CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / We investigated the protective effects of L-alanyl-Glutamine (L-ALN-GLN) through the immunohistochemical detection of proinflammatory mediators (IL-6, TNF, NF-kB e HO-1). This was achieved by means of a experimental and controlled study. Fifty-four male gerbils with a mean weight of 150g were used. They were divided randomly and equally into 3 groups: saline with ischemia and reperfusion (SIR), saline without ischemia and reperfusion (SSI) and with L-alanyl-glutamine and with ischemia and reperfusion (GIR). They were then redistributed into three subgroups according to time: T0 (maximum time of ischemia), T30 (30 minutes of reperfusion) and T60 (60 minutes of reperfusion), containing 6 animals each. They were pretreated with saline 2.0mL 0,9% intra-venously (iv) or L-ALN-GLN 0.75% G/KG (IV) 30 min before the start of the experiments. Cerebral ischemia was performed by bilateral occlusion of the common carotid arteries (CCAs) for a period of 15 minutes. After all surgical procedures and at the end of the three time periods the animals were sacrificed and brain tissue samples immediately obtained, which were fixed in 10% formalin solution for 24 hours in order being duly processed prior paraffin embedding. The samples (brain tissue) were collected at the end of each time periods. After this and through using a microtome, tissue sections were made at 4 microns and then placed on glass slides covered with L-polylysine, a proper way for performing immunohistochemistry. The streptavivin-biotin-peroxidase method was used. The quantitative analysis was made by means of counting immunocytochemically-stained cells, both neurons and glial cells, as observed at 400X magnification under an optical microscope Olympus model BX41. Ten fields were observed for each section, with a total of 4 sections per sub-group, always at start, looking for the internal pyramidal layer. Comparing the groups (SSI) and (SIR) a significant increase in NFkB-labeled cells in the subgroup SIR T30 (238,25Â218,437 versus 1234,75Â144,857); p=0,001 and SIR T60 (586,50Â141,210 versus 1286,25Â84,968); p=0,002 was observed. There was also a significant increase in the (SIR) subgroup, of the TNF immunostaining at T30 and T60 (263,75Â42,906 versus 1015,50Â102,796); p=0,01 in comparison to the SSI group. Regarding the cells expressing IL-6 positive labeling, comparing the groups (SIR) and (GIR), it was observed a significant decrease at T30 and T60 in the GIR group (536,25Â119,837 versus 9,00Â18,000); p=0,033. Comparing the groups (SIR) and (GIR), in relation to the NFkB immunolabeling, there was a significant decrease in group T30 in the GIR group (1234,75Â144,857 versus 247,50Â495); p=0,001 as well as at T60 (1286,25Â84,968 versus 217,75Â435,500); p=0,001. There was also a significant decrease, comparing the groups (SIR) and (GIR), of the TNF expression in (GIR) group at all time periods, chiefly at T60 (1015,50Â102,796 versus 3,50Â7,000); p=0,001. Regarding the immunostaining of the enzyme HO-1, there has been, comparing the groups (SSI) and (SIR), a significant decrease in the expression of this anti-oxidant protein in the SIR group. In the contrary, comparing the groups SIR and GIR, a substantial and significant expression of HO-1 was documented in the group treated previously by the L-alanil-glutamina, at times T30 and T60. In conclusion, the prior administration of L-alanil-glutamina in experiments of ischemia-reperfusion in Gerbils, disclosed a protective outcome, by promoting an in situ reduction in the expression of pro-inflammatory cytokines (TNF and IL-6) and nuclear transcription factor NFkB. At the same time, there has been an increase in the induction of the expression of the anti-oxidative enzyme HO-1, supporting a protective role of this preconditioning agent, chiefly against the inflammatory-oxidative stress. caused by the ischemia reperfusion brain injury model, in Gerbils / Foi investigado o efeito da administraÃÃo prÃvia de L-alanil-glutamina (L-ALN-GLN) nas lesÃes de isquemia e reperfusÃo em cÃrebros de gerbils atravÃs da detecÃÃo imunohistoquÃmica da expressÃo de mediadores prÃ-inflamatÃrios e da defesa anti-oxidante (IL-6, TNF, NF-kB e HO-1). Trata-se de um estudo experimental, controlado, utilizando 