O ATP é conhecido como um neurotransmissor e seus receptores são os da família P2X1-7. Foi demonstrada a presença de receptor purinérgico P2X2, 3, 7 no sistema nervoso entérico. AColite Ulcerativa e Doença de Crohn apresentam processos patofisiológicos, a necrose do intestino e efeitos nos neurônios entéricos e glias entéricas. Tem sido demonstrada a participação do receptor P2X7 no processo inflamatório e necrose de células. Este projeto visa estudar o efeito da colite ulcerativa no plexo mioentérico e célula glial entérica do intestino grosso de animais deficientes para o receptor P2X7 (P2X7 -/-, P2X7KO). Para isto, foi injetado 2, 4, 6, ácido trinitrobenzeno sulfônico (TNBS) no intestino grosso de animais C57BL/6 (WT) e deficientes do gene do receptor P2X7, no grupo sham foi injetado veículo. Foram coletados tecidos para análises 24h 4 dias após a administração. Foi usada técnica de duplas e triplas marcações de imunofluorescência para análises qualitativas e quantitativas que foram obtidas por microscópio de fluorescência Nikon 80i. Para a análise morfológica do intestino grosso foi usado o método de histologia convencional. Os resultados quantitativos demonstraram diminuição significante de 13,9% e 7,1% de células-ir ao receptor P2X7 nos grupos WT/Colite 24h e WT/Colite 4 dias, respectivamente. Não houve diminuição de neurônios-ir a NOS e ChAT do grupo KO/Colite de 4 dias. Não houve diminuição de neurônios-ir a PGP9.5 dos grupos KO/Colite de 24h e 4 dias. Houve diminuição de 19,3% de glias-ir a GFAP no grupo WT/Colite 24h e aumento de 19% no grupo WT/Colite 4 dias. Não houveram alterações morfológicas nos neurônios dos grupos de 24h. Houve aumento de 29,4% e 40,2% na área do perfil neuronal nos neurônios-ir a ChAT dos grupos WT/Colite 4 dias e KO/Colite 4 dias, respectivamente e aumento de 20,1% na área do perfil neuronal de neurônios-ir a PGP9.5 do grupo KO/Colite 4 dias. Análise histológica apresentou hiperemia e não úlceras, edema e infiltração celular nos grupos WT/Colite de 24h e 4 dias. Os grupos KO/Colite 24h e 4 dias foram menos afetados pela colite na análise histológica.
BACKGROUND The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases (IBDs) and that the P2X7 receptor triggers neuronal death. However, the mechanism by which enteric neurons are lost in IBDs is unknown. AIM To study the role of the caspase-3 and nuclear factor kappa B (NF-κB) pathways in myenteric neurons in a P2X7 receptor knockout (KO) mouse model of IBDs. METHODS Forty male wild-type (WT) C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid (colitis group). Mice in the sham groups were injected with vehicle. The mice were divided into eight groups ( n = 5): The WT sham 24 h and 4 d groups, the WT colitis 24 h and 4 d groups, the KO sham 24 h and 4 d groups, and the KO colitis 24 h and 4 d groups. The disease activity index (DAI) was analyzed, the distal colon was collected for immunohistochemistry analyses, and immunofluorescence was performed to identify neurons immunoreactive (ir) for calretinin, P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, and total NF-κB. We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion, the neuronal profile area (µm²), and corrected total cell fluorescence (CTCF). RESULTS Cells double labeled for calretinin and P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, or total NF-κB were observed in the WT colitis 24 h and 4 d groups. The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (2.10 ± 0.13 vs 3.33 ± 0.17, P < 0.001; 2.92 ± 0.12 vs 3.70 ± 0.11, P < 0.05), but was not significantly different between the KO groups. The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group (312.60 ± 7.85 vs 278.41 ± 6.65, P < 0.05), and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group (104.63 ± 2.49 vs 117.41 ± 1.14, P < 0.01). The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (19.49 ± 0.35 vs 22.21 ± 0.18, P < 0.001; 20.35 ± 0.14 vs 22.75 ± 0.51, P < 0.001), and no P2X7 receptor-ir neurons were observed in the KO groups. Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group. The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d...
The objective was to study the distal colon myenteric plexus and enteric glial cells (EGCs) in P2X7 receptor-de cient (P2X7-/-) animals after experimental ulcerative colitis on. 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) was injected into distal colon of C57BL/6 (WT) and P2X7 receptor gene-de cient (P2X7-/-, KO) animals. Distal colon tissues were analysed 24 h and 4 d after administration. Double immuno uorescence was used for analyses and histology was used for morphological analysis. Quantitative analysis demonstrated 13.9% and 7.1% decreases in the number/ganglia of P2X7 receptor-immunoreactive (ir) in the 24 h-WT/colitis group and 4 d-WT/colitis, respectively. There was no reduction in the number per ganglia of neuronal nitric oxide synthase (nNOS)-ir, choline acetyltransferase (ChAT)-ir and PGP9.5 (pan neuronal)-ir neurons in the 4 d-KO/colitis group. There was a reduction by 19.3% in the number of glial brillary acidic protein (GFAP, EGC)-ir in the 24 h-WT/colitis group and a 19% increase in the number of these cells in the 4 d-WT/colitis group. There were no pro le area changes in neurons in the 24 h groups. In the 4 d-WT/colitis and 4 d-KO/colitis groups, there was an increase in the pro le neuronal area of nNOS, ChAT and PGP9.5. Histological analysis showed hyperaemia, oedema or cellular in ltration in the 24 h-WT/colitis groups and 4 d-WT/colitis groups. The 4 d-KO/colitis groups showed no histological changes.
The objective was to study the distal colon myenteric plexus and enteric glial cells (EGCs) in P2X7 receptor-deficient (P2X7-/-) animals after experimental ulcerative colitis on. 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) was injected into distal colon of C57BL/6 (WT) and P2X7 receptor gene-deficient (P2X7-/-, KO) animals. Distal colon tissues were analysed 24 h and 4 d after administration. Double immunofluorescence was used for analyses and histology was used for morphological analysis. Quantitative analysis demonstrated 13.9% and 7.1% decreases in the number/ganglia of P2X7 receptor-immunoreactive (ir) in the 24 h-WT/colitis group and 4 d-WT/colitis, respectively. There was no reduction in the number per ganglia of neuronal nitric oxide synthase (nNOS)-ir, choline acetyltransferase (ChAT)-ir and PGP9.5 (pan neuronal)-ir neurons in the 4 d-KO/colitis group. There was a reduction by 19.3% in the number of glial fibrillary acidic protein (GFAP, EGC)-ir in the 24 h-WT/colitis group and a 19% increase in the number of these cells in the 4 d-WT/colitis group. There were no profile area changes in neurons in the 24 h groups. In the 4 d-WT/colitis and 4 d-KO/colitis groups, there was an increase in the profile neuronal area of nNOS, ChAT and PGP9.5. Histological analysis showed hyperaemia, oedema or cellular infiltration in the 24 h-WT/colitis groups and 4 d-WT/colitis groups. The 4 d-KO/colitis groups showed no histological changes.
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