Questo lavoro è stato sostenuto da una sovvenzione da parte del National Institutes of Health, R01CA142928.

1. Iniezione endovenosa di Evans Blue in the Vein laterale coda di topo <ol><li> Preparare una soluzione allo 0,5% sterile di blu di Evans in PBS. Se necessario, filtro sterilizzare la soluzione per rimuovere particelle che non è dissolto. </li><li> Aspirare 200 ul Evans soluzione di blu in una siringa. Evitare tutte le bolle d&#39;aria che possano esser sfuggite nella siringa. </li><li> Luogo topi che sono 8-12 settimane di vita in un dispositivo di ritenuta in modo che l&#39;animale non è liberamente mobile, ma la coda può essere gestito. </li><li> Posizionare il dispositivo di ritenuta del mouse sul fianco con la vena caudale laterale è facilmente visibile e sono rivolti verso l&#39;alto. </li><li> Tenere su la coda con la mano non dominante tra il pollice e l&#39;indice. </li><li> Inserire l&#39;ago (calibro piccolo, 27-30) in un anticipo 10-15 gradi, smussata, nella vena caudale laterale verso la direzione della testa. Tenere il parallelepipedo ago e la siringa alla coda. </li><li> Do non si applica la contropressione per confermare il corretto posizionamento in quanto ciò potrebbe comprimere la vena. </li><li> Iniettare lentamente 200 microlitri soluzione Evans blu nella vena della coda del topo. </li><li> Osservare la facilità con cui gli anticipi stantuffo, poiché questa è la prova di corretto posizionamento dell&#39;ago nella vena. </li><li> Ponga il mouse di nuovo nella sua gabbia e osservare per 30 minuti. </li></ol> 2. Organo Raccolta ed estrazione di Evans Blue dagli organi <ol><li> Sacrifica i topi tramite dislocazione cervicale. Ai fini del test Miles dislocazione cervicale è raccomandato in quanto limita la grave interferenza con la permeabilità vascolare. Sacrificare tutti i topi al tempo stesso, il più velocemente possibile. Lavora con coorti di 6 topi o meno, perché poco dopo la morte i vasi sanguigni diventano più permeabili. </li><li> Mettere i topi sulla schiena e il pin i piedi su un bordo bianco. </li><li> Aprire la cavità addominale e toracica per esporre thororgani ACIC e addominale. </li><li> Scatta foto rappresentative per mostrare le differenze di stravaso Evans Blue. Includere tutti i topi nello stesso campo in modo da avere condizioni di luce identiche per tutti i mouse. </li><li> Raccogliere gli organi di interesse e metterli in provette da 1,5 ml </li><li> Pesare un tubo vuoto e portare il valore di bilancio a zero. </li><li> Trasferire il campione di tessuto e peso. Ripetere l&#39;operazione per tutti i campioni di tessuto. Tessuti può essere essiccato per eliminare la variabilità contenuto di acqua tra diversi organi. </li><li> . Aggiungere 500 microlitri formammide in ogni provetta campione di tessuto. </li><li> Trasferire tutte le provette in un bagno d&#39;acqua ° 55 C o di un blocco di calore. Incubare per 24-48 ore per estrarre i Blu di Evans dal tessuto. </li></ol> 3. Quantificazione di Evans Blue travasato nel tessuto interstiziale <ol><li> Centrifugare la formammide / Evans miscela blu per far sedimentare i frammenti di tessuto rimanenti. </li><li> Misurare l&#39;assorbanza a 610nm. Usare 500 microlitri Formammide come bianco. </li><li> Calcola ng Evans Blue stravaso per mg di tessuto. </li><li> Tracciare tutti i dati in un grafico. </li><li> Eseguire l&#39;analisi statistica per determinare differenze significative. </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>

Gli autori non hanno alcun conflitto o in competizione interessi finanziari.

Permeabilità vascolare è un indicatore fondamentale per lo stato dei vasi sanguigni. Aumento della permeabilità vascolare ha dimostrato di essere presente in numerose malattie sistemiche, compreso il diabete, ipertensione e malattie autoimmuni 6,7,8. Aumento della permeabilità vascolare ha dimostrato di essere mediata da shear stress, fattori di crescita come il fattore di crescita vascolare endoteliale e fattore di crescita dei fibroblasti, mediatori infiammatori quali serotonina, istamina e bradichinina...

