Este trabalho foi apoiado por concessões do National Institutes of Health (R01-NS084941) e a Comissão de investigação biomédica do Arizona (ADHS16-162406) de PTR. WA recebeu passado o apoio de uma nomeação de doutorandos para um institutos nacionais de saúde formação Grant (T32-HL007249).

Todos os procedimentos descritos abaixo foram aprovados por um cuidado institucional do Animal e Comissão de utilização (IACUC) e estar de acordo com o National Institutes of Health (NIH) e pesquisa Animal: Reporting In Vivo experimentos (chegada) orientações. O fluxo processual para o protocolo é descrito na Figura 1.
1. set-up para o procedimento
<ol><li>Prepare o buffer de microvessel do cérebro (BMB). Comece por pesagem 54,66 g D-manitol, 1,90 g EGTA e base de 2-amino-2-(hydroxymethyl)-1,3-propanediol (ou seja, Tris) 1,46 g para um copo limpo. Adicione 1,0 L de água desionizada. Misture os componentes da reserva BMB utilizando um agitador magnético. Uma vez que a solução tem sido muito bem misturada, ajuste o pH para 7,4 usando 1,0 M de HCl.</li>
	<li>Prepare a solução de dextran (75.000 MW) de 26% (p/v) antes de anestesiar animais. Prepare a solução de dextran conforme descrito nas etapas a seguir. Nota: É criticamente importante preparar esta solução antes do tempo porque dextrano pode levar cerca de 1,5 a 2 h para dissolver em tampão aquosa.<ol>
<li>Pese o dextran em pó (26 g por 100 mL de tampão) em um copo limpo.</li>
		<li>Meça o volume apropriado de BMB e diluir em um copo separado, limpo.</li>
		<li>Lentamente despeje o copo contendo o pó dextrano BMB. Misture manualmente o dextran em pó durante o derramamento de BMB usando uma vareta de vidro celeuma.</li>
		<li>Ajuste o pH para 7,4 com 1,0 HCl M imediatamente antes de usar. Nota: Um isolamento típico do cérebro microvessels de 12 ratos de Sprague-Dawley individuais (masculino ou feminino; 3 meses de idade; 200-250 g cada) requer 200 mL da solução de dextran (i.e., dextrano 52 g em 200 mL de BMB).</li>
	</ol>
</li>
</ol>2. extração de tecido cerebral de ratos Sprague Dawley
<ol><li>Desejado no tratamento experimental, anestesiamos ratos Sprague-Dawley usando cetamina (50mg/kg; i.p. 2,5 mL/kg) e xilazina (10mg/mL; 2,5 mL/kg eu, p.). Dilua a ketamina e xilazina em soro fisiológico a 0,9% para concentrações finais de 20 mg/mL e 4,0 mg/mL, respectivamente. Eutanásia em animais por decapitação usando uma guilhotina afiada em conformidade com as diretrizes IACUC. Use o mesmo procedimento para masculinos e femininos ratos Sprague-Dawley.</li>
	<li>Ressecar a pele do crânio do rato, fazendo um único corte transversal com uma tesoura cirúrgica.</li>
	<li>Usando ruginas, Retire cuidadosamente a placa de crânio e expor o cérebro.</li>
	<li>Remova o cérebro com uma espátula. Desanexe o encéfalo e coloque o tecido cerebral isolada em um tubo cónico de 50 mL contendo 5 mL de BMB. Adicione 1,0 μL de cocktail por 1,0 mL de tampão BMB imediatamente antes da utilização do inibidor da protease.</li>
