Diese Arbeit wurde durch Zuschüsse aus dem American Heart Association Großraum Southeast Affiliate (09GRNT2060421), die American Medical Association, und von der University of Florida Clinical and Translational Science Institute unterstützt. Adam Mekka ist eine NIH / NINDS, NRSA Chemiefonds fellow (F30 NS-060.335). Robert Regenhardt erhalten Promotionsstipendium Unterstützung von der University of Florida Multidisziplinäre Training Program in Hypertension (T32 HL-083810).

Dieses Protokoll wurde von der Institutional Animal Care und Use Committee (IACUC) an der University of Florida zugelassen und ist in Übereinstimmung mit dem &quot;Leitfaden für die Pflege und Verwendung von Labortieren&quot; (achte Auflage National Academy of Sciences, 2011). Materialien <ol><li> Tiere: Acht-Wochen-alt, männlich, Sprague Dawley Ratten (Charles River Farms, Wilmington, MA, USA) mit einem Gewicht von 250-300 g zum Zeitpunkt der Operation. </li><li> Anästhesie <ol class="lalpha"><li> Inhalationsanästhesie System (VetEquip Inc., Pleasanton, CA, USA) </li><li> Isofluran Narkose (Baxter Pharmazie, Deerfield, IL, USA) </li></ol></li><li> Stereotaktische System (David Kopf Instruments, Tujunga, CA, USA) <ol class="lalpha"><li> Kleintier stereotaktischen Systems </li><li> Non-Ruptur Ohr Bars für Ratten </li><li> Gas Anästhesie Kopfhalter für Ratten </li></ol></li><li> Temperatur-Regelung <ol class="lalpha"><li> BAT-12 Mikrosonde Thermometer (WorldPrecision Instruments, Inc., Sarasota, FL, USA) </li><li> T / PUMP, TP600 Thermal Decke (Gaymar Industries, Inc., Orchard Park, NY, USA) </li></ol></li><li> Chirurgische Instrumente <ol class="lalpha"><li> Metzenbaumschere, Iris Pinzette, bulldog clamp Retraktoren, 10 ul Spritze mit 26 Gauge abgeschrägte Nadel, Bovie Kauter Kit (World Precision Instruments, Inc., Sarasota, FL, USA) </li><li> Mikromotor Bohrer (Stoelting, Wood Dale, IL, USA) </li><li> 0,8 mm Rosenbohrer bur (Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA) </li><li> STORZ Bonn Naht-Zange (Bausch und Lomb, Inc., Rochester, NY, USA) </li></ol></li><li> Surgical Supplies <ol class="lalpha"><li> 3,0 Nylonnaht (Oasis, Mettawa, IL, USA) </li><li> Wattestäbchen, Puralube Augensalbe (Fisher Scientific, Pittsburgh, PA, USA) </li><li> Chirurgische Punkte und Streifen (Medtronic Xomed, Inc., Jacksonville, FL, USA) </li><li> Haarschneidemaschine (Oster, Providence, RI, USA) </li></ol></li&gt; <li> Chemicals <ol class="lalpha"><li> Endothelin-1 (American Peptide, Sunnyvale, CA, USA) </li><li> Chlorhexidin 2% (Agrilabs, St. Joseph, MO, USA) </li></ol></li><li> Visualisierung Ausrüstung <ol class="lalpha"><li> Operationsmikroskop (Seiler Instrument and Manufacturing, St. Louis, MO, USA) </li><li> Sony Handycam HDR-SR12 (Sony, Minato, Tokio, Japan) </li><li> Fiber Optic Illuminator (TechniQuip Corp, Livermore, CA, USA) </li></ol></li><li> Messung der Gefäßdurchmesser <ol class="lalpha"><li> VLC media player (Paris, Frankreich) </li><li> Image J Software (ImageJ 1.42q Software, US National Institutes of Health, Bethesda, MA, USA) </li></ol></li></ol> Ein. Präoperative Steps <ol><li> Vor der Operation werden die Ratten unter einem 12:12 Hell / Dunkel-Zyklus mit freiem Zugang zu Wasser und Nagetierfutter untergebracht. </li><li> Narkose mit Isofluran