The work was supported by the intramural research program of the NIH, National Institute on Aging, and by a Priority Research Centers Program grant from the National Research Foundation (NRF-2009-0093812) funded by the Ministry of Science, Information and Communication Technology, &amp; Future Planning, the Republic of Korea (H.T.). We thank Dr. Han-sol Park for putting "material" part together for the manuscript.

HINWEIS: Die Verwendung von Ratten-Ballonverletzung Modell und die damit verbundenen Verfahren einschließlich der Injektion von rekombinanten sRAGE und Ultraschall sonographische Untersuchungen wurden von der Animal Care und Use Committee (ACUC) des National Institute on Aging, NIH genehmigt. 1. Pre-Chirurgie Vorbereitungen <ol><li> Instrument, OP-Plattform, und persönliche Schutzausrüstung. <ol><li> Siehe Materialien und Geräte der Tabelle für alle chirurgischen Instrumente und Reagenzien , die in diesem Verfahren verwendet. Autoklav alle chirurgischen Instrumente vor dem chirurgischen Eingriff. HINWEIS: Für mehrere Operationen am selben Tag durchgeführt, mit einem vorgeheizten Glasperle Sterilisator die Instrumente zwischen Operationen zu sterilisieren. </li><li> Sterilisieren Sie die Oberfläche von Betriebssystem-Plattform mit 70% Alkohol. Setzen Sie auf OP-Kittel, Haarabdeckung, chirurgische Maske, Schutzbrille und Handschuhe. Lassen Sie eine Schachtel mit sterilisierten Handschuhe in der Nähe der chirurgischen Plattform für zusätzliche unse. </li></ol></li><li> Vorbereitung des Ballonkatheters. <ol><li> Füllen Sie 1 ml Spritze mit sterilem Wasser und befestigen Sie die Spritze in ein steriles Wasser gefüllten Zweiwegehahn. Drücken Sie vorsichtig den Hahn das Wasser durch den Luer-Lock-Teil des Ballonkatheters zu füllen, und Luftblasen aus der Öffnung des Katheters zu entfernen. </li><li> Test-Ballon Inflation des Ballons, um sicherzustellen, können von 0,02 ml Wasser aufgepumpt werden. Speichern die Spitze des Ballons in einen Behälter mit sterilem Wasser vor der Operation Desikkation und Verunreinigung zu vermeiden. </li></ol></li><li> Anästhesie und Vorbereitung der Tiere. <ol><li> Wiegen männliche Wistar-Ratten (400-450 g) und überprüfen Sie die Ohrmarke die ID zu bestätigen. Legen Sie die Ratte in einer Induktionskammer, dann schalten Sie den Verdampfer und stellen Sie die Isofluran-Ebene auf 3,5-4,0% und der Sauerstoffgehalt auf 1,5-2,0 L. Überprüfen Sie die Tiefe der Sedierung durch Pedalrückzugreflex Überprüfung wie Kneifen der Zehe oder Schwanz des Tieres. </li><li> Nach der Anästhesie zu erreichen ( <em&gt; dh das Tier Pedal Rückzugsreflex) fehlt, das Tier auf die chirurgische Plattform übertragen und die Ratte mit einem Nasenkegel Haken, der mit einem Anästhesie Verdampfer verbindet. Stellen Sie Isofluran-Konzentration auf 1,5 bis 2,0 und Sauerstoff zu 0,6-1,0 L. in regelmäßigen Abständen die Tiefe der Sedierung überprüfen, indem Sie die Zehe oder Schwanz des Tieres kneifen, um sicherzustellen, dass die Ratte völlig entspannt und unbeweglich ist. </li><li> die chirurgische Ebene der Anästhesie, rasieren die ventrale Halsbereich vom Kinn bis zu knapp über dem Brustbein mit Tierhaarschneidemaschinen und Tupfer die rasierte Haut mit wechselnden 1:10 Verdünnung