</ol>3. cérebro processamento
<ol><li>Transferi o tecido cerebral do tubo cónico de 50 mL para um tubo de ensaio limpo.</li>
	<li>Usando fórceps, Rode suavemente o cérebro 12,5 cm de diâmetro, papel de filtro para remover o exteriores meninges, que são vagamente aderiu ao córtex cerebral. Delicadamente pressione o tecido cerebral contra o papel de filtro e rolar o tecido novo. Vire o papel de filtro com frequência durante esta etapa.</li>
	<li>Separe os hemisférios cerebrais usando fórceps do plexo coroide. Nota: O plexo coroide aparece como um tecido claro membranoso localizado na superfície dos ventrículos cerebrais.</li>
	<li>Delicadamente, achatar o tecido cerebral e remover restantes meninges e bulbos olfatórios usando fórceps.</li>
	<li>Coloque o tecido cortical cerebral em um almofariz de vidro refrigerados. Adicione 5,0 mL de cocktail contendo inibidor da protease BMB a argamassa.</li>
	<li>Usando um homogenizador eléctricas aéreas, homogeneizar o tecido cerebral usando 15 subindo e descendo traçados a 3.700 rpm. Realize a homogeneização usando um moedor de tecido de 10 mL almofariz e pilão. Entre a homogeneização de cada amostra individual, limpe o pilão usando etanol a 70%. Nota: Homogeneização traços devem ser consistentes em termos de ritmo e magnitude.</li>
	<li>Despeje o homogeneizado em tubos de centrífuga rotulada.</li>
</ol>4. centrifugação passos
<ol><li>Adicione 8,0 mL da solução de dextran de 26% para cada tubo de centrifugação rotulado que contém o cérebro homogeneizado.</li>
	<li>Inverter o tubo duas vezes e então cuidadosamente a amostra de vórtice. Realizar a utilização do Vortex de cada amostra usando vários ângulos para assegurar uma mistura completa da solução de homogeneizado de cérebro com solução de dextran de 26%.</li>
	<li>Centrifugar as amostras a 5.000 x g durante 15 min a 4 ° C. Nota: Para algumas análises, é útil comparar o cérebro microvessels com parênquima cerebral. Deve ser a avaliação da expressão da proteína em células de parênquima cerebral necessária, o sobrenadante desta etapa de centrifugação (ou seja, cérebro parenquimatosa fração) pode ser recolhido e armazenado a-80 ° C para uso futuro.</li>
	<li>Remover o sobrenadante usando um aspirador a vácuo e pipetas de vidro. Nota: O cuidado deve ser tomado para não perturbar a pelota. Caso contrário, a quantidade do cérebro microvessels coletados será reduzida, que diminuirá significativamente o rendimento de proteína deste procedimento.</li>
	<li>Resuspenda o pellet em 5,0 mL de BMB contendo inibidor da protease cocktail (ou seja, 1,0 μL inibidor da protease cocktail por 1,0 mL de tampão BMB). A pelota para assegurar uma mistura completa de vórtice.</li>
	<li>Adicione 8,0 mL de dextrano de 26% para cada tubo de centrifugação e vórtice, conforme descrito nas etapas 4.1-4.2 do presente protocolo.</li>
