A neurogênese induzida por crises no giro denteado não está relacionada ao brotamento de fibras musgosas, mas é dependente da idade, em ratos durante o desenvolvimento

Neurogenesis in the dentate gyrus (DG) has attracted attention since abnormal supragranular mossy fiber sprouting occurs in the same region, in temporal lobe epilepsy. Thus, we submitted developing rats to pilocarpine-induced status epilepticus (SE) to study the relationship between neurogenesis and mossy fiber sprouting. Groups were submitted to SE at: I-P9, II-P7, P8 and P9, III-P17 e IV-P21. Neurogenesis was quantified using BrdU protocol and confirmed through double staining, using neuronal pentraxin. Other animals were monitored by video system until P120 and their brain was studied (Timm and Nissl staining). The neurogenesis at P17 (p=0.007) and P21 (p=0.006) were increased. However, only P21 group showed recurrent seizures and the mossy fiber sprouting in the same region, during adult life, while P17 did not. Thus, our results suggest that neurogenesis is not related to mossy fiber sprouting neither to recurrent spontaneous seizures in pilocarpine model.A neurogênese no giro dentado tem atraído atenção já que ela ocorre na mesma região do hipocampo que o brotamento das fibras musgosas, na epilepsia do lobo temporal. Assim, submetemos ratos em desenvolvimento ao status epilepticus induzido (SE) por pilocarpine. Grupos foram submetidos em I-P9, II-P7, P8, P9; III-P17 e IV-P21. A neurogênese foi observada usando o protocolo do BrdU e confirmada por dupla marcação com pentraxina neuronal. Outros animais foram monitorados até P120 e seus cérebros analisados (Nissl e Timm). A neurogênese nos grupos P17 (p=0,007) e P21 (p=0,006) aumentaram. Entretanto, o P21 apresentou crises espontâneas e brotamento de fibras musgosas, na mesma região onde ocorreu a neurogênese, enquanto o grupo P17 apresentou somente aumento na neurogênese. Assim, nossos resultados sugerem que o fenômeno da neurogênese não está relacionado com o brotamento de fibras musgosas nem com o aparecimento de crises espontâneas e recorrentes no modelo da pilocarpina.Universidade Federal de São Paulo (UNIFESP) Escola Paulista de MedicinaUNIFESP, EPMSciEL

The role of extracelular matrix from stimulation of heparan sulfate antithrombotic syntesis promoted by heparin in the endothelial cells

BV UNIFESP: Teses e dissertaçõe

Heparin: interaction with endothelial cells matriz a antithrombin ativit

Heparina e drogas antitromboticas em geral estimulam, de forma especifica, a sintese do proteoglicano de heparam sulfato (PGHS) em celulas endoteliais. O efeito e composto e celula especificos. Ensaios cineticos com heparina mo que o estimulo e tempo e dose dependentes, e que esta se liga na superficie celular, sugerindo a existencia de possiveis receptores. Pelo presente trabalho, desenvolveu-se uma heparina marcada com biotina com o intuito de estudar a interacao com celulas endoteliais. A reacao de biotinilacao resultou num composto contendo uma biotina para cada residuo de acido urom'co. Essa heparina apresentou menor atividade anticoagulante (38U.I./mg), desprezivel atividade hemorragica, maior peso molecular (l8,6kDa), quando comparados com a heparina padrao, que possui 166U.I./mg, potente acao hemorragica e peso molecular de l3kDa. Essa heparina ainda, deixou de ser substrato para a heparinase e heparitinase H, porem manteve a capacidade de interacao com a Antitrombina III e a Fibronectina Os dados mostraram tambem que esta heparina e capaz de estimular a sintese de PGHS, como a heparina padrao. O(s) sitio(s) de ligacao para a heparina, nas celulas endoteliais, foram investigados utilizando-se como modelo, a heparina biotinilada em tecnicas de deteccao citoquimica e analise em microscopia confocal. Alem desta heparina, foram utilizados lectina WGA, que interage com a superficie celular e anticorpos especificos tanto para a superficie celular (anti-sindecam 4), como para a matriz extracelular (anti-fibronectina). Esses compostos foram ensaiados empregando-se tanto celulas recem-plaqueadas, sub-confluentes e confluentes, como em suspensao. Ainda a matriz extracelular livre de celulas foi investigada. Todos estes experimentos, revelaram que a ligacao da heparina biotinilada ocorreu somente nos componentes da matriz extracelular. Experimentos mantendo-se as celulas em presenca de heparina biotinilada por 22 horas, mostraram que ocorre o processo de internalizacao da mesma. Este conjunto de resultados sugere que o estimulo da sintese de PGHS, causado pela heparina possa ser independente da sua interacao com a superficie celularBV UNIFESP: Teses e dissertaçõe