von PVP-Jod und 70% Ethanol 3 mal Nach Erreichen. </li><li> Platzieren Sie ophthalmische Lösung auf den offenen Augen des Tieres mit einem Wattestäbchen zu verhindern, dass sie vor dem Austrocknen. Befeuchten Sie die Zunge und dem Mund des Tieres mit Trinkwasser Trocknen während des Betriebs zu vermeiden. </li><li> Decken Sie die Ratte mit einem sterilen OP-Blatt und schneiden Sie das Blatt, um den Halsbereich aus. Legen Sie das Tier in Rückenlage ona Heizkissen auf der chirurgischen Plattform mit dem Kopf in Richtung des Chirurgen. Befestigen Sie die Gliedmaßen des Tieres auf die chirurgische Plattform mit Klebeband. </li></ol></li></ol> 2. Chirurgische Verfahren <ol><li> Dissection und Isolierung der linken Arteria carotis <ol><li> Verwenden Sie ein Skalpell einen geraden Schnitt unter dem Kinn zu machen in einer Richtung auf den Schwanz den ganzen Weg an die Spitze des Brustbeins direkt über dem Brustkorb. Verwenden Sie eine 7S Zange unverblümt die Speicheldrüsengewebe unter der Haut sezieren die Muskelschicht zu belichten. Separate Muskeln Zugang zu Carotis Gefäßen und den Vagusnerv im Halsbereich zu gewinnen. </li><li> Unverblümt Gewebe sezieren die Arteria carotis umgibt, und trennen sorgfältig die Vagusnerv und Gefäß Faszie, um Schäden zu vermeiden. Kontinuierlich bis zur Bifurkation Arteria carotis sezieren die inneren und äußeren Zweige und die Arteria carotis communis bis zum Brustbein zu belichten. </li></ol></li><li> Herstellung von Arteriotomy <ol><li> Zum Einfahren und Hämostase, legen Sie eine Ligatur am meisten proximalen Stelle auf der linken Arteria carotis communis, und eine andere Ligatur an der Stelle sofort distal der Bifurkation. </li><li> Legen Sie eine 4.0 Seidennaht an der Arteria carotis externa so weit von der Bifurkation wie möglich die Arterie in Richtung auf den Kopf zurückzuziehen. Dauerhaft abzubinden kleinen Arterien 4,0 Seidensuturen entlang der A. carotis externa einschließlich der aufsteigende pharyngeal verwenden, Hinterhaupts und der überlegenen Schilddrüse arteriellen Leck zu vermeiden. </li><li> Legen Sie eine 4.0-Seidennaht um die Arteria carotis interna einen signifikanten Rückblutverlust zu vermeiden. Üblicherweise wird die zur Verfügung stehende Länge des internen Zweig ist minimal. Um die Arteria carotis interna sichtbar zu machen, setzen die Arteria carotis interna so viel wie möglich, indem Sie vorsichtig die darüber liegende Arteria carotis nach rechts zurückgezogen wird. </li></ol></li><li> Einführung des Ballonkatheters <ol><li> Ziehen Sie die proximale Naht auf der A. carotis communis einrtery und legen eine arterielle Klammer auf dem Gefäß vorübergehend den Blutfluss zu stoppen. Bilden einen kleinen (1/3 bis 1/4 des Umfangs der Arterie) Schnitt an der Arteria carotis externa an einer Stelle als distal der Naht wie möglich. </li><li> einen nicht aufgeblasenen Ballonkatheter in den Einschnitt und dann voraus, um den Katheter in das Lumen nahe der arteriellen Klemme an der Arteria carotis communis vorsichtig einsetzen. Entfernen Sie die Klemme, und weiter voran den Ballonkatheter auf den Aortenbogen, etwa 35-40 aus dem Einschnitt mm. </li></ol></li><li> Ballon-Verletzung <ol><li> aufzublasen manuell den Katheter zu einem Volumen