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 wordt gefinancierd door University of Illinois Chicago start-up fondsen.

Alle experimenten volgen de protocols van de Universiteit van Illinois, Chicago Institutional Animal Care and Use Committee. 1. Algemene Voorbereiding OPMERKING: De microvasculaire endotheliale cellijn van de hersenen (hCMEC / D3) is een extensief gekenmerkt immortalized human BEC lijn 14 , 15 , 16 , 18 , 19</...

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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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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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De beschreven methoden kunnen worden gebruikt om verschillende fundamentele biologische vragen aan te pakken die betrekking hebben op cerebrovasculaire dekking 24 . Specifiek kunnen zij identificeren welke receptoren en signaalwegen een rol spelen bij angiogenese, vaatdekking in kankerweefsel en perifere endotheliale cellen die relevant zijn voor de hersenen. Voorbeelden omvatten angiogene groeifactorreceptoren, stikstofoxide, mitogeen geactiveerde proteïne kinase signalering en calcium signaler...

<html> De bloed-hersenbarrière (BBB) ​​van hersenschapsvaten is de grootste interface van bloed-tot-hersencontact en speelt een centrale rol in de homeostase van het centrale zenuwstelsel (CNS) 1 , 2 . Dynamische processen bij de BBB verhinderen de opname van ongewenste moleculen uit het bloed, verwijderen afvalstoffen uit het CNS, leveren essentiële voedingsstoffen en signaalmoleculen aan het CNS, en moduleren neuro-inflammatie 1 , 2 , 3 , 4 , 5 . BBB-schade komt voor bij veroudering en verschillende neurodegeneratieve stoornissen waaronder de ziekte van Alzheimer (AD), multiple sclerose en beroerte 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Daarom kan BBB-disfunctie een sleutelrol spelen bij neurodegeneratieve stoornissen, ook als therapeutisch doel. Het behoud van de vaartuigdekking is belangrijk voor de homeostatische functies van de BBB. In vivo en in vitro gegevensconflicten echter of de processen die bij neurodegeneratieve stoornissen betrokken zijn, een hoger of lager BBB-dekking veroorzaken 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , met name in AD. Daarom is er een sterke reden voor de ontwikkeling van in vitro modellen met behulp van relevante celtypen om de dynamiek van BBB-dekking te beoordelen en beter te begrijpen. Serebrale capillairen zijn samengesteld uit astrocyten, pericyten en hersenendotheelcellen (BEC&#39;s) <sUp class = &quot;xref&quot;&gt; 3. Alle celtypes dragen bij tot de functie van de BBB door middel van structurele ondersteuning en via de afscheiding van effectormoleculen zoals angiogene groeifactoren, cytokinen en chemokines die op paracrine- en autocrine-achtige wijze optreden. De belangrijkste effectorcellen van de BBB zijn echter BEC&#39;s 3 . In het algemeen zijn de celcultuurtechnieken voor het beoordelen van BBB-functie permeabiliteitsbepalingen uitgevoerd op cellen die op filterinzetten worden gegroeid, of het beoordelen van niveaus van belangrijke BEC-eiwitten, beide na de toevoeging van stressors 14 , 15 , 16 . Hoewel belangrijk, concentreren deze analyses zich niet op de cerebrovasculaire dekking. Hier zijn onze vorige methoden 17 gedetailleerd om BEC-dekking en meshwork-achtige structuren te beoordelen met behulp van enkele culturen van onsterfelijke menselijke BEC&#39;s, enkele culturen van primaire muis-BEC&#39;s en een gehumaniseerde triple cultuurModel (BEC, astrocyten en pericytes) van de BBB. Het doel was om het schadelijke effect van oAβ te demonstreren, die wordt beschouwd als een belangrijke bijdrage aan AD progressie, op BEC dekking. Het beschermende effect van de epidermale groeifactor (EGF) benadrukt het potentieel van de techniek als therapeutisch screeningsinstrument. De techniek heeft verschillende brede toepassingen voor basis- en toegepast onderzoek, waaronder: 1) het bepalen van de rol van specifieke pathways bij angiogenese en dekking van het vaatstelsel; 2) het evalueren van de effecten van ziekte- en verouderingsrelevante factoren op angiogenese en vaatbescherming; en 3) het identificeren van farmacologische targets.

