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 est financé par des fonds de démarrage de l&#39;Université d&#39;Illinois à Chicago.

Toutes les expériences suivent les protocoles du Comité institutionnel d&#39;élevage et d&#39;utilisation de l&#39;Université de l&#39;Illinois, Chicago Institutional Animal Care and Use Committee. 1. Préparation générale REMARQUE: La lignée de cellules endothéliales microvasculaires du cerveau (hCMEC / D3) est une ligne 14 , 15 , 16 , <sup class="xref"...

<ol>
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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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Les méthodes décrites peuvent être utilisées pour répondre à plusieurs questions biologiques fondamentales entourant la couverture vasculaire 24 . Plus précisément, ils peuvent identifier quels récepteurs et voies de signalisation jouent un rôle dans l&#39;angiogenèse, la couverture vasculaire dans le tissu cancéreux et les cellules endothéliales périphériques pertinentes pour le cerveau. Les exemples comprennent les récepteurs du facteur de croissance angiogénique, l&#39;oxyde n...

<html> La barrière hémato-encéphalique (BBB) ​​des capillaires cérébraux est la plus grande interface de contact entre le sang et le cerveau et joue un rôle central dans l&#39;homéostasie du système nerveux central (SNC) 1 , 2 . Les processus dynamiques à la BBB empêchent l&#39;absorption de molécules indésirables du sang, éliminent les déchets du SNC, fournissent des nutriments essentiels et des molécules de signalisation au SNC, et modulent la neuroinflammation 1 , 2 , 3 , 4 , 5 . Le dommage de BBB est répandu pendant le vieillissement et plusieurs troubles neurodégénératifs, y compris la maladie d&#39;Alzheimer (AD), la sclérose en plaques et les accidents vasculaires cérébraux 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Par conséquent, la dysfonctionnement BBB peut jouer un rôle clé dans les troubles neurodégénératifs, y compris en tant que cible thérapeutique. Le maintien de la couverture des navires est important pour les fonctions homéostatiques du BBB. Cependant, les conflits de données in vivo et in vitro sur le fait que les processus impliqués dans les troubles neurodégénératifs causent une couverture BBB plus élevée ou inférieure à 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , en particulier en AD. Par conséquent, il existe une forte raison pour le développement de modèles in vitro utilisant des types cellulaires pertinents pour évaluer et comprendre de manière plus complète la dynamique de la couverture BBB. Les capillaires cérébraux sont composés d&#39;astrocytes, de pericytes et de cellules endothéliales du cerveau (BEC) <sUp class = &quot;xref&quot;&gt; 3. Tous les types de cellules contribuent à la fonction du BBB grâce au soutien structurel et à la sécrétion de molécules effectrices telles que les facteurs de croissance angiogéniques, les cytokines et les chimiokines qui agissent de manière paracrine et autocrine. Cependant, les principales cellules effectrices du BBB sont les BEC 3 . En général, les techniques de culture cellulaire pour évaluer la fonction BBB sont des essais de perméabilité effectués sur des cellules cultivées sur des inserts de filtre, ou l&#39;évaluation des niveaux de protéines BEC clés, à la fois après l&#39;addition de facteurs de stress 14 , 15 , 16 . Bien que important, ces tests ne se concentrent pas sur la couverture cérébrovasculaire. Ici, nos méthodes précédentes 17 sont détaillées pour évaluer la couverture de BEC et les structures en forme de maillage en utilisant des cultures simples de BEC humains immortalisés, des cultures uniques de BEC de souris primaires et une triple culture humaniséeModèle (BEC, astrocytes et pericytes) du BBB. L&#39;objectif était de démontrer l&#39;effet néfaste de l&#39;OAβ, qui est considéré comme un facteur important de la progression de l&#39;AD, sur la couverture de la BEC. L&#39;effet protecteur du facteur de croissance épidermique (EGF) souligne le potentiel de la technique en tant qu&#39;outil de dépistage thérapeutique. La technique a plusieurs applications générales pour la recherche fondamentale et appliquée, notamment: 1) délimiter le rôle des voies spécifiques sur l&#39;angiogenèse et la couverture des vaisseaux; 2) évaluer les effets de la maladie et les facteurs pertinents du vieillissement sur l&#39;angiogenèse et la couverture vasculaire; 3) Cibles.

