Diese Arbeit wurde von der JB Marshall Foundation unterstützt.

Ein. Bereiten Tier und Ausrüstung für Verfahren <ol><li> Vor Beginn der Operation, bereiten den OP-Bereich und das Tier. Machen Sie das Karotis-Katheter durch Einfügen eines 23-Gauge stumpfen Nadel in ein Ende der 12 &quot;PE50 Rohre. Schneiden Sie ein etwa 45 ° Schräge in die entgegengesetzte Ende des Katheters. Sterilisieren Geräte vor dem Eingriff. Tragen Sie ein Haar Motorhaube, OP-Maske und sterile Handschuhe. </li><li> Legen Sie ein Heizkissen auf der Oberfläche, wo die Operation durchgeführt werden soll. Aktivieren Sie es und lassen Sie es zu warm. Decken den Bereich mit einem saugfähigen Bank Pad. </li><li> Stellen Sie sicher, dass Sie 25% Mannitol in Lösung haben, ohne Kristalle. Wenn Kristalle vorhanden sind, lösen sie durch Platzieren des Gefäßes in einem heißen Wasserbad bei 80 ° C und Rühren periodisch. </li><li> Notieren Sie sich die Masse des Tieres. </li><li> Machen Sie eine Propofol Verdünnung Lieferung bei 0,8 mg / kg / min bei einer Geschwindigkeit von 0,1 ml / min. </li><li> Legen Sie die Ratte in der Induktion für 3 min. Einstellungen: 5% Isofluran, 2 L / minDurchfluss am Ventilator. </li><li> Auf der Spitze eines Einweg-Handtuch, rasieren den Hals und beide Innenseiten der Oberschenkel. </li></ol> 2. Intubieren den Rat <ol><li> Legen Sie das Tier wieder in der Induktion für 3 min. </li><li> Bereiten Sie Ihre Endotrachealtubus mit einem IV-Katheter. Für diese Größe und Belastung der Ratte, verwenden wir einen 18 ga Katheter. Das Rohr muss groß genug sein, um eine ausreichende Belüftung zu ermöglichen, aber klein genug, um in die Luftröhre zu passen, ohne dass Schäden. Ziehen Sie die Nadel aus der Kunststoff-Katheter und vorsichtig die Nadel abbrechen ca. 1 cm von der scharfen Spitze. Die bunted Nadel Stylus sein. Setzen Sie die Nadel wieder in den Katheter. </li><li> Hängen Sie die Ratte durch seine Zähne auf eine schräge Fläche. </li><li> Shine a light direkt gegen den Hals des Tieres und stellten sich zwischen den Ohren. </li><li> Setzen Sie einen kleinen Spatel ca. 3 cm in den Oropharynx. Heben Sie nach oben, während vorsichtig die Zunge aus dem Mund. </li><li> Visualisierung der Öffnung der Trachea. Sie werden seea kleines Loch öffnen und zu schließen mit jedem Atemzug. </li><li> Schieben Sie Katheter in das Loch. Sie können manchmal das Gefühl, der Kunststoff geben Sie die Knorpel der Luftröhre. Die Luftröhre ist empfindlich. Nicht mit Gewalt den Katheter. </li><li> Stellen Sie das Beatmungsgerät an 1 L / min und 60 bpm und Isofluran auf 2%. </li><li> Verbinden Sie den Katheter mit dem Beatmungsgerät und Band das Rohr auf die Arbeitsfläche. </li><li> Überprüfen Sie das Tier betäubt, indem eine Zehe Prise. </li></ol> 3. Stellen Propofol IV Anästhesie <ol><li> Sichern Sie die Ratte durch Abkleben aller vier Gliedmaßen und den Schwanz der Bedienoberfläche. </li><li> Scrub die rasierten Hals und Leiste Seiten mit Chlorhexidin Seife dreimal. Dann schrubben mit Chlorhexidin dreimal spülen. </li><li> Bringen Sie eine Spritze von Propofol zu einer IV Erweiterungs-Set. Montieren Sie die Spritze auf einer Infusionspumpe. </li><li> Mit einem Skalpell, durch die Haut des rasiert Oberschenkel geschnitten. Vermeiden Sie das Schneiden Muskel. </li><li> Öffnen Sie den Schnitt mit Gewaltps zur Visualisierung der femoralis. </li><li> Wegzuziehen Gewebe über der Arterie, um das Schiff aussetzen. </li><li> Legen Sie eine 26 ga MONOJECT Veterinär IV-Katheter in die Vene. Wenn die Nadel in die Wand der Vene punktiert werden Sie spüren es einfacher geworden zu schieben. Kanüle zurückziehen und drücken Sie den Katheter in das Gefäß. </li><li> Starten Sie die Propofol-Infusion bei 0,1 ml / min. </li><li> 3-5 min nach Beginn der Infusion Propofol, schalten Anästhesiegas zu einer Mischung von 50% O 2 und 50% N 2 O und Absperren der Isofluran. </li></ol> 4. Setzen Sie die Bifurkation der A. carotis communis <ol><li> Machen Sie eine Inzision am Hals mit Skalpell. </li><li> Schneiden Sie