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 finansiert av University of Illinois Chicago oppstartsfond.

Alle eksperimenter følger University of Illinois, Chicago Institutional Animal Care og Use Committee protokoller. 1. Generell forberedelse MERK: Hjernens mikrovaskulære endotelcellelinje (hCMEC / D3) er en omfattende karakterisert immortalisert human BEC linje 14 , 15 , 16 , 18 , 19 . Kultur hCMEC / D3...

<ol>
	<li>Abbott, N. J. <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+structure+and+function+and+the+challenges+for+CNS+drug+delivery.">Blood-brain barrier structure and function and the challenges for CNS drug delivery.</a> J Inherit Metab Dis. 36 (3), 437-449 (2013).</li><li>Engelhardt, B., Liebner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+insights+into+the+development+and+maintenance+of+the+blood-brain+barrier.">Novel insights into the development and maintenance of the blood-brain barrier.</a> Cell Tissue Res. 355 (3), 687-699 (2014).</li><li>Abbott, N. J., Patabendige, A. A., Dolman, D. E., Yusof, S. R., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+the+blood-brain+barrier.">Structure and function of the blood-brain barrier.</a> Neurobiol Dis. 37 (1), 13-25 (2010).</li><li>Zlokovic, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurovascular+pathways+to+neurodegeneration+in+Alzheimer's+disease+and+other+disorders.">Neurovascular pathways to neurodegeneration in Alzheimer's disease and other disorders.</a> Nat Rev Neurosci. 12 (12), 723-738 (2011).</li><li>Pardridge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+delivery+of+protein+and+gene+medicines+through+the+blood-brain+barrier.">Targeted delivery of protein and gene medicines through the blood-brain barrier.</a> Clin Pharmacol Ther. 97 (4), 347-361 (2014).</li><li>Tai, L. 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>Biron, K. E., Dickstein, D. L., Gopaul, R., Jefferies, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+triggers+extensive+cerebral+angiogenesis+causing+blood+brain+barrier+permeability+and+hypervascularity+in+Alzheimer's+disease.">Amyloid triggers extensive cerebral angiogenesis causing blood brain barrier permeability and hypervascularity in Alzheimer's disease.</a> PLoS One. 6 (8), e23789 (2011).</li><li>Cameron, D. J., 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=Alzheimer's-related+peptide+amyloid-beta+plays+a+conserved+role+in+angiogenesis.">Alzheimer's-related peptide amyloid-beta plays a conserved role in angiogenesis.</a> PLoS One. 7 (7), e39598 (2012).</li><li>Boscolo, E., 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=Beta+amyloid+angiogenic+activity+in+vitro+and+in+vivo.">Beta amyloid angiogenic activity in vitro and in vivo.</a> Int J Mol Med. 19 (4), 581-587 (2007).</li><li>Paris, D., 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=Impaired+angiogenesis+in+a+transgenic+mouse+model+of+cerebral+amyloidosis.">Impaired angiogenesis in a transgenic mouse model of cerebral amyloidosis.</a> Neurosci Lett. 366 (1), 80-85 (2004).</li><li>Kitaguchi, H., Ihara, M., Saiki, H., Takahashi, R., Tomimoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary+beds+are+decreased+in+Alzheimer's+disease,+but+not+in+Binswanger's+disease.">Capillary beds are decreased in Alzheimer's disease, but not in Binswanger's disease.</a> Neurosci Lett. 417 (2), 128-131 (2007).</li><li>Jantaratnotai, N., Ryu, J. 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. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid-beta-induced+occludin+down-regulation+and+increased+permeability+in+human+brain+endothelial+cells+is+mediated+by+MAPK+activation.">Amyloid-beta-induced occludin down-regulation and increased permeability in human brain endothelial cells is mediated by MAPK activation.</a> J Cell Mol Med. 14 (5), 1101-1112 (2010).</li><li>Tai, L. M., Loughlin, A. J., Male, D. K., Romero, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-glycoprotein+and+breast+cancer+resistance+protein+restrict+apical-to-basolateral+permeability+of+human+brain+endothelium+to+amyloid-beta.">P-glycoprotein and breast cancer resistance protein restrict apical-to-basolateral permeability of human brain endothelium to amyloid-beta.</a> J Cereb Blood Flow Metab. 29 (6), 1079-1083 (2009).</li><li>Tai, L. 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. V., Boroujerdi, A., Milner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+brain+endothelial+cells.">Isolation and culture of primary mouse brain endothelial cells.</a> Methods Mol Biol. 1135, 345-356 (2014).</li><li>Dahlgren, K. N., 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=Oligomeric+and+fibrillar+species+of+amyloid-beta+peptides+differentially+affect+neuronal+viability.">Oligomeric and fibrillar species of amyloid-beta peptides differentially affect neuronal viability.</a> J Biol Chem. 277 (35), 32046-32053 (2002).</li><li>Carpentier, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+Analyzer.">Angiogenesis Analyzer.</a> ImageJ News. , (2012).</li><li>Thomas, R., 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=Epidermal+growth+factor+prevents+APOE4+and+amyloid-beta-induced+cognitive+and+cerebrovascular+deficits+in+female+mice.">Epidermal growth factor prevents APOE4 and amyloid-beta-induced cognitive and cerebrovascular deficits in female mice.</a> Acta Neuropathol Commun. 4 (1), 111 (2016).</li><li>Tai, L. 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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+therapeutic+approach+for+senile+dementias:+neuroangiogenesis.">A therapeutic approach for senile dementias: neuroangiogenesis.</a> J Alzheimers Dis. 43 (1), 1-17 (2015).</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=The+Role+of+Capillaries+in+the+Lesser+Ailments+of+Old+Age+and+in+Alzheimer's+Disease+and+Vascular+Dementia:+The+Potential+of+Pro-Therapeutic+Angiogenesis.">The Role of Capillaries in the Lesser Ailments of Old Age and in Alzheimer's Disease and Vascular Dementia: The Potential of Pro-Therapeutic Angiogenesis.</a> J Alzheimers Dis. 54 (1), 31-43 (2016).</li></ol>

