Leon Tai é financiado por fundos de start-up da Universidade de Illinois Chicago.

Todas as experiências seguem os protocolos do Comitê de Consentimento Institucional de Animais e Instituições da Universidade de Illinois, Chicago. 1. Preparação geral NOTA: A linha de células endoteliais microvasculares do cérebro (hCMEC / D3) é uma linha BEC humana imortalizada amplamente caracterizada 14 , 15 , 16 , 18 , 19 . Cultive as células hCMEC / D3 em frascos de cultura de tecidos revestidos com colágeno tipo I (pele de bezerro, diluição 1:20 de solução a 0,1% na solução de sal equilibrada de Hank (HBSS) contendo Ca 2+ e Mg 2+ ) no meio basal de crescimento endotélial basal (EBM-2) contendo 2-5% de soro bovino fetal (FBS), 10% de ácido ascórbico, 10% de sulfato de gentamicina, 25% de hidrocortisona e 1/4 do volume total dos suplementos de fator de crescimento fornecidos por 500 mL de meio [ Endotélio vascular grO Factor Owth (VEGF), o fator de crescimento epidérmico (EGF), o fator de crescimento insulino-like 1 (IGF-1) eo fator de crescimento fibroblástico básico humano (bFGF), ver Tabela de Materiais ]. NOTA: O meio EBM-2 com FBS e fatores de crescimento é referido como &quot;EBM-2 completo&quot;. EBM-2 sem FBS e suplementos é referido como &quot;EBM-2 basal&quot;. Na confluência total, as células hCMEC / D3 são ~ 1 x 10 5 células / cm 2 . <ol><li> Passar as células hCMEC / D3 a uma proporção de 1: 5 pela primeira incubação com HBSS durante 3 min sem Ca 2+ e Mg 2+ seguido de desprendimento com 5 mL de tripsina / EDTA (0,25%) durante 5 min. Neutralize a tripsina utilizando EBM-2 completo (neutralização 1: 1) e centrifugue a 290 xg durante 5 minutos a 4 ° C; Descarte o sobrenadante. Ressuspender a pastilha em EBM-2 completa e re-chapa em proporção 1: 5. </li><li> Cultive os pericitos e astrócitos humanos primários de acordo com o protocolo do fornecedor [ Tabela de Materiais ]. CulturE os pericitos no meio basal pericyte com FBS e suplemento de crescimento pericítico. </li><li> Cultive os astrocitos humanos em meio de astrocito contendo fatores de crescimento de astrocitos. Cultive tanto os pericitos quanto os astrocitos em frascos de cultura de tecidos revestidos com 3 mL de poli-L-lisina (PLL) para a aderência celular e utilizem as células entre as passagens 2 e 5. </li><li> Eutanizar 7 camundongos e remover as hastes do cérebro e cerebella com fórceps; Desanexar as meninges com cuidado rolando o cérebro com gaze. Agite o tecido cerebral restante em uma placa de Petri em meio mínimo essencial (MEM) com HEPES (5 mL por cérebro) com uma lâmina de barbear estéril. Isolar os BEC de ratos primários de ratos C57BL / 6J de 2 meses de acordo com o protocolo 20 referenciado. </li><li> Transfira o tecido cerebral triturado para um tubo cônico de 15 mL. Centrifugar a 290 xg durante 5 min a 4 ° C. Remova o sobrenadante e incube o tecido em uma papaína (833,33 μL por cérebro) e DNase (41,7 μL por cérebro)Solução, em banho-maria a 37 ° C durante 1 h, para digerir o tecido. </li><li> Triturar o homogeneizado sequencialmente através de agulhas de calibre 19 e 21, misturar a uma proporção de 1: 1 com uma solução de albumina de soro bovino (BSA) a 22% e centrifugar a 1360 xg durante 10 min a 4 ° C. </li><li> Ressuspender o sedimento resultante em 1 mL de EBM-2 completo e centrifugar a 290 xg durante 5 min a 4 ° C. Ressuspender novamente em 1 mL de EBM-2 completo e prender as células (1 mL / poço) em placas de 6 poços revestidas com colágeno (~ 1 cérebro por poço). </li><li> Substitua a mídia 24 h mais tarde por EBM-2 completo contendo 4 μg / mL de cloridrato de puromicina; Substitua com o EBM-2 completo após 2 dias. Os BEC primários são cultivados quanto às células hCMEC / D3 e estão em confluência total em 1 x 10 5 células / cm 2 . </li><li> Prepare um 100 μM de oAβ, 24 h antes do ensaio. NOTA: OAβ é considerado a forma relevante para a doença de A. Para oAβ, use as preparações bem caracterizadas descritas por DahlgreN et al. 21 . </li><li> Destilar a solução de reserva da membrana no solo durante a noite a 4 ° C e uma alíquota em tiras de tubo de PCR estéril (8 tubos) a 140 μL de membrana basal por tubo. Refonte cada tira e armazene a -20 ° C. Descongelar uma tira por placa de 96 poços a 4 ° C, 24 h antes do ensaio. Pré-esfrie as pontas da pipeta para evitar a solidificação. NOTA: Todo o tratamento da matriz da membrana basal deve ser realizado em gelo para evitar solidificação rápida. Como 10 μL por poço de membrana basal é usado no dia do ensaio, cada tira é suficiente para uma placa de 96 poços. </li><li> Incubar BEC totalmente confluentes em EBM-2 basal, 24 h antes do ensaio. NOTA: O raciocínio é: o EBM-2 completo contém fatores suplementados e FBS com o objetivo de promover o crescimento celular ótimo; No entanto, as mesmas vias moleculares que promovem o crescimento celular também são importantes para a cobertura e dinâmica das células endoteliais cerebrais, tanto