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

León Tai es financiado por la Universidad de Illinois Chicago fondos de puesta en marcha.

Todos los experimentos siguen los protocolos del Comité Institucional de Cuidado de Animales y Uso de la Universidad de Illinois, Chicago. 1. Preparación General NOTA: La línea celular endotelial microvascular del cerebro (hCMEC / D3) es una línea BEC humana inmortalizada extensamente caracterizada 14 , 15 , 16 , 18 , <sup ...

<ol>
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Los métodos descritos pueden ser utilizados para abordar varias cuestiones biológicas fundamentales en torno a la cobertura cerebrovascular [ 24] . Específicamente, pueden identificar qué receptores y vías de señalización desempeñan un papel en la angiogénesis, la cobertura vascular en el tejido canceroso y las células endoteliales periféricas relevantes para el cerebro. Los ejemplos incluyen receptores del factor de crecimiento angiogénico, óxido nítrico, señalización de proteín...

<html> La barrera hematoencefálica (BBB) ​​de los capilares cerebrales es la mayor interfase de contacto entre sangre y desempeña un papel central en la homeostasis del sistema nervioso central (SNC) 1 , 2 . Los procesos dinámicos en el BBB evitan la absorción de moléculas no deseadas de la sangre, eliminan los productos residuales del SNC, suministran nutrientes esenciales y señalan moléculas al SNC, y modulan la neuroinflamación 1 , 2 , 3 , 4 , 5 . El daño a la BBB es frecuente durante el envejecimiento y varios trastornos neurodegenerativos incluyendo la enfermedad de Alzheimer (EA), la esclerosis múltiple y el accidente cerebrovascular 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Por lo tanto, la disfunción de la BBB puede desempeñar un papel clave en los trastornos neurodegenerativos, incluso como objetivo terapéutico. Mantener la cobertura del buque es importante para las funciones homeostáticas de la BBB. Sin embargo, los datos in vivo e in vitro contradicen si los procesos implicados en trastornos neurodegenerativos causan una cobertura BBB más alta o más baja 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , particularmente en AD. Por lo tanto, existe una fuerte justificación para el desarrollo de modelos in vitro utilizando tipos de células relevantes para evaluar y comprender de manera más completa la dinámica de la cobertura BBB. Los capilares cerebrales están compuestos de astrocitos, pericitos y células endoteliales cerebrales (BEC) <sUp class = &quot;xref&quot;&gt; 3. Todos los tipos de células contribuyen a la función de la BBB a través del soporte estructural ya través de la secreción de moléculas efectoras tales como factores de crecimiento angiogénicos, citoquinas y quimiocinas que actúan en forma parácrina y autocrina. Sin embargo, las principales células efectoras de la BBB son BECs 3 . En general, las técnicas de cultivo celular para evaluar la función de BBB son ensayos de permeabilidad realizados en células crecidas en insertos de filtro, o la evaluación de niveles de proteínas BEC claves, después de la adición de factores de estrés 14 , 15 , 16 . Aunque son importantes, estos ensayos no se centran en la cobertura cerebrovascular. Aquí, nuestros métodos anteriores 17 se detallan para evaluar la cobertura de BEC y estructuras de malla-como utilizando culturas únicas de BEC humanos inmortalizados, cultivos únicos de BECs de ratón primario, y una triple cultura humanizadaModelo (BECs, astrocitos y pericitos) de la BBB. El objetivo era demostrar el efecto perjudicial de oAβ, que se considera un importante contribuyente a la progresión de AD, en la cobertura de BEC. El efecto protector del factor de crecimiento epidérmico (EGF) pone de relieve el potencial de la técnica como una herramienta de cribado terapéutico. La técnica tiene varias aplicaciones amplias para la investigación básica y aplicada, incluyendo: 1) delinear el papel de las vías específicas en la angiogénesis y la cobertura de los vasos, 2) evaluar los efectos de la enfermedad y los factores relacionados con el envejecimiento sobre la angiogénesis y la cobertura vascular; Objetivos.

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

La cobertura de la barrera hematoencefálica (BBB) ​​desempeña un papel central en la homeostasis del sistema nervioso central (SNC). El BBB es mantenido dinámicamente por astrocitos, pericitos y células endoteliales cerebrales (CEC). Aquí se detallan métodos para evaluar la cobertura de BBB usando cultivos únicos de BECs humanos inmortalizados, cultivos únicos de BECs de ratón primario y un modelo de cultivo triple humanizado (BECs, astrocitos y pericitos) de la BBB. Para resaltar la aplicabilidad de los ensayos a estados de enfermedad, describimos el efecto de la amiloide oligomérica-β (oAβ), que es un contribuyente importante a la progresión de la enfermedad de Alzheimer (AD), en la cobertura de BBB. Además, utilizamos el factor de crecimiento epidérmico (EGF) para iluminar el potencial de detección de fármacos de las técnicas. Nuestros resultados muestran que los BECs cultivados individuales y triples forman estructuras tipo malla bajo condiciones basales y que oAβ altera esta formación de malla celular y degenera las estructuras de malla preformadas,Pero EGF bloquea esta interrupción. Por lo tanto, las técnicas descritas son importantes para diseccionar procesos fundamentales y relacionados con la enfermedad que modulan la cobertura de BBB.

En cultivos únicos, tanto las células hCMEC / D3 ( Figura 3A ) como las BECs primarias de ratón ( Figura 3B ) forman estructuras tipo malla en todo el pozo. Las estructuras se caracterizan por una red de nodos interconectados ( Figura 3 ). En todos los paradigmas descritos ( Figura 1 ), las estructuras tipo malla son similares después de 24 h en los grupos de contro...

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

El mantenimiento de la cobertura de la barrera hematoencefálica es clave para la homeostasis del sistema nervioso central. Este protocolo describe técnicas in vitro para delinear los procesos fundamentales y patológicos que modulan la cobertura de la barrera hematoencefálica.

Ensayos in vitro para evaluar la formación y la interrupción de los vasos sanguíneos de barrera hematoencefálica

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

Resonancia magnética guiada por disrupción de la barrera sangre-cerebro con ultrasonido transcraneal Enfocado en un modelo de rata

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

Investigación

Medicina

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

Modelo de rata de ruptura de la barrera sangre-cerebro para permitir dirigida Therapeutics neurovascular

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

Un<em&gt; In vivo</em&gt; Ensayo de prueba de permeabilidad Vasos Sanguíneos

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

Estirar en Brain células endoteliales microvasculares (CEND) como<em&gt; In Vitro</em&gt; Lesión cerebral traumática Modelo de la barrera hematoencefálica

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

Establecimiento de un<em&gt; In Vitro</em&gt; Modelo de Rata barrera hematoencefálica (BBB): Un Enfoque en la acreditación Impermeabilidad y transporte mediado por los receptores

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

Oxígeno-Glucosa Privación y Reoxigenación como<em&gt; In Vitro</em&gt; La isquemia-reperfusión modelo para el estudio de la disfunción barrera 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

El Método de Muestreo de 4 Barcos para Estudios Integrativos de la Fisiología Placentaria 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

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

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

Micro-disección del órgano del esmalte de incisivo de la mandíbula de ratas expuestas a tóxicos ambientales

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

La supresión conservadora de vasos y Anastomosis primaria

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