5. EFFECTIVENESS OF THE PROGRAMME
5.1. FARMER FIELD SCHOOLS AND IPM MAINSTREAMING Farmer field schools in comparison to other approaches used by donors
estudo pode indicar a preservação da vitalidade das estruturas
intestinais no transplante de intestino fetal, e ser capaz de reduzir
os riscos da síndrome de disfunção de múltiplos órgãos em outros
tipos de transplante.
As células caliciformes residentes no epitélio do intestino delgado e grosso são responsáveis pela produção e manutenção do muco protetor, secretando glicoproteínas de alto peso molecular, as mucinas 76,77. É atribuída às células caliciformes intestinais o papel de manutenção da barreira intestinal, seja pela produção de mucinas, seja pela expressão de proteínas de adesão (ocludina e claudina-3) 78. Isto pode ser comprovado pela cinética de reparação do epitélio após a lesão de IR 77. Em estudo experimental, Ikeda et al. 77 mostraram que, imediatamente após a lesão de IR, pode ser observado intenso descolamento dos enterócitos. Após cerca de 20 minutos, o epitélio começa a ser recoberto por células caliciformes e após 75 minutos, observa-se restituição completa do epitélio, sugerindo que as células caliciformes tem papel fundamental na reparação e na manutenção do epitélio intestinal após processo de IR.
Os resultados obtidos neste estudo mostram uma proteção do enxerto intestinal fetal pelo PCI-R, aumentando o desenvolvimento e o número de células caliciformes, além de diminuir a apoptose e atenuar o processo de rejeição. Ainda observou-se indução da proliferação celular no enxerto, o que permitiu o processo de manutenção do reparo celular após a lesão de IR.
46
5- Conclusões
O PCI-R mostrou efeito benéfico sobre a lesão de isquemia e reperfusão do enxerto intestinal fetal;
O PCI-R mostrou efeito benéfico no transplante alogênico, aumentando o desenvolvimento do enxerto intestinal fetal, contrário ao que ocorreu no transplante isogênico em que houve diminuição do grau de desenvolvimento do enxerto;
O PCI-R mostrou efeito benéfico nos transplantes isogênico e alogênico, aumentando o número de células caliciformes;
O PCI-R mostrou efeito benéfico no transplante alogênico, diminuindo o grau de rejeição aguda do enxerto intestinal na ausência de imunossupressão, porém não apresentou efeito sinérgico com o imunossupressor;
O PCI-R mostrou efeito benéfico nos transplantes isogênico e alogênico, aumentando a proliferação celular do enxerto intestinal fetal;
47
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ABSTRACT
Effect of remote ischemic preconditioning in the fetal small bowel transplant in mice. Morello RJ, Koike MK, Montero EFS
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.
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APÊNDICES
Apêndice 1 -Valores individuais da avaliação do desenvolvimento do enxerto intestinal fetal isogênico, obedecendo aos critérios de Auber et al., 1998. 26
Camundongos Tx PCI FK PCI-FK
1 8 5 10 13 2 10 4 12 11 3 7 6 12 8 4 10 4 13 11 5 7 6 9 10 6 12 6 11 8 Média 9,0±0,8 5,2±0,4 11,2 *±0,7 10,2 *±0,8
Tx, grupo transplante controle isogênico; FK, tratamento com Tacrolimo; PCI, submetido ao PCI-R; *, p<0,05 vs PCI.
Apêndice 2 -Valores individuais da avaliação do grau de desenvolvimento do enxerto intestinal fetal alogênico, obedecendo aos critérios de Auber et al., 1998.26
Camundongos Tx PCI FK PCI-FK
1 0 1 5 5 2 0 0 10 9 3 0 0 6 10 4 0 0 5 12 5 0 2 5 12 6 0 0 5 6 Média 0,0±0,0 0,5 ±0,3 6,0 ±0,8 9,0 ±1,2
Tx, grupo transplante controle alogênico; FK, tratamento com Tacrolimo; PCI, submetido ao PCI-R.
Apêndice 3 -Valores individuais da contagem de células caliciformes ao longo do epitélio intestinal fetal.
Camundongos Tx PCI FK PCI-FK
1 4,3 4,9 5,6 5,3 2 4,2 6,1 5,1 5,3 3 4,2 3,3 4,6 5,7 4 4,9 3,6 5,9 6,7 5 4,5 7,4 5,4 6,3 6 4,4 5,9 Média 4,4±0,1 5,1±0,8 5,3 ±0,2 5,9 ± 0,2
Tx, grupo transplante controle isogênico; FK, tratamento com Tacrolimo; PCI, submetido ao PCI-R; *, p<0,05 vs PCI.