Formazione e il mantenimento di barriere permeabili selettive sono essenziali per lo sviluppo di organi corretto e 1,2 prestazioni. Cellule endoteliali linea lume del vaso sanguigno e formano una barriera semipermeabile che è essenziale per il trasporto selettivo tra sangue e lo spazio interstiziale di tutti gli organi. Una barriera di permeabilità adeguata è mantenuta attraverso stretti cellula-cellula giunzioni che sono strettamente controllate da fattori di crescita, citochine e molecole correlate allo stress altri 3. Deterioramento della barriera endoteliale può causare aumentata permeabilità vascolare e perdite. Questi effetti sono visti in diversi stati patologici e la comprensione della segnalazione sottolineatura molecolare richiede metodi multidisciplinari 4,5. In questo articolo, si descrive un metodo in vivo per misurare la permeabilità nave che utilizza un modello di topo. Il saggio stiamo descrivendo, anche noto come saggio Miles, è una ben establmentata metodo per testare la permeabilità vascolare in vivo. Il saggio è basato sul fatto che, in condizioni fisiologiche basali, albumina non attraversa la barriera endoteliale. Evans blu, un colorante azoico con alta affinità per l&#39;albumina, viene iniettato nel flusso sanguigno di un animale sperimentale, e in condizioni fisiologiche, esso dovrebbe essere limitato entro vasi sanguigni. Quando uno stimolo permeabilità vascolare è aggiunto, sia a livello topico o per via sistemica, i vasi sanguigni iniziano a perdere proteine ​​e, quindi, anche il blu Evans che è legato all&#39;albumina. Ciò provoca una colorazione bluastra rapida dei tessuti che hanno vasi permeabili. Iniezione di successo del colorante nella vena laterale della coda del mouse è fondamentale per il buon esito dell&#39;esperimento. Vena tecnica Tail iniezione richiede pratica estesa e deve essere masterizzato prima di iniziare l&#39;esperimento. Permeabilità vasale è fortemente dipendente dall&#39;età e dal peso del l&#39;IMAL, così quando si confrontano diversi ceppi di topi è imperativo che i topi o dei soggetti di prova diversi da avere vicino date di nascita e peso identici. Altri fattori che influenzano la permeabilità della barriera endoteliale sono condizioni ambientali come temperatura, umidità, e molto importante, lo stress manipolazione del mouse. A causa del gran numero di fattori che possono influenzare l&#39;esito dell&#39;esperimento è sempre consigliabile che l&#39;esperimento viene ripetuto almeno tre volte e analisi statistica effettuata. Questo test può essere utilizzato o per confrontare permeabilità vasale di topi che sono stati geneticamente modificati, nonché topi con differenti background genetico. Permeabilità può essere valutata in presenza o assenza di uno stimolo, a seconda della funzione del gene che è modulato. Questo dosaggio può anche essere utilizzato o per testare l&#39;effetto di composti differenti permeabilità vasale.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REAGENTE </td></tr><tr><td> EVANS BLU </td><td> SIGMA </td><td> E2129 </td><td></td></tr><tr><td> FORMAMIDE </td><td> INVITROGEN </td><td> 15515-026 </td><td></td></tr><tr><td> PBS </td><td></td><td></td><td> Fosfato 0,2 M 1.5M NaCl pH 7,4 </td></tr><tr><td></td><td></td><td></td><td> ATTREZZATURE </td></tr><tr><td> SPETTROFOTOMETRO </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> MOUSE dispositivo di sicurezza per </td><td> Harvard Apparatus </td><td> 340012 </td><td></td></tr><tr><td> SIRINGA </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> AGHI </td><td> BD </td><td> 305106 </td><td> Il calibro dell&#39;ago dipende dalla taglia dell&#39;animale. </td></tr><tr><td> SALDO </td><td> DENVER STRUMENTO </td><td> TP-64 </td><td></td></tr></tbody></table>