zwischen den beiden großen Speicheldrüsen mit einer Schere oder Skalpell. </li><li> Dissect weg Gewebe lateral der Mittellinie, um die Halsschlagader freizulegen. Pinzette einsetzen Gewebeschichten zu öffnen. Sie können mit einer Schere Stücke, die Sie setzen mit der Pinzette zu entfernen und zu bestimmen minimall seiny vaskuläre. Blutstillung mit Gaze, Wattestäbchen und Kauter, wenn nötig. </li><li> Inject 3 ml Kochsalzlösung IP, wenn es zu mittelschweren bis schweren Blutungen. </li><li> Verwenden Retraktoren um Gewebe ziehen weg von der Halsschlagader. Finden Sie die Bifurkation. </li><li> Setzen Sie die A. occipitalis knapp distal der Bifurkation. Cauterize es zwischen Zange und trennen Sie es mit einem Elektrokauter. </li><li> Verwenden Pinzette vollständig aussetzen Karotisbifurkation und so viel von der ECA wie möglich. </li><li> Setzen Sie das überlegene Arteria thyreoidea Abzweigung des ECA distal der A. occipitalis. Cauterize es zwischen Zange und trennen Sie es mit einem Elektrokauter. </li></ol> 5. Katheterisierung der A. carotis externa <ol><li> Zeigen 2 Längen von 4/0 Seide unter der ECA. </li><li> Schnappen 1 Länge des Fadens mit Ihrem hemostat und Einfahren der ECA zurück kaudal (in Richtung der Ratte Fuß). </li><li> Nehmen Sie das andere Naht und binden so weit kranial (in Richtung der Ratte in den Mund) wie möglich with eines Chirurgen Knoten. Dies bildet eine dauerhafte Unterbindung der ECA zu opfern. </li><li> Nehmen Sie die erste hemostat (derjenige, der die Anwendung caudal Spannung wurde) und greifen das andere Naht (derjenige, der geknoteten um die ECA ist) und gelten sanfte Spannung kranial mit der Gefäßklemme. </li><li> Tie anderen Chirurgen Knoten locker (nicht zu sichern - lassen kaum gebunden) distal der Bifurkation und proximal der permanenten Ligatur. </li><li> Spülen mit 1%, um die Arterie entspannen Lidocain. </li><li> Füllen Sie Ihren Katheter mit Heparin Kochsalzlösung. </li><li> Platzieren eines temporären Behälters Clip so nahe wie möglich an der Bifurkation. Ort Gefäß Clip in einem Winkel, um Platz für Katheterisierung maximieren. Schieben Sie den lockeren Knoten in Richtung des Clips. </li><li> Machen Sie eine kleine Arteriotomie (cut) in ECA genau proximal der Ligatur mit Mikroschere. Es sollte keine Blutung, wenn der Clip und Ligatur sicher sind. </li><li> Mit einer Pinzette die ECA Wänden steuern, führen Sie das abgeschrägte Ende des Katheters in das Gefäß. </li&gt; <li> Sichern Sie den Katheter mit dem losen Faden Knoten. Binden Sie die Arterie um den Katheter. </li><li> Entfernen Sie den Clip und reinigen Sie den Bereich mit einem Wattestäbchen. </li><li> Vorzurücken den Katheter bis etwa 1 mm distal zur Bifurkation. Halten Sie die Arterie mit einer Pinzette nahe dem Knoten, um sicherzustellen, das Schiff immobilisiert ist. </li></ol> 6. Verwalten Mannitol <ol><li> Füllen Sie eine 8 &quot;IV Erweiterungs-Set und ein 6-ml-Spritze mit Mannitol ohne irgendwelche Luftblasen lagern diese im Brutschrank bei 37 ° C. Hinweis: Arbeit schnell mit Mannitol - es wird leicht aus der Lösung ausfällt bei Raumtemperatur.. </li><li> Zur Visualisierung BBBD können Sie verwalten Evans blauen Farbstoff und beobachten Sie es geben die Bereiche des Gehirns, wo die Barriere wurde unterbrochen. Dazu geben dem Tier 2 ml / kg 2% Evans Blue durch einen neuen Infusionsstelle am Bein kontralateral zur Propofol Infusion. </li><li> Bringen Sie die Erweiterung auf einen 5 um Filter und füllen es mit Mannit zu Luftblasen zu entfernenBles. Dann befestigen Sie diese an den Absperrhahn ohne Blasen zu erzeugen. </li><li> Verabreichen Mannit bei 0,09 ml / sec für 25 sek. Achten Sie auf die Mannitol Gleichgewicht mit der Strömung in carotis erreichen an einem Punkt unmittelbar proximal der ICA. Es sollte scheinen mit der Ratte Herzfrequenz pulsieren. Wenn die Mannitol fließt einfach nach unten in Richtung des Herzens haben Sie die Ratte den Blutdruck zu überwinden. Dies kann zu lebensbedrohlichen Komplikationen und schlechte BBBD. </li></ol>