na presença quanto no abseUm estressor. Portanto, soro e suplemento morrem de fome dos BECs para reduzir o efeito de confusão da ativação residual das vias de sinalização celular. De notar, as células são brevemente (5 min) expostas a FBS durante a neutralização de células tripsinizadas antes do ensaio. Em contraste com os BECs, os pericitos e astrócitos não são soro feridos, devido à exigência de diferentes fatores para o crescimento e a relativa instabilidade desses tipos de células em resposta à inanição de soro (laboratório de Tai, observações não publicadas). NOTA: O EBM-2 basal é utilizado durante o ensaio para os ensaios de formação e de interrupção da malha de cultura única e tripla. Isto é crítico para evitar a degradação do estressor ou sinalização dependente do tratamento. </li></ol> 2. Ensaios de Formação e Perturbação semelhantes a Meshwork <ol><li> Ensaios simples de cultura BEC: NOTA: Três paradigmas diferentes para culturas individuais de BECs são mostrados na Figura 1A </Forte&gt;. Cada paradigma é projetado para avaliar a resposta dos BEC em diferentes modelos de cobertura de embarcações. O paradigma 1 é a formação de malha. Este paradigma é projetado para examinar os efeitos de estressores e / ou tratamentos na angiogênese e formação de malha. BECs, oAβ e tratamentos são todos adicionados à placa no ponto horário de 0 h. Paradigma 2 é a prevenção da interrupção da malha. As células são plaqueadas na presença de um fator de crescimento ou fármaco e incubadas durante 4 h para formar estruturas tipo malha. Um estressor, oAβ neste caso, é adicionado após 4 h. Este paradigma avalia a capacidade de um tratamento para evitar danos induzidos pelo estressor a estruturas pré-formadas semelhantes a redes. Paradigma 3 é o tratamento simultâneo da interrupção da malha. As células são banhadas e permitidas formar estruturas tipo malha durante 4 h. Os tratamentos e oAβ são então adicionados simultaneamente no ponto de tempo de 4 h, avaliando a capacidade da rede celular pré-formada para responder a vários tratamentos. Todos os paradigmas paraEssas etapas comuns, exceto com diferenças no momento da adição do tratamento. <ol><li> No dia do ensaio, pipetar a matriz da membrana do porão a 10 μL / poço no fundo das placas de 96 poços e permitir que seja ajustado durante 1 h a 37 ° C. </li><li> Suspender o corante de rastreamento de células verdes liofilizado (ver Tabela de Materiais ) em 10 μL de DMSO para gerar uma solução de reserva de 10 mM e diluir para 10 μM (1: 1000) em EBM-2 basal. Remova a mídia do frasco totalmente confluente e substitua-a com base EBM-2 contendo o corante de rastreamento de células verdes (5 mL para um balão de 75 cm2). Preload BECs com o corante de rastreamento de células verdes, 20 min antes do início do ensaio. </li><li> Incube as células por 20-30 min a 37 ° C, remova o meio com corante de rastreamento celular e separe as células como descrito no Passo 1.1. Neutralize a mídia com EBM-2 contendo 10% de FBS e centrifugue a 240 xg durante 5 min a 4 ° C. Ressuspender o sedimento em 1 mL de EBM-2 basal. </li><li> Placa os BECs na placa de 96 poços em 10.000 células / poço em EBM-2 basal (0 h ponto de tempo em todos os paradigmas). NOTA: Dependendo do paradigma que está sendo utilizado, tratamentos, estressores ou controles do veículo são adicionados nos pontos de tempo especificados. Em todos os paradigmas, o meio é colhido para análise subsequente ( por exemplo, análise ELISA) e as células são fixadas às 24 h com paraformaldeído 4% recentemente preparado em Soro Tamponado de Fosfato (PBS). Como uma abordagem alternativa para determinar o termo efeitos de oAβ na formação de malha, o protocolo pode ser modificado para pré-incubar frascos de cultura de células confluentes com oAβ e, em seguida, realizar os paradigmas de formação de malha (+/- oAβ). NOTA: O volume final em cada poço para todos os paradigmas é de 70 μL. Todos os tratamentos (oAβ, EGF, etc. ) são adicionados aos respectivos poços com uma diluição de 1:20 ou seja, 3,5 μL por tratamento. Para o Paradigma 1, as células são adicionadas numa solução de suspensão celular a 63 μL / pEll. Imediatamente oAβ, tratamentos desejados e controles são adicionados a 3,5 μL / poço cada, dando um total de 70 μL. Para Paradigm 2, são adicionados 63 μL de suspensão celular e 3,5 μL de tratamento ( por exemplo, 100 ng / mL de EGF) no ponto de tempo de 0 h. Após 4 h de incubação, adicionam-se 3,5 μL de uma reserva de oAβ ( por exemplo, para uma concentração final de 100 μM de OAβ, 3,5 μL de 2 μM de reserva de OAβ). Para Paradigm 3, são adicionados 63 μL de suspensão celular no ponto de tempo de 0 h e às 4 h, oAβ e os tratamentos desejados são adicionados a 3,5 μL / poço cada, dando um total de 70 μL. Estes volumes podem ser ajustados de acordo com os tratamentos. Por exemplo, se apenas um tratamento for necessário, o volume da suspensão celular pode ser ajustado para 66,5 μL (mantendo 10 000 células / poço) e o volume do tratamento pode permanecer em 3,5 μL. </li></ol></li><li> Ensaio de cultura tripla: NOTA: Além dos ensaios de cultura única de BECs, desenvolvemosUm paradigma de ensaio de cultura tripla ( Figura 1B ) para determinar a resposta de BECs, pericítas e astrocitos em redes pré-formadas a estressores relevantes ( por exemplo, oAβ). Os BECs são chapeados na presença de tratamentos desejados às 0 h. Às 4 h, os pericitos são suavemente adicionados à placa. Às 7 h, os astrocitos são adicionados à placa, seguido da adição de um estressor às 11 h. As células são então incubadas até o ponto de tempo de 24 horas. NOTA: Para os ensaios de cultura tripla, é importante considerar o tempo para garantir que todas as células sejam confluentes ao mesmo tempo. Astrocitos e pericitos humanos devem ser cultivados 1 semana antes do ensaio, enquanto que as células hCMEC / D3 precisam ser banhadas ou passadas 4-5 dias antes da data do ensaio. Além disso, é importante usar células primárias de passagem baixa (2-5) para evitar deriva fenotípica. <ol><li> Adicione a solução de trabalho de corante de rastreamento de célula verde às células hCMEC / D3 20 min antes do início do ensaio. Placa as células hCMEC / D3 às 10, 000 células / poço em EBM-2 basal (ponto horário de 0 h). Os volumes utilizados são 45 μL de suspensão celular e 3,5 μL de tratamento ( ou seja, concentrações finais de EGF de 50 ng / mL, 100 ng / mL ou 1000 ng / mL de estoques 20 vezes mais concentrados). </li><li> Após a incubação de 3,5 h a 37 ° C, adicione a solução de trabalho de corante de rastreamento de células azuis (rastreador de células de 10 μM azul em EBM-2 basal) aos pericitos primários humanos. No ponto de tempo de 4 h (~ 30 min de incubação com corante de rastreamento celular), adicione suavemente 2,000 pericitos / poço à placa que contém o hCMEC / D3 num volume de 6 μL / poço. </li><li> Após uma incubação adicional de 2,5 h (total 6,5 h a partir do ponto de tempo de 0 h), adicione a solução de trabalho de corante de rastreamento de células de laranja (laranja de rastreador de células 10 μM em EBM-2 basal) aos astrócitos primários humanos. No ponto de tempo de 7 horas, adicione suavemente 10.000 astrocitos / bem à placa em 12 μL de volume. Portanto, a relação celular global para células endoteliais: pericítes: astrócitos iS 5: 1: 5. Além disso, o volume alcançado até este ponto é de 66,5 μL. </li><li> Adicione oAβ (3,5 μL de estoque de 100 μM) 4 h depois (total 11 h a partir do ponto de tempo de 0 h). </li><li> Corrija as células 13 h depois em paraformaldeído a 4% recentemente preparado em PBS. Cuidado: use roupas protetoras, luvas e óculos enquanto manipula paraformaldeído. </li></ol></li></ol> 3. Quantificação <ol><li> Capture imagens de fluorescência do poço inteiro da placa de 96 poços com uma ampliação de 1,6X com um tempo de exposição de 2 s com 50% de potência máxima usando um microscópio de dissecação. NOTA: Os microscópios equivalentes podem ser utilizados; No entanto, é fundamental capturar todo o poço para análise abrangente. </li><li> Para análise quantitativa, processe as imagens usando o plugin 22 do analisador de angiogênese ImageJ para quantificar o número de ramos, o número de malhas e o comprimento total da célula (essas leituras são tabuladas automaticamente pelo softwaRe) conforme descrito abaixo. Um protocolo visual detalhado desta etapa é fornecido na Figura 2 . <ol><li> Abra o fiji ImageJ clicando no ícone do software. </li><li> Clique nas duas setas diretas do lado direito localizadas no final da barra de ferramentas, depois clique em &quot;Angiogenesis Analyzer&quot;. </li><li> Selecione a pasta com arquivos de imagem, clique em &quot;Abrir&quot;. </li><li> Quando a caixa &quot;Configurações para análise&quot; aparecer, clique em &quot;OK&quot;. </li><li> Quando a caixa &quot;imagens de processamento&quot; for exibida, o que indica o número de imagens a serem processadas, clique em &quot;OK&quot;. </li><li> Quando aparece a caixa &quot;janela de progresso do lote&quot;, que indica o tempo de processamento estimado. Durante este tempo, o programa quantifica as saídas, incluindo o número de ramos, o número de malhas e o comprimento total da célula. NOTA: Uma vez que todas as imagens são quantificadas, o arquivo de resultados aparecerá na pasta de imagem original como um documento de planilha eletrônica. </li><li> SelecioneO arquivo da planilha contendo resultados e compare o número de ramos, malhas e o comprimento total da célula entre os grupos. NOTA: No ensaio de cultura tripla, o ImageJ é utilizado para analisar a cobertura e número de pericitos / astrocitos. As imagens são controladas por um experimentador cego e a função &quot;analisar partículas&quot; utilizada para gerar leituras. As células fixas e as estruturas semelhantes a tubos também podem ser imunossintérias para Aβ 17 ou outros marcadores. </li></ol></li></ol>

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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oligomeric+and+fibrillar+species+of+amyloid-beta+peptides+differentially+affect+neuronal+viability.">Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability.</a> J Biol Chem. 277 (35), 32046-32053 (2002).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+Analyzer.">Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li><li>Thomas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+prevents+APOE4+and+amyloid-beta-induced+cognitive+and+cerebrovascular+deficits+in+female+mice.">Epidermal growth factor prevents APOE4 and amyloid-beta-induced cognitive and cerebrovascular deficits in female mice.</a> Acta Neuropathol Commun. 4 (1), 111 (2016).</li><li>Tai, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+APOE+in+cerebrovascular+dysfunction.">The role of APOE in cerebrovascular dysfunction.</a> Acta Neuropathol. 131 (5), 709-723 (2016).</li><li>Ambrose, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroangiogenesis:+a+vascular+basis+for+Alzheimer's+disease+and+cognitive+decline+during+aging.">Neuroangiogenesis: a vascular basis for Alzheimer's disease and cognitive decline during aging.</a> J Alzheimers Dis. 32 (3), 773-788 (2012).</li><li>Ambrose, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+therapeutic+approach+for+senile+dementias:+neuroangiogenesis.">A therapeutic approach for senile dementias: neuroangiogenesis.</a> J Alzheimers Dis. 43 (1), 1-17 (2015).</li><li>Ambrose, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Capillaries+in+the+Lesser+Ailments+of+Old+Age+and+in+Alzheimer's+Disease+and+Vascular+Dementia:+The+Potential+of+Pro-Therapeutic+Angiogenesis.">The Role of Capillaries in the Lesser Ailments of Old Age and in Alzheimer's Disease and Vascular Dementia: The Potential of Pro-Therapeutic Angiogenesis.</a> J Alzheimers Dis. 54 (1), 31-43 (2016).</li></ol>

Os autores não têm nada a revelar.

Os métodos descritos podem ser utilizados para abordar várias questões biológicas fundamentais em torno da cobertura cerebrovascular 24 . Especificamente, eles podem identificar quais receptores e vias de sinalização desempenham um papel na angiogênese, na cobertura vascular no tecido cancerígeno e nas células endoteliais periféricas relevantes para o cérebro. Exemplos incluem receptores de fator de crescimento angiogênico, óxido nítrico, sinalização de proteína quinase ativada p...

<html> A barreira hematoencefálica (BBB) ​​dos capilares cerebrais é a maior interface de contato de sangue para cérebro e desempenha um papel central na homeostase do sistema nervoso central (SNC) 1 , 2 . Os processos dinâmicos no BBB impedem a absorção de moléculas indesejadas do sangue, eliminam os resíduos do SNC, fornecem nutrientes essenciais e moléculas de sinalização ao SNC e modulam a neuroinflamação 1 , 2 , 3 , 4 , 5 . O dano BBB é prevalente durante o envelhecimento e vários distúrbios neurodegenerativos, incluindo doença de Alzheimer (AD), esclerose múltipla e AVC 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Portanto, a disfunção BBB pode desempenhar um papel fundamental em distúrbios neurodegenerativos, inclusive como alvo terapêutico. Manter a cobertura do navio é importante para as funções homeostáticas da BBB. No entanto, os dados in vivo e in vitro conflitam se os processos envolvidos em distúrbios neurodegenerativos causam maior ou menor cobertura BBB 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , particularmente em AD. Portanto, há uma forte justificativa para o desenvolvimento de modelos in vitro usando tipos de células relevantes para avaliar e compreender de forma mais abrangente a dinâmica da cobertura BBB. Os capilares cerebrais são compostos de astrocitos, pericitos e células endoteliais do cérebro (BECs) <sUp class = &quot;xref&quot;&gt; 3. Todos os tipos de células contribuem para a função do BBB através do suporte estrutural e através da secreção de moléculas efectoras, como fatores de crescimento angiogênicos, citocinas e quimiocinas que atuam de forma paracrina e autocrina. No entanto, as principais células efetoras da BBB são BECs 3 . Em geral, as técnicas de cultura celular para avaliar a função BBB são os ensaios de permeabilidade realizados em células cultivadas em inserções de filtro, ou avaliar níveis de proteínas BEC chave, ambos após a adição de estressores 14 , 15 , 16 . Embora importantes, esses ensaios não se concentram na cobertura cerebrovascular. Aqui, nossos métodos anteriores 17 são detalhados para avaliar a cobertura de BEC e as estruturas semelhantes a redes, usando culturas únicas de BEC humanas imortalizadas, culturas únicas de BEC primários de mouse e uma cultura tripla humanizadaModelo (BECs, astrocitos e pericitos) do BBB. O objetivo foi demonstrar o efeito prejudicial da OAβ, que é considerado um contribuinte importante para a progressão do AD, na cobertura da BEC. O efeito protetor do fator de crescimento epidérmico (EGF) destaca o potencial da técnica como ferramenta de triagem terapêutica. A técnica tem várias aplicações amplas para pesquisa básica e aplicada, incluindo: 1) delineando o papel de caminhos específicos sobre angiogênese e cobertura de vasos, 2) avaliação dos efeitos da doença e fatores relevantes ao envelhecimento na angiogênese e cobertura vascular e 3) identificação de farmacologia Alvos.

<table border="0" cellpadding="0" cellspacing="0" width="847">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">hCMEC/D3 cells</td>
			<td style="width:183px;">Milipore</td>
			<td style="width:119px;">SCC066</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">EBM-2 basal media&#160;</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-3156</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Collagen Type 1</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">A1064401&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, calcium, magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14025092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">HBSS, no calcium, no magnesium, no phenol red</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Trypsin-EDTA (0.25%)</td>
			<td style="width:183px;">ThermoFisher</td>
			<td style="width:119px;">25200056</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:379px;">Final concentrations of the SingleQuot growth factor supplements for EBM2 media</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5% FBS</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Ascorbic acid</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10% Gentamycin sulphate</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25% Hydrocortisone</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="78">
			<td height="78" style="height:78px;width:379px;">1/4 volume of the supplied growth factors: fibroblast growth factor, epidermal growth factor, insulin-like growth factor, vascular endothelial growth factor</td>
			<td style="width:183px;">Lonza</td>
			<td>CC-4147</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Puromycin hydrochloride</td>
			<td style="width:183px;">VWR</td>
			<td style="width:119px;">80503-312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">MEM-HEPES&#160;</td>
			<td style="width:183px;">Thermo Scientific&#160;</td>
			<td style="width:119px;">12360-038</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Papain cell dissociation system (papain and DNase1)</td>
			<td style="width:183px;">Worthington Biochemical</td>
			<td style="width:119px;">LK003150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human pericytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte basal media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Pericyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1252</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Astrocytes</td>
			<td style="width:183px;">Sciencell</td>
			<td>1800</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte media</td>
			<td style="width:183px;">Sciencell</td>
			<td>1801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Astrocyte growth supplement</td>
			<td style="width:183px;">Sciencell</td>
			<td>1852</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Basement membrane (Matrigel Growth Factor Reduced)&#160;</td>
			<td style="width:183px;">Corning</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Angiogenesis m-plates (96-well)</td>
			<td style="width:183px;">ibidi</td>
			<td style="width:119px;">89646</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Human Epidermal growth factor&#160;</td>
			<td style="width:183px;">Shenendoah Biotechnology</td>
			<td style="width:119px;">100-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker green&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C7025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker orange</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C34551</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTracker blue&#160;</td>
			<td style="width:183px;">ThermoFisher</td>
			<td>C2110</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Poly-l-lysine</td>
			<td style="width:183px;">Sciencell</td>
			<td>0403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">10% Neutral Buffered Formalin</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td>HT5012-60ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">C57BL mice</td>
			<td style="width:183px;">Jackson Laboratory</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">PCR tube strips</td>
			<td style="width:183px;">GeneMate</td>
			<td style="width:119px;">T-3014-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Zeiss stereo discover v.8 dissecting microscope</td>
			<td style="width:183px;">Zeiss</td>
			<td style="width:119px;">na</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption

A cobertura da barreira hematoencefálica (BBB) ​​desempenha um papel central na homeostase do sistema nervoso central (SNC). O BBB é mantido dinamicamente por astrocitos, pericitos e células endoteliais cerebrais (BECs). Aqui, detalhamos métodos para avaliar a cobertura BBB usando culturas únicas de BEC humanas imortalizadas, culturas únicas de BEC de ratos primários e um modelo de cultura triplicada humanizado (BECs, astrocitos e pericitos) do BBB. Para destacar a aplicabilidade dos ensaios em estados de doença, descrevemos o efeito da amilóide-β (oAβ) oligomérica, que é um contribuinte importante para a progressão da doença de Alzheimer (AD), na cobertura de BBB. Além disso, utilizamos o fator de crescimento epidérmico (EGF) para iluminar o potencial de rastreamento de drogas das técnicas. Nossos resultados mostram que os BEC cultivados de um único e triplo formam estruturas semelhantes a malhas em condições basais, e que oAβ interrompe essa formação de malha celular e degenera as estruturas de malha pré-formadas,Mas EGF bloqueia essa interrupção. Assim, as técnicas descritas são importantes para a dissecação de processos fundamentais e relevantes para a doença que modulam a cobertura BBB.