von 0,02 ml über die Spritze befestigt und dann sperren den Hahn zwischen der Spritze und dem Katheter. zurückziehen langsam den Katheter mit Drehungen. Wenn der Katheter in die arteriotomic Einschnitt nahe ist, abzulassen, den Ballon. Schieben Sie den Katheter zurück zum ursprünglichen Speicherort. </li><li> Wiederholen Sie 2.4.1 für insgesamt 3 mal. Entfernen Sie vorsichtig den Katheter von der Arterie Lumen. </li></ol></li><li> Close Up der Operationswunde <ol><li> Schließen Sie die Arteriotomiestelle mit Nähten. Lassen Sie die A. carotis interna, um den Blutfluss wiederherzustellen. Überprüfen Sie für die arterielle Blutungen oder Leck. Wenn die Blutung auftritt, mit ein wenig Druck ein Stück Gaze anwenden, die Blutung zu stoppen. </li><li> Entfernen Sie den Rest von Nähten und Klammern für das Verfahren verwendet. Schließen Sie das Drüsengewebe mit 6,0 Nähten. Schließen Sie die Haut mit Hautnähte oder Wundklammern. Schalten Sie Isofluran und lassen Sie das Tier mit reinem Sauerstoff für ein paar Minuten. </li></ol></li></ol> 3. Die Verabreichung von Therapeutika, Analgetika, Post-Chirurgie Pflege und Euthanasie <ol><li> Die Verabreichung von Therapeutika und Analgetika. <ol><li> Nach der Operation injiziert intramuskulär Buprenorphin (0,03 mg / kg). Spritzen 5 ml vorgewärmt steriler Kochsalzlösung subkutan den Blutverlust während der Operation zu kompensieren. Injizieren sRAGE (0,5 ng / g) (Therapie) in das Peritoneum. </li><li> Befeuchten Sie die Augen wieder mit ophthalmic Salbe. Legen Sie die Ratte auf dem Heizkissen und überwachen das Tier, bis es vollständig das Bewusstsein wiedererlangt Brustlage zu halten. </li><li> Bringen Sie die Ratte auf einem Käfig mit sauberen Betten und bringen Sie den Käfig zurück in das Vivarium. Legen Sie eine spezielle Beobachtungskarte auf dem Käfig die anwesenden Tierärzte und Pfleger zu alarmieren. </li></ol></li><li> Post-Chirurgie Cleanup <ol><li> Setzen Sie gebrauchte chirurgisches Material einschließlich Pads, Tupfer, Nadeln et al entweder in den Müll oder einem speziellen Abfallbehälter einbringen. Desinfizieren Sie die Operation Plattform und reinigen Sie die chirurgischen Instrumenten. </li></ol></li><li> Post-Chirurgie Pflege <ol><li> Verabreichen Buprenorphin (0,03 mg / kg) zweimal täglich (BID) während der 48 Stunden nach der Operation Zeitraum. Überwachen Sie das Tier genau während der 48 Stunden nach der Operation Zeitraum für die gemeinsamen Anzeichen von Leiden wie Immobilität, die Unfähigkeit zu ernähren, hutched Haltung, gekräuselte Fell und Grimasse. Überwachen Wund Bedingungen für Rötung, Schwellung, Aufspringen, und Infektionen. HINWEIS:Zusätzliche Behandlungen können nach behandelnde Tierarzt Empfehlung vorgeschrieben werden. </li><li> Entfernen Sie die Naht oder Wundklammern nach behandelnde Tierarzt Empfehlung. </li></ol></li><li> Euthanasie <ol><li> Zwei Wochen nach der Operation, einschläfern die Ratten die Arteria carotis für post-mortem histo-morphologischen zu isolieren , analysiert 22. Legen Sie die Ratte in einer Kammer mit 5-20% Isofluran Luft gesättigt für mindestens 2 min bis zur vollständigen Einstellung der Atmung. Überprüfen Sie den Tod vor der Präparation der Organe und Gewebe. </li></ol></li></ol>