in 4% 100% O 2-Gasgemisch in einer Induktionskammer induziert. </li><li> Die Krähe<html> n des Kopfes mit elektrischen Haarschneidemaschinen rasiert. </li><li> Die Ratte wird in einer Bauchlage auf einem saugfähigen Kissen, das auf einer Temperatur-gesteuerten Bedienfläche (Wärmedecke) und der Kopf wird in der stereotaktischen Vorrichtung beginnend mit Plazierung des Gases Anästhetikum Gesichtsmaske platziert. </li><li> Anschließend werden die Stäbe Ohr eingesetzt und verschlossen. </li><li> Während des Verfahrens Anästhesie mit 2% Isofluran in 100% O 2-Gasgemisch aufrechterhalten wird. </li><li> Ophthalmischen Schmiermittels Salbe wird auf beide Augen aufgetragen und die Augenlider geschlossen sind, um Austrocknung Auges während des chirurgischen Verfahrens zu verhindern. </li><li> Rektale Temperatursonde eingeführt wird, um eine konstante tierischen Kerntemperatur von 37 ± 0,5 ° C zu halten </li><li> Mit dem Kopf der narkotisierten Ratte fest in der Vorrichtung gehalten stereotaxischen der chirurgische Umgebung gereinigt mit alternierenden Chlorhexidin und Kochsalzlösung dreimal. </li></ol> 2. Pre-kranialen Fenster Vorbereitung nhalt &quot;&gt; Vor Erstellung einer kranialen Fenster sollte der Ratte für jegliche Experimente durch Implantation von anderen benötigten Hardware hergestellt werden sollten und alle notwendigen chirurgischen Eingriffe erhalten. Aus diesem Protokoll, wir zuvor eine Führungskanüle für Endothelin-1 (ET-implantierten 1) induzierte MCAO wie in einem Begleiter Publikation mit dem Titel angezeigt &quot;Endothelin-1 induzierte Middle Cerebral Artery Occlusion Modell für ischämischen Schlaganfall mit Laser Doppler Flowmetrie Guidance in Ratte.&quot; 3. Schaffung des Cranial Fenster Nach Platzierung einer Führungskanüle oder Einrichtungen für die Experimente erforderlich, wird eine Schädelknochen Fenster erzeugt, um direkt visualisieren proximalen Abschnitte der mittleren zerebralen Arterie während eines Hubes Prozedur. <ol><li> Zunächst werden Schere verwendet, um die Haut über dem M. temporalis ab medial und Arbeitsbedingungen seitlich einschneiden. </li><li> Der M. temporalis wird halbiert mit Elektrokauter und dann zurückgezogen mit 3,0 Nylonnaht zu visua<html> kristallisieren die Plattenepithelkarzinome Teil des Schläfenbein. </li><li> Ein ca. 3-4 mm Platz ist auf dem Plattenepithel Teil des Schläfenbein kaudal der Umlaufbahn und überlegen der Basis des Processus zygomaticus gezogen, wie es reflektiert der zeitlichen Knochen. </li><li> Ein Bohrer wird verwendet, um allmählich schneiden die skizzierten Stück Knochen frei von Schläfenbein. Es ist darauf zu vermeiden, dass zu viel Druck auf die Bohrmaschine da es möglich, die Dura oder Großhirnrinde beschädigen ist. </li><li> Häufige Spülungen mit steriler Kochsalzlösung durchgeführt werden, um die Visualisierung des OP-Feld zu verbessern und verhindern eine Überhitzung des Schädels. </li><li> Ab einer losen Ecke wird das Stück temoporal Knochen vorsichtig entfernt mit feinen rat-gezahnten Pinzette gleichzeitig dafür, dass keine Schiffe mit der Dura verbunden reißen. </li><li> Die Dura intakt bleibt und Schutt weg mit steriler Kochsalzlösung gespült. </li></ol> 4. Aufzeichnung von Cerebral Artery Constriction Zelt &quot;&gt; Um zu demonstrieren, wie