	<li>Centrifugar as amostras a 5.000 x g durante 15 min a 4 ° C.</li>
	<li>Usando um balão de vácuo e pipeta de vidro, aspirar o sobrenadante e garantir aquela pelota contendo microvessel do cérebro não é interrompida. Nota: Excesso material que está aderido à parede do tubo não contém microvessels e deve ser cuidadosamente limpos e removido.</li>
	<li>Repita as etapas 4.5 a 4.8 um adicional duas vezes.</li>
	<li>Uma vez 4 dextrano centrifugação as etapas foram concluídas, adicionar 5,0 mL de BMB cada pellet e vórtice para Ressuspender a amostra. Nota: Após a conclusão da etapa 4.10, toda microvessels podem ser coletados para a análise da localização da proteína. Isso pode ser feito tomando um 50 μL alíquota de pelota microvessel re-suspensas e manchas em um vidro de microscópio. Microvessels são, então, calor-corrigido a 95 ° C por 10 min em um bloco de aquecimento seguido de fixação em etanol gelado para um adicional de 10 min. de Slides podem ser armazenados a 4 ° C até necessária para estudos de imagem.</li>
</ol>5. ultracentrifugação para preparar amostras de cérebro Total Microvascular membranas
<ol><li>Transferi as amostras para o homogeneizador copo gelado...</li>
	<li>Usando um homogenizador eléctricas aéreas, homogeneizar o tecido cerebral usando 8-10 e descer traçados a 3.000 rpm. Homogeneização é realizada usando um moedor de tecido de 10 mL almofariz e pilão. Limpe o pilão usando etanol a 70% após a homogeneização de cada amostra individual.</li>
	<li>Transferi as amostras para limpar os tubos se. Número e pesar cada tubo individual. Equilíbrio e emparelhar os tubos previamente ao seu embarque para o rotor se. Nota: Todos os pesando emparelhamento dos tubos se devem proceder e com as tampas para garantir uma medição precisa de pesos de tubo.</li>
	<li>Centrifugar as amostras a 150.000 x g durante 1 h a 4 ° C.</li>
	<li>Usando um balão de vácuo e vidro pipeta Pasteur, aspirar o sobrenadante usando o cuidado para não perturbar a pelota de membrana capilar.</li>
	<li>Baseado no tamanho da pelota, adicione um volume adequado de buffer de armazenamento, que é composta de água desionizada e BMB na proporção de 1:1 (v/v). Normalmente, as pelotas são resuspended em 400-500 μL de tampão de armazenamento. Adicione o cocktail de inibidor de protease para o buffer de armazenamento na proporção de 1,0 μL por 1,0 mL de buffer de armazenamento.</li>
	<li>Amostras de vórtice para resuspenda o pellet em buffer de armazenamento.</li>
	<li>Usando um pipeta Pasteur de vidro, transferi amostras de membrana capilar para um tubo de microcentrifugadora rotulado de 1,5 mL.</li>
	<li>Amostra de passagem através de uma agulha seringa 5 x para garantir que a amostra fique bem misturada e que não agregados celulares existem.</li>
	<li>Armazenar as amostras a-80 ° C até necessária para análise. Nota: O conteúdo de proteína de cada amostra de membrana microvessel é medido usando o método de Bradford.</li>
</ol>

