânglios/patologia , Doença de Hirschsprung/diagnóstico , Doença de Hirschsprung/patologia , Humanos , Ileostomia , Recém-Nascido , Obstrução Intestinal/patologia , Plexo Mientérico/patologia , Estudos RetrospectivosRESUMO
Tumour necrosis factor alpha (TNFα) is essential in neuroinflammatory modulation. Therefore, the goal of this study is to reveal the effects of chronic hyperglycaemia and insulin treatment on TNFα expression in different gut segments and intestinal wall layers. TNFα expression was mapped by fluorescent immunohistochemistry and quantitative immunogold electron microscopy in myenteric ganglia of duodenum, ileum and colon. Tissue TNFα levels were measured by enzyme-linked immunosorbent assays in muscle/myenteric plexus-containing (MUSCLE-MP) and mucosa/submucosa/submucous plexus-containing (MUC-SUBMUC-SP) homogenates. Increasing density of TNFα-labelling gold particles is observed in myenteric ganglia from proximal to distal segments and TNFα tissue levels are much more elevated in MUSCLE-MP homogenates than in MUC-SUBMUC-SP samples in healthy controls. In the diabetics, the number of TNFα gold labels is significantly increased in the duodenum, decreased in the colon and remained unchanged in the ileal ganglia, while insulin does not prevent these diabetes-related TNFα changes. TNFα tissue concentration is also increased in MUSCLE-MP homogenates of diabetic duodenum, while decreased in MUC-SUBMUC-SP samples of diabetic ileum and colon. These findings support that type 1 diabetes has region-specific and intestinal layer-dependent effects on TNFα expression, contributing to the regional damage of myenteric neurons and their intestinal milieu.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Trato Gastrointestinal/metabolismo , Hipoglicemiantes/administração & dosagem , Insulina/administração & dosagem , Intestinos/fisiologia , Plexo Mientérico/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/patologia , Trato Gastrointestinal/efeitos dos fármacos , Trato Gastrointestinal/patologia , Intestinos/efeitos dos fármacos , Masculino , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/patologia , Ratos , Ratos WistarRESUMO
Vincristine is widely used in treatment of various malignant tumors. The clinical application of vincristine is accompanied by peripheral neurotoxicity which might not be strictly related to the mechanism of anti-tumor action. There are several possible mechanisms but the effect of vincristine on enteric neurons and the underlying mechanism are still unclear. C57BL6/J mice were systematically treated with vincristine for 10 days, and macrophages were depleted using clodronate liposomes. The colonic myenteric plexus neurons were extracted and cultured in vitro. Macrophages from different parts were extracted in an improved way. In the current study, we demonstrated that system treatment of vincristine resulted in colonic myenteric neurons injury, pro-inflammatory macrophages activation and total gastrointestinal transport time increase. Vincristine promoted the pro-inflammatory macrophages activation individually or in coordination with LPS and increased the expression of pro-inflammatory factors IL-1ß, IL-6, TNF-α via increasing the phosphorylation of ERK1/2 and p38. In addition, pro-inflammatory macrophages led to colonic myenteric neurons apoptosis targeting on SGK1-FOXO3 pathway. These effects were attenuated by inhibitors of the ERK1/2 and p38-MAPK pathways. Importantly, macrophages depletion alleviated colonic myenteric neurons injury and the delay of gastrointestinal motility caused by system treatment of vincristine. Taken together, system treatment of vincristine led to colonic myenteric neurons injury via pro-inflammatory macrophages activation which was alleviated by depletion of macrophages.
