Name: in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption
Uploaded: 2017-06-20T14:00:00
Description: Watch this Scientific Journal Video about In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption at JoVE.com

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 ,<...

<ol>
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A., Couraud, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hCMEC/D3+cell+line+as+a+model+of+the+human+blood+brain+barrier.">The hCMEC/D3 cell line as a model of the human blood brain barrier.</a> Fluids Barriers CNS. 10 (1), 16 (2013).</li><li>Weksler, B. B., 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=Blood-brain+barrier-specific+properties+of+a+human+adult+brain+endothelial+cell+line.">Blood-brain barrier-specific properties of a human adult brain endothelial cell line.</a> FASEB J. 19 (13), 1872-1874 (2005).</li><li>Welser-Alves, J. 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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. 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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...

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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 ...

Research

JoVE Journal

Medicine

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

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

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

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

An in vivo Assay to Test Blood Vessel Permeability

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 ...

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

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

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

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

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

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

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

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

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

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) ...

Micro-dissection of Enamel Organ from Mandibular Incisor of Rats Exposed to Environmental Toxicants

Microdissecação do órgão do esmalte do incisivo de ratos expostos a tóxicos ambientais

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...

Vessel-sparing Excision and Primary Anastomosis

Excisão de poupadores de navio e anastomose primária

Urethroplasty is considered to be the standard treatment for urethral strictures since it provides excellent long-term success rates. For isolated short ...