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Neste artigo, é descrito um método simples e eficaz de preparação de amostras de proteínas de membrana de microvessels recentemente isolada do tecido de cérebro de rato. Várias abordagens para isolamento de microvessels de cérebro de rato e/ou geração de preparações de membrana da microvasculatura isolada têm sido relatadas na literatura13,20,21,22 , 24...

A barreira hemato - encefálica (BBB) existe na interface entre o sistema nervoso central (SNC) e a circulação sistêmica e desempenha um papel essencial na manutenção da homeostase do cérebro. Especificamente, as funções BBB para precisamente controle soluto concentrações no fluido extracelular de cérebro e eficientemente fornecer os nutrientes necessários para atender as demandas metabólicas consideráveis do CNS1pelo tecido cerebral. Essas funções implicam que o BBB, que existe principalmente no nível da célula endotelial microvascular, deve possuir mecanismos discretos que permitem que algumas substâncias acessar o parênquima cerebral, garantindo simultaneamente que xenobióticos potencialmente prejudiciais não pode se acumulam. Com efeito, as células endoteliais microvascular do cérebro não são fenestradas e exibem pinocitose limitado, que garante uma falta de permeabilidade não-seletivo2. Além disso, as células endoteliais do cérebro microvessel expressam apertado junção aderente junção proteínas e que agem para formar um físico "selar" entre células endoteliais adjacentes e restringir consideravelmente paracellular difusão de substâncias pelo sangue para o cérebro parênquima. Com efeito, permeabilidade seletiva de substâncias endógenas e exógenas requer expressão funcional de captação e efluxo de transportadores3. Globalmente, apertados cruzamentos, entroncamentos adherens e transportadores trabalham em conjunto para manter as propriedades de barreira exclusivo do BBB.
O BBB é uma barreira dinâmica que responde a estímulos fisiológicos, fisiopatológicos e farmacológicos. Por exemplo, estresse de hipóxia/reoxigenação foi mostrado para modular a expressão de proteínas críticas junção apertada (i.e., occludin, zonulae occluden-1 (ZO-1)), que está associado com aumento paracellular permeabilidade para marcadores vasculares tais como sacarose4,5,6. Observações semelhantes foram feitas no BBB no cenário do de lesão cerebral traumática7 e dor inflamatória periférica8,9. Essas mesmas doenças também podem modular os mecanismos de transporte para o BBB10,11,12,13,14. De fato, lesão hipóxia/reoxigenação aumenta a expressão funcional de ânion orgânico transportando polipeptídeo 1a4 (Oatp1a4) no BBB, que pode levar a aumentos significativos no transporte de sangue-cérebro de substratos de transporte Oatp específicos tais como taurocholate e atorvastatina13. Propriedades BBB também podem ser alteradas por farmacoterapia em si, um mecanismo que pode servir de base para as duas mudanças profundas na eficácia da droga no cérebro e para interações medicamentosas. Por exemplo, paracetamol alvos receptor nuclear mecanismos de sinalização nas células endoteliais microvascular cerebral, aumenta a expressão funcional do transportador efluxo crítico P-glicoproteína (P-gp) e modifica a analgesia dependente do tempo conferidos pela morfina, uma droga analgésica opioide e P-gp estabelecida transportam substrato15. Uma compreensão completa das alterações BBB, que pode ser induzida por doenças ou drogas, também requer a identificação e caracterização de mecanismos regulamentares específicos que controlam estas modificações. Com efeito, as vias de sinalização discretas foram identificadas nas células endoteliais microvascular do cérebro que controlam a expressão molecular de junção apertada proteínas16,17 e transportadores15, 18,19. Tomados em conjunto, estas observações indicam que vias moleculares complexas estão envolvidas na regulação de junções apertadas de BBB e transportadores na saúde e na doença.
Um desafio significativo no estudo do BBB é a exigência absoluta de um método simples e eficaz para isolamento de microvessels de derivados de animais experimentais e posterior preparação de amostras de membrana de tecido cerebral. Estas amostras devem estar preparadas para que eles são ambos enriquecidos em células endoteliais microvascular do cérebro e limitados na presença de outros tipos de células. Ao longo dos últimos anos, várias metodologias para isolamento da microvasculatura do cérebro de roedor têm sido relatadas na literatura científica13,20,21,22. Este artigo descreve um simples, robusto e o método reprodutível para isolamento de microvessels de cérebro de rato e para preparação das amostras endoteliais de membrana-enriquecido que pode ser usado para a análise da expressão da proteína. Uma vantagem deste protocolo de isolamento de microvessel é a capacidade de obter preparações de amostras de alta qualidade e com rendimento de proteína suficiente de um animal experimental individual. Isto permite a consideração de animal para animal variabilidade na expressão da proteína. Tal adiantamento neste protocolo melhorou extremamente a robustez dos estudos do BBB porque sobreavaliação (ou Under estimativa) a verdadeira amplitude das alterações de proteína no BBB agora pode ser evitada. Além disso, a inclusão de várias etapas de centrifugação com dextran permite melhor enriquecimento de microvessels em amostras experimentais facilitando a remoção dos constituintes celulares indesejados como os neurônios.