Establishment of experimental conditions for preserving samples of fish blood for analysis with both comet assay and flow cytometry

When environmental analysis is performed, the high number of samples required and handling conditions during the transport of these samples to the laboratory are common problems. The comet assay is a useful, highly sensitive tool in biomonitoring. Some studies in the literature aim to preserve slides in lysis solution for use in the comet assay. Until now, however, no efficient methodology for preserving blood samples for this assay has been described. Because of this, the present report aimed to establish the proper conditions for samples maintenance prior to comet assay analysis. Samples were conserved in three different solutions: a high protein concentration solution (fetal bovine serum-FBS), an anticoagulant agent (a calcium chelator - ethylenediaminetetracetic acid - EDTA), and a salt buffered solution (phosphate buffered saline-PBS). Therefore, peripheral blood samples of Rhamdia quelen specimens were collected and maintained in these solutions until testing at 72 h. Analyses of DNA fragmentation via the comet assay and cell viability via flow cytometry were performed at intervals of 24 h. The results showed that samples maintained in FBS were preserved better; this was followed by those preserved in PBS and then last by those preserved in EDTA. In conclusion, blood samples from freshwater fish can be preserved up to 48 h in fetal bovine serum at 4 °C in the absence of light. In this period, no DNA fragmentation occurs. We thus describe an excellent method of sample conservation for subsequent analysis in the laboratory

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid

Lauryl gallate loaded in superparamagnetic poly (methyl methacrylate) nanoparticles surface modified with folic acid were synthesized by miniemulsion polymerization in just one step. In vitro biocompatibility and cytotoxicity assays on L929 (murine fibroblast), human red blood, and HeLa (uterine colon cancer) cells were performed. The effect of folic acid at the nanoparticles surface was evaluated through cellular uptake assays in HeLa cells. Resultsshowed that the presence of folic acid did not affect substantially the polymer particle size (~120 nm), the superparamagnetic behavior, the encapsulation efficiency of lauryl gallate (~87 %), the Zeta potential (~38 mV) of thepolymeric nanoparticles or the release profile of lauryl gallate. The release profile of lauryl gallate from superparamagnetic poly(methyl methacrylate) nanoparticles presented an initial burst effect (0?1 h) followed by a slow andsustained release, indicating a biphasic release system.Lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles with folic acid did not present cytotoxicity effects on L929 and human red blood cells. However, free lauryl gallate presented significant cytotoxic effects on L929 and human red blood cells at all tested concentrations. The presence of folic acid increased the cytotoxicity of lauryl gallate loaded in nanoparticles on HeLa cells due to a higher cellular uptake when HeLa cells were incubated at 37 °C. On the other hand, when the nanoparticles were incubated at low temperature (4 °C) cellular uptake was not observed, suggesting that the uptake occurred by folate receptor mediated energy-dependent endocytosis. Based on presented results our work suggeststhat this carrier system can be an excellent alternative in targeted drug delivery by folate receptor.Fil: Feuser, Paulo Emilio. Universidade Federal de Santa Catarina; BrasilFil: Carpio Arévalo, Juan Marcelo. Universidade Federal do Paraná; BrasilFil: Lima, Enio Junior. Comisión Nacional de Energía Atómica. Gerencia del Área de Investigación y Aplicaciones No Nucleares. Gerencia de Física. Laboratorio de Resonancias Magnéticas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte; ArgentinaFil: Rodrigues Rossi, Gustavo. Universidade Federal do Paraná; BrasilFil: Trindade, Edvaldo da Silva. Universidade Federal do Paraná; BrasilFil: Merlin Rocha, Maria Eliane. Universidade Federal do Paraná; BrasilFil: Virtuoso Jacques, Amanda. Universidade Federal de Santa Catarina; BrasilFil: Ricci Júnior, Eduardo. Universidade Federal do Rio de Janeiro; BrasilFil: Santos Silva, Maria Claudia. Universidade Federal de Santa Catarina; BrasilFil: Sayer, Claudia. Universidade Federal de Santa Catarina; BrasilFil: Hermes de Araújo, Pedro H.. Universidade Federal de Santa Catarina; Brasi