Diese Arbeit wurde durch ein Stipendium der National Institutes of Health, R01CA142928 unterstützt.

Ein. Intravenöse Injektion von Evans Blue in der Maus laterale Schwanzvene <ol><li> Bereiten Sie eine 0,5% sterile Lösung von Evans-Blau in PBS. Falls notwendig, Filter-sterilisiert die Lösung, um Partikel, die nicht aufgelöst hat, zu entfernen. </li><li> Absaugen 200 ul Evans Blue-Lösung in einer Spritze. Vermeiden Sie alle Luftblasen, die in die Spritze entgangen sein könnte. </li><li> Ort Mäusen, 8-12 Wochen alt sind in eine Rückhaltevorrichtung so dass das Tier nicht frei beweglich, sondern mit dem Schwanz gehandhabt werden kann. </li><li> Die Maus Rückhaltevorrichtung auf die Seite, so dass die laterale Schwanzvene gut sichtbar und sind nach oben gerichtet. </li><li> Halten auf das Endstück mit der nicht dominanten Hand zwischen dem Daumen und dem Zeigefinger. </li><li> Sie mit der Nadel (kleine Spurweite, 27-30) bei einer 10 bis 15 Grad Winkel, Abschrägung, voraus in die laterale Schwanzvene in die Richtung des Kopfes. Halten Sie die Nadel und Spritze parallel zum Schwanz. </li><li> Do nicht anwendbar Gegendruck auf die richtige Platzierung zu bestätigen, da dies die Vene zusammenbrechen könnte. </li><li> Langsam spritzen 200 ul Evans-Blau-Lösung in die Schwanzvene der Maus. </li><li> Beachten Sie die Leichtigkeit, mit der der Kolben Fortschritte, denn dies ist der Beweis für die korrekte Platzierung der Nadel in der Vene. </li><li> Legen Sie die Maus wieder in seinen Käfig und beobachten Sie es für 30 min. </li></ol> 2. Orgel Sammlung und Extraktion von Evans Blue von den Organen <ol><li> Opfert die Mäuse durch Genickbruch. For Miles Assayzwecke Genickbruch wird empfohlen, da es wesentliche Beeinflussung begrenzt mit vaskulären Permeabilität. Opfern alle Mäuse gleichzeitig, so schnell wie möglich. Arbeiten Sie mit Kohorten von 6 Mäusen oder weniger, denn kurz nach dem Tod Blutgefäße durchlässiger geworden. </li><li> Platzieren Sie die Maus auf den Rücken und setzen ihre Füße auf einer weißen Tafel. </li><li> Öffnen Sie die Bauch-und Brusthöhle zu thor aussetzenACIC und Bauchorgane. </li><li> Nehmen repräsentative Bilder von Unterschieden in Evans Blue Extravasation zeigen. Fügen Sie alle Mäuse in dem selben Gebiet, um identische Lichtverhältnisse für alle Mäuse haben. </li><li> Sammle Organe von Interesse und steckte sie in 1,5 ml Röhrchen </li><li> Wiegen Leerhülse und bringen den Saldowert Null. </li><li> Übertragen der Gewebeprobe und gewichtet sie. Wiederholen Sie dies für alle Gewebeproben. Gewebe können luftgetrocknet werden, um den Wassergehalt Variabilität zwischen verschiedenen Organen zu eliminieren. </li><li> . Fügen Sie 500 ul Formamid zu jeder Gewebeprobe Röhre. </li><li> Übertragen Sie alle Rohre mit einem 55 ° C Wasserbad oder Heizblock. Inkubieren 24-48 h bis Evans Blue aus dem Gewebe zu extrahieren. </li></ol> 3. Quantifizierung von Evans Blue extravasierten in interstitiellen Gewebe <ol><li> Zentrifugieren Formamid / Evans Blue Mischung Pellet alle verbleibenden Gewebefragmente. </li><li> Absorption bei 610nm aufweist. Verwenden Sie 500 ul Formamid als leer. </li><li> Calculate ng Evans Blue pro mg Gewebe ausgetretenes. </li><li> Zeichnen Sie alle Daten in einer Grafik. </li><li> Statistische Analysen durchzuführen, um signifikante Unterschiede festzustellen. </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>

Die Autoren haben keinen Konflikt oder konkurrierende finanziellen Interessen.

