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 è finanziato da fondi d&#39;avvio di Chicago University of Illinois.

Tutti gli esperimenti seguono i protocolli del Comitato di istruzione e tutela degli animali istituzionali dell&#39;Università dell&#39;Illinois, Chicago. 1. Preparazione generale NOTA: La linea cerebrale endoteliale (hCMEC / D3) è una linea umana immortalizzata ampiamente caratterizzata dalla linea BEC 14 , 15 , 16 , 18 , <s...

<ol>
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K., Schwab, C., McGeer, P. L., McLarnon, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Vascular+Perturbations+in+an+Abeta-Injected+Animal+Model+and+in+AD+Brain.">Comparison of Vascular Perturbations in an Abeta-Injected Animal Model and in AD Brain.</a> Int J Alzheimers Dis. 2011, 918280 (2011).</li><li>Donnini, S., 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=Abeta+peptides+accelerate+the+senescence+of+endothelial+cells+in+vitro+and+in+vivo,+impairing+angiogenesis.">Abeta peptides accelerate the senescence of endothelial cells in vitro and in vivo, impairing angiogenesis.</a> FASEB J. 24 (7), 2385-2395 (2010).</li><li>Tai, L. M., Holloway, K. A., Male, D. K., Loughlin, A. J., Romero, I. 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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=Polarized+P-glycoprotein+expression+by+the+immortalised+human+brain+endothelial+cell+line,+hCMEC/D3,+restricts+apical-to-basolateral+permeability+to+rhodamine+123.">Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123.</a> Brain Res. 1292, 14-24 (2009).</li><li>Koster, K. P., Thomas, R., Morris, A. W., Tai, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+prevents+oligomeric+amyloid-beta+induced+angiogenesis+deficits+in+vitro.">Epidermal growth factor prevents oligomeric amyloid-beta induced angiogenesis deficits in vitro.</a> J Cereb Blood Flow Metab. 36 (11), 1865-1871 (2016).</li><li>Weksler, B., Romero, I. 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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I metodi descritti possono essere utilizzati per affrontare diverse questioni biologiche fondamentali che riguardano la copertura cerebrovascolare24. In particolare, essi possono identificare quali recettori e vie di segnalazione svolgono un ruolo in angiogenesi, la copertura dei recipienti nei tessuti del cancro e cellule endoteliali periferiche rilevanti per il cervello. Gli esempi includono i recettori del fattore di crescita angiogenico, l&#39;ossido nitrico, la mitogen attivata dalla proteina chinasi e la segnalazi...

<html> La barriera emato-cerebrale (BBB) ​​dei capillari cerebrali è la più grande interfaccia del contatto ematico-to-cerebrale e svolge un ruolo centrale nell&#39;omeostasi del sistema nervoso centrale (CNS) 1 , 2 . I processi dinamici del BBB impediscono l&#39;assorbimento di molecole indesiderate dal sangue, rimuovono i prodotti di scarto dal CNS, forniscono nutrienti essenziali e molecole di segnalazione al CNS e modulano la neuroinflammazione 1 , 2 , 3 , 4 , 5 . Il danno di BBB è prevalente durante l&#39;invecchiamento e diversi disturbi neurodegenerativi tra cui la malattia di Alzheimer (AD), la sclerosi multipla e l&#39;ictus 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Pertanto, la disfunzione di BBB può svolgere un ruolo chiave nei disturbi neurodegenerativi, anche come obiettivo terapeutico. Il mantenimento della copertura dei recipienti è importante per le funzioni homeostatiche del BBB. Tuttavia, i dati in vivo e in vitro confligano sul fatto che i processi coinvolti nei disturbi neurodegenerativi causino una copertura BBB maggiore o minore 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , particolarmente in AD. Pertanto, vi è una forte logica per lo sviluppo di modelli in vitro utilizzando tipi di cellule per valutare e comprendere in modo più completo le dinamiche della copertura BBB. I capillari cerebrali sono composti da astrociti, periciti e cellule endoteliali cerebrali (BECs) <sUp class = &quot;xref&quot;&gt; 3. Tutti i tipi di cellule contribuiscono alla funzione del BBB attraverso il supporto strutturale e attraverso la secrezione di molecole effector come fattori di crescita angiogenici, citochine e chemiokine che agiscono in modo paracrino e autocrino. Tuttavia, le principali cellule efficaci del BBB sono BECs 3 . In generale, le tecniche di coltura cellulare per la valutazione della funzione BBB sono i test di permeabilità eseguiti su cellule coltivate su inserti di filtri o valutando i livelli di proteine ​​chiave BEC, sia dopo l&#39;aggiunta di stress 14 , 15 , 16 . Sebbene importanti, questi dosaggi non riguardano la copertura cerebrovascolare. Qui, i nostri precedenti metodi 17 sono dettagliati per valutare la copertura BEC e le strutture di tipo reticolo utilizzando singole colture di BEC umani immortalizzati, singole colture di BEC primari del mouse e una cultura tripla umanizzataModello (BEC, astrociti e periciti) del BBB. L&#39;obiettivo era dimostrare l&#39;effetto dannoso di oAβ, che è considerato un importante contributore alla progressione dell&#39;AD, sulla copertura BEC. L&#39;effetto protettivo del fattore di crescita epidermico (EGF) evidenzia il potenziale della tecnica come strumento di screening terapeutico. La tecnica presenta diverse applicazioni per la ricerca di base e applicata, tra cui: 1) definire il ruolo di percorsi specifici in materia di angiogenesi e copertura dei vasi, 2) valutare gli effetti delle malattie e dei fattori rilevanti sull&#39;invecchiamento sull&#39;angiogenesi e sulla vasca e 3) individuare le attività farmacologiche obiettivi.