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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Es wurden zwei Methoden verwendet worden, um den Ballon, um aufzublasen die Schädigung zu erzeugen, die die Wand Endothel in der Arteria carotis Lumen entfernt. Eine davon ist die beigefügten Spritze mit Flüssigkeit 20 zu füllen, und der andere Luftdruck 21 zu verwenden. Wir bevorzugen es mit Flüssigkeit gefüllte Spritze zu verwenden, da die genaue Flüssigkeitsvolumen (0,02 ml) wird für jedes Verfahren verwendet werden. Dies macht eine genaue und reproduzierbare Aufblasen des Ballons, auf e...

Angioplastie ist ein endovaskulären Verfahren verwendet, um die verengt oder verstopft Arterien wie Atherosklerose ergeb von pathologischen Zuständen zu erweitern. Eine häufige Komplikation der Angioplastie ist die postoperative Neointimalhyperplasie oder Restenose, die auf chirurgische Verletzungen und die anschließende Entzündung induzierte vaskuläre Remodeling tritt wegen. Diese Bedingungen führen zu einer Proliferation der glatten Gefäßzellen und mehrere pathophysiologische Konsequenzen 1-3. Neointimalhyperplasie Wieder dickt das Schiff und tritt in bis zu 60% Post-Angioplastie-Patienten innerhalb des ersten Jahres. Daher ist die Restenose ein schwerer Rückschlag der weit verbreiteten Angioplastie - Verfahren 4. Obwohl die Implantation des medikamenten Elution Stent - Restenose zu verhindern , kann helfen, können nur ausgewählte Bewerber dieses kostspieliges Verfahren 5 unterziehen. Sowohl Tier- und klinische Studien haben gezeigt, dass die chronische Entzündung, die durch Vascular Verletzungen und / oder Operationswunde dient als Hauptreiz für die Post-Angioplastie neointimale Wachstum 2,4. Die Ratten - Karotis - Verletzungsmodell Ballon ahmt die klinische Situation und dient daher als ein wertvolles Modell System zellulären Faktoren zu identifizieren , die 6-9 in dem vaskulären Remodelling und Gefäßzellproliferation beteiligt. Dieses Modellsystem ist auch ein sehr nützliches Instrument zur Bewertung und / oder Bildschirm für Arzneimittel und therapeutische Reagenzien , die neointimale Wachstum in der präklinischen translationale Studien 10-14 unterdrücken. Im Vergleich zu dem murinen carotis Drahtverletzungsmodell 15 und der murine Femoralarterie Drahtverletzungsmodell 16, die Ratte carotis Ballon - Verletzungsmodell hat die Vorteile der in der Größe ausreichend groß ist für die Leichtigkeit des chirurgischen Eingriffs , die Reproduzierbarkeit der zugefügten Verletzungen erleichtert. Es kann eine größere Anzahl von primären Zellen bereitzustellen (zB vaskulären Glattmuskelzellen, Endothelzellen) zur additional in - vitro - Studien , den molekularen Mechanismus regeln vaskuläre Remodeling zu beschreiben. Wichtig ist , dass im Vergleich zu Mäusen, Ratten sind auch 17 ein besseres Modell zu sein für physiologische und toxikologische Studien bekannt. Obwohl ein Nachteil oder Begrenzung des Rattenmodell der Mangel an genetisch veränderte und Gen-Knockout-Modelle ist, kann dieser Nachteil durch die Verfügbarkeit der Ratte genomischen Sequenz und die jüngste Entwicklung von leistungsfähigen genomischen Editing-Tools wie CRISPR-CAS-Technologie überwunden werden, die macht mögliche Manipulation von weiten Bereichen von genomischen Sequenzen in verschiedenen Modellsystemen 18,19. Obwohl die Ratte Ballonverletzung Modell durch mehrere Labore und verschiedene umfassende Protokolle verwendet wurde , 20,21 veröffentlicht wurden, zielt darauf ab , dieses Protokoll an mehr Details zu Vorchirurgischer Vorbereitungen bietet und neue Forscher zu diesem Verfahren führen können diese chirurgischen Praxis einzurichten. Wir betonen auch die postoperative Pflege von ter Tiere , die nicht nur postmortalen pathologischen und histomorphologische Analysen der therapeutischen Wirkungen auf die arterielle Remodeling ermöglicht, aber auch Ultraschall sonographische Untersuchungen in lebenden Tieren 13,22.