Gefäßpermeabilität ist ein kritischer Marker für Blutgefäß-Status. Erhöhte Gefäßpermeabilität hat sich gezeigt, dass in mehreren systemischen Erkrankungen, wie Diabetes, Hypertension, und Autoimmunerkrankungen 6,7,8 vorliegt. Erhöhte Gefäßpermeabilität ist gezeigt worden, durch Scherbeanspruchung vermittelt werden, Wachstumsfaktoren wie vaskulären endothelialen Wachstumsfaktor und Fibroblasten-Wachstumsfaktor, Entzündungsmediatoren wie Serotonin, Histamin und Bradykinin 9. Extravasa...

Bildung und Erhaltung der selektiven permeablen Barrieren für die richtige Entwicklung der Organe und Leistung 1,2 unerlässlich. Endothelzellen Linie das Blutgefäßlumen und bilden eine semi-permeable Barriere, die wesentlich in den selektiven Transport zwischen Blut und dem interstitiellen Raum aller Organe ist. Eine ausreichende Permeabilitätsbarriere wird durch enge Zell-zu-Zell-Verbindungen, die ausschließlich durch Wachstumsfaktoren, Zytokine und andere stressbedingte Moleküle 3 gesteuert werden beibehalten. Störung der Endothelzelle Barriere kann in erhöhter Permeabilität und vaskuläre Leckage führen. Diese Effekte sind in verschiedenen Krankheitsstadien gesehen und das Verständnis der Unterstreichung molekularen Signalwegen erfordert multidisziplinäre Methoden 4,5. In diesem Artikel beschreiben wir ein Verfahren zur In-vivo-Verwendung eines Gefäßpermeabilität Mausmodell messen. Der Assay wir beschreiben, auch als Miles-Assay bezeichnet, ist ein gut establIshed Methode, um vaskuläre Permeabilität in vivo zu testen. Der Assay basiert auf der Tatsache, dass unter basalen physiologischen Bedingungen, Albumin passiert nicht die Endothelbarriere basiert. Evans Blue, ein Azofarbstoff mit hoher Affinität für Albumin, wird in den Blutstrom eines Versuchstiers injiziert werden, und unter physiologischen Bedingungen, so wird erwartet, dass sie innerhalb von Blutgefäßen beschränkt werden. Wenn ein Gefäßpermeabilität Reiz hinzugefügt wird, entweder lokal oder systemisch, beginnen die Blutgefäße zu Protein auslaufen und damit auch die Evans Blau, das an Albumin gebunden ist. Dies führt zu einem raschen bläuliche Färbung von Geweben durchlässigen Behälter haben. Erfolgreiche Injektion des Farbstoffes in der Maus laterale Schwanzvene ist entscheidend für die gute Ergebnis des Experiments. Schwanzveneninjektion Technik erfordert umfangreiche Praxis und sollte vor dem Start des Experiments gemeistert werden. Gefäßpermeabilität ist stark abhängig von dem Alter und dem Gewicht des eineimal, so beim Vergleich verschiedener Mausstämmen ist es unerlässlich, dass die Mäuse oder andere Probanden nahezu identisch Geburtsdaten und Gewicht haben. Andere Faktoren, die die Durchlässigkeit der Endothelbarriere beeinflussen Umgebungsbedingungen wie Temperatur, Feuchtigkeit, was sehr wichtig, die Handhabung Stress der Maus. Aufgrund der Vielzahl von Faktoren, die das Ergebnis des Experiments beeinflussen kann es immer ratsam, dass das Experiment wiederholt wird mindestens dreimal und statistische Analysen durchgeführt. Dieser Assay kann oder verwendet werden, um Gefäßpermeabilität von Mäusen, die gentechnisch verändert wurden, sowie Mäuse mit unterschiedlichen genetischen Hintergründen vergleichen. Permeabilität kann in Gegenwart oder Abwesenheit eines Reizes bewertet werden, in Abhängigkeit von der Funktion des Gens, das moduliert ist. Dieser Assay kann auch verwendet werden, oder um die Wirkung von unterschiedlichen Verbindungen auf Gefäßpermeabilität testen.

