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 Illinois Üniversitesi Chicago başlangıç ​​fonlarıyla finanse edilir.

Tüm deneyler Illinois Üniversitesi, Chicago Kurumsal Hayvan Bakımı ve Kullanım Komitesi protokollerini takip etmektedir. 1. Genel Hazırlık NOT: Beyin mikrovasküler endotel hücresi hattı (hCMEC / D3), yoğun şekilde karakterize edilmiş, ölümsüzleştirilmiş insan BEC hattı 14 , 15 , 16 , 18 , 19...

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Tarif edilen yöntemler, serebrovasküler kapsama alanı ile ilgili çeşitli temel biyolojik soruları çözmek için kullanılabilir 24 . Spesifik olarak hangi reseptörlerin ve sinyal yollarının anjiyogenez, kanser dokusunda gemi kapsama alanı ve beynin ilgili olduğu periferik endotel hücrelerinde rol oynadığını tespit edebilirler. Örnekler, anjiyojenik büyüme faktörü reseptörleri, nitrik oksit, mitojen aktive protein kinaz sinyali ve kalsiyum sinyali 25 ...

<html> Serebral kılcal damarların kan-beyin bariyeri (BBB), kan-beyin temasının en büyük arayüzüdür ve merkezi sinir sistemi (SSS) 1 , 2&#39;nin homeostazında merkezi bir rol oynar. BBB&#39;deki dinamik işlemler, istenmeyen moleküllerin kandan alınmasını önler, atık ürünleri MSS&#39;den uzaklaştırır, gerekli besin maddelerini ve CNS&#39;ye sinyal moleküllerini gönderir ve nöroinflamasyonu 1 , 2 , 3 , 4 , 5 modüle eder. Yaşlanma sırasında BBB hasarı ve Alzheimer hastalığı (AD), multipl skleroz ve inme 1 , 2 , 3 , 4 , 5 ve 6&#39;yı içeren çeşitli nörodejeneratif bozukluklar görülür.Ass = &quot;xref&quot;&gt; 6. Bu nedenle, BBB disfonksiyonu terapötik bir hedef de dahil nörodejeneratif bozukluklarda önemli bir rol oynayabilir. Gemi kapsama alanının korunması BBB&#39;nin homeostatik işlevleri için önemlidir. Bununla birlikte, in vivo ve in vitro veriler, nörodejeneratif bozukluklardaki süreçlerin özellikle AD&#39;de 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 yüksek veya düşük BBB kapsama alanına sahip olup olmadığı konusunda çatışma oluşturmaktadır. Bu nedenle, BBB kapsama alanının dinamiklerini daha kapsamlı olarak anlamak ve değerlendirmek için ilgili hücre tiplerini kullanan in vitro modellerin geliştirilmesi için güçlü bir gerekçe vardır. Beyin kılcal damarları, astrositler, perisitler ve beyin endotel hücrelerinden (BEC&#39;ler) oluşur. <sUp class = &quot;xref&quot;&gt; 3. Tüm hücre tipleri yapısal destek vasıtasıyla ve parakrin ve otokrin benzeri modda hareket eden anjiyojenik büyüme faktörleri, sitokinler ve kemokinler gibi efektör moleküllerin salgılanması yoluyla BBB&#39;nin işlevine katkıda bulunur. Bununla birlikte, BBB&#39;nin efektör hücreleri BEC&#39;lerdir3. Genel olarak, BBB fonksiyonunu değerlendirmek için hücre kültürü teknikleri, hem stresörlerin 14 , 15 , 16 eklenmesinden sonra, filtre ekleri üzerinde büyütülen hücreler üzerinde gerçekleştirilen geçirgenlik testleri veya anahtar BEC proteinlerinin seviyelerini değerlendirmektir. Önemli olmasına rağmen, bu testler serebrovasküler kapsama odaklanmaz. Burada önceki yöntemlerimiz, ölümsüzleştirilen insan BEC&#39;lerinin tek kültürleri, birincil fare BEC&#39;lerin tek kültürleri ve bir hümanize üçlü kültür kullanılarak BEC kapsamı ve ağ-yapı benzeri yapıları değerlendirmek için ayrıntılı olarak verilmektedirModel (BEC&#39;ler, astrositler ve perisitler). Amaç, AD progresyonuna önemli katkıda bulunan oAβ&#39;nın zararlı etkisini BEC kapsamı üzerine göstermekti. Epidermal büyüme faktörünün (EGF) koruyucu etkisi, tekniğin terapötik bir tarama aracı olarak potansiyelini vurgular. Teknik, temel ve uygulamalı araştırmalar için çeşitli geniş uygulamalara sahiptir: 1) anjiyogenez ve gemi kapsama alanındaki spesifik yolların rolünün belirlenmesi, 2) hastalık ve yaşlanmayla ilişkili faktörlerin anjiyogenez ve gemi kapsama alanındaki etkilerinin değerlendirilmesi ve 3) farmakolojik hedefler.