an in vivo assay to test blood vessel permeability

Questo metodo si basa sulla iniezione endovenosa di Evans blu in topi come modello animale di prova. Blu di Evans è un colorante che si lega all&#39;albumina. In condizioni fisiologiche l&#39;endotelio è impermeabile all&#39;albumina, in modo da Evans blu resti albumina legata restrizioni all&#39;interno dei vasi sanguigni. In condizioni patologiche che promuovere un aumento della permeabilità delle cellule endoteliali vascolari parzialmente perdono i loro contatti stretti e l&#39;endotelio diventa permeabile alle proteine ​​di piccole dimensioni come l&#39;albumina. Questa condizione consente per lo stravaso di Blu di Evans nei tessuti. Un endotelio sano impedisce stravaso del colorante nei tessuti circostanti vascolarizzati. Organi con aumento della permeabilità mostrerà significativamente aumentata colorazione blu rispetto a organi con endotelio intatto. Il livello di permeabilità vascolare può essere valutata mediante semplice visualizzazione o misurazione quantitativa del colorante incorporato per milligrammo di tessuto di controllo contro animale sperimentale / tessuto. Due PoweRFUL aspetti di questo test è la sua semplicità e le caratteristiche quantitative. Evans blu colorante può essere estratto dai tessuti incubando una quantità specifica di tessuto in formammide. Evans Blue è massima assorbanza a 620 nm e minimo assorbimento è a 740 nm. Utilizzando una curva standard per Evans blu, misure di densità ottica può essere convertito in colorante milligrammo catturato per milligrammo di tessuto. Analisi statistica dovrebbe essere utilizzato per valutare le differenze significative nella permeabilità vascolare.

Abbiamo utilizzato un saggio di permeabilità in vivo per verificare perdite nave in topi 8-12 settimane di vita. Questo test è utile per confrontare la relativa permeabilità vascolare tra animali di diverso background genetico o in un singolo ceppo di topi sottoposti a trattamenti che colpiscono il sistema vascolare. I nostri risultati mostrano che un ceppo di topi geneticamente modificati che abbiamo creato nel nostro laboratorio ha un endotelio più permeabile rispetto ai topi wild-type. Questi ca...

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

Stiamo presentando un<em&gt; In vivo</em&gt; Saggio per testare vaso sanguigno permeabilità. Questo saggio è basato sulla iniezione endovenosa di un colorante e la successiva visualizzazione della sua diffusione negli spazi interstiziali.

Un<em&gt; In vivo</em&gt; Test per la verifica della nave Permeabilità Sangue

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 Views.  Fox Chase Cancer Center. Stiamo presentando un In vivo Saggio per testare vaso sanguigno permeabilità. Questo saggio è basato sulla iniezione endovenosa di un colorante e la successiva visualizzazione della sua diffusione negli spazi interstiziali.

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.

In vitro dosaggi per valutare la formazione e la rottura del vaso come la barriera emato-cerebrale

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

Ricerca

Medicina

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.

In Vivo barriera emato - encefalica permeabilità saggio nei topi utilizzando fluorescente etichettati traccianti

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

Neuroscienze

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.

Un metodo In Vivo per valutare la Gut-barriera e metabolismo epatico del Microbiota prodotti

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.

Quantificare singolo microvasi Permeabilità in Isolata Blood-perfusione Rat Lung Preparazione

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

Biologia

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.

Microperfusione Tecnica per Indagare regolamento di microvasi permeabilità a Rat mesentere

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.

Studio in vivo di Human Interaction endoteliale-pericyte Utilizzando la matrice di gel Plug Assay nel topo

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.

Saggio di transcitosi delle cellule endoteliali come modello in vitro per valutare la permeabilità della barriera emato-retinica interna

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.

Valutazione vascolari agenti d'induzione Hyperpermeability nella pelle con il dosaggio di Miles

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.

In vivo Rilevamento della permeabilità vascolare nella ghiandola sottomandibolare del topo

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

Semplificazione degli studi di angiogenesi con un test Matrix Gel Plug

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.

Autore in primo piano: Studio dell'angiogenesi e della permeabilità dei vasi attraverso un saggio di tappo in gel a matrice modificata

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.

Saggio modificato in vivo del gel plug della matrice per gli studi sull'angiogenesi

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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