Diese Arbeit wurde von der Europäischen Gemeinschaft Siebten Rahmenprogramms (FP7/2007-2013) unter Finanzhilfevereinbarung Nr. 201024 und Nr. 202213 (European Stroke Network) finanziert. Zusätzliche Mittel wurden vom Bundesministerium für Bildung und Forschung (Centrum für Schlaganfall-Forschung Berlin) und die Deutsche Forschungsgemeinschaft (Exzellenzcluster NeuroCure) gegeben. Die Autoren bedanken sich bei Mareike Thielke (Dep. für Experimentelle Neurologie, Charité Berlin) für die Unterstützung danken während der Operation und Elke Ludwig (Charité Video Services) für die Herstellung der Animation.

Um eine hohe Qualität und Reproduzierbarkeit, empfehlen wir die Verwendung von Standard Operating Procedures (SOP). In diesem Video werden veröffentlicht SOPs als entwickelt und in unserem Labor angewandt. 1 1. Middle Zerebralarterie <ol><li> Mäuse sind betäubt mit einem entsprechenden Betäubungsmittel Regime in Rücksprache mit Tierärzten. (ZB Induktion mit 1,5 bis 2% Isofluran und Wartung mit 1,0 bis 1,5% Isofluran in 2 / 3 N2O und 1 / 3 O2 mit einem Verdampfer). <ol><li> Die Körpertemperatur der Mäuse ist mit 36,5 ° C ± 0,5 ° C während der Operation mit einer Heizplatte gehalten. Ein Feedback-kontrollierte Heizkissen, die nach der rektalen Temperatur der Maus erwärmt, wird dringend empfohlen. </li><li> Desinfizieren Sie die Haut und die umliegenden Fell mit einem geeigneten Mittel (zB 70% Ethylalkohol) und trocknen Sie ihn danach. </li></ol></li><li> Eine Mittellinie Hals Schnitt gemacht und die Weichteile sind auseinander gezogen. </li><li> Die linke Arteria carotis communis (LCCA) wird vorsichtig frei präpariert aus den umliegenden Nerven (ohne Schädigung der Vagus-Nerv) und einer Ligatur erfolgt über 6.0/7.0 String. 5,0 String kann auch verwendet werden. </li><li> Die linke A. carotis externa (LECA) wird dann abgetrennt und ein zweiter Knoten gemacht wird. </li><li> Als nächstes wird die linke A. carotis interna (LICA) isoliert und ein Knoten ist mit einem 6,0 Wendel vorbereitet. </li><li> Nach Erhalt einer guten Ansicht der linken Arteria carotis interna (LICA) und die linke pterygopalatina Arterie (LPA), sind beide Arterien abgeschnitten, mit einem mikrovaskulären Clips. </li><li> Ein kleines Loch ist in der LCCA geschnitten, bevor sie an die LECA und die LICA gabelt. Ein Monofilament von 8,0-Nylon mit Silizium-Härter-Gemisch (siehe unten) beschichtet wird dann in den LICA eingeführt, bis es an den Clip anhält. Ist darauf zu achten nicht auf die A. occipitalis einzugeben. (Abbildung 1) </li><li> Der abgeschnittene Arterien sind geöffnet, während das Filament in die LICA eingeführt wird, um die Herkunft der LMCA in den Kreis der Willis verschließen. </li><li> Der dritte Knoten auf der LICA geschlossen wird, um das Filament in Position zu fixieren. </li><li> Die Mäuse erhalten Kochsalzlösung 0,5 ml subkutan als Volumen Nachschub. Zur Schmerzlinderung ist Lidocain-Gel topisch in die Wunde aufgetragen. </li><li> Wenn Reperfusion soll, Mäuse für 30 bleiben - 90 min Okklusion in einem beheizten Käfig, konnte die Wunde mit einer kleinen Naht-Clip verschlossen werden. Danach eine zweite Betäubung durchgeführt wird, ist der dritte Knoten auf der ICA momentanen geöffnet und der Faden zurückgezogen. </li><li> Die restlichen Nähte werden verkürzt und die Haut mit einer chirurgischen Naht adaptiert. </li><li> Alle Tiere erhalten einen zweiten Band Nachschub wie oben beschrieben. </li><li> Die Tiere werden in einem beheizten Käfig für zwei Stunden gesetzt, um die Körpertemperatur zu kontrollieren. <ol><li> Die Tiere müssen täglich nach der Operation für Anzeichen von Beschwerden überprüft werden. Die Mäuse konnten zeigen einige postoperative Gewichtsabnahme. Sie erhalten pürierte Nahrung in einer Petrischale auf den Boden zu ermutigen, zu essen gegeben. Das Essen wird täglich für sieben Tage ersetzt. </li></ol></li></ol> 2. Sham Betrieb <ol><li> Für Schein-Operationen der Faden eingelegt, um LMCA verschließen und sofort Instant Reperfusion (1,8) ermöglichen zurückgezogen. Die anschließende Operation ist identisch mit der auf die Tiere unterzogen zerebrale Ischämie (1,9-1,14) durchgeführt, einschließlich einer zweiten Narkose. </li></ol> 3. Vorbereitung der Filament <ol><li> Die Sterilität der Faden sollte in Betracht gezogen werden. Die Verwendung von sterilisierten Geräten sowie eine angemessene Behandlung des Filaments danach ist eine Voraussetzung für eine sterile Operation. Desinfektion des Filaments ist schwierig, da viele der gängigen Sterilisationsverfahren, die Qualität des Fadens verschlimmern kann. Allerdings Methoden wie Strahlung, zum Beispiel mit UV-Licht oder γ-Strahlen oder chemische Sterilisation, z. B. mit hoch reaktiven Gasen wie Ethylenoxid, anwendbar sind. </li><li> 8,0 Nylonfaden ist in Längen von 11 mm unter dem Mikroskop geschnitten </li><li> Das Filament Spitze muss vollständig und gleichmäßig über eine Länge von 8 mm beschichtet werden mit einem Härter Mischung aus Xantopren M Mucosa und Activator NF Optosil </li></ol> 4. Repräsentative Ergebnisse Je nach Dauer des Blutflusses Einschränkung, verschiedene Motor-und Verhaltenswissenschaften Defizite führen. Sowohl nach 30 und 60 min von zerebraler Ischämie, Tiere in den meisten Fällen zeigen verringerte Widerstand gegen seitliche push und kreisenden aufgrund von Störungen in Fortbewegung. Milder Läsionen manifestieren als flexor Position in der vorderen limbs. Diese leicht zu beobachtenden Anzeichen kann als ein grundlegendes Ergebnis für den Erfolg der Operation verwendet. 2. Morphologisch der Läsion kann unter Verwendung entweder Histologie oder Magnetic Resonance Imaging (MRI) werden. Sechzig Minuten Verschluss der mittleren Hirnarterie produziert Gewebe pannecrosis in einem Gebiet, darunter sowohl das Striatum und dem Neokortex, während 30 Minuten der Ischämie hauptsächlich neuronalen Zelltod begrenzt auf das Striatum verursachen. 3 (Abbildung 2) In Bezug auf Infarktvolumen erwarten wir, dass einer Standardabweichung von weniger als 30% in eine Reihe von Operationen. Die Sterblichkeit ist abhängig von der Okklusion Zeit mit rund 5% nach 30 min der Ischämie und 10 bis 20% nach 60 min. Ein weiterer minimal invasive Möglichkeit ist der Einsatz von Laser-Doppler-Flussmessung (LDF) während der Operation, die eine direkte Kontrolle über seinen Erfolg ermöglicht. In einem einzelnen Tier, Reduktion auf 10 bis 20% der preocclusion Werte zeigt deutlich erfolgreicher Induktion fokaler zerebraler Ischämie 4 Allerdings LDF nicht als eine Methode für interindividuelle Vergleiche verwendet werden, da LDF nur messen kann quantitative Veränderungen (in Prozent) des Blutes. flow in einem kleinen und begrenzten Gewebeprobe Volumen. Es gibt keine Informationen über die räumliche Ausdehnung der verminderte Durchblutung. 5 Es gibt mehrere Tests, um Verhaltensweisen nach einem Schlaganfall zu beurteilen, einschließlich Ganganalyse 6,7, Rotarod 8, Pole-Test 9,10, Klebstoff Entfernung test 11,12, Treppenhaus-Test 13,14, Leitersprosse test 15,16 und Morris Water Maze 17. In all diesen Tests, Mäuse unterzogen MCAo weniger erfolgreich als die Kontrolltiere durchzuführen. <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Scheme des Schiffes Architektur der hirnversorgenden (dargestellt im Hintergrund) in der Maus. Verschiedene Stämme können Abweichungen zeigen, zum Beispiel die A. occipitalis manchmal Blätter aus der Arteria carotis interna. <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Schematische Darstellung des typischen Läsion Größen nach verschiedenen Zeitpunkten der Reperfusion in der proximalen MCAo Modell. In der Mitte sind die typischen Verlauf der funktionellen Aktivität und der zerebralen Durchblutung nach MCAo dargestellt. (MCAo: Middle Zerebralarterie, LDF: Laser-Doppler-Messung)