<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

Kan-beyin bariyeri (BBB) ​​kapsama merkezi sinir sisteminin (CNS) homeostazında merkezi bir rol oynamaktadır. BBB, astrositler, perisitler ve beyin endotel hücreleri (BEC&#39;ler) tarafından dinamik olarak muhafaza edilir. Burada, ölümsüzleştirilmiş insan BEC&#39;lerinin tek kültürleri, birincil fare BEC&#39;lerin tek kültürleri ve BBB&#39;nin bir hümanize üçlü kültür modeli (BEC&#39;ler, astrositler ve perisitler) kullanarak BBB kapsama alanını değerlendirecek yöntemleri ayrıntılı olarak inceliyoruz. Tahlillerin hastalık durumlarına uygulanabilirliğini vurgulamak için, Alzheimer hastalığının (AD) ilerlemesine önemli katkıda bulunan oligomerik amiloid-β&#39;nın (oAβ) BBB kapsamı üzerindeki etkisini tanımlıyoruz. Ayrıca, tekniklerdeki ilaç tarama potansiyelini aydınlatmak için epidermal büyüme faktörünü (EGF) kullanıyoruz. Sonuçlarımız, tek ve üçlü kültürlenmiş BEC&#39;lerin temel koşullar altında ağ gibi yapılar oluşturduğunu ve oAβ&#39;nın bu hücre ağ oluşumunu bozar ve önceden oluşturulmuş örgü yapılarını dejenere ettiğini,Ancak EGF bu aksama engeller. Bundan dolayı, tarif edilen teknikler, BBB kapsama alanını modüle eden temel ve hastalığa bağlı süreçleri ayırmada önemlidir.

Tek kültürlerde hem hCMEC / D3 hücreleri ( Şekil 3A ) hem de birincil fare BEC&#39;ler ( Şekil 3B ), kuyu boyunca ağ örgüsü benzeri yapılar oluşturmaktadır. Yapılar, birbirine bağlı düğümlerin ağ tarafından karakterize edilmiştir ( Şekil 3 ). Açıklanan tüm paradigmalarda ( Şekil 1 ), ağ örgüsü benzeri yapılar kontrol gruplarında 24 saat s...

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

Kan-beyin bariyerinin korunması, merkezi sinir sisteminin homeostazı için kilit unsurdur. Bu protokol, kan-beyin bariyerinin kapsamını düzenleyen temel ve patolojik süreçleri tanımlamak için in vitro teknikleri tanımlamaktadır.

Kan-beyin Bariyeri Mesh Benzeri Gemi Oluşumu ve Kesintisini Değerlendiren İn Vitro Tahliller

