Efeito do precondicionamento isquêmico remoto no transplante de intestino fetal em camundongos
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Arquivos
Data
2009
Autores
Morello, Ricardo José [UNIFESP]
Orientadores
Montero, Edna Frasson de Souza [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Objetivo: avaliar os efeitos de precondicionamento isquêmico remoto (PCI-R)
no modelo de transplante de intestino delgado fetal. Métodos: foram
constituídos dois grupos: transplante isogênico (Iso, camundongos C57BL/6,
n=24) e transplante alogênico (Alo, camundongos BALB/c, n=24). Em cada
grupo, distribuíram-se os animais com e sem PCI-R, que foi realizado por
oclusão da artéria femoral esquerda da fêmea prenhe durante 10 minutos,
seguida por tempo igual de reperfusão. O imunossupressor utilizado foi
Tacrolimo (Fk, 5 mg/kg/dia v.o.). Ao final obteve-se os seguintes subgrupos:
Alo-Tx, Alo-Pci, Alo-Fk, Alo-Pci-Fk, Iso-Tx, Iso-Pci, Iso-Fk e Iso-Pci-Fk. O
enxerto foi transplantado no espaço entre o músculo reto-abdominal e pré-
peritoneal dos receptores a meio centímetro do apêndice xifóide, à esquerda da
linha mediana. Após o sétimo dia de seguimento, o enxerto foi removido, fixado
e embebido em parafina para avaliação histomorfológica (desenvolvimento e
rejeição) e análise imunohistoquímica (anti-PCNA e anti-caspase-3 clivada). Os
dados foram analisados usando ANOVA e testes complementares e foi
considerado significante quando p <0.05. Resultados: A avaliação do
desenvolvimento do enxerto no grupo de Iso mostrou que o PCI-R reduziu o
desenvolvimento comparado com Iso-Tx (5,2±0,4 vs 9,0±0,8), o Fk e sua
associação com PCI-R aumentaram o desenvolvimento do enxerto comparado
com PCI-R (11,2±0,7 e 10,2±0,8, respectivamente). No grupo Alo, o Fk e/ou
sua associação com PCI-R aumentaram o desenvolvimento comparado com
Alo-Tx e Alo com PCI-R (6,0±0,8, 9,0±1,2, 0,0±0,0, 0,5±0,3, respectivamente).
A expressão de PCNA foi maior no grupo ISO em animais tratados com Fk e
PCI-R comparados a outros grupos (12,2±0,8 vs Tx: 8,8±0,9, PCI-R: 8,0±0,4 e
Fk: 9,0±0,6). No grupo Alo, a expressão de PCNA não diferiu entre grupos. A
rejeição do enxerto foi menor nos grupos tratados com PCI-R (-18%), Fk (-
68%) ou ambos (-61%) comparados com Alo-Tx. A expressão de caspase-3
clivada foi menor no grupo Iso em animais tratados com associação de PCI-R e
Fk (6,2 ±0,9 vs Tx: 8,6±0,5; PCI-R: 5,8 ±0,9 e Fk: 6,0 ±0,3). Conclusão: O PCIR
mostrou efeito benéfico sobre a lesão de isquemia e reperfusão do enxerto
intestinal fetal nos transplantes isogênico e alogênico, aumentando o número
de células caliciformes e a proliferação celular. No transplante alogênico,
aumentou o desenvolvimento do enxerto, diminuiu o grau de rejeição aguda na
ausência de imunossupressão, porém não apresentou efeito sinérgico com o
imunossupressor. No transplante isogênico houve diminuição do grau de
desenvolvimento do enxerto, porém foi efetivo na redução da apoptose.
