Bu çalışma Ulusal Sağlık Enstitüleri, R01CA142928 bir hibe ile desteklenmiştir.

1. Fare Yanal Tail Ven içinde Evans Blue İntravenöz Enjeksiyon <ol><li> PBS içinde Evans mavisi% 0.5 &#39;lik steril çözelti hazırlayın. Gerekirse, çözünmüş henüz herhangi bir partikül madde kaldırmak için çözüm filtre sterilize. </li><li> Bir enjektöre 200 ul Evans Blue çözüm aspire. Şırıngaya kaçmış olabilir tüm hava kabarcıkları kaçının. </li><li> Hayvan serbestçe mobil değil ama kendi kuyruğunu ele alınabilir böylece bir kısıtlama cihaza 8-12 hafta eski il fareler. </li><li> Böylece lateral kuyruk ven kolayca görülebilir ve yukarı bakacak şekilde yan fare emniyet cihazı yerleştirin. </li><li> Başparmak ve işaret parmağı arasında baskın olmayan elle Kuyruğunu tut. </li><li> Baş yönünde yanal kuyruk ven içine 10-15 derecelik açı, eğim yukarı, önceden de iğne (küçük ölçü, 27-30) yerleştirin. Kuyruk iğne ve şırınga paralel tutun. </li><li> Do Bu damar çökebilir olarak uygun yerleştirme onaylamak için basınç geri geçerli değildir. </li><li> Yavaşça fare kuyruk ven 200 ul Evans Blue enjekte çözüm. </li><li> Piston gelişmeler, bu gibi damar iğnenin doğru yerleştirme kanıtıdır kolaylığını gözlemleyin. </li><li> Onun kafesine geri fare koyun ve 30 dakika için bunu gözlemlemek. </li></ol> 2. Organ Organ Toplama ve Evans Blue Ekstraksiyon <ol><li> Servikal dislokasyon yoluyla farelerin Kurban. Bu damar geçirgenliği ile önemli girişim sınırlar gibi Miles tahlil amacıyla servikal dislokasyon önerilir. Mümkün olduğunca hızlı ve aynı zamanda tüm fareler kurban edin. Ölüm kan damarlarının daha geçirgen hale kısa bir süre sonra, çünkü 6 fareler veya daha az tabur ile çalışın. </li><li> Sırtlarında fareler yerleştirin ve bir beyaz tahta üzerinde kendi ayakları pin. </li><li> Thor maruz karın ve göğüs boşluğu açınacic ve karın organları. </li><li> Evans Blue ekstravazasyonu farklılıkları göstermek için temsili fotoğraflar çekin. Tüm fareler için aynı aydınlatma koşullarına sahip olmak için aynı alanda tüm fareler ekleyin. </li><li> Ilgi organları toplayın ve 1.5 ml tüpler koydu </li><li> Boş tüp tartılır ve sıfır bakiye değer getirmek. </li><li> Doku örneği aktarın ve kilo. Tüm doku örnekleri için yineleyin. Dokular hava farklı organlar arasında su içeriği değişkenlik ortadan kaldırmak için kurutulabilir. </li><li> . Her doku örneği tüpüne 500 ul formamid ekleyin. </li><li> 55 ° C su banyosu veya ısıtma bloğu tüm tüpler aktarın. Dokudan Evans Blue ayıklamak için 24-48 saat süreyle inkübe edin. </li></ol> 3. Evans Blue Kantitasyonu İnterstisyel Doku ekstravaze <ol><li> Pelet kalan doku parçaları için Formamid / Evans Blue karışımı santrifüjleyin. </li><li> 610 absorbans ölçülürnm. Boş olarak 500 ul Formamid kullanın. </li><li> Hesapla ng Evans Blue mg doku başına ekstravaze. </li><li> Bir grafikte tüm veriler çizilir. </li><li> Önemli farklılıklar belirlemek için istatistiksel analiz gerçekleştirin. </li></ol>