Assuntos
Colo/metabolismo , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/fisiologia , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Vincristina/toxicidade , Animais , Antineoplásicos Fitogênicos/toxicidade , Células Cultivadas , Colo/efeitos dos fármacos , Colo/patologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Células RAW 264.7RESUMO
Congenital myenteric hypoganglionosis is a rare developmental disorder characterized clinically by severe and persistent neonatal intestinal pseudoobstruction. The diagnosis is established by the prevalence of small myenteric ganglia composed of closely spaced ganglion cells with sparse surrounding neuropil. In practice, the diagnosis entails familiarity with the normal appearance of myenteric ganglia in young infants and the ability to confidently recognize significant deviations in ganglion size and morphology. We review clinical, histologic, and immunohistochemical findings from 12 patients with congenital myenteric hypoganglionosis in comparison with similar data from age-matched controls and clearly delineate the diagnostic features of the condition. Practical guidelines are provided to assist surgical pathologists, who are likely to encounter this condition only infrequently. The diagnosis typically requires full-thickness intestinal biopsy as the abnormality is confined to the myenteric plexus in many patients. Immunohistochemistry for Hu C/D may be used to confirm hypoganglionosis. Reduced staining for calretinin and NeuN implicates a selective deficiency of intrinsic primary afferent neurons in this disease.
Assuntos
Doenças do Colo/congênito , Doenças do Colo/patologia , Anormalidades do Sistema Digestório/patologia , Plexo Mientérico/patologia , Neurônios/patologia , Criança , Pré-Escolar , Anormalidades do Sistema Digestório/complicações , Feminino , Humanos , Lactente , Pseudo-Obstrução Intestinal/etiologia , Pseudo-Obstrução Intestinal/patologia , MasculinoRESUMO
Plexitis in the proximal margin of intestinal resections are associated with post-operative recurrence of Crohn's disease. To understand their formation, in vitro analyzes were performed. T cells adhered preferentially to neuron and glial cells in mixed primary cultures of enteric nervous system and T cell activation increased their adhesion capacity. Higher number of T lymphocytes in close proximity to enteric glial cells was also observed in the myenteric ganglia of Crohn's patients as compared to control. These data show that close proximity between lymphocytes and enteric neural cells exists and may contribute to the formation of plexitis.
Assuntos
Adesão Celular/fisiologia , Doença de Crohn/metabolismo , Gânglios/metabolismo , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Linfócitos T/metabolismo , Animais , Células Cultivadas , Técnicas de Cocultura , Doença de Crohn/patologia , Sistema Nervoso Entérico/metabolismo , Sistema Nervoso Entérico/patologia , Feminino , Gânglios/patologia , Humanos , Plexo Mientérico/patologia , Neurônios/patologia , Gravidez , Ratos , Ratos Sprague-Dawley , Linfócitos T/patologiaRESUMO
Considering the antioxidant, neuroprotective, inflammatory and nitric oxide modulatory actions of quercetin, the aim of this study was to test the effect of quercetin administration in drinking water (40 mg/day/rat) on neuronal nitric oxide synthase (nNOS), vasoactive intestinal peptide (VIP), overall population of myenteric neurons (HuC/D) and nitric oxide (NO) levels in the jejunal samples from diabetic rats. Male Wistar rats were distributed into four groups (8 rats per group): euglycemic (E), euglycemic administered with quercetin (E+Q), diabetic (D) and diabetic administered with quercetin (D+Q). Rats were induced to diabetes with streptozotocin (35mg/kg/iv) and, after 120 days, the proximal jejunum were collected and processed for immunohistochemical (VIP, nNOS and HuC/D) and chemiluminescence (quantification of tissue NO levels) techniques. Diabetes mellitus reduced the number of nNOS-IR (immunoreactive) (p <0.05) and HuC/D-IR (p <0.001) neurons, however, promoted an increased morphometric area of nNOS-IR neurons (p <0.001) and VIP-IR varicosities (p <0.05). In D+Q group, neuroplasticity effects were observed on HuC/D-IR neurons, accompanied by a reduction of cell body area of neurons nNOS- and VIP-IR varicosities (p <0.05). The NO levels were increased in the E+Q (p <0.05) and D+Q group (p <0.001) compared to the control group. In conclusion, the results showed that quercetin supplementation increased the bioavailability of NO in the jejunum in euglycemic and mitigate the effects of diabetes on nNOS-IR neurons and VIP-IR varicosities in the myenteric plexus of diabetic rats.