De beskrevne fremgangsmåter kan benyttes for å adressere flere grunnleggende biologiske spørsmål rundt cerebrovaskulær dekning 24 . Spesifikt kan de identifisere hvilke reseptorer og signalveier som spiller en rolle i angiogenese, fartøyets dekning i kreftvev og perifere endotelceller som er relevante for hjernen. Eksempler inkluderer angiogene vekstfaktorreceptorer, nitrogenoksyd, mitogenaktivert proteinkinase-signalering og kalsiumsignalering 25 , <sup c...

<html> Blodhjernebarrieren (BBB) ​​av cerebrale kapillærer er det største grensesnittet mellom blod-til-hjernekontakt og spiller en sentral rolle i hemostasen i sentralnervesystemet (CNS) 1 , 2 . Dynamiske prosesser på BBB forhindrer opptak av uønskede molekyler fra blodet, fjerner avfallsprodukter fra CNS, leverer essensielle næringsstoffer og signalmolekyler til CNS, og modulerer nevrologi 1 , 2 , 3 , 4 , 5 . BBB-skade er utbredt ved aldring og flere nevrodegenerative forstyrrelser, inkludert Alzheimers sykdom (AD), multippel sklerose og slag 1 , 2 , 3 , 4 , 5 ,Ass = &quot;xref&quot;&gt; 6. Derfor kan BBB-dysfunksjon spille en nøkkelrolle i neurodegenerative lidelser, inkludert som et terapeutisk mål. Opprettholdelse av fartøyets dekning er viktig for de homeostatiske funksjonene til BBB. Imidlertid er in vivo og in vitro datakonflikt om hvorvidt prosessene involvert i nevrodegenerative forstyrrelser forårsaker høyere eller lavere BBB-dekning 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , spesielt i AD. Derfor er det en sterk begrunnelse for utviklingen av in vitro- modeller ved bruk av relevante celletyper for å vurdere og forståere dynamikken til BBB-dekning. Serebral kapillærer består av astrocytter, pericytter og hjerne endotelceller (BEC) <sOpp klasse = &quot;xref&quot;&gt; 3. Alle celletyper bidrar til funksjonen til BBB gjennom strukturell støtte og via sekresjon av effektormolekyler som angiogene vekstfaktorer, cytokiner og kjemokiner som virker på parakrin- og autokrin-lignende måte. De viktigste effektorcellene til BBB er imidlertid BEC 3 . Generelt er cellekulturteknikkene for å vurdere BBB-funksjonen permeabilitetsanalyser utført på celler dyrket på filterinnlegg, eller vurdering av nivåer av sentrale BEC-proteiner, både etter tilsetning av stressorer 14 , 15 , 16 . Selv om det er viktig, fokuserer disse analysene ikke på cerebrovaskulær dekning. Her er våre tidligere metoder 17 detaljert for å vurdere BEC-dekning og meshwork-lignende strukturer ved bruk av enkeltkulturer av immortaliserte humane BEC&#39;er, enkeltkulturer av primære mus-BEC&#39;er og en humanisert trippelkulturModell (BEC, astrocyter og pericytter) av BBB. Målet var å demonstrere den skadelige effekten av oAβ, som anses som en viktig bidragsyter til AD-progresjon, på BEC-dekning. Den beskyttende effekten av epidermal vekstfaktoren (EGF) fremhever potensialet av teknikken som et terapeutisk screeningsverktøy. Teknikken har flere brede anvendelser for grunnleggende og anvendt forskning, herunder: 1) avgrense rollen av spesifikke veier angiogenese og fartøy dekning, 2) evaluere effektene av sykdom og aldringsrelevante faktorer på angiogenese og fartøy dekning, og 3) identifisere farmakologiske 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