Sie Bilder in Echtzeit, eine Ratte, die durchlaufen ET-1-induzierten MCAO ist zu erfassen ist für dieses Protokoll verwendet. <ol><li> Vor dem Aufbringen des vasoaktiven Verbindungen sollten eine Grundlinie Video für mindestens 1 min aufgezeichnet. Für ET-1-induzierten MCAO wird die Basislinie Aufzeichnung erfolgt, wenn die Nadel in das Hirnparenchym abgesenkt worden, aber vor der ET-1 Injektion. </li><li> Die Spritzenpumpe wird zur Injektion begonnen und aufgezeichnet für 1 Stunde oder bis zum gewünschten Endpunkt. Die Nadel wird in die während Videoaufzeichnung belassen, um Störungen der Fokalebene zu verhindern. </li><li> Die Ratte sollte tief betäubt und eingeschläfert nach einer genehmigten Protokoll des folgenden Verfahrens. </li></ol> 5. Image Analysis Gefäßdurchmesser kann für einen beliebigen Teil der visualisierten MCA bestimmt werden. Als Beispiel verwenden wir ein Zweig der MCA zu Behälterdurchmesser bei Zeit-Punkten messen vor und nach ET-1 Injektion. Still Frames aus dem Video werden auf 1 min Abständen mit VLC media player (VideoLAN) erfasst. <ol><li> VLC Player installiert und geöffnet. </li><li> Durch Auswahl von Werkzeugen und Vorlieben, wird die Einstellung auf &quot;All&quot; unter veränderten &quot;Einstellungen anzeigen&quot;. </li><li> Der &quot;Video-Menü&quot; in der linken Navigationsleiste erweitert und &quot;Output Module&quot; wird dann erweitert. &quot;Scene Filter&quot; ausgewählt wird, um die Szene Videofilter Menü. </li><li> &quot;Szene&quot; in den Kasten für Dateinamenpräfix getippt. </li><li> Ein Verzeichnispfad Präfix definiert. Dies ist, wo Standbilder gespeichert werden. </li><li> Aufzeichnungs-Verhältnis wird auf der Basis der gewünschten Geschwindigkeit der Standbild-Capture ausgewählt. Wenn ein Video mit 29 Frames pro Sekunde, dann &quot;1749&quot; (29 Bilder / sec x 60 sec / min) erfasst wurde, sollte in der Box eingegeben werden, um zu sparen ein Standbild alle 1 min. Alle Änderungen werden dann gespeichert. </li><li> Die gewünschte Video wird dann im VLC-Player geöffnet werden, um automatisch noch retten Frames einmal pro 1 min. </li><li> Gefäßdurchmesser wird dann mit diesenBilder. Zuerst wird ImageJ Software (NIH) geöffnet. </li><li> Dann werden die Standbilder, die Sie messen in ImageJ geöffnet. </li><li> Durch die Auswahl der &quot;analysieren&quot; Menü &quot;gesetzt Messungen&quot; wird dann geöffnet und alle Boxen deaktiviert sind. </li><li> Als nächstes wird die gerade Linie Werkzeug ausgewählt. </li><li> Die Tastenkombination &quot;Strg +&quot; wird verwendet, um in nach Bedarf vergrößern und eine Linie senkrecht zur Schiffes Weg zum Gefäßdurchmesser zu messen. </li><li> Schließlich wird Gefäßlänge, indem Sie die &quot;analysieren&quot; Menü und dann &quot;Maßnahme&quot; (Strg + M) in den Behälter Länge zu erhalten erhalten. </li><li> Dieser Vorgang wird mindestens dreimal wiederholt und gemittelt für jede gemessene Gefäß in jedem Standbild gemessen. </li><li> Gefäßdurchmessers zu jedem Zeitpunkt an der Grundlinie Gefäßdurchmesser normiert, so dass Vergleiche können unter Verwendung mehrerer Ratten werden. Um dies zu tun, verwenden Sie die Formel aktuellen Durchmesser / Baseline Durchmesser x 100% die% Baseline Durchmesser jedes Schiff zu berechnen. </li></ol>