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M., Prass, K., Meisel, A., Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+injury-induced+immune+deficiency+syndrome.">Central nervous system injury-induced immune deficiency syndrome.</a> Nat Rev Neurosci. 6, 775-786 (2005).</li><li>Engel, O., Meisel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+Infection+Before+and+After+Stroke:+Investigating+New+Targets.">Models of Infection Before and After Stroke: Investigating New Targets.</a> Infect Disord Drug Targets. 9, (2010).</li><li>Dirnagl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bench+to+bedside:+the+quest+for+quality+in+experimental+stroke+research.">Bench to bedside: the quest for quality in experimental stroke research.</a> J Cereb Blood Flow Metab. 26, 1465-1478 (2006).</li><li>Macleod, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Good+laboratory+practice:+preventing+introduction+of+bias+at+the+bench.">Good laboratory practice: preventing introduction of bias at the bench.</a> Stroke. 40, 50-52 (2009).</li><li>Group, S. T. A. I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recommendations+for+standards+regarding+preclinical+neuroprotective+and+restorative+drug+development.">Recommendations for standards regarding preclinical neuroprotective and restorative drug development.</a> Stroke. 30, 2752-2758 (1999).</li></ol>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her, indem Landesamt fuer Gesundheit und Soziales, Berlin, Deutschland gesetzt durchgeführt.