Assuntos
Antioxidantes/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Jejuno/efeitos dos fármacos , Plexo Mientérico/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Óxido Nítrico Sintase Tipo I/efeitos dos fármacos , Óxido Nítrico/metabolismo , Quercetina/farmacologia , Peptídeo Intestinal Vasoativo/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Masculino , Plexo Mientérico/patologia , Quercetina/administração & dosagem , Ratos , Ratos WistarRESUMO
Colorectal cancer (CRC) invasion within the large intestine wall results in the replacement of normal tissue architecture by tumour mass. Cancer cells digest the extracellular matrix (ECM) by the release of proteolytic enzymes. The disintegration of matrix ground substance activates several deposited growth factors which stimulate cell proliferation. Stromal (mainly fibroblasts), immune and cancer cells dominate in this area and become involved in a network of multimodal interactions which significantly induce proliferation of colon cancer cells, inhibit their apoptosis and promote their spreading within the local tumour microenvironment. Cancer invasion destroys nerve fibres and neurons of the local enteric nervous system (ENS) and induces subsequent atrophy of the submucosal and myenteric plexuses in areas adjacent to the cancer boundary. Interestingly, the reduction of plexuses' size is accompanied by the increased number of galanin-immunoreactive neurons and increased galanin content in parts of the colon located close to the tumour. Galanin, a neuroprotective peptide, may inhibit the extrinsic pathway of apoptosis and in this way promote cancer cell survival. The possible role of acetylcholine and some ENS neuropeptides was also discussed. Invasion of cancer cells spreads along nerve fibres with the involvement of locally-released neutrophins which promote, via their specific receptors, cancer cell proliferation and pro-survival signalling pathways. Thus, during CRC development cancer cells and neurons of the ENS release many neurotransmitters/neuropeptides which affect key cellular signalling pathways promoting cancer cell proliferation and pro-survival phenotype. The multiple interactions between ENS neurons, cancer cells and other cell types present in the colon wall increase cancer cell invasiveness and have a negative impact on the course of CRC.
Assuntos
Neoplasias Colorretais/imunologia , Sistema Nervoso Entérico/imunologia , Plexo Mientérico/imunologia , Microambiente Tumoral/imunologia , Animais , Atrofia/imunologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/fisiopatologia , Progressão da Doença , Sistema Nervoso Entérico/fisiopatologia , Retroalimentação Fisiológica , Humanos , Plexo Mientérico/patologia , Plexo Mientérico/fisiopatologia , Invasividade NeoplásicaRESUMO
Intestinal inertia is a severe form of gut dysmotility that may require surgical resection. Loss of myenteric ganglion cells has been proposed as a possible etiology. Preclinical models have also suggested that virus infection-associated ganglionitis may be an alternative pathogenic factor. We determined to the extent intestinal inertia is associated with the lack of myenteric ganglion cells or ganglionitis using resection specimens from 27 intestinal inertia and 28 colon cancer patients. A hot spot approach with 5 HPFs was used for quantifying inflammatory cells. CD3, CD8, and CD20 immunohistochemistry was used to quantify T and B lymphocytes, along with subtyping the T-lymphocyte population by CD8. None of the intestinal inertia nor control cases showed the absence of myenteric ganglion cells. A total of 15 (55.6%) of the intestinal inertia cases showed inflammatory cell infiltration in the myenteric ganglion cells, compared with only 1 of 28 (3.6%) control cases (P<0.0001 by Fisher exact test). The inertia cases with inflammatory infiltrates were all associated predominantly with lymphocytes, including 3 cases (11.1%) with concurrent eosinophil infiltration, and 1 case (3.7%) with concurrent neutrophil infiltration. Furthermore, all 15 inertia cases with myenteric lymphocytic ganglionitis were associated with T lymphocytes (100%), including 1 case with a subset of concurrent B lymphocytes. The average CD3 count was 3.8 cells/HPF. CD8 immunohistochemical stain showed positive staining in 12 of the 15 cases (80%) with CD8-positive cells ranging from 1 to 8/HPF. In contrast, the only control case with lymphocytic ganglionitis showed mixed B and T lymphocytes and eosinophils. The high prevalence of T-lymphocyte infiltration in the myenteric ganglion in intestinal inertia cases suggests a possible pathogenic role.