<ol>
	<li>Levasseur, J. E., Wei, E. P., Raper, A. J., Kontos, A. A., Patterson, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+description+of+a+cranial+window+technique+for+acute+and+chronic+experiments.">Detailed description of a cranial window technique for acute and chronic experiments.</a> Stroke. 6, 308-317 (1975).</li><li>Baumbach, G. L., Dobrin, P. B., Hart, M. N., Heistad, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+cerebral+arterioles+in+hypertensive+rats.">Mechanics of cerebral arterioles in hypertensive rats.</a> Circ. Res. 62, 56-64 (1988).</li><li>Regrigny, O., 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=Effects+of+melatonin+on+rat+pial+arteriolar+diameter+in+vivo.">Effects of melatonin on rat pial arteriolar diameter in vivo.</a> Br. J. Pharmacol. 127, 1666-1670 (1999).</li><li>Mecca, A. P., O'Connor, T. E., Katovich, M. J., Sumners, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candesartan+pretreatment+is+cerebroprotective+in+a+rat+model+of+endothelin-1-induced+middle+cerebral+artery+occlusion.">Candesartan pretreatment is cerebroprotective in a rat model of endothelin-1-induced middle cerebral artery occlusion.</a> Exp. Physiol. 94, 937-946 (2009).</li><li>Mecca, A. P., 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=Cerebroprotection+by+angiotensin-(1-7)+in+endothelin-1-induced+ischaemic+stroke.">Cerebroprotection by angiotensin-(1-7) in endothelin-1-induced ischaemic stroke.</a> Exp. Physiol. 96, 1084-1096 (2011).</li></ol>