<ol>
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A., 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=Primary+CNS+lymphoma+treated+with+osmotic+blood-brain+barrier+disruption:+prolonged+survival+and+preservation+of+cognitive+function.">Primary CNS lymphoma treated with osmotic blood-brain barrier disruption: prolonged survival and preservation of cognitive function.</a> Journal of clinical oncology. 9, 1580 (1991).</li><li>Remsen, L. G., Trail, P. A., Hellstr&#246;m, I., Hellstr&#246;m, K. E., Neuwelt, E. 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Es gibt einige Mittel zur Maximierung der Wirksamkeit BBBD. Es ist wichtig, um eine Blutung während der Cut-Down-Phase zu minimieren. Blutdruck und Herzfrequenz kann durch erhebliche Blutungen berührt und diese Faktoren sind bekannt, den Grad der BBBD 13 beeinflussen. Ausbluten kann mithilfe Nähten großen Gefäße, wie den überlegenen Schilddrüse und okzipitalen Arterien, die aufgeteilt werden müssen ligieren reduziert werden. Darüber hinaus können Elektrokauter verwendet, um Schiffe zu teilen und zu...

<table border="1"><tbody><tr><td> Material Name </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentar </td></tr><tr><td> Long Evans Ratten </td><td> Harlan Laboratories </td><td></td><td> 210-250 g, männlich </td></tr><tr><td> PE 50 Rohre </td><td> Beckton-Dickinson </td><td></td><td></td></tr><tr><td> 18 Gauge x 2,5 &quot;IV-Katheter </td><td> Terumo </td><td></td><td> Für ET-Tubus </td></tr><tr><td> 30 &quot;IV-Erweiterung setzt </td><td> Abbott </td><td></td><td></td></tr><tr><td> 26 Gauge Veterinär IV-Katheter </td><td> Monoject </td><td></td><td></td></tr><tr><td> Evans blauen Farbstoff </td><td> Sigma </td><td> E2129 </td><td></td></tr><tr><td> Zweipolig </td><td> Codman </td><td></td><td></td></tr><tr><td> Filter, 5 mm </td><td> Braun </td><td></td><td></td></tr></tbody></table>

rat model of blood-brain barrier disruption to allow targeted neurovascular therapeutics