<ol>
	<li>Beck, K. F., 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=Inducible+NO+synthase:+role+in+cellular+signaling.">Inducible NO synthase: role in cellular signaling.</a> J. Exp. Biol. 202, 645-653 (1999).</li><li>Bertglia, S., Giusti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+nitric+oxide+in+capillary+perfusion+and+oxygen+delivery+regulation+during+systemic+hypoxia.">Role of nitric oxide in capillary perfusion and oxygen delivery regulation during systemic hypoxia.</a> Am. J. Physiol. Heart Circ. Physiol. 288, H525-H531 (2005).</li><li>Miles, A. A., Miles, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+reactions+to+histamine,+histamine-liberator+and+leutaxine+in+the+skin+of+guinea+pigs.">Vascular reactions to histamine, histamine-liberator and leutaxine in the skin of guinea pigs.</a> J. Physiol. (London). 118, 228-257 (1952).</li><li>Weis, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+permeability+in+cardiovascular+disease+and+cancer.">Vascular permeability in cardiovascular disease and cancer.</a> Curr. Opin. Hematol. 15, 243-249 (2008).</li><li>Kumar, P., Shen, Q., Pivetii, C. D., Lee, E. S., We, M. H., Yuan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+endothelial+hyperpermeability:+implications+in+inflammation.">Molecular mechanisms of endothelial hyperpermeability: implications in inflammation.</a> Expert Rev. Mol. Med. 30, 11-19 (2009).</li><li>Viazzi, F., 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=Vascular+permeability,+blood+pressure,+and+organ+damage+in+primary+hypertension.">Vascular permeability, blood pressure, and organ damage in primary hypertension.</a> Hypertens. Res. 31, 873-879 (2008).</li><li>Scheppke, ., 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=Retinal+vascular+permeability+suppression+by+topical+application+of+a+novel+VEGFR2/Src+kinase+inhibitor+in+mice+and+rabbits.">Retinal vascular permeability suppression by topical application of a novel VEGFR2/Src kinase inhibitor in mice and rabbits.</a> J. Clin. Invest. 118, 2337-2346 (2008).</li><li>Blanchet, M. 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=Loss+of+CD34+Leads+To+Exacerbated+Autoimmune+Arthritis+through+Increased+Vascular+Permeability.">Loss of CD34 Leads To Exacerbated Autoimmune Arthritis through Increased Vascular Permeability.</a> J. Immunol. 184, 1292-1299 (2010).</li><li>Dvorak, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mast+cell-derived+mediators+of+enhanced+microvascular+permeability,+vascular+permeability+factor/vascular+endothelial+growth+factor,+histamine,+and+serotonin,+cause+leakage+of+macromolecules+through+a+new+endothelial+cell+permeability+organelle,+the+vesiculo-vacuolar+organelle.">Mast cell-derived mediators of enhanced microvascular permeability, vascular permeability factor/vascular endothelial growth factor, histamine, and serotonin, cause leakage of macromolecules through a new endothelial cell permeability organelle, the vesiculo-vacuolar organelle.</a> Chem. Immunol. Allergy. 85, 185-204 (2005).</li><li>Le Guelte, A., Gavard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+endothelial+cell-cell+junctions+in+endothelial+permeability.">Role of endothelial cell-cell junctions in endothelial permeability.</a> Methods Mol. Biol. 763, 265-279 (2011).</li><li>Martins-Green, M., Petreaca, M., Yao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+for+in+vitro+detection+of+permeability+in+human+"endothelium".">An assay system for in vitro detection of permeability in human "endothelium".</a> Methods Enzymol. 443, 137-153 (2008).</li></ol>

Yazarlar herhangi bir çatışma veya rekabet mali çıkarları var.

Damar geçirgenliği kan damarı durumu için kritik bir göstergesidir. Artan vasküler permeabilite diyabet, hipertansiyon ve otoimmün hastalıklar 6,7,8 dahil olmak üzere çeşitli sistemik hastalıklar, mevcut olduğu gösterilmiştir. Artan vasküler permeabilite kayma gerilmesi aracılık gösterilmiştir, vasküler endotelyal büyüme faktörü ve fibroblast büyüme faktörü, serotonin, histamin ve 9 bradikinin gibi yangı benzeri büyüme faktörleri. Su ve küçük moleküller ekstrava...