Das Modell der transienten, proximalen MCA-Okklusion 18,19 hier vorgestellten imitiert eine der häufigsten Arten von ischämischen Schlaganfällen bei Patienten. 20 Basierend auf variable Reperfusion mal, das Modell verschiedene Sorten von Schäden von transitorische ischämische Attacke (TIA), große Angebote Infarkte darunter große Teile der ischämischen Hemisphäre. Dies ermöglicht dem Forscher zu verschiedenen pathophysiologischen Mechanismen Studie nach einem Schlaganfall. 20,21 Die Operation kann in kurzer Zeit durchgeführt werden und produziert mit einer hohen reproduzierbaren Läsionen. Dennoch ist dies eine gründliche Kontrolle von Störfaktoren erfordert. 22 Kleine Unterschiede in der Bedienung Technik kann für unterschiedliche Auswirkungen auf die Infarkt-Konto. 23,24 Außerdem ist es aufgrund Varianzen in zerebrale vaskuläre Anatomie, zeigen verschiedene Mausstämme ein anderes Ergebnis. 25,26 Körper Temperatur wirkt sich neurologische Schäden, mit Hypothermie führt zu kleineren Läsionen 27 und Hyperthermie zu schweren Defiziten. 28 Folglich Temperaturregelung und Wartung ist sehr relevant in diesem Modell. 29 Darüber hinaus, Blutdruck und Blutgase wichtige Confounder des Ergebnisses werden und müssen überwacht werden. 30,31 Der Einsatz von schnellen und minimal invasive Methoden (nicht-invasive Blutdruckmessung, geeignete und leicht zugängliche Blutentnahme Sites) wird empfohlen. Die Wahl der Anästhesie ist auch sehr wichtig, da einige neuroprotektive Effekte haben können, und / oder Vasodilatatoren, wie zB Isofluran. 32 Daher sollte die Exposition auf die Anästhesie so kurz wie möglich und genormt. Wir schließen Tiere, die operiert wurden länger als 15 min. Rasieren der chirurgischen Stelle produziert microabrasions und Entzündung und löst Haar-Fragmente. Dies kann weiter erhöhen Entzündungen und fördern lokale Infektion, die auf Schlaganfall Pathophysiologie auswirken können. Wohnverhältnisse, insbesondere die Verwendung von Bereicherung, kann sich auf Schlaganfall Ergebnis und sollten standardisiert und beschrieben in Research-Berichten. 6,33,34 Die Nutzung von Umweltressourcen Bereicherung und ihre Auswirkung auf die Reproduzierbarkeit ist eine Frage der Diskussion. 35 Ein weiterer wichtiger Confounder ist die Schlaganfall induziert Infektionsrisiko vor allem nach längeren Zeiten der Ischämie 36, was zu zusätzlichen Morbidität und erhöhter Mortalität führt. 37,38 Da Infektionen werden bei rund Tag 3 bis 5 symptomatisch, hat dies wichtige Konsequenzen für die langfristige Follow-up in solche Modelle. Um zu Ergebnissen führen, die für die Entwicklung neuartiger Behandlungsmöglichkeiten für Schlaganfall, Standardisierung und Qualitätskontrolle ist sehr wichtig in der präklinischen Translational Stroke Research 39 &quot;Good Laboratory Practice&quot; 40,41 Mandate.: <ol><li> eine angemessene und detaillierte Beschreibung der verwendeten Tiere; </li><li> Berechnung der Stichprobengröße und die Berichterstattung über den erwarteten Effekt Größe; </li><li> Ein-und Ausschlusskriterien, die vor der Studie festgelegt; </li><li> Randomisierung der Aufteilung in Gruppen; </li><li> Zuteilungsverdeckung in Bezug auf die Ermittler; </li><li> Berichterstattung Tiere aus der Analyse ausgeschlossen; </li><li> geblendet Bewertung der Ergebnisse; </li><li> Berichterstattung potentielle Interessenkonflikte und Studienfinanzierung. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Surgical scissors (skin cut)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>14028-10</td>
<td>&nbsp;</td>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>Spring scissors (according to Vannas)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>15000-00</td>
<td>&nbsp;</td>
<tr>
<td>Applying forceps for micro clamps</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00072-14</td>
<td>&nbsp;</td>
<tr>
<td>Micro vascular clamp (e.g. S&amp;G B1-V)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>00396-01</td>
<td>Also Serrefine possible</td>
</tr>
<tr>
<td>2 Hemostats according to Hartmann</td>
<td>&nbsp;</td>
<td>FST</td>
<td>13002-10</td>
<td>&nbsp;</td>
<tr>
<td>Needle holder (according Olsen-Hegar or other)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>12002-14</td>
<td>&nbsp;</td>
<tr>
<td>Standard anatomical forceps (for wound closure)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11000-12</td>
<td>&nbsp;</td>
<tr>
<td>5/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for vascular ligatures</td>
</tr>
<tr>
<td>6/0 sutures </td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>&nbsp;</td>
<td>for skin suture, 5/0 also possible</td>
</tr>
<tr>
<td>Lidocaine (e.g. Xylocain Gel 2%)</td>
<td>&nbsp;</td>
<td>Astra Zeneca</td>
<td>&nbsp;</td>
<td>or other local pain relief</td>
</tr>
<tr>
<td>Dissecting microscope (stereo microscope), magnification 6x to max. 40x</td>
<td>&nbsp;</td>
<td>Leica Zeiss</td>
<td>MZ6 Stemi2000C</td>
<td>&nbsp;</td>
<tr>
<td>Cold light source</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>KL1500</td>
<td>&nbsp;</td>
<tr>
<td>Temperature feedback controlled heating pad system</td>
<td>&nbsp;</td>
<td>FST</td>
<td>21052-00</td>
<td>With mouse pad and small probe</td>
</tr>
<tr>
<td>Anaethesia system for isoflurane</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane</td>
<td>&nbsp;</td>
<td>Abott</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Veterinary Recovery Chamber</td>
<td>&nbsp;</td>
<td>Peco Services</td>
<td>V1200</td>
<td>Heated cage</td>
</tr>
<tr>
<td>8.0 nylon filament</td>
<td>&nbsp;</td>
<td>Suprama</td>
<td>TEL181005</td>
<td>for coating</td>
</tr>
<tr>
<td>Scalpel</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For cutting filament in pieces</td>
</tr>
<tr>
<td>Ruler</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>To cut correct length of filament</td>
</tr>
<tr>
<td>Xantopren M Mucosa</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Activator Universal Plus</td>
<td>&nbsp;</td>
<td>Heraeus Kulzer</td>
<td>&nbsp;</td>
<td>Optosil - Xantopren</td>
</tr>
<tr>
<td>2 Dumont forceps (Medical #5 straight tip)</td>
<td>&nbsp;</td>
<td>FST</td>
<td>11253-20</td>
<td>&nbsp;</td>
<tr>
<td>A soft underlay for storing and grasping the uncoated filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. swabs, foam, &#x2026;</td>
</tr>
<tr>
<td>Receptacle for new build filaments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>e.g. petri dish, flexible foam,&#x2026;</td>
</tr>		</tbody>
		</table>
		</div>

modeling stroke in mice - middle cerebral artery occlusion with the filament model