Purpose: to evaluate the effect of remote ischemic preconditioning (R-IPC) in the fetal small bowel transplantation model. Methods: two groups were constituted: the Isogenic transplant (Iso, C57BL/6 mice, n=24) and the allogeneic transplant (Alo, BALB/c mice, n=24). In each group, the animals were distributed with and without R-IPC, that was accomplished by occlusion of the left femoral artery of the pregnant female during 10 minutes, followed for similar time of reperfusion. The imunossupressor used was Tacrolimo (Fk, 5 mg/kg/day v.o.) It was obtained the following subgrups: Alo-Tx, Alo-IPC, Alo-Fk, Alo- IPC - Fk, Iso-Tx, Iso- IPC, Iso-Fk and Iso- IPC-Fk. The graft was transplanted in the space between the straight-abdominal muscle and preperitoneal of the receivers to half centimeter of the xiphoid appendix, left of the medium line. After seven days follow-up, the graft of small bowel was removed and embedded in paraffin to histomorphological evaluation (the graft development and rejection) and immunohistochemical analysis (anti-PCNA and anti-caspase- 3 cleaved). Data were analyzed using ANOVA and post-hoc tests, and it was considered significant when p<0.05. Results: The graft development evaluation in Iso group showed that R-IPC reduced the development compared with Iso-Tx (5.2±0.4 vs 9.0±0.8) and Fk and its association with R-IPC increased the graft development compared with R-IPC (11.2±0.7 and 10.2±0.8, respectively). In Alo group, Fk and its association with R-IPC increased the development compared with Alo-Tx and Alo with R-IPC (6.0±0.8, 9.0±1.2, 0.0±0.0, 0.5±0.3, respectively). The PCNA expression was increased in Iso group only in animals treated with Fk and R-IPC compared to other groups (12.2±0.8 vs Tx: 8.8±0.9, R-IPC: 8.0±0.4 and FK: 9.0±0.6). In Alo group, PCNA expression did not differ among groups. The graft rejection was lower in groups treated with R-IPC (- 18%), Fk (-68%) or both (-61%) compared with Alo-Tx. The caspase-3 cleaved expression was lower in Iso group in animals treated with association of R-IPC and/or Fk (Tx: 8.6±0.5 vs R-IPC: 5.8 ±0.9; Fk: 6.0 ±0.3; R-IPC-Fk: 6.2 ±0.9). Conclusion: R-IPC showed benefic effect on the ischemy and reperfusion lesion of the fetal intestinal graft in the Isogenic and Allogeneic transplants, increasing the number of goblet cells and the cellular proliferation. In the Allogeneic transplant, it increased the development of the graft, it reduced the degree of acute rejection without Immunosuppression, however it didn't present synergic effect with the imunossupressor. In the Isogenic transplant there was decrease of the degree of development of the graft, however it was effective in the apoptosis reduction.
Purpose: to evaluate the effect of remote ischemic preconditioning (R-IPC) in the fetal small bowel transplantation model. Methods: two groups were constituted: the Isogenic transplant (Iso, C57BL/6 mice, n=24) and the allogeneic transplant (Alo, BALB/c mice, n=24). In each group, the animals were distributed with and without R-IPC, that was accomplished by occlusion of the left femoral artery of the pregnant female during 10 minutes, followed for similar time of reperfusion. The imunossupressor used was Tacrolimo (Fk, 5 mg/kg/day v.o.) It was obtained the following subgrups: Alo-Tx, Alo-IPC, Alo-Fk, Alo- IPC - Fk, Iso-Tx, Iso- IPC, Iso-Fk and Iso- IPC-Fk. The graft was transplanted in the space between the straight-abdominal muscle and preperitoneal of the receivers to half centimeter of the xiphoid appendix, left of the medium line. After seven days follow-up, the graft of small bowel was removed and embedded in paraffin to histomorphological evaluation (the graft development and rejection) and immunohistochemical analysis (anti-PCNA and anti-caspase- 3 cleaved). Data were analyzed using ANOVA and post-hoc tests, and it was considered significant when p<0.05. Results: The graft development evaluation in Iso group showed that R-IPC reduced the development compared with Iso-Tx (5.2±0.4 vs 9.0±0.8) and Fk and its association with R-IPC increased the graft development compared with R-IPC (11.2±0.7 and 10.2±0.8, respectively). In Alo group, Fk and its association with R-IPC increased the development compared with Alo-Tx and Alo with R-IPC (6.0±0.8, 9.0±1.2, 0.0±0.0, 0.5±0.3, respectively). The PCNA expression was increased in Iso group only in animals treated with Fk and R-IPC compared to other groups (12.2±0.8 vs Tx: 8.8±0.9, R-IPC: 8.0±0.4 and FK: 9.0±0.6). In Alo group, PCNA expression did not differ among groups. The graft rejection was lower in groups treated with R-IPC (- 18%), Fk (-68%) or both (-61%) compared with Alo-Tx. The caspase-3 cleaved expression was lower in Iso group in animals treated with association of R-IPC and/or Fk (Tx: 8.6±0.5 vs R-IPC: 5.8 ±0.9; Fk: 6.0 ±0.3; R-IPC-Fk: 6.2 ±0.9). Conclusion: R-IPC showed benefic effect on the ischemy and reperfusion lesion of the fetal intestinal graft in the Isogenic and Allogeneic transplants, increasing the number of goblet cells and the cellular proliferation. In the Allogeneic transplant, it increased the development of the graft, it reduced the degree of acute rejection without Immunosuppression, however it didn't present synergic effect with the imunossupressor. In the Isogenic transplant there was decrease of the degree of development of the graft, however it was effective in the apoptosis reduction.