Endothelzellen mit tight junctions zusammen mit der Basalmembran und Astrozyten Ende Füßen Surround zerebralen Blutgefäße, die Blut-Hirn-Schranke 1 zu bilden. Die Barriere selektiv schließt Moleküle vor der Kreuzung zwischen dem Blut und dem Gehirn von ihrer Größe und Ladung basiert. Diese Funktion kann behindern die Abgabe von Therapeutika für neurologische Erkrankungen. Eine Reihe von chemotherapeutischen Arzneimitteln, zum Beispiel, wird nicht effektiv die Blut-Hirn-Schranke zu erreichen Tumorzellen 2. So, die Verbesserung der Abgabe von Medikamenten durch die Blut-Hirn-Schranke ist ein Gebiet von Interesse. Die häufigsten Methoden zur Verbesserung der Abgabe von Medikamenten in das Gehirn sind direkte zerebrale Infusions-und Blut-Hirn-Schranke Störung 3. Direkte intrazerebrale Infusion garantiert, dass Therapien das Gehirn erreichen, jedoch hat diese Methode eine begrenzte Fähigkeit zu zerstreuen die Droge 4. Blut-Hirn-Schranke Störungen (BBBD) alTiefs Medikamente fließen direkt aus dem Kreislaufsystem in das Gehirn und damit effektiver zu erreichen dispergiert Tumorzellen. Drei Methoden der Barrierestörung gehören osmotische Barriere Störung, pharmakologische Barrierestörung und fokussierten Ultraschall mit Mikrobläschen. Osmotische Störungen, Pionierarbeit von Neuwelt, verwendet eine hypertone Lösung von 25% Mannitol, die die Zellen der Blut-Hirn-Schranke wodurch sie zu schrumpfen und stören ihren Tight Junctions entwässert. Barrier Störungen können auch pharmakologisch mit vasoaktiven Verbindungen wie Histamin 5 und Bradykinin 6 erreicht werden. Dieses Verfahren ist jedoch in erster Linie für das Gehirn-Schranke 7 Tumor selektiv. Zusätzlich wurde RMP-7, ein Analogon des Peptids Bradykinin, sich als minderwertig im Vergleich Kopf-an-Kopf mit osmotischen BBBD mit 25% Mannitol 8. Ein anderes Verfahren, fokussierter Ultraschall (FUS) in Verbindung mit Mikroblasen Ultraschall-Kontrastmitteln, wurde auch gezeigt,reversibel öffnen die Blut-Hirn-Schranke 9. Im Vergleich zu FUS hat allerdings 25% Mannitol eine längere Geschichte der Sicherheit bei menschlichen Patienten, die es ein bewährtes Instrument für die translationale Forschung 10-12 macht. Zur Erfüllung BBBD muss Mannit mit einer hohen Rate direkt in das Gehirn die arterielle Zirkulation verschickt werden. Beim Menschen wird eine endovaskuläre Katheter zum Gehirn, wo schnelle und direkte Strömung erreicht werden führbar. Dieses Protokoll Modelle menschlichen BBBD so eng wie möglich. Nach einem cut-down bis zur Bifurkation der A. carotis communis wird ein Katheter eingeführt in die ECA retrograde und verwendet, um Mannit liefern direkt in die Arteria carotis interna (ICA) Umlauf. Propofol und N 2 O Anästhesie sind für ihre Fähigkeit, die Wirksamkeit der Schrankenstörung 13 zu maximieren. Wenn richtig ausgeführt, hat dieses Verfahren die Möglichkeit, sicher, effektiv und reversibel zu öffnen, die Blut-Hirn-Schranke und Improe die Lieferung von Medikamenten, die gewöhnlich nicht das Gehirn erreichen 8,13,14.