Em culturas individuais, tanto as células hCMEC / D3 ( Figura 3A ) como os BEC primários do mouse ( Figura 3B ) formam estruturas semelhantes a redes em todo o poço. As estruturas são caracterizadas por uma malha de nós interligados ( Figura 3 ). Em todos os paradigmas descritos ( Figura 1 ), as estruturas semelhantes às malhas são semelhantes após 24 h nos grup...

Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

A manutenção da cobertura da barreira hematoencefálica é fundamental para a homeostase do sistema nervoso central. Este protocolo descreve técnicas in vitro para delinear os processos fundamentais e patológicos que modulam a cobertura da barreira hematoencefálica.

Ensaios In Vitro para Avaliar Formação e Perturbação de Embarque de Barreira de Sangue-Cérebro

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by ...

in-vitro-assays-to-assess-blood-brain-barrier-mesh-like-vessel

Research

JoVE Journal

Medicine

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

8.4K visualizações.  University of Illinois at Chicago.  A manutenção da cobertura da barreira hematoencefálica é fundamental para a homeostase do sistema nervoso central. Este protocolo descreve técnicas in vitro para delinear os processos fundamentais e patológicos que modulam a cobertura da barreira hematoencefálica. 

Vídeo: In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Guiada por MRI rompimento da barreira hemato-encefálica utilizando Ultrassom Focalizado transcraniana em um modelo de rato

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Medicina

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

A Medição e Tratamento de Supressão de ambliopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

Modelo de ratos de sangue-cérebro Rompimento de barreira para permitir alvejado Neurovascular Therapeutics

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

An in vivo Assay to Test Blood Vessel Permeability

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under physiologic conditions the endothelium is impermeable to albumin, so Evans blue bound albumin remains restricted within blood vessels. In pathologic conditions that promote increased vascular permeability endothelial cells partially lose their close contacts and the endothelium becomes permeable to small proteins such as albumin. This condition allows for extravasation of Evans Blue in tissues. A healthy endothelium prevents extravasation of the dye in the neighboring vascularized tissues. Organs with increased permeability will show significantly increased blue coloration compared to organs with intact endothelium. The level of vascular permeability can be assessed by simple visualization or by quantitative measurement of the dye incorporated per milligram of tissue of control versus experimental animal/tissue. Two powerful aspects of this assay are its simplicity and quantitative characteristics. Evans Blue dye can be extracted from tissues by incubating a specific amount of tissue in formamide. Evans Blue absorbance maximum is at 620 nm and absorbance minimum is at 740 nm. By using a standard curve for Evans Blue, optical density measurements can be converted into milligram dye captured per milligram of tissue. Statistical analysis should be used to assess significant differences in vascular permeability.

An in vivo Assay to Test Blood Vessel Permeability

We are presenting an in vivo assay to test blood vessel permeability. This assay is based on intravenous injection of a dye and subsequent visualization of its diffusion into interstitial spaces.

Um<em&gt; In vivo</em&gt; Ensaio para ensaio de permeabilidade dos vasos sangüíneos

This method is based on the intravenous injection of Evans Blue in mice as the test animal model. Evans blue is a dye that binds albumin. Under ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanente oclusão vascular cerebral<em&gt; Via</em&gt; Dupla ligadura e transecção

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Estique em células cerebrais endoteliais microvasculares (cEnd) como um<em&gt; In Vitro</em&gt; Modelo de lesão cerebral traumática do cérebro Barreira Hemato

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Criação de um<em&gt; In Vitro</em&gt; Modelo de Rato barreira hemato-encefálica (BBB): A Focus on BBB impermeabilidade e Transportes Receptor mediada

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Oxigênio-Glucose Privação e reoxigenação como um<em&gt; In Vitro</em&gt; Isquemia e reperfusão Lesão modelo para estudar Disfunção Barreira Hematoencefálica

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

A abordagem de amostragem de 4 embarcações para estudos integrativos de fisiologia placentária humana<em&gt; In Vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Melhor método para o estabelecimento de uma In Vitro barreira hemato - encefálica modelo baseado em células endoteliais do cérebro de suínos

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Ensaios In Vitro para Avaliar Formação e Perturbação de Embarque de Barreira de Sangue-Cérebro

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