<table><tbody><tr><td>Protease Inhibitor Cocktail</td><td>Sigma-Aldrich</td><td>#P8340</td><td>Component of brain microvessel buffer</td></tr><tr><td>D-mannitol</td><td>Sigma-Aldrich</td><td>#M4125</td><td>Component of brain microvessel buffer</td></tr><tr><td>EGTA</td><td>Sigma-Aldrich</td><td>#E3889</td><td>Component of brain microvessel buffer</td></tr><tr><td>Trizma Base</td><td>Sigma-Aldrich</td><td>#T1503</td><td>Component of brain microvessel buffer</td></tr><tr><td>Dextran (MW 75,000)</td><td>Spectrum Chemical Mftg Corp</td><td>#DE125</td><td>Dextran used in centrifugation steps to separate microvessels from brain parenchyma</td></tr><tr><td>Zetamine</td><td>MWI Animal Health</td><td>#501072</td><td>General anesthetic</td></tr><tr><td>Xylazine</td><td>Western Medical Supply</td><td>#5530</td><td>General anesthetic</td></tr><tr><td>0.9% saline solution</td><td>Western Medical Supply</td><td>N/A</td><td>General anesthetic diluent</td></tr><tr><td>Filter Paper (12.5 cm diameter)</td><td>VWR</td><td>#28320-100</td><td>Used for removal of meninges from brain tissue</td></tr><tr><td>Centrifuge Tubes</td><td>Sarstedt</td><td>#60.540.386</td><td>Disposable tubes used for dextran centrifugation steps</td></tr><tr><td>Pierce&amp;trade; Coomassie Plus (Bradford) Assay</td><td>ThermoFisher Scientific</td><td>#23236</td><td>Measurement of protein concentration in membrane preparations</td></tr><tr><td>Wheaton Overhead Power Homogenizer</td><td>DWK Life Sciences</td><td>#903475</td><td>Required for homogenization of samples</td></tr><tr><td>10.0ml glass mortar and pestle tissue grinder</td><td>DWK Life Sciences</td><td>#358039</td><td>Required for homogenization of samples</td></tr><tr><td>Hydrochloric Acid</td><td>Sigma-Aldrich</td><td>#H1758</td><td>Required for pH adjustment of buffers</td></tr><tr><td>Bovine Serum Albumin</td><td>ThermoFisher Scientific</td><td>#23210</td><td>Protein standard for Bradford Assay</td></tr><tr><td>Standard Forceps</td><td>Fine Science Tools</td><td>#91100-12</td><td>Used for dissection of brain tissue</td></tr><tr><td>Friedman-Pearson Rongeurs</td><td>Fine Science Tools</td><td>#16020-14</td><td>Used for opening skull to isolate brain</td></tr><tr><td>50 ml conical centrifuge tubes</td><td>ThermoFisher Scientific</td><td>#352070</td><td>Used for collection of brain tissue following isolation</td></tr><tr><td>Glass Pasteur Pipets</td><td>ThermoFisher Scientific</td><td>#13-678-20C</td><td>Used for aspiration of cellular debris following dextran spins</td></tr><tr><td>Ethanol, anhydrous</td><td>Sigma-Aldrich</td><td>#459836</td><td>Used for cleaning tissue grinder; diluted to 70% with distilled water</td></tr><tr><td>Ultracentrifuge tubes</td><td>Beckman-Coulter</td><td>#41121703</td><td>Used for ultracentrifugation of samples</td></tr></tbody></table>

a simple and reproducible method to prepare membrane samples from freshly isolated rat brain microvessels

A barreira hemato - encefálica (BBB) é um tecido dinâmico barreira que responde a vários estímulos fisiopatológicos e farmacológicos. Tais alterações resultantes destes estímulos podem grandemente modulam a entrega da droga para o cérebro e, por extensão, causam consideráveis desafios no tratamento do sistema nervoso central (CNS) doenças. Muitas mudanças BBB que afetam a farmacoterapia, envolvem as proteínas que são localizadas e expresso a nível das células endoteliais. Com efeito, tal conhecimento sobre fisiologia do BBB na saúde e na doença gerou considerável interesse no estudo destas proteínas de membrana. Do ponto de vista de pesquisa de ciência básica, isto implica uma exigência para um método simples mas robusto e reprodutível para isolamento de microvessels de tecido cerebral, colhido em animais experimentais. Para preparar amostras de membrana de microvessels recentemente isolada, é essencial que os preparativos da amostra ser enriquecidos em células endoteliais mas limitados na presença de outros tipos de células da unidade neurovascular (i.e., astrócitos, micróglia, neurônios, pericitos). Um benefício adicional é a capacidade para preparar amostras de animais individuais, a fim de capturar a verdadeira variabilidade da expressão da proteína em uma população experimental. Neste manuscrito, detalhes a respeito de um método que é utilizado para isolamento de microvessels de cérebro de rato e preparação de amostras de membrana são fornecidos. Enriquecimento de Microvessel, de amostras derivadas, é conseguido usando quatro etapas de centrifugação onde dextrano é incluído no buffer de amostra. Este protocolo pode facilmente ser adaptado por outros laboratórios para suas próprias aplicações específicas. Amostras geradas a partir deste protocolo foram mostradas para produzir robustos dados experimentais de experimentos de análise de proteína que podem ajudar muito a compreensão das respostas BBB a estímulos fisiológicos, fisiopatológicos e farmacológicos.