<img alt="Abbildung 1" src="/files/ftp_upload/50019/50019fig1.jpg" /> Abbildung 1. Visualisierung Blut-Hirn-Schranke Störungen via Evans blauen Farbstoff Extravasation. Evans blauen Farbstoff ist ein Pigment, das an Albumin bindet und nicht in das Gehirn unter physiologischen Bedingungen extravasiert. Störung der Blut-Hirn-Schranke auf der einen Seite des Gehirns ermöglicht Evans Blau zu geben und Flecken auf der gestörten Hemisphäre blau, während die nic...

Watch this Scientific Journal Video about Rat Model of Blood-brain Barrier Disruption to Allow Targeted Neurovascular Therapeutics at JoVE.com

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

Blut-Hirn-Schranke Störungen hilft die Lieferung von bestimmten Medikamenten an das Gehirn. Mannitol geliefert intraarteriell schrumpft umgebenden Zellen die Blutgefäße, um physisch stören die Barriere.

Rat Modell der Blut-Hirn-Schranke Störung zu Neurovascular Therapeutics Gezielte zulassen

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain ...

rat-model-blood-brain-barrier-disruption-to-allow-targeted

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19.3K Aufrufe.  Vanderbilt University School of Medicine. Blut-Hirn-Schranke Störungen hilft die Lieferung von bestimmten Medikamenten an das Gehirn. Mannitol geliefert intraarteriell schrumpft umgebenden Zellen die Blutgefäße, um physisch stören die Barriere.

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

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

MRI-geführten Störung der Blut-Hirn-Schranke mit Hilfe der transkraniellen fokussiertem Ultraschall in einem Rattenmodell

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

Forschung

Medizin

Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.
Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.

Stretch in Brain Microvascular Endothelial Cells cEND as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier

In vitro traumatic brain injury models are being developed to reproduce in vivo brain deformation. Stretch-induced injury has been employed for astrocytes, neurons, glial cells, aortic, and brain endothelial cells. However, our system uses a blood brain barrier (BBB) model that possesses properties constituting a legitimate model of the BBB to establish an in vitro&#160;TBI model.

Stretchlimousine in Gehirn mikrovaskulären Endothelzellen (CEND) als<em&gt; In Vitro</em&gt; Traumatische Hirnverletzung Modell der Blut-Hirn-Schranke

Due to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying ...

Setting-up an In Vitro Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm&#183;cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 &#177;&#160;0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an In Vitro Model of Rat Blood-brain Barrier BBB: A Focus on BBB Impermeability and Receptor-mediated Transport

The aim of the present study was to validate the reproducibility of an in vitro BBB model involving a rat syngeneic co-culture of endothelial cells and astrocytes. The endothelial cell monolayer presented high TEER and low LY permeability. Expression of specific TJ proteins, functional responses to inflammation and functionality of transporters and receptors were assessed.

Einrichten einer<em&gt; In-vitro-</em&gt; Modell der Ratte Blut-Hirn-Schranke (BBB): Ein Fokus auf BBB-Undurchlässigkeit und Rezeptor-vermittelten Transport

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

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic cerebrovascular accidents account for 80% of all strokes. A common cause of IR injury is the rapid inflow of fluids following an acute/chronic occlusion of blood, nutrients, oxygen to the tissue triggering the formation of free radicals.
Ischemic stroke is followed by blood-brain barrier (BBB) dysfunction and vasogenic brain edema. Structurally, tight junctions (TJs) between the endothelial cells play an important role in maintaining the integrity of the blood-brain barrier (BBB). IR injury is an early secondary injury leading to a non-specific, inflammatory response. Oxidative and metabolic stress following inflammation triggers secondary brain damage including BBB permeability and disruption of tight junction (TJ) integrity.
Our protocol presents an in vitro example of oxygen-glucose deprivation and reoxygenation (OGD-R) on rat brain endothelial cell TJ integrity and stress fiber formation. Currently, several experimental in vivo models are used to study the effects of IR injury; however they have several limitations, such as the technical challenges in performing surgeries, gene dependent molecular influences and difficulty in studying mechanistic relationships. However, in vitro models may aid in overcoming many of those limitations. The presented protocol can be used to study the various molecular mechanisms and mechanistic relationships to provide potential therapeutic strategies. However, the results of in vitro studies may differ from standard in vivo studies and should be interpreted with caution.