<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

La copertura del sangue-cervello (BBB) ​​svolge un ruolo centrale nell&#39;omeostasi del sistema nervoso centrale (CNS). Il BBB è mantenuto dinamicamente da astrociti, periciti e cellule endoteliali cerebrali (BECs). Qui metodi dettagliati per valutare la copertura BBB utilizzando singole colture di BEC umani immortalizzati, singole colture di BEC primari del mouse e un modello di coltura triplicata umanizzata (BECs, astrociti e periciti) del BBB. Per evidenziare l&#39;applicabilità dei dosaggi agli stati di malattia, descriviamo l&#39;effetto dell&#39;amyloid oligomerico (oAβ), che rappresenta un importante contributo alla progressione della malattia di Alzheimer (AD), sulla copertura BBB. Inoltre, utilizziamo il fattore di crescita epidermico (EGF) per illuminare il potenziale di screening delle droghe delle tecniche. I nostri risultati mostrano che singole e triple coltivate BECs formano strutture a maglia in condizioni basali e che oAβ interferisce con questa formazione di mesh e degenerizza le strutture preformate di maglie,Ma EGF blocca questa rottura. Quindi le tecniche descritte sono importanti per la dissezione di processi fondamentali e rilevanti per la malattia che modulano la copertura BBB.

In singole colture, entrambe le cellule hCMEC / D3 ( Figura 3A ) e il BEC primario del mouse ( Figura 3B ) formano strutture a maglie simili a tutto il pozzo. Le strutture sono caratterizzate da un reticolo di nodi interconnessi ( Figura 3 ). In tutti i paradigmi descritti ( Figura 1 ), le strutture simili a maglie reticolari sono simili dopo 24 ore nei gruppi di 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

Il mantenimento della copertura della barriera emato-sangue è fondamentale per l&#39;omeostasi del sistema nervoso centrale. Questo protocollo descrive tecniche in vitro per delineare i processi fondamentali e patologici che modulano la copertura della barriera emato-sangue.

In vitro dosaggi per valutare la formazione e la rottura del vaso come la barriera emato-cerebrale

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

MRI-guidato Interruzione della barriera emato-encefalica transcranica con ultrasuoni focalizzati in un modello murino

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

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Medicina

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

Modello Rat di emato-encefalica Turbativa Barriera per consentire mirato Therapeutics neurovascolare

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; Test per la verifica della nave Permeabilità Sangue

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

Stretch in microvascolari cerebrali cellule endoteliali (CEND) come<em&gt; In Vitro</em&gt; Trauma cranico modello della barriera ematoencefalica

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

Istituzione di un<em&gt; In Vitro</em&gt; Modello di Rat barriera emato-encefalica (BBB): A Focus on BBB impermeabilità e Trasporti recettore-mediata

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

L&#39;ossigeno-glucosio Deprivazione e riossigenazione come<em&gt; In Vitro</em&gt; Ischemia-riperfusione Injury modello per lo studio barriera sanguigna del cervello Erettile

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;approccio di campionamento a 4 nave per studi integrativi della fisiologia placentare umana<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

Migliorato il metodo per la creazione di un In Vitro barriera emato - encefalica modello basato sulle cellule endoteliali Porcine cervello

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-dissezione dell'organo dello smalto da incisivo dei ratti esposti a tossici ambientali

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

Nave-con parsimonia l'asportazione e l'anastomosi primaria

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