<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

Bloed-hersenbarrière (BBB) ​​dekking speelt een centrale rol in de homeostase van het centrale zenuwstelsel (CNS). De BBB wordt dynamisch onderhouden door astrocyten, pericytes en hersenendotheelcellen (BEC&#39;s). Hier worden details beschreven om BBB-dekking te beoordelen met behulp van enkele culturen van onsterfelijke menselijke BEC&#39;s, enkele culturen van primaire muis-BEC&#39;s en een gehumaniseerd triple culture-model (BEC, astrocyten en pericyten) van de BBB. Om de toepasbaarheid van de analyses op ziektetoestanden te benadrukken beschrijven we het effect van oligomere amyloïde-ß (oAβ), die een belangrijke bijdrage levert aan de ziekte van Alzheimer (AD), op BBB-dekking. Verder gebruiken we de epidermale groeifactor (EGF) om het drugscreeningspotentieel van de technieken te verlichten. Uit onze resultaten blijkt dat enkelvoudige en drievoudige gecultiveerde BEC&#39;s meshwork-achtige structuren vormen onder basale omstandigheden en dat oAβ deze celwerkvorming vormt en de voorgevormde maasstructuren ontaardt,Maar EGF blokkeert deze verstoring. Zo zijn de beschreven technieken belangrijk voor het analyseren van fundamentele en ziekte-relevante processen die de BBB-dekking moduleren.

In enkele culturen vormen zowel de hCMEC / D3-cellen ( Figuur 3A ) en de primaire muis-BEC&#39;s ( Figuur 3B ) meshwerkvormige structuren in de put. De structuren worden gekenmerkt door een netwerkwerk van onderling verbonden knooppunten ( Figuur 3 ). In alle beschreven paradigma&#39;s ( Figuur 1 ) zijn de meshwork-achtige structuren gelijk na 24 uur in de controlegroep...

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

Het behoud van de bloeddrukbarriere dekking is de sleutel voor de homeostase van het centrale zenuwstelsel. Dit protocol beschrijft in vitro technieken om de fundamentele en pathologische processen te definiëren die de bloeddrukbeperking moduleren.

In Vitro- analyses voor het beoordelen van bloed-hersenbarrière Mesh-like Vessel Formation and Disruption

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

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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-geleide Verstoring van de bloed-hersen barrière met behulp van Transcraniële Focused Ultrasound in een ratmodel

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

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

Rat Model van bloed-hersen barrière Verstoring toestaan ​​Gerichte Neurovasculaire 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

Een<em&gt; In vivo</em&gt; Assay naar Blood Vessel Permeabiliteit Test

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

Strekken in Brain microvasculaire endotheelcellen (CEND) als een<em&gt; In Vitro</em&gt; Traumatisch Hersenletsel Model van de Blood Brain Barrier

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

Oprichting van een<em&gt; In Vitro</em&gt; Model van Rat bloed-hersenbarrière (BBB): een focus op BBB Ondoordringbaarheid en receptor-gemedieerd 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 ...

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

Zuurstof-Glucose beroving en reoxygenatie als een<em&gt; In Vitro</em&gt; Ischemie-reperfusie letsel model voor de studie bloed-hersenbarrière Dysfunction

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

De 4-vaartuig Steekproef Aanpak bij Integratieve Studies van Menselijke Placentale Fysiologie<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

Verbeterde methode voor de vaststelling van een In Vitro bloed - hersenbarrière Model gebaseerd op varkens Endothelial hersencellen

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-dissectie van glazuur orgel van snijtand van ratten blootgesteld aan toxische stoffen in het milieu

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

Vaartuig-sparend excisie en primaire anastomose

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