Der Schlaganfall ist eine der häufigsten Ursachen für Tod und Invalidität bei Erwachsenen, vor allem in den hoch entwickelten Ländern. Allerdings sind die Behandlungsmöglichkeiten bisher sehr begrenzt. Um den Bedarf an neuen Therapieansätzen, experimentellen Schlaganfall-Forschung häufig beschäftigt Nagermodellen fokaler zerebraler Ischämie. Die meisten Forscher nutzen dauerhafte oder vorübergehende Okklusion der mittleren zerebralen Arterie (MCA) in Mäusen oder Ratten. Proximalen Verschluss der mittleren Hirnarterie (MCA) über die intraluminale Nahttechnik (so genannte Filament oder Faden-Modell) ist wahrscheinlich die am häufigsten verwendete Modell in der experimentellen Schlaganfall-Forschung. Die intraluminale MCAO Modell bietet den Vorteil der Induktion reproduzierbare vorübergehende oder dauerhafte Ischämie der MCA Gebiet in einer relativ nicht-invasive Art und Weise. Intraluminale Ansätze unterbrechen die Durchblutung des gesamten Territoriums von dieser Arterie. Filament Okklusion damit Verhaftungen flow proximal des lenticulo-gestreifte Arterien, die den Basalganglien zu versorgen. Filament Verschluss der MCA Ergebnisse in reproduzierbaren Läsionen im Kortex und Striatum und kann entweder dauerhaft oder vorübergehend. Im Gegensatz dazu Modellen induzieren distalen (an der Verzweigung der lenticulo-gestreifte Arterien) MCA-Okklusion in der Regel ersparen das Striatum und in erster Linie um den Neocortex. Darüber hinaus sind diese Modelle erfordern Kraniektomie. In das Modell in diesem Artikel gezeigt, ist ein Silizium-beschichteten Faden, der in der Arteria carotis communis eingeführt und entlang der A. carotis interna in den Kreis der Willis, wo es blockiert den Ursprung der Arteria cerebri media. Bei Patienten, sind Verschlüsse der A. cerebri media zu den häufigsten Ursachen für einen ischämischen Schlaganfall. Da unterschiedliche ischämische Abständen frei in diesem Modell gewählt werden, je nach Zeitpunkt der Reperfusion, ischämische Läsionen, die mit unterschiedlichen Schweregraden hergestellt werden können. Reperfusion durch Entfernen der Okklusion Filament zumindest teilweise Modelle der Wiederherstellung der Durchblutung nach spontaner oder therapeutischer (tPA) Lyse einer thromboembolischen Blutgerinnsel in den Menschen. In diesem Video stellen wir Ihnen die grundlegende Technik sowie die wichtigsten Fallstricke und Störfaktoren, die den prädiktiven Wert dieses Modells beschränken können.

Watch this Scientific Journal Video about Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model at JoVE.com

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

Filamentous Verschluss der mittleren Hirnarterie ist ein gemeinsames Modell für die Untersuchung einen ischämischen Schlaganfall bei Mäusen.

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 ...

modeling-stroke-mice-middle-cerebral-artery-occlusion-with-filament

Research

JoVE Journal

Medicine

91.5K Aufrufe.  Center for Stroke Research Berlin, Charité Universitätsmedizin. Filamentous Verschluss der mittleren Hirnarterie ist ein gemeinsames Modell für die Untersuchung einen ischämischen Schlaganfall bei Mäusen.

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

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 ...

Forschung

Medizin

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminale Nahen Zerebralarterie (MCAO) Modell des ischämischen Schlaganfalls mit Laser-Doppler-Flussmessung Guidance in Mäuse

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

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 ...

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

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 performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

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 ...

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 ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Modellierung Schlaganfall bei Mäusen: Permanent Koagulation der distalen A. cerebri media

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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, ...