54 gerbils machos, com peso mÃdio de 150g, distribuÃdos aleatoriamente em 3 grupos: salina sem isquemia e reperfusÃo (SSI), salina com isquemia e reperfusÃo (SIR), L-alanil-glutamina com isquemia e reperfusÃo (GIR), redistribuÃdos em 3 subgrupos: T0 (tempo mÃximo de isquemia), T30 (30 min de reperfusÃo) e T60 (60 min de reperfusÃo), com 6 animais por subgrupo. Foram prÃ-tratados com soluÃÃo salina 2,0 mL 0,9% via endovenosa (e.v.) ou L-ALN-GLN 0,75g/Kg (e.v.), 30 min antes do inÃcio dos experimentos. A isquemia cerebral foi induzida pela oclusÃo bilateral das artÃrias carÃtidas comuns (ACCs), por um perÃodo de 15 minutos. ApÃs todos os procedimentos cirÃrgicos e ao final dos trÃs tempos 0, 30, 60 minutos, os animais foram sacrificados e tiveram os tecidos cerebrais removidos e fixados em formol a 10% por 24 horas, a fim de serem devidamente processados para inclusÃo em parafina. ApÃs esse procedimento, foram feitos cortes de 4 Âm em micrÃtomo e colocados em lÃminas de L-polilisina, apropriadas para a realizaÃÃo da imunohistoquÃmica. Foi utilizado o mÃtodo de estreptavidina-biotina-peroxidase. A avaliaÃÃo quantitativa foi feita por meio da contagem das cÃlulas nervosas imunomarcadas, tanto neurÃnios quanto cÃlulas da neuroglia, observadas com aumento de 400X em microscÃpio Ãptico modelo Olympus BX41. Foram observados dez campos de cada corte, com um total de 4 cortes por subgrupo, procurando-se sempre iniciar pela camada piramidal interna. Nas comparaÃÃes entre os grupos (SSI) e (SIR) houve um aumento significante nas cÃlulas NFkB-posittivas no grupo SIR nos tempos T30 (238,3Â218,4 versus 1234,8Â144,9); p=0,000 e T60 (586,5Â141,2 versus 1286,3Â85,0); p=0,000. Verificou-se tambÃm um aumento significante do TN no grupo SIR nos tempos T30 (214,0Â17,3 versus 333,0Â395,6); p=0,999 e fortemente, em T60 (263,8Â42,9 versus 1015,5Â102,8); p=0,00 e observou-se um aumento significante da IL-6 no tempo T60 no grupo SIR em comparaÃÃo ao grupo SSI, (206,8Â248,3 versus 316,0Â365,4); p=0,999. Entretanto, as cÃlulas marcadas pela HO-1, no grupo SIR, comparativamente ao grupo SSI, diminuÃram significativamente nos tempos T30 (25,8Â3,9 versus 11,3Â13,0) p=1,000 e T60 (18,5Â7,6 versus 9,8Â13,7) p=1,000. Para as cÃlulas expressando marcaÃÃo para as citocinas IL-6 em T0 (311,0Â246,5 versus 14,8Â29,5); p=0,817. T30 (536,3Â119,8 versus 9,0,3Â18,0); p=0,033. T60 (316,0Â365,4 versus 28,5Â57,0); p=0,591. E TNF em T0 (30,3Â36,0 versus 3,5Â7,0); p=1,000 e T30 (333,0Â395,6 versus 3,5Â7,0); p=0,503, T60 (1015,5Â102,8 versus 3,5Â7,0); p=0,000 comparando-se os grupos (SIR) e (GIR), observou-se uma diminuiÃÃo significante, em todos os tempos, no grupo GIR, contrariamente à imunomarcaÃÃo da HO-1, que aumentou significativamente no grupo (GIR) tratado pela L-alanil-glutamina em T0 (32,5Â9,7 versus 0,0+0,0); p=1,000. T30 (11,3Â13,0 versus 6,8Â13,5); p=1,000 e T60 (9,8Â13,7 versus 7,5Â15,0); p=1,000. Nas comparaÃÃes entre os grupos (SIR) e (GIR), em relaÃÃo ao NFkB, houve uma diminuiÃÃo significante no grupo GIR em T30 (1234,8Â144,9 versus 247,5Â495,0); p=0,000 e T60 (1286,3Â85 versus 217,8Â435,5); p=0,000. Em conclusÃo, a administraÃÃo prÃvia de L-alanil-glutamina, no presente experimento de isquemia/reperfusÃo cerebral, em gerbils, revelou efeito protetor da mesma, ao promover reduÃÃo in situ na expressÃo de citocinas e fator de transcriÃÃo de genes prÃ-inflamatÃrios: TNF, IL-6 e NFkB. Concomitantemente, houve induÃÃo aumentada da expressÃo da enzima antioxidante HO-1, corroborando a aÃÃo protetora deste nutracÃutico na injÃria de isquemia/reperfusÃo, mormente do estresse inflamatÃrio-oxidativo, em gerbils
| Identifer | oai:union.ndltd.org:IBICT/oai:www.teses.ufc.br:6232 |
| Date | 23 October 2012 |
| Creators | Leidelamar RosÃrio Alves de Oliveira |
| Contributors | Paulo Roberto LeitÃo de Vasconcelos, Vilma Leite Sousa Pires, Antonio Wilson Vasconcelos |
| Publisher | Universidade Federal do CearÃ, Programa de PÃs-GraduaÃÃo em Cirurgia, UFC, BR |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | application/pdf |
| Source | reponame:Biblioteca Digital de Teses e Dissertações da UFC, instname:Universidade Federal do Ceará, instacron:UFC |
| Rights | info:eu-repo/semantics/openAccess |
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