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			<td height="21" style="height:21px;width:287px;">2 F Fogarty balloon embolectomy catheter&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Edwards Lifesciences&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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			<td height="21" style="height:21px;width:287px;">Standard scalpel</td>
			<td style="width:163px;">Fine Science Tools</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:287px;">Small&#160; curved forceps (Large radius Dumont#7shanks curved)&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Large, medium and small micro-scissors</td>
			<td style="width:163px;">Roboz</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Needles (20 G)&#160;&#160;</td>
			<td style="width:163px;">TycoHealthcare</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Micro-surgery forceps with micro-blunted atraumatic tips</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Atraumatic straight small arterial clamps&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Retractor&#160; with maximum spread 5.5 cm long blunt teeth</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Silk suture (4.0 and&#160; 6.0 )</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Syringe (1.0 ml)&#160;</td>
			<td style="width:163px;">BD&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Curity gauze sponges</td>
			<td style="width:163px;">AllegroMedical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cotton tip applicators sterile and non-sterile</td>
			<td style="width:163px;">Puritan Medical Products</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Compact hot bead sterilizer</td>
			<td style="width:163px;">Fine Science Tools</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Self-regulating heating pad</td>
			<td style="width:163px;">Fine Science Tools&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">ADS200 anesthesia system/ventilator</td>
			<td style="width:163px;">Paragon Medical</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Isoflurane (forane), liquid form</td>
			<td style="width:163px;">Baxter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Sodium chloride 0.9% (Saline)&#160;</td>
			<td style="width:163px;">Hospira</td>
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		<tr height="41">
			<td height="41" style="height:41px;width:287px;">Buprenex (buprenorphine)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:163px;">Reckitt Benckiser Healthcare (UK) Ltd.&#160;</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:287px;">70% alcohol</td>
			<td style="width:163px;">Fisher</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">1: 10 Betadine</td>
			<td style="width:163px;">Fisher</td>
		</tr>
	</tbody>
</table>

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.

Zwei Wochen nach der Ballonverletzung, wird die Ratte getötet und die Carotis-Arterien für histo-morphologische Analyse isoliert. Beide betrieben linke Arteria carotis und un-betriebenen rechten Arterie sind im Querschnitt, verarbeitet und in Paraffin eingebettet. Die Paraffinproben werden dann weiter Dünn geschnitten und gefärbt mit Hämatoxylin-Eosin (H &amp; E). Histo-morphologische Analysen werden unter Verwendung eines digitalen Bildanalyse-System durchgeführt. Die Einzelheiten...

Watch this Scientific Journal Video about A Rat Carotid Balloon Injury Model to Test Anti-vascular Remodeling Therapeutics at JoVE.com

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

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 ...

a-rat-carotid-balloon-injury-model-to-test-anti-vascular-remodeling

Research

JoVE Journal

Medicine

8.6K Aufrufe.  National Institute on Aging. 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. 

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

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasale Verabreichung von CNS Therapeutics Awake Mäuse

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Forschung

Medizin

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

Endothelial cells with tight junctions along with the basement membrane and astrocyte end feet surround cerebral blood vessels to form the blood-brain barrier1. The barrier selectively excludes molecules from crossing between the blood and the brain based upon their size and charge. This function can impede the delivery of therapeutics for neurological disorders. A number of chemotherapeutic drugs, for example, will not effectively cross the blood-brain barrier to reach tumor cells2. Thus, improving the delivery of drugs across the blood-brain barrier is an area of interest.
The most prevalent methods for enhancing the delivery of drugs to the brain are direct cerebral infusion and blood-brain barrier disruption3. Direct intracerebral infusion guarantees that therapies reach the brain; however, this method has a limited ability to disperse the drug4. Blood-brain barrier disruption (BBBD) allows drugs to flow directly from the circulatory system into the brain and thus more effectively reach dispersed tumor cells. Three methods of barrier disruption include osmotic barrier disruption, pharmacological barrier disruption, and focused ultrasound with microbubbles. Osmotic disruption, pioneered by Neuwelt, uses a hypertonic solution of 25% mannitol that dehydrates the cells of the blood-brain barrier causing them to shrink and disrupt their tight junctions. Barrier disruption can also be accomplished pharmacologically with vasoactive compounds such as histamine5 and bradykinin6. This method, however, is selective primarily for the brain-tumor barrier7. Additionally, RMP-7, an analog of the peptide bradykinin, was found to be inferior when compared head-to-head with osmotic BBBD with 25% mannitol8. Another method, focused ultrasound (FUS) in conjunction with microbubble ultrasound contrast agents, has also been shown to reversibly open the blood-brain barrier9. In comparison to FUS, though, 25% mannitol has a longer history of safety in human patients that makes it a proven tool for translational research10-12.
In order to accomplish BBBD, mannitol must be delivered at a high rate directly into the brain's arterial circulation. In humans, an endovascular catheter is guided to the brain where rapid, direct flow can be accomplished. This protocol models human BBBD as closely as possible. Following a cut-down to the bifurcation of the common carotid artery, a catheter is inserted retrograde into the ECA and used to deliver mannitol directly into the internal carotid artery (ICA) circulation. Propofol and N2O anesthesia are used for their ability to maximize the effectiveness of barrier disruption13. If executed properly, this procedure has the ability to safely, effectively, and reversibly open the blood-brain barrier and improve the delivery of drugs that do not ordinarily reach the brain 8,13,14.

