Die chirurgische Technik wurde ursprünglich im Labor von Dr. William D. Hill am Medical College of Georgia erworben. Die Autoren möchten auch Dr. David A. Rempe und Landa Prifti für die Nutzung der Dissektion Kamera danken. Diese Arbeit wurde vom NIH NS041744, NS051279, F31 NS064700 und AHA 30815697D unterstützt.

Dieses Protokoll wurde von der University of Rochester Ausschuss sich mit der ethischen Verwendung von Tieren in der Forschung (UCAR) zugelassen. Aseptische Techniken sollten in das Protokoll befolgt werden. Die Verwendung von sterilen Handschuhen und einer Maske erforderlich ist. Alle Geräte, Materialien, Chemikalien und Werkzeuge, die während des Protokolls verwendet werden, sind in Tabelle 1 beschrieben. 1. Präoperative Vorbereitung <ol><li> Inject Mäusen subkutan mit Buprenorphin (0.05mg/kg) 2 h vor der Operation, unmittelbar nach der Operation und dann alle 3-5 h während der ersten 24 h postoperativen Phase. </li><li> Sterilisieren aller chirurgischen Instrumente, Gaze Schwämme und Wattestäbchen Applikatoren von Autoklaven. Halten Sie chirurgische Instrumente in 70% igem Ethanol während der Operation und an der Luft trocknen auf steriler Gaze rechts vor dem Gebrauch. Sprühen Sie den Arbeitsbereich mit 70% Ethanol. </li><li> Anesthetize der Maus mit 3% Isofluoran-20% Sauerstoff-Gasgemisch durch ein Betäubungsmittel Verdampfer. Passen Sie die Sauerstoffmenge mit dem Durchflussmesser. Testen Sie das Niveau der Anästhesie durch Zehe oder Schwanz kneifen (sie sollten nicht mehr reagiert). Halten Sie das Niveau der Anästhesie mit 2% (v / v) Isofluoran. </li><li> Bewerben künstliche Tränen, um die Maus in die Augen und seien Sie vorsichtig, um eine Beschädigung des Auges während der Operation. Platzieren Sie die Maus auf der rechten Seite in eine seitliche Position. </li><li> Shave Bereich auf der linken Seite zwischen dem linken Auge und die Basis des linken Ohres mit dem Tierschermaschinen. Reinigen Sie die Fläche durch den Wechsel zwischen einem betadine-Lösung und 70% Ethanol mit Wattestäbchen Applikatoren und leichtes Reiben der Gegend. Bei Bedarf wiederholen. </li><li> Legen Sie die Maus auf ein Heizkissen an eine rektale Sonde auf Körpertemperatur bei 37 ° C zu halten Legen Sie die rektale Sonde mit Mineralöl. </li><li> Bewegen Sie die Maus auf der rechten Seite unter dem Mikroskop und sicher mit Klebeband. Schneiden Sie ein Fenster in einem Gaze-Schwamm und Abdeckung OP-Bereich. </li></ol> 2. Chirurgische Verfahren und MCA Ligation <ol><li> Machen Sie einen vertikalen Einschnitt zwischen dem linken Auge und die Basis des linken Ohres mit feinen geraden Schere. Verwenden Sie gebogenen Hämostatika zu halten OP-Bereich zu öffnen. </li><li> Machen Sie ein horizontaler Schnitt auf der Schläfenmuskels mit Feder Schere und etwas trennen Schläfenmuskels aus dem Schädel durch langsames Ziehen mit der Pinzette. </li><li> Machen Sie eine kleine subtemporal Kraniotomie an der Kreuzung der Jochbogen und Squamosum durch Verwendung einer 18G Nadel, während der Kieferknochen mit einer gebogenen Pinzette. </li><li> Expose der MCA durch das Entfernen der kleine Schädel mit Knochen Rongeure. Der Jochbogen und Orbital Inhalte sollten dabei nicht beschädigt werden. Wenn übermäßige Trocknung des Gewebes auftritt, während dieses Prozesses, steriler PBS mit Wattestäbchen Applikatoren. </li><li> Ligieren des distalen Teil der MCA mit einem kleinen Schiff cauterizer. </li><li> Legen Sie die Schläfenmuskels wieder in seine ursprüngliche Position und schließen Sie die Inzisionsstelle mit chirurgischen 5-0 Nylonnaht. </li><li> Unterbrechen Sie Anästhesie und entfernen Sie die rektale Sonde. Spritzen Sie die Maus mit einer 2. Dosis von Buprenorphin (0.05mg/kg) subkutan. Bringen Sie die Maus, um den Käfig, der auf 37 ° C wurde mit einer Heizplatte. </li><li> Genau beobachten, mit