Oxygen-Glucose Deprivation and Reoxygenation as an In Vitro Ischemia-Reperfusion Injury Model for Studying Blood-Brain Barrier Dysfunction

Ischemia-Reperfusion (IR) injury is associated with a high rate of morbidity and mortality. The goal of the in vitro model of oxygen-glucose deprivation and reoxygenation (OGD-R) described here is to assess the effects of ischemia reperfusion injury on a variety of cells, particularly in blood-brain barrier (BBB) endothelial cells.

Sauerstoff-Glukose Deprivation und Reoxygenierung als<em&gt; In-vitro-</em&gt; Ischämie-Reperfusion Injury Modell zum Studium der Blut-Hirnschranke Dysfunction

Ischemia-Reperfusion (IR) injury is known to contribute significantly to the morbidity and mortality associated with ischemic strokes. Ischemic ...

A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is also a valuable model system to test, and to evaluate therapeutics and drugs that negate maladaptive remodeling in the vessel. The injury, or barotrauma, in the vessel lumen caused by an inflated balloon via an inserted catheter induces subsequent neointimal growth, often leading to hyperplasia or thickening of the vessel wall that narrows, or obstructs the lumen. The method described here is sufficiently sensitive, and the results can be obtained in relatively short time (2 weeks after the surgery). The efficacy of the drug or therapeutic against the induced-remodeling can be evaluated either by the post-mortem pathological and histomorphological analysis, or by ultrasound sonography in live animals. In addition, this model system has also been used to determine the therapeutic window or the time course of the administered drug. These studies can leadto the development of a better administrative strategy and a better therapeutic outcome. The procedure described here provides a tool for translational studies that bring drug and therapeutic candidates from bench research to clinical applications.

The rat carotid balloon injury model described below allows researchers to evaluate drugs or therapeutics that negate injury-induced arterial hyperplasia. Detailed pre-surgical preparation, surgical procedure, and post-surgical cares of the animal are described.

Eine Ratte Karotis-Ballon-Verletzung Modell Anti-vaskuläre Remodeling Therapeutics zu Test

The rat carotid balloon injury is a well-established surgical model that has been used to study arterial remodeling and vascular cell proliferation. It is ...

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Retroductal Submandibulardrüse Einträufeln und Lokalisierte fraktionierten Bestrahlung in einem Rattenmodell von Speichel- Unterfunktion

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

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 ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Verbesserte Methode für die Einrichtung einer In-vitro- Blut - Hirn-Schranke Modell auf porcinen Endothelzellen Gehirnzellen basiert

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Delivery of Cardioactive Therapeutics in a Porcine Myocardial Infarction Model

Myocardial infarction is one of the leading causes of death and disability worldwide, and there is an urgent need for novel cardioprotective or regenerative strategies. An essential component of drug development is determining how a novel therapeutic is to be administered. Physiologically relevant large animal models are of critical importance in assessing the feasibility and efficacy of various therapeutic delivery strategies. Due to their similarities to humans in cardiovascular physiology, coronary vascular anatomy, and heart weight to body weight ratio, swine is one of the preferred species in the preclinical evaluation of new therapies for myocardial infarction. The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. After percutaneously induced myocardial infarction, female landrace swine received treatment with novel agents through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump. The procedures employed for each technique are reproducible, resulting in reliable cardioactive drug delivery. These models can be easily adapted to suit individual study designs, and each of these delivery techniques can be used to investigate a variety of possible interventions. Therefore, these methods are a useful tool for translational scientists pursuing novel biological approaches in cardiac repair following myocardial infarction.

The present protocol describes three methods of administering cardioactive therapeutic agents in a porcine model. Female landrace swine received treatment through either: (1) thoracotomy and transepicardial injection, (2) catheter-based transendocardial injection, or (3) intravenous infusion via jugular vein osmotic minipump.