Seçici geçirgen engellerin Oluşumu ve bakım uygun organ gelişimi ve performansı 1,2 için gereklidir. Endotel hücreleri hattı damar lümeni ve kan ve tüm organları interstisyel boşluk arasında seçici ulaşım esastır yarı geçirgen bir bariyer oluşturur. Yeterli bir geçirgenlik bariyer sıkı bir şekilde büyüme faktörleri, sitokinler ve diğer stres bağlantılı moleküllerin 3 tarafından kontrol edilmektedir sıkı hücre-hücre birleşme yoluyla korunur. Endotel hücre bariyerin bozulması artmış geçirgenlik ve vasküler sızmasına neden olabilir. Bu etkiler, çeşitli hastalık durumlarında görülür ve altı çizili olan moleküler sinyalizasyonun anlayış multidisipliner yöntemlerin 4,5 gerektirir edilir. Bu yazıda, bir fare modeli kullanılarak damar geçirgenliğini ölçmek için in vivo yöntemi bir tarif. Ayrıca Miles test olarak bilinir tanımlamakta olduğumuz testi, iyi establ olduğunuin vivo vasküler permeabilite test yöntemi ished. Tahlil bazal fizyolojik koşullar altında, albümin endotel bariyer çapraz değil, gerçeğine dayanır. Evans Mavisi, albümin için yüksek afinitesi olan bir azo boyası, deneysel bir hayvan, kan dolaşımında enjekte edilir, ve fizyolojik koşullar altında, bu kan damarları içinde sınırlı olması beklenmektedir. Bir vasküler geçirgenlik uyarıcı eklendiği zaman, ya da topikal veya sistemik olarak, kan damarlarını albümin bağlı Evans mavisi da, böylece protein sızdırmaya başlar ve. Geçirgen damarlar vardır dokuların hızlı bir mavi renk elde edilir. Fare yanal kuyruk damarı içinde boya başarılı bir şekilde enjeksiyon deney iyi sonuç için kritik önem taşır. Kuyruk ven enjeksiyon tekniği kapsamlı uygulama gerektirir ve deney başlamadan önce hakim olmalıdır. Gemi geçirgenliği bir yaşına ve ağırlığına son derece bağlıdırolmuş bölgeler, çok farklı fare suşları karşılaştırırken bu fareler veya diğer deneklere aynı doğum tarihleri ​​ve kilosuna yakın olması şarttır. Endotel bariyer geçirgenliğini etkileyen diğer faktörler, sıcaklık, nem gibi çevresel koşullar ve çok önemlisi, fare kullanımı stres. Deney sonucunu etkileyebilecek faktörlerin çokluğu nedeniyle bu deney en az üç kez ve istatistiksel analiz yapılır tekrarlanır olduğunu her zaman tavsiye edilir. Bu tahlil, damar genetik olarak modifiye edilmiş farelerin geçirgenlik yanı sıra, farklı genetik geçmişe sahip fareler veya karşılaştırmak için kullanılabilir. Geçirgenlik modüle edilen gen fonksiyonu bağlı olarak, bir uyarıcı varlığında ya da yokluğunda bir biçimde tespit edilebilir. Bu tahlil, ayrıca damar geçirgenliğini farklı bileşiklerin etkisini test etmek için ya da kullanılabilmektedir.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REAKTİF </td></tr><tr><td> Evans Mavisi </td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td> Formamid </td><td> Invitrogen </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> 0.2M Fosfat 1,5 M NaCl pH 7.4 </td></tr><tr><td></td><td></td><td></td><td> EKİPMAN </td></tr><tr><td> SPEKTROFOTOMETRESİNDE </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> FARE GÜVENLİK CİHAZI </td><td> HARVARD APARATI </td><td> 340012 </td><td></td></tr><tr><td> SYRINGE </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> İĞNE </td><td> BD </td><td> 305106 </td><td> İğnenin göstergesi hayvanın büyüklüğüne bağlıdır. </td></tr><tr><td> BALANCE </td><td> DENVER INSTRUMENT </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

Bu yöntem, deney hayvan modeli olarak, farelerde Evans Blue intravenöz enjeksiyon esas alınmaktadır. Evans mavisi albümin bağlayan bir boyadır. Fizyolojik koşullarda endotel albümin geçirmez, yani Evans mavisi ciltli albümini kalıntıları kan damarları içinde sınırlı. Artmış vasküler permeabilite endotel hücreleri teşvik patolojik koşullarda kısmen onların yakın temas kaybetmek ve endotel albumin gibi küçük proteinlerin geçirgen hale gelir. Bu durum dokularda Evans Mavisi ekstravazasyonu sağlar. Sağlıklı bir endotelin komşu damarlı dokularda boya ekstravazasyonu önler. Artan geçirgenlik sahip organlar sağlam endotel ile organlara kıyasla önemli ölçüde artmıştır mavi rengi gösterecektir. Vasküler permeabilite seviyesi basit görselleştirme veya deneysel hayvan / doku karşı kontrol doku miligram başına dahil boya kantitatif ölçümü ile değerlendirilebilir. İki poweBu testin rful yönlerini sadeliği ve kantitatif özellikleri vardır. Evans mavisi formamid doku belirli bir miktarda bekletilmeleri ile ekstrakte edilebilir. Evans Blue absorbans maksimum 620 nm&#39;de ve absorbans asgari 740 nm&#39;de olduğunu. Evans Mavisi için bir standart eğri kullanılarak, optik yoğunluk ölçümleri doku başına miligram çekilen miligram boya haline dönüştürülebilir. İstatistiksel analiz vasküler geçirgenliği önemli farklılıkları değerlendirmek için kullanılmalıdır.