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

La couverture de la barrière hémato-encéphalique (BBB) ​​joue un rôle central dans l&#39;homéostasie du système nerveux central (SNC). Le BBB est maintenu dynamiquement par les astrocytes, les pericytes et les cellules endothéliales du cerveau (BEC). Ici, nous détaillons les méthodes pour évaluer la couverture BBB en utilisant des cultures individuelles de BEC humains immortalisés, des cultures simples de BEC de souris primaires et un modèle de culture triple humanisé (BEC, astrocytes et pericytes) du BBB. Pour mettre en évidence l&#39;applicabilité des tests aux états pathologiques, nous décrivons l&#39;effet de l&#39;amyloide-β (oAβ) oligomère, qui est un facteur important de la progression de la maladie d&#39;Alzheimer (AD), sur la couverture BBB. De plus, nous utilisons le facteur de croissance épidermique (EGF) pour éclairer le potentiel de dépistage des techniques des médicaments. Nos résultats montrent que les BEC cultivés à une ou à trois fois forment des structures en forme de maille dans des conditions basales et que oAβ perturbe cette formation de maillage cellulaire et dégénère les structures de maille préformées,Mais EGF bloque cette perturbation. Ainsi, les techniques décrites sont importantes pour la dissection de processus fondamentaux et pathologiques qui modulent la couverture BBB.

Dans les cultures uniques, les cellules hCMEC / D3 ( Figure 3A ) et les BEC de souris primaires ( Figure 3B ) forment des structures semblables à des mailles dans le puits. Les structures sont caractérisées par un maillage de noeuds interconnectés ( Figure 3 ). Dans tous les paradigmes décrits ( figure 1 ), les structures en forme de maillage sont similaires après...

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

Le maintien de la couverture de la barrière hémato-encéphalique est la clé de l&#39;homéostasie du système nerveux central. Ce protocole décrit des techniques in vitro pour délimiter les processus fondamentaux et pathologiques qui modulent la couverture de la barrière hémato-encéphalique.

Essais in vitro pour évaluer la formation et la perturbation du vaisseau en forme de barrière de sang-cerveau

