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 finansieras av University of Illinois Chicago startfonder.

Alla experiment följer University of Illinois, Chicago Institutional Animal Care and Use Committee protokoll. 1. Allmänna preparat OBS: Hjärnens mikrovaskulära endotelcellinje (hCMEC / D3) är en omfattande karaktäriserad odödlig human BEC-linje 14 , 15 , 16 , 18 , 19 . Kultur hCMEC / D3-cellerna p...

<ol>
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De beskrivna metoderna kan användas för att adressera flera grundläggande biologiska frågor kring cerebrovaskulär täckning 24 . Specifikt kan de identifiera vilka receptorer och signalvägar som spelar en roll vid angiogenes, kärltäckning i cancervävnad och perifera endotelceller som är relevanta för hjärnan. Exempel innefattar angiogena tillväxtfaktorreceptorer, kväveoxid, mitogenaktiverad proteinkinassignalering och kalciumsignalering 25 , <sup c...

<html> Blocket-hjärnbarriären (BBB) ​​av cerebrala kapillärer är det största gränssnittet mellan blod och hjärnans kontakt och spelar en central roll i hemostasen i centrala nervsystemet (CNS) 1 , 2 . Dynamiska processer vid BBB hindrar upptag av oönskade molekyler från blodet, tar bort avfallsprodukter från CNS, levererar väsentliga näringsämnen och signalmolekyler till CNS och modulerar neuroinflammation 1 , 2 , 3 , 4 , 5 . BBB-skada är utbredd under åldrande och flera neurodegenerativa störningar inklusive Alzheimers sjukdom (AD), multipel skleros och stroke 1 , 2 , 3 , 4 , 5 ,Röv = &quot;xref&quot;&gt; 6. Därför kan BBB-dysfunktion spela en nyckelroll i neurodegenerativa störningar, inklusive som ett terapeutiskt mål. Att behålla fartygets täckning är viktigt för BBB: s hemostatiska funktioner. In vivo och in vitro data konflikt om huruvida processerna som är involverade i neurodegenerativa störningar orsakar högre eller lägre BBB-täckning 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , särskilt i AD. Därför finns det en stark motivering för utvecklingen av in vitro- modeller med användning av relevanta celltyper för att bedöma och förstå djupare BBB-täckningens dynamik. Cerebral kapillärer är sammansatta av astrocyter, pericytes och hjärnendotelceller (BEC) <sUp class = &quot;xref&quot;&gt; 3. Alla celltyper bidrar till BBB-funktionen genom strukturellt stöd och genom sekretion av effektormolekyler, såsom angiogena tillväxtfaktorer, cytokiner och kemokiner som verkar på parakrin-och autokrinliknande sätt. De viktigaste effektorcellerna hos BBB är emellertid BEC 3 . I allmänhet är cellodlingsteknikerna för att bedöma BBB-funktionen permeabilitetsanalyser utförda på celler odlade på filterinsatser, eller bedömning av nivåer av nyckel-BEC-proteiner, båda efter tillsats av stressorer 14 , 15 , 16 . Även om viktiga, fokuserar dessa analyser inte på cerebrovaskulär täckning. Här är våra tidigare metoder 17 detaljerade för att bedöma BEC-täckning och meshworkliknande strukturer med användning av enskilda kulturer av odödliga humana BEC, enskilda kulturer av primära mus-BEC och en humaniserad trippel kulturModell (BEC, astrocyter och pericytes) av BBB. Målet var att demonstrera skadlig effekt av oAβ, som anses vara en viktig bidragsyter till AD-progression, på BEC-täckning. Den skyddande effekten av epidermal tillväxtfaktorn (EGF) framhäver potentialen hos tekniken som ett terapeutiskt screeningsverktyg. Tekniken har flera brett tillämpningar för grundläggande och tillämpad forskning, bland annat: 1) avgränsa rollen av specifika vägar för angiogenes och kärlskydd, 2) utvärdera effekterna av sjukdomar och åldringsrelevanta faktorer vid angiogenes och kärltäckning, och 3) identifiera farmakologiska 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>
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in vitro assays to assess blood-brain barrier mesh-like vessel formation and disruption

Blood-brain barrier (BBB) ​​täckning spelar en central roll i hemostas i centrala nervsystemet (CNS). BBB hålls dynamiskt av astrocyter, pericytes och hjärnendotelceller (BEC). Här beskriver vi metoder för att bedöma BBB-täckning med användning av enskilda kulturer av odödliga humana BEC, enskilda kulturer av primära mus-BEC och en humaniserad trippelkulturmodell (BEC, astrocyter och pericyt) hos BBB. För att lyfta fram användbarheten av analyserna till sjukdomstillstånd beskriver vi effekten av oligomer amyloid-P (oAβ), vilket är en viktig bidragsyter till Alzheimers sjukdom (AD) -framsteg, på BBB-täckning. Vidare använder vi den epidermala tillväxtfaktorn (EGF) för att belysa läkemedelsscreeningspotentialen hos teknikerna. Våra resultat visar att enkla och tredubblade odlade BEC bildar meshworkliknande strukturer under basala förhållanden och att oAβ stör denna cellnätverksbildning och degenererar de förformade maskestrukturerna,Men EGF blockerar denna störning. Således är de beskrivna teknikerna viktiga för att dissekera grundläggande och sjukdomsrelaterade processer som modulerar BBB-täckning.

I enskilda kulturer bildar både hCMEC / D3-cellerna ( Figur 3A ) och primära mus-BEC ( Figur 3B ) meshworkliknande strukturer i hela brunnen. Strukturerna kännetecknas av ett nätverk av interlänkade noder ( figur 3 ). I alla de paradigm som beskrivits ( Figur 1 ) liknar de mesh-liknande strukturerna efter 24 timmar i kontrollgrupperna, vilket bildar ~ 20 meshworkli...

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

Att upprätthålla blod-hjärnbarriär täckning är nyckeln till hemostasen i centrala nervsystemet. Detta protokoll beskriver in vitro- tekniker för att avgränsa de grundläggande och patologiska processerna som modulerar blod-hjärnbarriär täckning.

In vitro- analyser för att bedöma blod-hjärnbarriären Mesh-liknande fartygsformation och störning

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

MR-guidad Rubbning av blod-hjärnbarriären med hjälp Transkraniell fokuserat ultraljud i en råttmodell

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

Råttmodell av blod-hjärnbarriären avbrott Tillåt riktade Neurovaskulär 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; Analys för att testa Permeabilitet blodkärl

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 i Brain mikrovaskulära endotelceller (cEND) som en<em&gt; In Vitro</em&gt; Traumatisk hjärnskada Modell av blodhjärnbarriären

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

Ställa upp en<em&gt; In Vitro</em&gt; Modell av Rat blod-hjärnbarriären (BBB): Fokus på BBB Impermeabilitet och Receptor-medierad 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

Syre Glukos Deprivation och ny syresättning som en<em&gt; In Vitro</em&gt; Ischemi-reperfusion skada modell för att studera blodhjärnbarriären 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-kärlprovtagningsstrategin för integrationsstudier av human placentalfysiologi<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

Förbättrad metod för inrättandet av ett In Vitro blod - hjärnbarriären modell baserat på svin hjärnan endotelceller

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-dissektion av emalj Organ från Mandibular framtand hos råttor exponerade för miljögifter

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

Fartyg-sparing Excision och primär anastomos

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