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

The goals of this assay are to dissect the role of disease-relevant processes in blood brain-barrier coverage disruption, identify the underlying mechanisms, and test potential treatments in vitro.This method can answer key basic and applied research questions in the blood-brain barrier and neuro-degeneration fields.For example, by addressing whether disease-relevant stresses disrupt preformed network structures and identifying the underlying pathways.Through further screening, treatments can be identified that prevent stress-induced blood-brain barrier disruption for future in vivo testing.The implications of this technique extend towards mechanistic understanding of and treatment for Alzheimer's disease, because of the importance of the homeostatic functions of the blood-brain barrier on cognition.Though this method can provide insight into the blood-brain barrier dysfunction in Alzheimer's disease, it can also be applied to other neurodegenerative disorders with blood-brain barrier dysfunction, such as stroke.Begin by passaging hCMEC/D3 immortal endothelial cells at a ratio of one to five by incubating the cell sheet with calcium and magnesium-free HBSS for three minutes.Then detach the cells using five milliliters of 0.25%trypsin EDTA for five minutes.Neutralize the trypsin by adding five milliliters of EBM-2 complete medium.Collect the cells, and centrifuge at 290 times g for five minutes at four degrees Celsius.Following centrifugation, discard the supernatant, and resuspend the pellet in EBM-2 complete medium.Then, replate the cells at a one to five ratio.To prepare endothelial cells from the mouse brain, remove the brain stems and cerebella from seven euthanized mice with forceps.Then, detach the meninges by carefully rolling the brains on gauss.Then, place the brain tissue into a petri dish containing minimal essential medium with HEPES, and use a sterile razor blade to mince the remaining brain tissue.Then transfer the minced brain tissue to a 15 milliliter conical tube.And centrifuge at 290 times g for five minutes at four degrees Celsius.After removing the supernatant, add papain and DNase solution, and incubate in a 37 degree Celsius water bath for one hour to digest the tissue.Following the incubation, triturate the homogenate using a 19-gauge needle, and then a 21-gauge needle.Once triturated, add one volume of 22%bovine serum albumin solution.Then centrifuge at 1, 360 times g for 10 minutes at four degrees Celsius.Resuspend the resulting pellet in one milliliter of EBM-2 complete, and then centrifuge at 290 times g for five minutes at four degrees Celsius.After the centrifugation, again use one milliliter of EBM-2 complete medium to resuspend the cells, and plate the cells at one milliliter per well in collagen-coated, six-well plates.24 hours later, replace the medium with EBM-2 complete medium containing four micrograms per milliliter of puromycin hydrochloride, and continue to culture the cells until they reach full confluence at one times 10 to the fifth cells per square centimeter.Lastly, 24 hours prior to the assay, incubate the fully confluent brain endothelial cells in EBM-2 basal medium.On the day of the assay, pipette 10 microliters of basement membrane matrix into each well of a 96-well plate, and allow to set for one hour at 37 degrees Celsius.Suspend the lyophilized green cell tracking dye in 10 microliters of DMSO to generate a 10-millimolar stock solution.Dilute this solution to 10-micromolar in EBM-2 basal medium.Remove the medium from the six-well plate containing brain endothelial cells, and replace with EBM-2 basal containing the green cell tracking dye.Incubate the cells for 20 to 30 minutes at 37 degrees Celsius.Following the incubation, remove the medium with cell tracking dye, wash the cell sheet, and attach the cells with trypsin.Neutralize the trypsin with EBM-2 containing 10%FBS, and transfer the cells to centrifuge tubes.Centrifuge at 240 time g for five minutes at four degrees Celsius.Following centrifugation, resuspend the cell pellets in one milliliter of EBM-2 basal medium, plate the labeled cells in multiple 96-well plates at 10, 000 cells per well, and return the cells to the incubator until oligomeric oa

Research

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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

Sıçan Modelinde Transkraniyal Odaklanmış Ultrason kullanarak Kan-beyin bariyeri MRG eşliğinde bozulması

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

Tıp

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

Kan-beyin bariyer bozulması arasında Rat Modeli Nörovasküler Therapeutics Hedefli izin ver için

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

An<em&gt; In vivo</emKan Gemi Geçirgenlik Test&gt; 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

Bir olarak beyin mikrovasküler endotel hücreleri (cend) olarak gerin<em&gt; In Vitro</emKan Beyin Bariyer&gt; Travmatik Beyin Hasarı Modeli

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

Ayarlama-up bir<em&gt; In Vitro</emSıçan Kan-beyin bariyerinin&gt; Model (BBB): BBB geçirimsizliği ve Reseptör aracılı Ulaştırması Bir Odak

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Bir Roman<em&gt; In vivo</em&gt; Gen Transferi Tekniği ve<em&gt; In vitro</emİskelet Hastalıkları Kemik Kaybı Çalışmaları&gt; Hücre Tabanlı Tahliller

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

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

Bir oksijen-Glukoz Yoksunluk ve reoksijenizasyonun<em&gt; In Vitro</em&gt; İskemi-Reperfüzyon Modeli incelenmesi Kan-Beyin Bariyeri Disfonksiyon için

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

İnsan vücudunun fizyolojisini bütünleştiren 4 damarlı Örnekleme Yaklaşımı<emIn 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

Yöntem bir Vitro kan - beyin bariyerini modeli domuz beyin endotel hücreleri temel alan kurulması için geliştirilmiş

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-diseksiyon emaye Organ dan çene kemiğinde kesici sıçan için çevre Toxicants maruz

Enamel defects resulting from environmental conditions and ways of life are public health concerns because of their high prevalence. These defects result ...