Assuntos
Linfócitos T CD8-Positivos/patologia , Constipação Intestinal/patologia , Defecação , Gânglios Autônomos/patologia , Motilidade Gastrointestinal , Intestinos/inervação , Plexo Mientérico/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/imunologia , Doença Crônica , Constipação Intestinal/imunologia , Constipação Intestinal/fisiopatologia , Constipação Intestinal/cirurgia , Eosinófilos/imunologia , Eosinófilos/patologia , Feminino , Gânglios Autônomos/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Plexo Mientérico/imunologia , Infiltração de Neutrófilos , Estudos RetrospectivosRESUMO
BACKGROUND: Hirschsprung's disease (HD) is a congenital disorder affecting neonates that presents with distal intestinal obstruction. It is the most common type of anorectal malformation. Treatment of HD consists of surgical removal of the distal colon including the most distal aganglionic segment, the transitional zone, and a prudent length of proximal colon that is determined during the surgical procedure to be normally ganglionated by intraoperative demonstration of normal ganglion cells up to and including the surgical resection margin. METHODS: In a retrospective study of formalin-fixed paraffin-embedded colon tissue from the proximal resection margin (PRM) of 209 HD patients, we made morphometric measures and detected immature ganglion cells defined as dysmorphic by immunohistochemical demonstration of cytoplasmic neurofilament (NF). RESULTS: The majority of NF-positive ganglion cells in HD patients appeared immature, with less cytoplasm. Occasional positive ganglion cells in the same patients appeared mature with abundant eosinophilic cytoplasm, Nissl bodies, prominent nucleoli, and adjacent glial cells. Patients with NF-positive ganglion cells in the myenteric plexuses at the PRM may have poor postoperative recovery. CONCLUSION: We propose that NF expression in dysmorphic ganglion cells at the PRM may predict poor outcome after pull-through surgery for HD.
Assuntos
Doença de Hirschsprung/patologia , Doença de Hirschsprung/cirurgia , Filamentos Intermediários/metabolismo , Plexo Mientérico/patologia , Neurônios/patologia , Biomarcadores/análise , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Margens de Excisão , Plexo Mientérico/metabolismo , Neurônios/metabolismo , PrognósticoRESUMO
Gastrointestinal symptoms appear in Parkinson's disease patients many years before motor symptoms, suggesting the implication of dopaminergic neurones of the gut myenteric plexus. Inflammation is also known to be increased in PD. We previously reported neuroprotection with progesterone in the brain of mice lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and hypothesised that it also has neuroprotective and immunomodulatory activities in the gut. To test this hypothesis, we investigated progesterone administered to adult male C57BL/6 mice for 10 days and treated with MPTP on day 5. In an additional experiment, progesterone was administered for 5 days following MPTP treatment. Ilea were collected on day 10 of treatment and microdissected to isolate the myenteric plexus. Dopaminergic neurones were reduced by approximately 60% and pro-inflammatory macrophages were increased by approximately 50% in MPTP mice compared to intact controls. These changes were completely prevented by progesterone administered before and after MPTP treatment and were normalised by 8 mg kg-1 progesterone administered after MPTP. In the brain of MPTP mice, brain-derived neurotrophic peptide (BDNF) and glial fibrillary acidic protein (GFAP) were associated with progesterone neuroprotection. In the myenteric plexus, increased BDNF levels compared to controls were measured in MPTP mice treated with 8 mg kg-1 progesterone started post MPTP, whereas GFAP levels remained unchanged. In conclusion, the results obtained in the present study show neuroprotective and anti-inflammatory effects of progesterone in the myenteric plexus of MPTP mice that are similar to our previous findings in the brain. Progesterone is non-feminising and could be used for both men and women in the pre-symptomatic stages of the disease.
Assuntos
Encéfalo/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Transtornos Parkinsonianos/tratamento farmacológico , Progesterona/uso terapêutico , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Proteína Glial Fibrilar Ácida/metabolismo , Imunomodulação/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Fármacos Neuroprotetores/farmacologia , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/patologia , Progesterona/farmacologiaRESUMO
BACKGROUND: Hirschprung's disease is characterized by aganglionic bowel and often requires surgical resection. Cell-based therapies have been investigated as potential alternatives to restore functioning neurons. Skin-derived precursor cells (SKPs) differentiate into neural and glial cells in vitro and generate ganglion-like structures in rodents. In this report, we aimed to translate this approach into a large animal model of aganglionosis using autologous transplantation of SKPs. METHODS: Juvenile pigs underwent skin procurement from the shoulder and simultaneous chemical denervation of an isolated colonic segment. Skin cells were cultured in neuroglial-selective medium and labeled with fluorescent dye for later identification. The cultured SKPs were then injected into the aganglionic segments of colon, and the specimens were retrieved within seven days after transplantation. SKPs in vitro and in vivo were assessed with histologic samples for various immunofluorescent markers of multipotency and differentiation. SKPs from the time of harvest were compared to those at the time of injection using PCR. RESULTS: Prior to transplantation, 72% of SKPs stained positive for nestin and S100b, markers of neural and glial precursor cells of neural crest origin, respectively. Markers of differentiated neurons and gliocytes, TUJ1 and GFAP, were detected in 47% of cultured SKPs. After transplantation, SKPs were identified in both myenteric and submucosal plexuses of the treated colon. Nestin co-expression was detected in the SKPs within the aganglionic colon in vivo. Injected SKPs appeared to migrate and express early neuroglial differentiation markers. CONCLUSIONS: Autologous SKPs implanted into aganglionic bowel demonstrated immunophenotypes of neuroglial progenitors. Our results suggest that autologous SKPs may be potentially useful for cell-based therapy for patients with enteric nervous system disorders. TYPE OF STUDY: Basic science.
Assuntos
Diferenciação Celular , Doença de Hirschsprung/terapia , Pele/citologia , Transplante de Células-Tronco , Células-Tronco/metabolismo , Animais , Células Cultivadas , Colo , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida/metabolismo , Doença de Hirschsprung/induzido quimicamente , Plexo Mientérico/patologia , Nestina/metabolismo , Neurônios/metabolismo , Subunidade beta da Proteína Ligante de Cálcio S100/metabolismo , Células-Tronco/fisiologia , Plexo Submucoso/patologia , Suínos , Transplante Autólogo , Tubulina (Proteína)/metabolismoRESUMO
Gastrointestinal (GI) symptoms can originate from severe dysmotility due to enteric neuropathies. Current methods used to demonstrate enteric neuropathies are based mainly on classic qualitative histopathological/immunohistochemical evaluation. This study was designed to identify an objective morphometric method for paraffin-embedded tissue samples to quantify the interganglionic distance between neighboring myenteric ganglia immunoreactive for neuron-specific enolase, as well as the number of myenteric and submucosal neuronal cell bodies/ganglion in jejunal specimens of patients with severe GI dysmotility. Jejunal full-thickness biopsies were collected from 32 patients (22 females; 16-77 yr) with well-characterized severe dysmotility and 8 controls (4 females; 47-73 yr). A symptom questionnaire was filled before surgery. Mann-Whitney U test, Kruskal-Wallis coupled with Dunn's posttest and nonparametric linear regression tests were used for analyzing morphometric data and clinical correlations, respectively. Compared with controls, patients with severe dysmotility exhibited a significant increase in myenteric interganglionic distance (P = 0.0005) along with a decrease in the number of myenteric (P < 0.00001) and submucosal (P < 0.0004) neurons. A 50% reduction in the number of submucosal and myenteric neurons correlated with an increased interganglionic distance and severity of dysmotility. Our study proposes a relatively simple tool that can be applied for quantitative evaluation of paraffin sections from patients with severe dysmotility. The finding of an increased interganglionic distance may aid diagnosis and limit the direct quantitative analysis of neurons per ganglion in patients with an interganglionic distance within the control range.NEW & NOTEWORTHY Enteric neuropathies are challenging conditions characterized by a severe impairment of gut physiology, including motility. An accurate, unambiguous assessment of enteric neurons provided by quantitative analysis of routine paraffin sections may help to define neuropathy-related gut dysmotility. We showed that patients with severe gut dysmotility exhibited an increased interganglionic distance associated with a decreased number of myenteric and submucosal neurons, which correlated with symptoms and clinical manifestations of deranged intestinal motility.
Assuntos
Motilidade Gastrointestinal/fisiologia , Enteropatias , Intestinos , Plexo Mientérico , Proteínas do Tecido Nervoso , Manejo de Espécimes/métodos , Plexo Submucoso , Correlação de Dados , Feminino , Humanos , Imuno-Histoquímica , Enteropatias/imunologia , Enteropatias/patologia , Enteropatias/fisiopatologia , Intestinos/inervação , Intestinos/patologia , Intestinos/fisiopatologia , Masculino , Pessoa de Meia-Idade , Plexo Mientérico/imunologia , Plexo Mientérico/patologia , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/imunologia , Plexo Submucoso/imunologia , Plexo Submucoso/patologiaRESUMO
BACKGROUND: Fabry disease (FD) is a hereditary X-linked metabolic storage disorder characterized by deficient or absent lysosomal α-galactosidase A (α-Gal A) activity. This deficiency causes progressive accumulation of glycosphingolipids, primarily globotriaosylceramide (Gb3), in nearly all organ systems. Gastrointestinal (GI) symptoms can be very debilitating and are among the most frequent and earliest of the disease. As the pathophysiology of these symptoms is poorly understood, we carried out a morphological and molecular characterization of the GI tract in α-Gal A knockout mice colon in order to reveal the underlying mechanisms. METHODS: Here, we performed the first morphological and biomolecular characterization of the colon wall structure in the GI tract of the α-Gal A knock-out mouse (α-Gal A -/0), a murine model of FD. KEY RESULTS: Our data show a greater thickness of the gastrointestinal wall in α-Gal A (-/0) mice due to enlarged myenteric plexus' ganglia. This change is paralleled by a marked Gb3 accumulation in the gastrointestinal wall and a decreased and scattered pattern of mucosal nerve fibers. CONCLUSIONS AND INFERENCES: The observed alterations are likely to be a leading cause of gut motor dysfunctions experienced by FD patients and imply that the α-Gal A (-/0) male mouse represents a reliable model for translational studies on enteropathic pain and GI symptoms in FD.
Assuntos
Colo/metabolismo , Colo/patologia , Doença de Fabry/metabolismo , Doença de Fabry/patologia , Fibras Nervosas/patologia , Triexosilceramidas/metabolismo , Animais , Citocinas/sangue , Feminino , Masculino , Camundongos , Camundongos Knockout , Plexo Mientérico/metabolismo , Plexo Mientérico/patologia , Triexosilceramidas/genética , Ubiquitina Tiolesterase/genética , alfa-Galactosidase/genéticaRESUMO
BACKGROUND AND AIMS: Diagnosis of GI neuromuscular diseases is mostly on the basis of symptomatology and is often unreliable. Pathologic analysis of affected tissue (eg, the myenteric plexus and muscle) is a potentially valuable method for both diagnosis and advancement of our knowledge about the biologic basis for these syndromes. However, until now access to the deeper layers of the GI tract has been limited, generally requiring invasive surgical techniques. METHODS: We report a "close-then-resect" endoscopic full-thickness biopsy sampling (EFTB) technique using an over-the-scope clip and telescope for rectal muscle biopsy sampling in patients with suspected severe neuromuscular gut disorders. The main outcome measures were technical success and adverse events. RESULTS: Thirteen patients (11 women; mean age 27 ± 5.4 years) with diffusely delayed colonic transit underwent EFTB. The mean (± standard deviation) procedure time was 30 ± 5.2 minutes. The mean size of the resected specimen was 18 ± 3.5 mm. Histologic full-thickness tissue samples were achieved for all patients. Postprocedural adverse events were reported in 2 patients, and both were graded as mild (1 self-limited bleeding and 1 with rectal pain). Hematoxylin and eosin staining of tissue samples confirmed adequate cross-sectional imaging of muscularis propria in all patients with excellent demonstration of the myenteric plexus and both layers of muscle. Two patients demonstrated a decrease in interstitial cells of Cajal as demonstrated by CD117 staining. No cases demonstrated appreciable inflammation involving myenteric ganglia. CONCLUSIONS: Diagnostic EFTB with modified over-the-scope clip for the close-then-resect method appears to be a safe and effective technique to obtain adequate full-thickness rectal specimens, allowing for both quantitative and qualitative analysis for the diagnosis of neuromuscular GI dysmotility.