Biz 8-12 hafta Yaşlı farelerde damar kaçaklarının tespitinde in vivo geçirgenliği tayininde kullanılır. Bu test, farklı genetik arka plan hayvanlar arasında veya damar sistemi etkileyen işlemlere tabi fareler tek bir gerilme içinde göreli damar geçirgenliğini karşılaştıran yararlıdır. Bizim sonuçlar bizim laboratuarda yaratılmış genetiği değiştirilmiş farelerin bir gerginlik yabani tip farelere göre daha geçirgen endotele sahip olduğunu göstermektedir. Bu değişiklikl...

Watch this Scientific Journal Video about An in vivo Assay to Test Blood Vessel Permeability at JoVE.com

An in vivo Assay to Test Blood Vessel Permeability

Biz sunuyoruz<em&gt; In vivo</em&gt; Damar geçirgenliği test etmek için test. Bu tahlil, interstisiyel boşluklar içine difüzyonu bir boya ve daha sonraki görüntüleme intravenöz enjeksiyon dayanmaktadır.

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 ...

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Research

JoVE Journal

Medicine

An in vivo Assay to Test Blood Vessel Permeability

64.4K Görüntüleme.  Fox Chase Cancer Center. Biz sunuyoruz In vivo Damar geçirgenliği test etmek için test. Bu tahlil, interstisiyel boşluklar içine difüzyonu bir boya ve daha sonraki görüntüleme intravenöz enjeksiyon dayanmaktadır.

Video: An in vivo Assay to Test Blood Vessel Permeability

In Vitro Assays to Assess Blood-brain Barrier 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 astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-&#946; (oA&#946;), which is an important contributor to Alzheimer&#39;s disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oA&#946; disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

In Vitro Assays to Assess Blood-brain Barrier Mesh-like Vessel Formation and Disruption

Maintaining blood-brain barrier coverage is key for the homeostasis of the central nervous system. This protocol describes in vitro techniques to delineate the fundamental and pathological processes that modulate blood-brain barrier coverage.

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 ...

Tıp

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

Vivo kan - beyin bariyerini geçirgenliği tahlil Fluorescently kullanarak farelerde izleyiciler etiketli

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Nörobilim

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

Gut-kan bariyerini ve karaciğer metabolizması Microbiota ürünlerin değerlendirilmesi için In Vivo yöntemi

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

İzole Kan-perfüze Sıçan Akciğer Hazırlık Tek Mikrodamar Geçirgenliği miktarının

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Biyoloji

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Şekilde, mikro Tekniği Sıçan mezenter mikrodamar geçirgenliği Yönetmelik Araştırma

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Fare Matrix Jel Plug Test kullanma İnsan endotel-pericyte Etkileşim Vivo Çalışmasında

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and subsequent vision loss. The inner blood-retinal barrier (iBRB) is mainly composed of retinal vascular endothelium with low permeability under physiological conditions. This feature of low permeability is tightly regulated and maintained by low rates of paracellular transport between adjacent retinal microvascular endothelial cells, as well as transcellular transport (transcytosis) through them. The assessment of retinal transcellular barrier permeability may provide fundamental insights into iBRB integrity in health and disease. In this study, we describe an endothelial cell (EC) transcytosis assay, as an in vitro model for evaluating iBRB permeability, using human retinal microvascular endothelial cells (HRMECs). This assay assesses the ability of HRMECs to transport transferrin and horseradish peroxidase (HRP) in receptor- and caveolae-mediated transcellular transport processes, respectively. Fully confluent HRMECs cultured on porous membrane were incubated with fluorescent-tagged transferrin (clathrin-dependent transcytosis) or HRP (caveolae-mediated transcytosis) to measure the levels of transferrin or HRP transferred to the bottom chamber, indicative of transcytosis levels across the EC monolayer. Wnt signaling, a known pathway regulating iBRB, was modulated to demonstrate the caveolae-mediated HRP-based transcytosis assay method. The EC transcytosis assay described here may provide a useful tool for investigating the molecular regulators of EC permeability and iBRB integrity in vascular pathologies and for screening drug delivery systems.