Blood-brain barrier disruption aids the delivery of certain drugs to the brain. Mannitol delivered intra-arterially shrinks cells surrounding blood vessels in order to physically disrupt the barrier.

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 ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

Halsschlagader Infusionen für pharmakokinetischen und pharmakodynamischen Analyse von Taxanen in Mäusen

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Vascular Balloon Injury and Intraluminal Administration in Rat Carotid Artery

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and cellular mechanisms involved in vascular smooth muscle dedifferentiation, neointima formation and vascular remodeling. Male Sprague-Dawley rats are the most frequently employed animals for this model. Female rats are not preferred as female hormones are protective against vascular diseases and thus introduce a variation into this procedure. The left carotid is typically injured with the right carotid serving as a negative control. Left carotid injury is caused by the inflated balloon that denudes the endothelium and distends the vessel wall. Following injury, potential therapeutic strategies such as the use of pharmacological compounds and either gene or shRNA transfer can be evaluated. Typically for gene or shRNA transfer, the injured section of the vessel lumen is locally transduced for 30 min with viral particles encoding either a protein or shRNA for delivery and expression in the injured vessel wall. Neointimal thickening representing proliferative vascular smooth muscle cells usually peaks at 2 weeks after injury. Vessels are mostly harvested at this time point for cellular and molecular analysis of cell signaling pathways as well as gene and protein expression. Vessels can also be harvested at earlier time points to determine the onset of expression and/or activation of a specific protein or pathway, depending on the experimental aims intended. Vessels can be characterized and evaluated using histological staining, immunohistochemistry, protein/mRNA assays, and activity assays. The intact right carotid artery from the same animal is an ideal internal control. Injury-induced changes in molecular and cellular parameters can be evaluated by comparing the injured artery to the internal right control artery. Likewise, therapeutic modalities can be evaluated by comparing the injured and treated artery to the control injured only artery.

This protocol uses a balloon catheter to cause an intraluminal injury on the rat carotid artery and henceforth elicit neointimal hyperplasia. This is a well-established model for studying the mechanisms of vascular remodeling in response to injury. It is also widely used to determine the validity of potential therapeutic approaches.

Gefäßballon Verletzung und intraluminale Verwaltung in Rattenhalsschlagader

The carotid artery balloon injury model in rats has been well established for over two decades. It remains an important method to study the molecular and ...

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. ...

The Rabbit Model of Accelerated Atherosclerosis: A Methodological Perspective of the Iliac Artery Balloon Injury

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the industrialized world. New Zealand White (NZW) rabbits are widely used as an animal model for the study of atherosclerosis. They develop spontaneous lesions when fed with atherogenic diet; however, this requires long time of 4 - 8 months. To further enhance and accelerate atherogenesis, a combination of atherogenic diet and mechanical endothelial injury is often employed. The presented procedure for inducing atherosclerotic plaques in rabbits uses a balloon catheter to disrupt the endothelium in the left iliac artery of NZW rabbits fed with atherogenic diet. Such mechanical damage caused by the balloon catheter induces a chain of inflammatory reactions initiating neointimal lipid accumulation in a time dependent fashion. Atherosclerotic plaque following balloon injury show neointimal thickening with extensive lipid infiltration, high smooth muscle cell content and presence of macrophage derived foam cells. This technique is simple, reproducible and produces plaque of controlled length within the iliac artery. The whole procedure is completed within 20 - 30 min. The procedure is safe with low mortality and also offers high success in obtaining substantial intimal lesions. The procedure of balloon catheter induced arterial injury results in atherosclerosis within two weeks. This model can be used for investigating the disease pathology, diagnostic imaging and to evaluate new therapeutic strategies.

Animal models of atherosclerosis are essential to understand the mechanism and to investigate newer approaches to prevent plaque development or rupture, a leading cause of death in the industrialized world. This protocol uses a combination of balloon injury and cholesterol rich diet to induce atherosclerotic plaques in rabbit iliac artery.

Das Kaninchen-Modell der beschleunigten Atherosklerose: methodischer Sicht der Beckenkamm Arterie Ballon Verletzung

Acute coronary syndrome resulting from coronary occlusion following atherosclerotic plaque development and rupture is the leading cause of death in the ...

