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 er finansieret af University of Illinois Chicago startfonde.

Alle eksperimenter følger University of Illinois, Chicago Institutional Animal Care og Use Committee protokoller. 1. Generel forberedelse BEMÆRK: Hjernens mikrovaskulære endotelcellelinie (hCMEC / D3) er en omfattende karakteriseret immortaliseret human BEC linje 14 , 15 , 16 , 18 , 19 . Kultur hCMEC ...

<ol>
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De beskrevne fremgangsmåder kan anvendes til at adressere flere grundlæggende biologiske spørgsmål omkring cerebrovaskulær dækning 24 . Specifikt kan de identificere hvilke receptorer og signalveje der spiller en rolle i angiogenese, fartøjsdækning i cancervæv og perifere endotelceller, der er relevante for hjernen. Eksempler indbefatter angiogene vækstfaktorreceptorer, nitrogenoxid, mitogenaktiveret proteinkinase-signalering og calciumsignalering 25 ,...

<html> Blodhjernebarrieren (BBB) ​​af cerebrale kapillærer er den største grænseflade af kontakt mellem blod og hjerne og spiller en central rolle i homeostasen i centralnervesystemet (CNS) 1 , 2 . Dynamiske processer på BBB forhindrer optagelse af uønskede molekyler fra blodet, fjerner affaldsprodukter fra CNS, leverer essentielle næringsstoffer og signalmolekyler til CNS og modulerer neuroinflammation 1 , 2 , 3 , 4 , 5 . BBB-skade er udbredt under aldring og flere neurodegenerative lidelser, herunder Alzheimers sygdom (AD), multipel sklerose og slagtilfælde 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Derfor kan BBB dysfunktion spille en central rolle i neurodegenerative lidelser, herunder som et terapeutisk mål. Vedligeholdelse af skibsdækning er vigtigt for BBB&#39;s homeostatiske funktioner. Imidlertid er in vivo og in vitro datakonflikt om, hvorvidt processerne involveret i neurodegenerative lidelser forårsager højere eller lavere BBB-dækning 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , især i AD. Derfor er der en stærk begrundelse for udviklingen af in vitro- modeller ved brug af relevante celletyper til at vurdere og forståelsen af ​​BBB-dækningsdynamikken dynamisk. Cerebral kapillærer er sammensat af astrocytter, pericytter og hjerne endotelceller (BEC) <sUp class = &quot;xref&quot;&gt; 3. Alle celletyper bidrager til BBB&#39;s funktion gennem strukturel understøttelse og via sekretion af effektormolekyler, såsom angiogene vækstfaktorer, cytokiner og kemokiner, der virker i parakrin og autokrinlignende mode. De vigtigste effektorceller af BBB er imidlertid BEC 3 . Cellekultursteknikkerne til vurdering af BBB-funktion er generelt permeabilitetsassays udført på celler dyrket på filterindsatser eller vurdering af niveauer af centrale BEC-proteiner, begge efter tilsætning af stressorer 14 , 15 , 16 . Selv om det er vigtigt, fokuserer disse analyser ikke på cerebrovaskulær dækning. Her er vores tidligere metoder 17 detaljerede for at vurdere BEC dækning og meshwork-lignende strukturer ved brug af enkeltkulturer af udødeliggjorte humane BEC&#39;er, enkeltkulturer af primære mus-BEC&#39;er og en humaniseret tredobbelt kulturModel (BEC, astrocytter og pericytter) af BBB. Målet var at demonstrere den skadelige virkning af oAβ, som betragtes som en vigtig bidragyder til AD-fremskridt på BEC-dækning. Den beskyttende virkning af den epidermale vækstfaktor (EGF) fremhæver potentialet ved teknikken som et terapeutisk screeningsværktøj. Teknikken har flere brede anvendelser til grundlæggende og anvendt forskning, herunder: 1) afgrænsning af rollen af ​​specifikke veje angiogenese og dækningsdækning, 2) evaluering af virkningerne af sygdomme og aldringsrelevante faktorer ved angiogenese og dækningsdækning og 3) identificering af farmakologisk mål.

<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

Blood-brain barrier (BBB) ​​dækning spiller en central rolle i homeostasen af ​​centralnervesystemet (CNS). BBB holdes dynamisk af astrocytter, pericytter og hjerneendotelceller (BEC&#39;er). Her beskriver vi metoder til vurdering af BBB-dækning ved anvendelse af enkeltkulturer af immortaliserede humane BEC&#39;er, enkeltkulturer af primære mus-BEC&#39;er og en humaniseret tripelkulturmodel (BEC, astrocytter og pericytter) af BBB. For at fremhæve anvendeligheden af ​​analyserne til sygdomstilstande beskriver vi virkningen af ​​oligomer amyloid-β (oAβ), hvilket er en vigtig bidragyder til Alzheimers sygdom (AD) progression på BBB-dækning. Derudover udnytter vi den epidermale vækstfaktor (EGF) for at belyse teknikkens lægemidlets screeningspotentiale. Vores resultater viser, at single og triple cultured BEC&#39;er danner meshwork-lignende strukturer under basale forhold, og at oAβ forstyrrer denne celle meshwork formation og degenererer de præformede mesh strukturer,Men EGF blokerer for denne forstyrrelse. Således er de beskrevne teknikker vigtige for dissektering af grundlæggende og sygdomsrelevante processer, som modulerer BBB-dækning.

I enkeltkulturer danner både hCMEC / D3-cellerne ( Figur 3A ) og de primære mus-BEC&#39;er ( Figur 3B ) meshworklignende strukturer i hele brønden. Strukturerne er kendetegnet ved et netværk af indbyrdes forbundne knuder ( figur 3 ). I alle de beskrevne paradigmer ( figur 1 ) er de meshwork-lignende strukturer ens efter 24 timer i kontrolgrupperne, der danner ~ 20 m...

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

Vedligeholdelse af blod-hjerne barriere dækning er nøglen til homeostasen i centralnervesystemet. Denne protokol beskriver in vitro teknikker til at afgrænse de grundlæggende og patologiske processer, der modulerer blod-hjerne barriere dækning.

I Vitro Assays at vurdere blod-hjerne barriere mesh-lignende fartøj dannelse og afbrydelse

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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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

MR-guided Forstyrrelse af blod-hjerne barrieren ved hjælp af Transcranial fokuseret ultralyd i en rottemodel

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

Rottemodel af blod-hjerne barrieren Disruption at muliggøre målrettede neurovaskulær 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

En<em&gt; In vivo</em&gt; Assay til Test Blood Vessel Permeabilitet

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

Stræk i Brain mikrovaskulære endotelceller (cEND) som et<em&gt; In Vitro</em&gt; Traumatisk Hjerneskade Model af blod-hjerne barrieren

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

Etablering af en<em&gt; In Vitro</em&gt; Model of Rat blod-hjerne barrieren (BBB): Fokus på BBB Uigennemtrængelighed og receptor-medieret 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

Ilt-Glucose afsavn og reoxygenering som en<em&gt; In vitro</em&gt; Iskæmireperfusion Skade Model for studere blod-hjerne barrieren dysfunktion

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

4-fartøjets prøveudtagning til integrerende studier af human placental fysiologi<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

Forbedret metode til etablering af et In Vitro blod - hjerne barrieren Model baseret på svin hjernen Endothelial Cells

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

Mikro-dissektion af emalje orgel fra Mandibular chopper rotter udsat for miljømæssige giftige stoffer

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

Fartøj-besparende Excision og primær anastomose

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