<table border="1"><tbody><tr><td></td><td></td><td></td><td> REAGENT </td></tr><tr><td> EVANS BLUE </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> 0,2 M Phosphat 1,5 M NaCl pH 7,4 </td></tr><tr><td></td><td></td><td></td><td> EQUIPMENT </td></tr><tr><td> SPEKTRALPHOTOMETER </td><td> EPPENDORF </td><td> 952000006 </td><td></td></tr><tr><td> MOUSE Rückhaltevorrichtung </td><td> Harvard Apparatus </td><td> 340012 </td><td></td></tr><tr><td> SPRITZE </td><td> BD </td><td> 309659 </td><td></td></tr><tr><td> NADELN </td><td> BD </td><td> 305106 </td><td> Die Spurweite der Nadel hängt von der Größe des Tieres. </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

Dieses Verfahren basiert auf der intravenösen Injektion von Evans Blue in Mäusen als Versuchstiere Modell. Evans Blau ist ein Farbstoff, der Albumin bindet. Unter physiologischen Bedingungen das Endothel ist undurchlässig für Albumin, so Evans blue gebundene Albumin Überreste innerhalb von Blutgefäßen beschränkt. In pathologischen Bedingungen, die erhöhte Gefäßpermeabilität Endothelzellen fördern teilweise verlieren ihren engen Kontakt und das Endothel durchlässig wird, um kleine Proteine ​​wie Albumin. Dieser Zustand ermöglicht Extravasation von Evans Blue in den Geweben. Eine gesunde Endothel verhindert Extravasation des Farbstoffs in den benachbarten vaskularisierten Geweben. Organe mit einer erhöhten Permeabilität deutlich erhöht Blaufärbung Vergleich zu Organen mit intaktem Endothel zeigen. Das Niveau der Gefäßpermeabilität kann durch einfache Visualisierung oder durch quantitative Messung der Farbstoff pro Milligramm Gewebe Kontrolle gegenüber Versuchstier / Gewebe eingebaut beurteilt werden. Zwei powerful Aspekte dieses Assays sind seine Einfachheit und quantitativen Merkmalen. Evans Blau-Farbstoff kann aus Geweben, die durch Inkubieren eines bestimmten Menge an Gewebe in Formamid extrahiert werden. Evans Blue Absorptionsmaximum liegt bei 620 nm und Absorption Minimum bei 740 nm auf. Durch die Verwendung einer Standardkurve für Evans Blue, können Messungen der optischen Dichte in Milligramm Farbstoff pro Milligramm Gewebe eingefangen umgewandelt werden. Statistische Analyse sollte verwendet werden, um signifikante Unterschiede in Gefäßpermeabilität zu beurteilen.

Wir haben eine in vivo Durchlässigkeit Assay zum Schiff Leck in Mäusen 8-12 Wochen alt zu testen. Dieser Test ist nützlich beim Vergleich der relativen Gefäßpermeabilität zwischen Tieren unterschiedlicher genetischer Hintergrund oder in einem einzelnen Stamm von Mäusen unterworfen Behandlungen, die das Gefäßsystem beeinflussen. Unsere Ergebnisse zeigen, dass ein Stamm von genetisch veränderten Mäusen wir in unserem Labor erstellt eine durchlässige Endothel im Vergleich zu Wildtyp-Mäusen hat. Diese ...

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An in vivo Assay to Test Blood Vessel Permeability

Wir präsentieren ein<em&gt; In vivo</em&gt; Assay Blutgefäß Durchlässigkeit zu testen. Dieser Assay basiert auf intravenöse Injektion eines Farbstoffes und nachfolgender Visualisierung dessen Diffusion in Zwischenräume basiert.

Ein<em&gt; In vivo</em&gt; Assay Blood Vessel Durchlässigkeitsprüfung

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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An in vivo Assay to Test Blood Vessel Permeability

63.9K Aufrufe.  Fox Chase Cancer Center. Wir präsentieren ein In vivo Assay Blutgefäß Durchlässigkeit zu testen. Dieser Assay basiert auf intravenöse Injektion eines Farbstoffes und nachfolgender Visualisierung dessen Diffusion in Zwischenräume basiert.

Serie: An in vivo Assay to Test Blood Vessel Permeability

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapie-induzierte vaskuläre Toxizität - Echtzeit-<em&gt; In-vivo-</em&gt; Imaging von Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

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Medizin

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

Ex vivo-Darm-Sacs zu beurteilen Durchlässigkeit der Schleimhaut in Modelle von Magen-Darm-Krankheit

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

Depletion von Maus-Zellen aus menschlichem Tumorxenografte deutlich verbessert Downstream-Analyse von Zielzellen

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

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- Assays zur Beurteilung der Blut-Hirn-Schranke Mesh-artigen Gefäßbildung und -störung