Balloon-based Injury to Induce Myointimal Hyperplasia in the Mouse Abdominal Aorta

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine balloon denudation model, which is comparable with established vessel injury models in large animals. The aorta denudation model with balloon catheters mimics the clinical setting and leads to comparable pathobiological and physiological changes. Briefly, after performing a horizontal incision in the aorta abdominalis, a balloon catheter will be inserted into the vessel, inflated, and introduced retrogradely. Inflation of the balloon will lead to intima injury and overdistension of the vessel. After removing the catheter, the aortic incision will be closed with single stiches. The model shown in this article is reproducible, easy to perform, and can be established quickly and reliably. It is especially suitable for evaluating expensive experimental therapeutic agents, which can be applied in an economical fashion. By using different knockout-mouse strains, the impact of different genes on MH development can be assessed.

This article demonstrates a murine model to study the development of myointimal hyperplasia (MH) after aortic balloon injury.

Ballon-basierte Verletzungen induzieren Myointimal Hyperplasie in der Bauchschlagader Maus

The use of animal models is essential for a better understanding of MH, one major cause for arterial stenosis.In this article, we demonstrate a murine ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

Eine Drosophila In Vivo Verletzungen Modell für das Studium Neuroregeneration in den peripheren und zentralen Nervensystems

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, we have translated a vascular injury model from a small animal into a large animal (canine), with slight modifications that can be used for pre-clinical screening of prophylactic and thrombolytic agents. In addition to the surgical methods, the modified protocol describes the step-by-step methods to assess carotid artery canalization by angiography, detailed instructions to process both the brain and carotid artery for histological analysis to verify carotid canalization and cerebral hemorrhage, and specific parameters to complete an assessment of downstream thromboembolic events by utilizing magnetic resonance imaging (MRI). In addition, specific procedural changes from the previously well-established small animal model necessary to translate into a large animal (canine) vascular injury are discussed.

Here, we present the modifications necessary to a well characterized and commonly used small animal ferric chloride-induced (FeCl3) carotid artery injury model for use in a large animal vascular injury model. The resulting model can be utilized for pre-clinical trial assessment of both prophylactic and thrombolytic pharmacological and mechanical interventions.

Eisenchlorid-induzierte eckzahn Halsschlagader Thrombosen: Eine große Tiermodell der Gefäßverletzung

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

A Rat Carotid Artery Pressure-Controlled Segmental Balloon Injury with Periadventitial Therapeutic Application

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the luminal surface area in arteries thereby reducing adequate blood flow to organs and distal tissues. Clinically, revascularization procedures such as balloon angioplasty with or without stent placement aim to restore blood flow. Although these procedures reestablish blood flow by reducing plaque burden, they damage the vessel wall, which initiates the arterial healing response. The prolonged healing response causes arterial restenosis, or re-narrowing, ultimately limiting the long-term success of these revascularization procedures. Therefore, preclinical animal models are integral for analyzing the pathophysiological mechanisms driving restenosis, and provide the opportunity to test novel therapeutic strategies. Murine models are cheaper and easier to operate on than large animal models. Balloon or wire injury are the two commonly accepted injury modalities used in murine models. Balloon injury models in particular mimic the clinical angioplasty procedure and cause adequate damage to the artery for the development of restenosis. Herein we describe the surgical details for performing and histologically analyzing the modified, pressure-controlled rat carotid artery balloon injury model. Additionally, this protocol highlights how local periadventitial application of therapeutics can be used to inhibit neointimal hyperplasia. Lastly, we present light sheet fluorescence microscopy as a novel approach for imaging and visualizing the arterial injury in three-dimensions.

The rat carotid artery balloon injury mimics the clinical angioplasty procedure performed to restore blood flow in atherosclerotic vessels. This model induces the arterial injury response by distending the arterial wall, and denuding the intimal layer of endothelial cells, ultimately causing remodeling and an intimal hyperplastic response.

Eine Ratte Carotis Arterie Druck-kontrollierte segmentale Ballonverletzung mit periadventitialen therapeutischen Anwendung

Cardiovascular disease remains the leading cause of death and disability worldwide, in part due to atherosclerosis. Atherosclerotic plaque narrows the ...