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

The goals of this assay are to dissect the role of disease-relevant processes in blood brain-barrier coverage disruption, identify the underlying mechanisms, and test potential treatments in vitro.This method can answer key basic and applied research questions in the blood-brain barrier and neuro-degeneration fields.For example, by addressing whether disease-relevant stresses disrupt preformed network structures and identifying the underlying pathways.Through further screening, treatments can be identified that prevent stress-induced blood-brain barrier disruption for future in vivo testing.The implications of this technique extend towards mechanistic understanding of and treatment for Alzheimer's disease, because of the importance of the homeostatic functions of the blood-brain barrier on cognition.Though this method can provide insight into the blood-brain barrier dysfunction in Alzheimer's disease, it can also be applied to other neurodegenerative disorders with blood-brain barrier dysfunction, such as stroke.Begin by passaging hCMEC/D3 immortal endothelial cells at a ratio of one to five by incubating the cell sheet with calcium and magnesium-free HBSS for three minutes.Then detach the cells using five milliliters of 0.25%trypsin EDTA for five minutes.Neutralize the trypsin by adding five milliliters of EBM-2 complete medium.Collect the cells, and centrifuge at 290 times g for five minutes at four degrees Celsius.Following centrifugation, discard the supernatant, and resuspend the pellet in EBM-2 complete medium.Then, replate the cells at a one to five ratio.To prepare endothelial cells from the mouse brain, remove the brain stems and cerebella from seven euthanized mice with forceps.Then, detach the meninges by carefully rolling the brains on gauss.Then, place the brain tissue into a petri dish containing minimal essential medium with HEPES, and use a sterile razor blade to mince the remaining brain tissue.Then transfer the minced brain tissue to a 15 milliliter conical tube.And centrifuge at 290 times g for five minutes at four degrees Celsius.After removing the supernatant, add papain and DNase solution, and incubate in a 37 degree Celsius water bath for one hour to digest the tissue.Following the incubation, triturate the homogenate using a 19-gauge needle, and then a 21-gauge needle.Once triturated, add one volume of 22%bovine serum albumin solution.Then centrifuge at 1, 360 times g for 10 minutes at four degrees Celsius.Resuspend the resulting pellet in one milliliter of EBM-2 complete, and then centrifuge at 290 times g for five minutes at four degrees Celsius.After the centrifugation, again use one milliliter of EBM-2 complete medium to resuspend the cells, and plate the cells at one milliliter per well in collagen-coated, six-well plates.24 hours later, replace the medium with EBM-2 complete medium containing four micrograms per milliliter of puromycin hydrochloride, and continue to culture the cells until they reach full confluence at one times 10 to the fifth cells per square centimeter.Lastly, 24 hours prior to the assay, incubate the fully confluent brain endothelial cells in EBM-2 basal medium.On the day of the assay, pipette 10 microliters of basement membrane matrix into each well of a 96-well plate, and allow to set for one hour at 37 degrees Celsius.Suspend the lyophilized green cell tracking dye in 10 microliters of DMSO to generate a 10-millimolar stock solution.Dilute this solution to 10-micromolar in EBM-2 basal medium.Remove the medium from the six-well plate containing brain endothelial cells, and replace with EBM-2 basal containing the green cell tracking dye.Incubate the cells for 20 to 30 minutes at 37 degrees Celsius.Following the incubation, remove the medium with cell tracking dye, wash the cell sheet, and attach the cells with trypsin.Neutralize the trypsin with EBM-2 containing 10%FBS, and transfer the cells to centrifuge tubes.Centrifuge at 240 time g for five minutes at four degrees Celsius.Following centrifugation, resuspend the cell pellets in one milliliter of EBM-2 basal medium, plate the labeled cells in multiple 96-well plates at 10, 000 cells per well, and return the cells to the incubator until oligomeric oa

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

Guidée par IRM rupture de la barrière hémato-encéphalique en utilisant transcrânienne d&#39;ultrasons focalisés dans un modèle de rat

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

Recherche

Médecine

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

Modèle Rat de rupture de la barrière hémato-encéphalique pour permettre ciblée Therapeutics neurovasculaire

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</emTest&gt; Tester la perméabilité des vaisseaux sanguins

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

Étirer dans le cerveau cellules endothéliales microvasculaires (CEND) comme<em&gt; In Vitro</em&gt; Traumatisme modèle du cerveau de la barrière hémato-encéphalique

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

Mise en place d&#39;un<em&gt; In Vitro</em&gt; Modèle de Rat barrière hémato-encéphalique (BHE): un regard sur le Bureau imperméabilité et de transport médié par le récepteur

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

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Novel<em&gt; In vivo</em&gt; Technique Gene Transfer et<em&gt; In vitro</em&gt; Essais cellule sur la base de l&#39;étude de la perte osseuse dans les troubles musculo-squelettiques

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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

Oxygène-glucose privation et réoxygénation comme un<em&gt; In Vitro</em&gt; Ischémie-reperfusion modèle pour étudier la dysfonction barrière hémato-encéphalique

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

L&#39;approche d&#39;échantillonnage à 4 navires pour les études intégratives de la physiologie du plaidoyer humain<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

Procédé amélioré pour la création d’un In Vitro barrière hémato - encéphalique modèle basé sur les cellules endothéliales du cerveau porcin

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-dissection de l’organe de l’émail des incisives mandibulaires de Rats exposés à des substances toxiques environnementales

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