O fluxo experimental para isolamento de microvessels de cérebro de rato e para a preparação de amostras de membrana de microvessel é mostrado na Figura 1. Usando o procedimento aqui apresentado, é demonstrado sucesso isolamento de microvessels intacto de cérebro de rato (Figura 2A). Estes navios foram obtidos após a conclusão de centrifugação com dextran e imediatamente antes de iniciar a ultracentrifugação para prepa...

Watch this Scientific Journal Video about A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels at JoVE.com

A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

Aqui, um método para isolamento de microvessels de cérebro de rato e para a preparação de amostras de membrana é descrito. Este protocolo tem a clara vantagem de produzir microvessel enriquecido amostras com proteína aceitável de rendimento de animais individuais. Amostras podem ser usadas para as análises de proteína robusto no endotélio microvascular cerebral.

Um método simples e reprodutível para preparar amostras de membrana de Microvessels de cérebro de rato recém isoladas

The blood-brain barrier (BBB) is a dynamic barrier tissue that responds to various pathophysiological and pharmacological stimuli. Such changes resulting ...

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Research

JoVE Journal

Neuroscience

10.1K visualizações.  University of Arizona, Tucson. Aqui, um método para isolamento de microvessels de cérebro de rato e para a preparação de amostras de membrana é descrito. Este protocolo tem a clara vantagem de produzir microvessel enriquecido amostras com proteína aceitável de rendimento de animais individuais. Amostras podem ser usadas para as análises de proteína robusto no endotélio microvascular cerebral.

Vídeo: A Simple and Reproducible Method to Prepare Membrane Samples from Freshly Isolated Rat Brain Microvessels

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 ...

Medicina

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Usando fluorescência celular ativado Classificando a Examine-Cell-tipo específico Expressão Gênica em Tissue cérebro de um rato

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Neurociência

Purification of Mouse Brain Vessels

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and immune cells between the brain and the blood. Moreover, the huge neuronal metabolic demand requires a moment-to-moment regulation of blood flow. Notably, abnormalities of these regulations are etiological hallmarks of most brain pathologies; including glioblastoma, stroke, edema, epilepsy, degenerative diseases (ex: Parkinson&#8217;s disease, Alzheimer&#8217;s disease), brain tumors, as well as inflammatory conditions such as multiple sclerosis, meningitis and sepsis-induced brain dysfunctions. Thus, understanding the signaling events modulating the cerebrovascular physiology is a major challenge. Much insight into the cellular and molecular properties of the various cell types that compose the cerebrovascular system can be gained from primary culture or cell sorting from freshly dissociated brain tissue. However, properties such as cell polarity, morphology and intercellular relationships are not maintained in such preparations. The protocol that we describe here is designed to purify brain vessel fragments, whilst maintaining structural integrity. We show that isolated vessels consist of endothelial cells sealed by tight junctions that are surrounded by a continuous basal lamina. Pericytes, smooth muscle cells as well as the perivascular astrocyte endfeet membranes remain attached to the endothelial layer. Finally, we describe how to perform immunostaining experiments on purified brain vessels.

We describe a protocol allowing the purification of the mouse brain's vascular compartment. Isolated brain vessels include endothelial cells linked by tight junctions and surrounded by a continuous basal lamina, pericytes, vascular smooth muscle cells, as well as perivascular astroglial membranes.