Blod-hjernebarriere (BBB) ​​dekning spiller en sentral rolle i homeostasen i sentralnervesystemet (CNS). BBB holdes dynamisk av astrocytter, pericytter og hjerne endotelceller (BEC). Her beskriver vi metoder for å vurdere BBB-dekning ved bruk av enkeltkulturer av immortaliserte humane BEC, enkeltkulturer av primære mus-BEC, og en humanisert triple-kulturmodell (BEC, astrocyter og pericytter) av BBB. For å markere anvendelighet av analysene til sykdomstilstander, beskriver vi effekten av oligomer amyloid-β (oAβ), som er en viktig bidragsyter til Alzheimers sykdom (AD) -progresjon, på BBB-dekning. Videre benytter vi den epidermale vekstfaktoren (EGF) for å belyse stoffets screeningspotensial av teknikkene. Våre resultater viser at single og triple cultured BECs danner meshwork-lignende strukturer under basale forhold, og at oAβ forstyrrer denne celle meshwork formasjonen og degenererer de preformed mesh strukturer,Men EGF blokkerer denne forstyrrelsen. Således er de beskrevne teknikkene viktige for å dissekere grunnleggende og sykdomsrelevante prosesser som modulerer BBB-dekning.

I enkelt kulturer danner både hCMEC / D3-celler ( figur 3A ) og primære mus-BEC ( figur 3B ) mesh-lignende strukturer gjennom brønnen. Strukturene er preget av et nettverk av sammenknyttede noder ( figur 3 ). I alle de beskrevne paradigmene ( figur 1 ) er de meshwork-lignende strukturer like etter 24 timer i kontrollgruppene, og danner ~ 20 mesh-lignende strukturer 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

Opprettholdelse av blod-hjerne barriere dekning er nøkkelen til homeostasis av sentralnervesystemet. Denne protokollen beskriver in vitro teknikker for å avgrense de grunnleggende og patologiske prosessene som modulerer blod-hjerne barriere dekning.

I Vitro- analyser for å vurdere blod-hjernebarriere 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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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-veiledet Forstyrrelse av blod-hjerne-barrieren ved hjelp Transkraniell fokusert ultralyd i en rotte Model

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

Rotte modell av blod-hjerne barrieren avbrudd i Tillat Målrettet nevrovaskulære 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

Strekk i Brain mikrovaskulær endotelceller (cend) som en<em&gt; In Vitro</em&gt; Traumatic Brain Injury Modell av 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

Sette opp en<em&gt; In Vitro</em&gt; Modell av Rat blod-hjerne barrieren (BBB): Et fokus på BBB Ugjennomtrengelighet og Receptor-mediert 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

Oxygen-Glukose deprivasjon og reoksygenering som en<em&gt; In Vitro</em&gt; Iskemi-reperfusjonskade modell for å studere blod-hjernebarrieren 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

4-karet prøvetaking til integrative studier av 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 metoden for etablering av en In Vitro blod - hjerne barrieren modell basert på svin hjernen 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

Mikro-Disseksjon av emalje orgel fra Mandibular helikopteret rotter utsatt for miljømessig giftstoffer

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

Fartøyet-sparing Excision og primære anastomose

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