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 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore largely based on animal studies, despite the high diversity among species in placental anatomy, hemodynamics and duration of the pregnancy. The vast majority of human placenta studies are ex vivo perfusion studies or in vitro trophoblast studies. Although in vitro studies and animal models are essential, extrapolation of the results from such studies to the human placenta in vivo is uncertain. We aimed to study human placenta physiology in vivo at term, and present a detailed protocol of the method. Exploiting the intraabdominal access to the uterine vein just before the uterine incision during planned cesarean section, we collect blood samples from the incoming and outgoing vessels on the maternal and fetal sides of the placenta. When combining concentration measurements from blood samples with volume blood flow measurements, we are able to quantify placental and fetal uptake and release of any compound. Furthermore, placental tissue samples from the same mother-fetus pairs can provide measurements of transporter density and activity and other aspects of placental functions in vivo. Through this integrative use of the 4-vessel sampling method we are able to test some of the current concepts of placental nutrient transfer and metabolism in vivo, both in normal and pathological pregnancies. Furthermore, this method enables the identification of substances secreted by the placenta to the maternal circulation, which could be an important contribution to the search for biomarkers of placenta dysfunction.

The 4-vessel Sampling Approach to Integrative Studies of Human Placental Physiology In Vivo

We present a detailed method to study human placental physiology in vivo at term. The method combines blood sampling from the incoming and outgoing vessels on the maternal and fetal sides of the placenta with ultrasound measurements of volume blood flow and placental tissue sampling.

Der 4-Gefäß-Stichprobenansatz für integrative Studien der menschlichen Plazenta-Physiologie<em&gt; In vivo</em

The human placenta is highly inaccessible for research while still in utero. The current understanding of human placental physiology in vivo is therefore ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Re Arterialized Ratte teilweise Leber Transplantation mit einem in Vivo Schiff-orientierte 70 % Hepatektomie

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...

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.

Eine In Vivo -Methode zur Bewertung der Darm-Blut-Schranke und LEBERSTOFFWECHSEL Mikrobiota Produkte

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

Using an Automated Hirschberg Test App to Evaluate Ocular Alignment

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing the difference in corneal reflection generated by the phone camera flash relative to the iris center based on high resolution images, the app can measure misalignment with a much higher precision than the naked eye performing the Hirschberg test. It has been validated in a previous clinical evaluation study by comparing to the clinical gold standard-prism and alternate cover test. The goal of this article is to describe the testing techniques regarding how to use the app to measure ocular alignment for different fixation distances, without or with cover to break fusion, as well as angle kappa, so that users can use the app to perform equivalent tests typically done in clinic using prisms.

We present a protocol of using a smartphone app to perform Hirschberg test for measuring manifest and intermittent ocular misalignment (strabismus) under near and far fixation conditions.

Verwenden einer automatisierten Hirschberg-Test-App zur Bewertung der Augenausrichtung

A smartphone app has been developed to perform the automated photographic Hirschberg test for objective measurement of ocular misalignment. By computing ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Messung des restrischen Blutflusses in Maus-Cochlea mittels offenem Gefäßfenster und intravitaler Fluoreszenzmikroskopie

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, the preservation of pulmonary vessels that truly represents physiological conditions still presents challenges. In addition, the delicate configuration of murine lungs result in technical challenges preparing samples for high-quality images that preserve both cellular composition and architecture. Similarly, cellular contractility assays can be performed to study the potential of cells to respond to vasoconstrictors in vitro, but these assays do not reproduce the complex environment of the intact lung. In contrast to these technical issues, the precision-cut lung slice (PCLS) method can be applied as an efficient alternative to visualize lung tissue in three dimensions without regional bias and serve as a live surrogate contractility model for up to 10 days. Tissue prepared using PCLS has preserved structure and spatial orientation, making it ideal to study disease processes ex vivo. The location of endogenous tdTomato-labeled cells in PCLS harvested from an inducible tdTomato reporter murine model can be successfully visualized by confocal microscopy. After exposure to vasoconstrictors, PCLS demonstrates the preservation of both vessel contractility and lung structure, which can be captured by a time-lapse module. In combination with the other procedures, such as western blot and RNA analysis, PCLS can contribute to the comprehensive understanding of signaling cascades that underlie a wide variety of disorders and lead to a better understanding of the pathophysiology in pulmonary vascular diseases.

Precision Cut Lung Slices as an Efficient Tool for Ex vivo Pulmonary Vessel Structure and Contractility Studies

Presented here is a protocol for preserving the vascular contractility of PCLS murine lung tissue, resulting in a sophisticated three-dimensional image of the pulmonary vasculature and airway, which can be preserved for up to 10 days that is susceptible to numerous procedures.

Präzisionsschnitt-Lungenschnitte als effizientes Werkzeug für Ex-vivo-Studien zur Struktur und Kontraktilität von Lungengefäßen

The visualization of murine lung tissue provides valuable structural and cellular information regarding the underlying airway and vasculature. However, ...