Purificação de embarcações do cérebro do rato

In the brain, most of the vascular system consists of a selective barrier, the blood-brain barrier (BBB) that regulates the exchange of molecules and ...

Preparation of Newborn Rat Brain Tissue for Ultrastructural Morphometric Analysis of Synaptic Vesicle Distribution at Nerve Terminals

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial organization of synaptic vesicles. In order to obtain high-quality electron micrographs that can yield the degree of morphological detail necessary for quantitative analysis of pre-synaptic vesicle distribution, optimal specimen preparation is critical. Chemical fixation is the first step in the process of specimen preparation, and of utmost importance to preserve fine ultrastructure. Vascular fixation with a glutaraldehyde-formaldehyde solution, followed by treatment of vibratome-sectioned specimens with osmium tetroxide, stabilizes the maximum number of molecules, especially proteins and lipids, and results in superior conservation of ultrastructure. Tissue is then processed with counterstaining, sequential dehydration and resin-embedding. En bloc staining with uranyl acetate (i.e., staining of vibratome-sectioned tissue before resin embedding) enhances endogenous contrast and stabilizes cell components against extraction during specimen processing. Contrast can be further increased by applying uranyl acetate as a post-stain on ultrathin sections. Double-staining of ultrathin sections with lead citrate after uranyl acetate treatment also improves image resolution, by intensifying electron-opacity of nucleic acid-containing structures through selective binding of lead to uranyl acetate. Transmission electron microscopy is a powerful tool for characterization of the morphological details of synaptic vesicles and quantification of their size and spatial organization in the terminal bouton. However, because it uses fixed tissue, transmission electron microscopy can only provide indirect information regarding living or evolving processes. Therefore, other techniques should be considered when the main objective is to study dynamic or functional aspects of synaptic vesicle trafficking and exocytosis.

We describe procedures for processing newborn rat brain tissue to obtain high-resolution electron micrographs for morphometric analysis of synaptic vesicle distribution at nerve terminals. The micrographs obtained with these methods can also be used to study the morphology of a number of other cellular components and their dimensional structural relationships.

Preparação do tecido cerebral de rato recém-nascido para Análise morfométrica ultraestrutural da distribuição de vesículas sinápticas em terminais nervosos

Our laboratory and many others have exploited the high resolving power of transmission electron microscopy to study the morphology and spatial ...

Simultaneous Measurements of Intracellular Calcium and Membrane Potential in Freshly Isolated and Intact Mouse Cerebral Endothelium

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the lumen of blood vessels and command changes in vascular diameter as needed to meet the metabolic demand of neurons. Primary endothelial-dependent signaling pathways of hyperpolarization of membrane potential (Vm) and nitric oxide typically operate in parallel to mediate vasodilation and thereby increase blood flow. Although integral to coordinating vasodilation over several millimeters of vascular length, components of endothelium-derived hyperpolarization (EDH) have been historically difficult to measure. These components of EDH entail intracellular Ca2+ [Ca2+]i increases and subsequent activation of small- and intermediate conductance Ca2+-activated K+ (SKCa/IKCa) channels.
Here, we present a simplified illustration of the isolation of fresh endothelium from mouse cerebral arteries; simultaneous measurements of endothelial [Ca2+]i and Vm using Fura-2 photometry and intracellular sharp electrodes, respectively; and a continuous superfusion of salt solutions and pharmacological agents under physiological conditions (pH 7.4, 37 &#176;C). Posterior cerebral arteries from the Circle of Willis are removed free of the posterior communicating and the basilar arteries. Enzymatic digestion of cleaned posterior cerebral arterial segments and subsequent trituration facilitates removal of adventitia, perivascular nerves, and smooth muscle cells. Resulting posterior cerebral arterial endothelial &#34;tubes&#34; are then secured under a microscope and examined using a camera, photomultiplier tube, and one to two electrometers while under continuous superfusion. Collectively, this method can simultaneously measure changes in endothelial [Ca2+]i and Vm in discrete cellular locations, in addition to the spreading of EDH through gap junctions up to millimeter distances along the intact endothelium. This method is expected to yield a high-throughput analysis of the cerebral endothelial functions underlying mechanisms of blood flow regulation in the normal and diseased brain.