Endothelial Cell Transcytosis Assay as an In Vitro Model to Evaluate Inner Blood-Retinal Barrier Permeability

This protocol illustrates an in vitro endothelial cell transcytosis assay as a model to evaluate inner blood-retinal barrier permeability by measuring the ability of human retinal microvascular endothelial cells to transport horseradish peroxidase across cells in caveolae-mediated transcellular transport processes.

İç Kan-Retinal Bariyer Geçirgenliğini Değerlendirmek için In Vitro Model Olarak Endotel Hücre Transsitozu Testi

Dysfunction of the blood-retinal barrier (BRB) contributes to the pathophysiology of several vascular eye diseases, often resulting in retinal edema and ...

Evaluating Vascular Hyperpermeability-inducing Agents in the Skin with the Miles Assay

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby the permeability of the endothelium to blood cells, plasma macromolecules, and water can be adapted according to the physiological need. In certain diseases, cytokines and growth factors are released that target the endothelial barrier to transiently increase vascular permeability; however, their prolonged presence may cause chronic vascular hyperpermeability and thereby tissue-damaging edema. The Miles assay is an in vivo technique that allows researchers to study vascular hyperpermeability through the proxy measurement of vascular leakage. Here, we provide a detailed protocol on how to perform this procedure in the mouse, which is the most widely used model organism to study mammalian physiology and pathology. The procedure involves the intravenous injection of Evans blue dye to label the circulating albumin followed by multiple intradermal injections of permeability-inducing agents and vehicle control solutions into opposing flanks of the mouse. Consequently, Evans blue dye gradually leaks into the dermis, where it accumulates and can be extracted for quantification as leakage induced by the permeability-inducing agent relative to the vehicle. The Miles assay can be performed in wild type or genetically modified mouse models and may be combined with drug administration to study molecular mechanisms that regulate vascular permeability and identify agents/targets capable of inducing or blocking hyperpermeability.

Here, we present a protocol to measure the vascular leakage induced by intradermal administration of permeability promoting agents into the murine skin. This technique can be used to determine the ability of molecules to promote or inhibit vascular leakage or to study the molecular mechanisms that regulate vascular permeability.

Vasküler Hyperpermeability indükleyici ajanlar cilt mil tahlil ile değerlendirilmesi

The primary function of the vascular endothelium in vertebrate organisms is to serve as a barrier between the blood and each tissue of the body, whereby ...

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the endothelial barrier dysfunction is related to many salivary gland secretory disorders. The present protocol describes an in vivo paracellular permeability detection method to evaluate the function of endothelial tight junctions (TJs) in mouse submandibular glands (SMG). First, fluorescence-labeled dextrans with different molecular weights (4 kDa, 40 kDa, or 70 kDa) were injected into the angular veins of mice. Afterward, the unilateral SMG was dissected and fixed in the customized holder under a two-photon laser-scanning microscope, and then images were captured for blood vessels, acini, and ducts. Utilizing this method, the real-time dynamic leakage of the different-sized tracers from blood vessels into the basal sides of the acini and even across the acinar epithelia into the ducts was monitored to evaluate the alteration of the endothelial barrier function under physiological or pathophysiological conditions.

In Vivo Vascular Permeability Detection in Mouse Submandibular Gland

In the present protocol, the endothelial barrier function of the submandibular gland (SMG) was evaluated by injecting different molecular weighted fluorescent tracers into the angular veins of test animal models in vivo under a two-photon laser-scanning microscope.

İn Vivo Fare Submandibuler Bezinde Vasküler Geçirgenlik Tespiti

Saliva plays an important role in oral and overall health. The intact endothelial barrier function of blood vessels enables saliva secretion, whereas the ...

Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez Çalışmalarının Basitleştirilmesi

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Yazar Spot Işığı: Modifiye Matris Jel Tıkaç Testi ile Anjiyogenez ve Damar Geçirgenliğinin Araştırılması

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Anjiyogenez Çalışmaları için Modifiye In Vivo Matrix Jel Plug Testi

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...

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