Keine Interessenskonflikte erklärt.

Zusammenfassend ist das kraniale Fenster Präparationstechnik sehr vielseitig wie sie kann geändert werden, um den Anforderungen vieler Versuche mit geringfügigen Modifikationen 4, 5 erfüllen. Zum Beispiel haben wir erfolgreich zerebralen Blutflusses in bestimmten MCA Niederlassungen mittels Laser-Doppler-Flussmessung direkt konzentrieren sich auf eine Hirnarterie visualisiert durch ein Schädel-Fenster (Mekka AP 2009 und 2011) überwacht. Zusätzlich kann eine ähnliche Zubereitung mit der Dura mit einges...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Inhalationsanästhesie System </td><td> VetEquip Inc., Pleasanton, CA, USA </td><td> 901806 </td><td></td></tr><tr><td> Isofluran Narkose </td><td> Baxter Pharmazie, Deerfield, IL, USA </td><td> 1001936060 </td><td></td></tr><tr><td> Kleintier stereotaktischen Systems </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 900 </td><td></td></tr><tr><td> Non-Ruptur Ohr Bars, Ratte </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 957 </td><td></td></tr><tr><td> Rat Gas Anästhesie Kopfhalter </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 1929 </td><td></td></tr><tr><td> BAT-12 Mikrosonde Thermometer </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> BAT-12 </td><td&gt; </td></tr><tr><td> T / PUMP, Thermal Decke </td><td> Gaymar Industries, Inc., Orchard Park, NY, USA </td><td> T / PUMP, TP600 </td><td></td></tr><tr><td> Metzenbaumschere </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> 501254 </td><td></td></tr><tr><td> Iris Pinzette </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> 15915 </td><td></td></tr><tr><td> Bulldog Klemme Retraktoren </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> 14.119-G </td><td></td></tr><tr><td> 10 &amp;mgr; l-Spritze 26-gaugue </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> SGE010RNS </td><td></td></tr><tr><td> Bovie, Hochtemperatur Kauter Kit </td><td> World Precision Instruments, Inc., Sarasota, FL, USA </td><td> 500392 </td><td></td></tr><tr><td> Rat Zahn-Zange 0,12 </td><td> Stotz </td><td> E1811 </td><td></td></tr><tr><td> Mikromotor Bohrer </td><td> Stoelting,Wood Dale, IL, USA </td><td> 51449 </td><td></td></tr><tr><td> 0,8 mm Rosenbohrer bur </td><td> Roboz Surgical Instrument Co., Inc., Gaithersburg, MD, USA </td><td> RS-6280C-1 </td><td></td></tr><tr><td> STORZ Bonn Naht Pinzette </td><td> Bausch &amp; Lomb, Inc., Rochester, NY, USA </td><td></td><td></td></tr><tr><td> Nylon Naht, Größe 3,0 </td><td> Oasis, Mettawa, IL, USA </td><td> MV-663 </td><td></td></tr><tr><td> Wattestäbchen </td><td> Fisher Scientific, Pittsburgh, PA, USA </td><td> 22-029-488 </td><td></td></tr><tr><td> Puralube Augensalbe </td><td> Fisher Scientific, Pittsburgh, PA, USA </td><td> NC0138063 </td><td></td></tr><tr><td> Haarschneidemaschine </td><td> Oster, Providence, RI, USA </td><td> 78005-301 </td><td></td></tr><tr><td> ET-1 bis 80 μ M Konzentration in PBS verdünnt </td><td> American Peptide, Sunnyvale, CA, USA </td><td> 88-1-10A </td><td></td></tr><tr><td> Chlorhexidin, 2% </td&gt; <td> Agrilabs, St. Joseph, MO, USA </td><td> 1040, Rev. 6.6, NAC-Nr: 10580322 </td><td></td></tr><tr><td> Operationsmikroskop </td><td> Seiler Instrument and Manufacturing, St. Louis, MO, USA </td><td> Evolution XR6 </td><td></td></tr><tr><td> Sony Handycam </td><td> Sony, Minato, Tokio, Japan </td><td> HDR-SR12 </td><td></td></tr><tr><td> Faseroptik-Lichtquelle </td><td> TechniQuip Corp, Livermore, CA, USA </td><td> FO1-150 </td><td></td></tr><tr><td> VLC Media Player </td><td> (Paris, Frankreich) </td><td></td><td></td></tr><tr><td> Image J Software </td><td> US National Institutes of Health, Bethesda, MA, USA </td><td></td><td></td></tr></tbody></table>

utilizing a cranial window to visualize the middle cerebral artery during endothelin-1 induced middle cerebral artery occlusion

Erstellung eines Schädel-Fenster ist eine Methode, die eine direkte Visualisierung der Strukturen ermöglicht auf der kortikalen Oberfläche des Gehirns 1-3. Diese Technik kann an verschiedenen Orten, die über der Ratte Cerebrum durchgeführt werden, sondern ist am einfachsten durch die Schaffung eines Kraniektomie über den leicht zugänglichen frontalen oder Scheitelbeine durchgeführt. Am häufigsten haben wir diese Technik in Kombination mit dem Endothelin-1 cerebri Arterienverschluss Modell eines ischämischen Schlaganfalls, um die Änderungen in der mittleren zerebralen Arterie Gefäßdurchmesser, der mit Einspritzung von Endothelin-1 treten in das Hirnparenchym angrenzend an das proximale MCA quantifizieren 4, 5. Um den proximalen Abschnitt des MCA während Endothelin induzierten -1 MCAO visualisieren, verwenden wir eine Technik, um ein Fenster durch die kraniale Schläfenbein auf der lateralen Seite des Schädels Ratte (Abbildung 1) zu schaffen. Hirnarterien können entweder mit der Dura intakt oder mit der Dura eingeschnitten und retra visualisiert werdenCTED. Am häufigsten, verlassen wir die Dura intakt während der Visualisierung, da Endothelin-1-induzierten MCAO beinhaltet Lieferung der gefäßverengende Peptids in das Hirnparenchym. Dies umgeht die Notwendigkeit, die Dura direkt über einzuschneiden visualisierter Behältern zur Medikamentenabgabe. Dieses Protokoll wird beschrieben, wie ein Schädel Fenster Hirnarterien in einer stufenweisen Weise visualisieren, und wie viele der möglichen Gefahren im Zusammenhang mit dieser Methode zu vermeiden erstellen.

Standbilder von der Videoaufnahme entnommen wurden, zeigen, dass die Veränderung in Hirnarterie Durchmesser nach ET-1 Injektion kann leicht erkannt werden, mit dieser kranialen Fenster-Technik (Abbildung 2). Innerhalb von Minuten nach ET-1 Injektion wird das Schiff beginnt zu verengen. Schließlich die Gefäße schwierig sein wird, zu visualisieren und das Gehirngewebe wird bleich. Nach ca. 20 min die Auswirkungen von ET-1 wird abnehmen und die Gefäße beginnen zu erweitern, allmählich wieder auf den...

Watch this Scientific Journal Video about Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion at JoVE.com

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Dieser Artikel beschreibt eine Methode zur Visualisierung rat Hirnarterien durch ein Schädel-Fenster mit zeitlicher Kraniektomie um proximalen Abschnitte der mittleren Hirnarterie anzusehen (<strong&gt; Abbildung 1</strong&gt;). Diese Methode lässt sich mit verschiedenen Techniken von Drug-Delivery kombiniert werden, um Hirnarterie Reaktivität zu messen<em&gt; In vivo</em&gt;.

Mit Hilfe eines Cranial Fenster, um den Nahen Cerebral Artery Während Endothelin-1 induzierte Middle Cerebral Artery Occlusion Visualisieren

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

utilizing-cranial-window-to-visualize-middle-cerebral-artery-during

Research

JoVE Journal

Medicine

14.8K Aufrufe.  University of Florida. Dieser Artikel beschreibt eine Methode zur Visualisierung rat Hirnarterien durch ein Schädel-Fenster mit zeitlicher Kraniektomie um proximalen Abschnitte der mittleren Hirnarterie anzusehen ( Abbildung 1). Diese Methode lässt sich mit verschiedenen Techniken von Drug-Delivery kombiniert werden, um Hirnarterie Reaktivität zu messen In vivo.

Serie: Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modeling Stroke in Mice - Middle Zerebralarterie mit dem Filament-Modell

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Forschung

Medizin

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Mouse Model of Middle Zerebralarterie

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Die Anwendung des dauerhaften mittleren Hirnarterie Ligation in der Maus

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Endothelin-1 induzierte Middle Cerebral Artery Occlusion Modell für ischämischen Schlaganfall mit Laser Doppler Flowmetrie Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Optimierte System for Cerebral Perfusion Überwachung im Rat Stroke Modell Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Embolischen mittleren Hirnarterie Okklusion (MCAO) für ischämischen Schlaganfall mit homologen Blutgerinnsel in Ratten

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Die Quantifizierung der neurovaskulären Schutz Nach Repetitive hypoxische Präkonditionierung und Transient mittleren Hirnarterie Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Darstellende Permanent Distal cerebri mit Arteria carotis communis Occlusion bei alten Ratten zu studieren kortikale Ischämie mit anhaltenden Behinderungs

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Chirurgische Ansatz für Arteria cerebri media Occlusion und Reperfusion fokaler zerebraler Ischämie bei Mäusen

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transient Middle Cerebral Artery Occlusion Modell von Neugeborenen Stroke in P10 Ratten

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...