Demonstrated here are protocols for (1) freshly isolating intact cerebral endothelial "tubes" and (2) simultaneous measurements of endothelial calcium and membrane potential during endothelium-derived hyperpolarization. Further, these methods allow for pharmacological tuning of endothelial cell calcium and electrical signaling as individual or interactive experimental variables.

Medições simultâneas de cálcio intracelular e potencial de membrana no endotélio Cerebral rato recém isoladas e intacta

Cerebral arteries and their respective microcirculation deliver oxygen and nutrients to the brain via blood flow regulation. Endothelial cells line the ...

Biologia

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Um método de alta saída para isolar pericites cerebrais do rato

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

Endothelial Cell Culture from Rat Brain Microvessels: A Procedure to Isolate and Culture Endothelial Cells from Microvessels of Rat Brain

Source: Molino, Y. et al. <a href="https://www.jove.com/v/51278/setting-up-an-vitro-model-rat-blood-brain-barrier-bbb-focus-on-bbb">Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport.</a> J. Vis. Exp.&#160;(2014)
This video presents the isolation of microvessels from a rat brain using a combination of density gradient centrifugation and enzymatic digestion mehods. The isolated microvessels can be cultured in a culture flask pre-coated with collagen and fibronectin matrix to obtain endothelial cell colonies.

Cultura de células endoteliais de microvasos cerebrais de ratos: um procedimento para isolar e cultivar células endoteliais de microvasos de cérebro de ratos

Source: Molino, Y. et al. Setting-up an&#160;In Vitro&#160;Model of Rat Blood-Brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated ...

JoVE Encyclopedia of Experiments

Pesquisa do câncer

Câncer do Sistema Nervoso

Estudos in vitro

Isolation and Culture of Microglia from Rat Brains using Serum-Free Media

Source: Collins, H. Y., et al., <a href="https://app.jove.com/v/57122">Isolation and Culture of Rodent Microglia to Promote a Dynamic Ramified Morphology in Serum-free Medium.</a> J. Vis. Exp. (2018)
This video demonstrates a method for isolating and culturing microglia from rat brains. It entails separating microglia from other neuronal and non-neuronal cells using plates coated with a monoclonal antibody specific to microglia.

Isolamento e cultura de microglia de cérebros de ratos usando meios sem soro

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Prepare a Petri Dish for CD11b ...

Neuronal Culture Techniques

Primary Neuronal Culture

Isolating Mouse Brain Vessels: A Protocol to Isolate Intact Vessels from Murine Brain Sample

Source: Boulay, A. C. et al. <a href="https://www.jove.com/video/53208" target="_blank">Purification of Mouse Brain Vessels.</a> J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a mouse&#39;s brain that constitute the blood-brain barrier (BBB) along with neurons and glial cells. The isolated vessels can be used as an in vitro model to study their role in maintaining brain homeostasis.

Isolando vasos cerebrais de camundongos: um protocolo para isolar vasos intactos de amostras de cérebro murino

Source: Boulay, A. C. et al. Purification of Mouse Brain Vessels. J. Vis. Exp. (2015)
This video demonstrates the isolation of intact vessels from a ...

Estudos com modelo animal

Obtaining a Single-Cell Suspension from Frozen Rat Brain Tissue

Source: Rubio, F. J., et. al. <a href="https://app.jove.com/v/54358">Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue.</a> J. Vis. Exp. (2016)
This video demonstrates the dissociation of frozen rat brain tissue into a single-cell suspension using enzymatic digestion and mechanical trituration. The prepared cells are fixed and permeabilized with ethanol for downstream applications like flow cytometry or gene expression analysis.

Source: Rubio, F. J., et. al. Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat ...