Caracterização histomorfométrica e imunohistoquímica do sistema nervoso entérico e das células intersticiais de Cajal no cólon distal de camundongos SOX 10 DOM
Data
2021-11-30
Autores
Teixeira, Luciana Cristina [UNIFESP]
Orientadores
Montero, Edna Frasson de Souza [UNIFESP]
Tipo
Dissertação de mestrado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Introdução: A doença de Hirschsprung (DH) é uma aganglionose congênita caracterizada pela ausência de gânglios entéricos em variáveis comprimentos no intestino distal. Ocorre devido à uma falha das células da crista neural ao colonizar a região distal intestino. Fatores ambientais e genéticos coordenam a diferenciação e manutenção das células do trato gastro intestinal. O camundongo Dominante megacólon (SoxDom) representa um dos modelos genéticos da Doença de Hirschsprung. A mutação do gene que codifica o fator de transcrição Sox10, causa aganglionose intestinal do cólon distal e hipopigmentação, no abdome, membros inferiores e ponta da cauda. Objetivo: Correlacionar aspectos histomorfométricos (espessura) das camadas musculares longitudinal, circular, submucosa e distância entre os plexos e imunohistoquímica dos neurônios entéricos (enolase neurônio específica), células da glia (proteína S100) e das células intersticiais de Cajal (CD117) do cólon distal do camundongo Sox-10Dom. Métodos: 18 camundongos, divididos em três grupos: 6 C57BL/ 6J, 6 Dom/+ e 6 Wild type +/+. A genotipagem foi feita por sequenciamento. A espessura das camadas musculares foi realizada através das medidas em µm. As frações das áreas da expressão de enolase neurônio específica, proteína S100 e CD117, foram analisados por fração de área marcada. Para as análises estatísticas foram aplicados os testes ANOVA unicaudal e de TUKEY para comparações múltiplas. Nível de significância de p< 0,05. Resultados: a mutação na linhagem Dom/+ foi confirmada na genotipagem por sequenciamento. A hipertrofia foi observada nas camadas longitudinal, circular e submucosa da linhagem Dom/+ em relação às linhagens C57BL/ 6J e Wild type +/+. No plexo mioentérico foi encontrada diminuição da ENE, no Dom/+ em relação ao C57BL/ 6J e Wild type +/+ e no plexo submucoso não foi observada diferença estatística. Foi encontrada diminuição da proteína S-100, no plexo mioentérico no Dom/+em relação às linhagens C57BL/ 6J e Wild type +/+, no plexo submucoso não houve diferença estatística entre os grupos. Quanto à distribuição da CIC, não houve diferença nas camadas mioentérica e intermuscular. Conclusões: O sequenciamento genético confirmou a mutação dos camundongos heterozigotos Sox 10, ou seja, a inserção de resíduo de guanina, reforçando as características fenotípicas da linhagem, quais sejam a despigmentação no abdome e membros inferiores. Na caracterização do SNE pela imunohistoquímica, observou-se hipoganglionose nos camundongos Dom/+, por meio do anticorpo enolase neurônio-específica, na identificação dos neurônios entéricos, e da proteína S-100, na marcação das células da glia entérica. No modelo Sox Dom, na região aganglionar, a distribuição das CIC não foi afetada pela mutação do gene SOX10.
Introduction: Hirschsprung's disease (HD) is a congenital aganglionosis characterized by the absence of enteric ganglia in varying lengths in the distal gut. It occurs due to the failure of the neural crest cells to colonize a distal region of the gut. Environmental and genetic factors coordinate the differentiation and maintenance of the cells of the gastrointestinal tract. The dominant megacolon mouse (SoxDom) represents one of the genetic models of Hirschsprung's disease. The mutation of the gene encoding the SRY-related transcription factor Sox10, causing intestinal aganglionosis of the distal colon and hypopigmentation, white spot belly, feet and tail tip. Objective: To correlate histomorphometric aspects (thickness) of longitudinal, circular, submucosal muscle layers and distance between plexuses and immunohistochemistry of enteric neurons (specific neuron enolase), glial cells (S100 protein), and interstitial cells of Cajal (CD117) of the colon distal of the SoxDommouse. Methods: 18 mice are divided in three groups: 6 C57BL / 6J, 6 Dom/+ and Wild type +/+. Genotyping was done by sequencing. The thickness of the muscle layers was measured using µm measurements. The fractions of the areas of expression of specific neuron enolase (ENE), protein S100 and CD117, were analyzed by the fraction of stained area. For statistical analysis, the one-tailed ANOVA and TUKEY tests had been applied for multiple comparisons. The significance level of p <0.05. Results: the mutation in the Dom/+ strain was confirmed in genotyping by sequencing. Hypertrophy was observed in the longitudinal, circular, and submucosa layers of the Dom/+ line in relation to the C57BL / 6J and Wild type +/+ lines. No decrease was found in the myenteric plexus, in the Dom/+ in relation to the C57BL / 6J and Wild type +/+, and in the submucosal plexus, no statistical difference was observed. Although, a decline in protein S-100 was verified in the myenteric plexus in the Dom/+ in relation to the C57BL / 6J and Wild type +/+ lines, in the submucosal plexus there was no statistical difference between the groups. Regarding the distribution of interstitial cells of Cajal, no difference was seen in the myenteric and intermuscular layers. Conclusions: Genetic sequencing confirmed the mutation of the heterozygous SOX 10 mice, i.e., the insertion of a guanine residue, reinforcing the phenotypic characteristics of the strain, which are depigmentation in the abdomen and lower limbs. In the characterization of the SNE by immunohistochemistry, hypoganglionosis was observed in the Dom/+ mice, by using the neuron-specific enolase antibody, in the identification of enteric neurons, and the S-100 protein, in the labeling of enteric glia cells. In the Sox Dom model, in the aganglionic region, the distribution of CICs was not affected by mutation of the SOX10 gene.
Introduction: Hirschsprung's disease (HD) is a congenital aganglionosis characterized by the absence of enteric ganglia in varying lengths in the distal gut. It occurs due to the failure of the neural crest cells to colonize a distal region of the gut. Environmental and genetic factors coordinate the differentiation and maintenance of the cells of the gastrointestinal tract. The dominant megacolon mouse (SoxDom) represents one of the genetic models of Hirschsprung's disease. The mutation of the gene encoding the SRY-related transcription factor Sox10, causing intestinal aganglionosis of the distal colon and hypopigmentation, white spot belly, feet and tail tip. Objective: To correlate histomorphometric aspects (thickness) of longitudinal, circular, submucosal muscle layers and distance between plexuses and immunohistochemistry of enteric neurons (specific neuron enolase), glial cells (S100 protein), and interstitial cells of Cajal (CD117) of the colon distal of the SoxDommouse. Methods: 18 mice are divided in three groups: 6 C57BL / 6J, 6 Dom/+ and Wild type +/+. Genotyping was done by sequencing. The thickness of the muscle layers was measured using µm measurements. The fractions of the areas of expression of specific neuron enolase (ENE), protein S100 and CD117, were analyzed by the fraction of stained area. For statistical analysis, the one-tailed ANOVA and TUKEY tests had been applied for multiple comparisons. The significance level of p <0.05. Results: the mutation in the Dom/+ strain was confirmed in genotyping by sequencing. Hypertrophy was observed in the longitudinal, circular, and submucosa layers of the Dom/+ line in relation to the C57BL / 6J and Wild type +/+ lines. No decrease was found in the myenteric plexus, in the Dom/+ in relation to the C57BL / 6J and Wild type +/+, and in the submucosal plexus, no statistical difference was observed. Although, a decline in protein S-100 was verified in the myenteric plexus in the Dom/+ in relation to the C57BL / 6J and Wild type +/+ lines, in the submucosal plexus there was no statistical difference between the groups. Regarding the distribution of interstitial cells of Cajal, no difference was seen in the myenteric and intermuscular layers. Conclusions: Genetic sequencing confirmed the mutation of the heterozygous SOX 10 mice, i.e., the insertion of a guanine residue, reinforcing the phenotypic characteristics of the strain, which are depigmentation in the abdomen and lower limbs. In the characterization of the SNE by immunohistochemistry, hypoganglionosis was observed in the Dom/+ mice, by using the neuron-specific enolase antibody, in the identification of enteric neurons, and the S-100 protein, in the labeling of enteric glia cells. In the Sox Dom model, in the aganglionic region, the distribution of CICs was not affected by mutation of the SOX10 gene.