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Apêndice 4 -Valores individuais da contagem de células caliciformes ao longo do epitélio intestinal fetal no transplante alogênico.
Camundongos FK PCI-FK 1 2,6 3,9 2 2,8 4,2 3 3,0 3,5 4 4,3 4,4 5 3,5 5,1 6 2,6 5,3 Média 3,1 ±0,4 4,4 ± 0,3
PCI, tratamento prévio de PCI-R; FK, tratamento com Tacrolimo. *. P<0,05 vs ALO-FK.
Apêndice 5 -Valores individuais da avaliação da positividade para marcação de PCNA em células da vilosidade, criptais e da camada muscular do intestino fetal transplantado.
Camundongos Tx PCI FK PCI + FK
1 9 6 8 10 2 5 5 6 11 3 7 8 5 12 4 5 5 7 8 5 7 9 6 10 7 7 Média 7,1±0,7 6,0 ±0,7 7,0 ±0,7 10,3 ±0,9
Tx, grupo transplante controle isogênico; FK, grupo isogênico tratado com Tacrolimo; PCIR, grupo; p<0,05 vs ISO; p<0,05 vs PCIR e, p<0,05 vs FK.
Apêndice 6 -Valores individuais da avaliação da positividade para marcação de PCNA em células da vilosidade, criptais e da camada muscular do intestino fetal no transplante alogênico.
Camundongos FK PCI + FK 1 11 5 2 7 12 3 6 9 4 0 10 5 12 5 6 7 Média 7,2 ±0,9 8,2 ±1,4 PCI, tratamento prévio de PCI-R; FK, tratamento com Tacrolimo.
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Apêndice 7 - Valores da avaliação da positividade para marcação de PCNA na comparação entre os grupos de transplante isogênico (ISO) e alogênico (ALO) tratados com Tacrolimo (FK).
Camundongos ISO-FK ALO-FK ISO- PCI-FK ALO- PCI-FK
1 9 11 12 5 2 7 7 13 12 3 5 6 15 10 4 8 0 9 9 5 11 12 5 5 6 7 Média 8,0±1,7 7,2 ±2,3 10,8 ±1,7 8,2 ±1,4 FK, grupo tratado com Tacrolimo; PCI, tratamento prévio de PCI-R.
Apêndice 8 -Valores individuais da avaliação da apoptose celular no intestino fetal transplantado.
Camundongos Tx PCI FK PCIR - FK
1 7 9 7 7 2 8 4 5 9 3 9 6 6 6 4 7 4 6 5 5 9 6 6 4 6 9 7 11 Média 8,6±0,5 5,8 ±0,9 6,0 ±0,3 6,2 ±0,9
Tx, grupo transplante controle isogênico; PCI, tratamento prévio de PCI-R; FK, tratamento com Tacrolimo. *, p<0,05 vs ISO-Tx.
Apêndice 9 -Valores individuais da avaliação da rejeição do intestino fetal transplantado.
Camundongos Tx PCI FK PCI - FK
1 0 0 0 0 2 0 4 0 0 3 0 0 0 0 4 0 0 0 0 5 0 0 0 0 6 0 0 4 0 7 0 Média 0,0±0,0 0,7 ±0,7 0,7 ±0,7 0,0 ±0,0
Tx, grupo transplante controle isogênico; FK, grupo isogênico tratado com Tacrolimo; PCI, tratamento prévio de PCI-R.
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Apêndice 10 -Valores individuais da avaliação do grau de rejeição do enxerto intestinal fetal, obedecendo aos critérios de Auber et al., 1998.
Camundongos ALO-Tx PCIR FK PCIR + FK
1 16 14 4 5 2 16 16 5 10 3 22 14 5 7 4 16 16 8 5 5 16 12 7 7 6 22 16 6 8 Média 18,0±1,3 14,7 ±0,7 5,8 ±0,6 7,0 ±0,8
ALO-Tx, transplantado sem tratamento; PCI, transplantado submetido ao PCI-R prévio; FK, transplantado tratado com Tacrolimo. *, P<0,05 vs. ALO-Tx; #, P<0,05 vs ALO-PCI.
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61