der Maus für die nächsten 24 h für alle Beschwerden (Appetitlosigkeit / Wasserverbrauch, gekrümmte Haltung, beschleunigte Atmung, Pilo-errichtet Haare). Spritzen Sie die Maus mit Buprenorphin subkutan alle 3-5 h nach der Operation bis zu 24 Stunden. Geben Sie die Maus mit Recovery-Gel in diesem Zeitraum. </li></ol> 3. TTC-Färbung und Bestimmung der Stroke Volume <ol><li> Vierundzwanzig Stunden nach der Operation tief betäuben die Maus, transkardial perfuse der Maus mit 4% TTC (w / v) in Phosphat-gepufferter Kochsalzlösung (PBS) für 15 min und dann mit einer 4% Paraformaldehyd-Lösung für 10 min mit einem Mini-Schlauchpumpe bei mittlerer Strömungsgeschwindigkeit. Entfernen Sie das Gehirn und legen Sie sie in die 4% Paraformaldehyd-Lösung über Nacht. </li><li> Position des Gehirns in die Schnitte zu blockieren. Scheiben des Gehirns in 1mm dicke Scheiben schneiden mit Rasierklingen. </li><li> Legen Sie die Scheiben neben einer Millimeter-Skala Lineal. Fotografieren Sie die Scheiben mit einer Digitalkamera mit dem Binokular. </li><li> Berechnen Sie den Hubbereich durch Subtraktion der nicht-Infarktareal der ipsilateralen Seite von der Gesamtfläche der kontralateralen Seite mit Image J Software (http://rsbweb.nih.gov/ij/). Berechnen Sie das Hubvolumen durch Stapeln der Hubbereich der Scheiben 22. </li></ol> 4. Anfahrt zu nutzen Image J Software <ol><li> Öffnen Sie die Bilddatei in Image J Software, indem Sie auf das Menü Datei analysiert werden. </li><li> Klicken Sie auf den &quot;geraden Linie&quot; Tab im Programm. Zeichnen einer geraden Linie zwischen den beiden Rändern auf dem Lineal. Klicken Sie auf &quot;Analyse&quot;-Menü und wählen Sie &#39;set scale&#39;. Set dem bekannten Abstand als 1, Einheit der Länge als mm. </li><li> Klicken Sie auf &quot;Freihand Kreis-Register. Zeichne einen Kreis um die kontralateralen Hemisphäre zu skizzieren. Klicken Sie auf &quot;Analyse&quot;-Menü und wählen Sie &quot;Maßnahme&quot;. Die Berechnung Fenster öffnet sich, welche die Fläche des Kreises gezogen. </li><li> Draw noch ein Kreis um den ipsilateralen Hemisphäre ohne Hub (weiss) Bereich. Man misst die Fläche, wie in Schritt 4.3 angegeben. Der neue Wert wird auf die Berechnung Fenster hinzugefügt werden. Der Unterschied zwischen der 1. und 2. Werte stellt den Bereich des Infarktes, die als A in der folgenden Formel angegeben ist. </li><li> Berechnen Sie den Hubbereich in jeder Scheibe durch Wiederholen der Schritte 4,2-4,4. Nehmen Summation der Hubbereich in jede Scheibe (ΣA n) berechnet. Dies entspricht Hubvolumen Kenntnis der Tatsache, dass die Dicke jeder Scheibe 1mm ist. ΣA n x 1mm (Dicke jeder Scheibe) = Hubvolumen </li></ol> 5. Repräsentative Ergebnisse: Die Infarkte durch permanente MCA Ligation in der Maus gewonnen werden meist kortikalen. Allerdings ist es möglich, subkortikale Läsionen zu erhalten, wenn der MCA ist ligiert proximal des lentikulostriatalen Branche. Das Hubvolumen nach MCA Ligation könnte von 10mm bis 35mm ³ ³ 19, 23 variieren. Das Hubvolumen mit TTC-Färbung in Moyanova et al. 19 sind zwischen 10mm bis 22mm ³ ³, während die Hubvolumen von MRT-Bildern in Filiano et al 23 sind zwischen 20 mm ³ bis 35mm ³ bestimmt. Ein möglicher Grund für diese Unterschiede vielleicht die genaue Lage der Ligation und die verschiedenen Methoden verwendet, um die Hubvolumen zu messen. In Abbildung 1 ist ein TTC gefärbt Gehirn 24 Stunden nach dauerhaften MCA Ligation in einem Wildtyp-C57/BL6 Maus gezeigt. Der Hub Site ist weiß in Erscheinung (Abb. 1A, B). Abbildung 1A und B zeigen die Infarkt Ort aus verschiedenen Blickwinkeln. Abbildung 2 zeigt 1mm dicke Scheiben des TTC gefärbt Schlaganfall Gehirn von vorne nach hinten (Abb. 2A-G). Infarktvolumen berechnet wurde 24 h nach der Operation. Das Volumen des Infarkts ist ~ 23mm ³. <img src="/files/ftp_upload/3039/3039fig1.jpg" alt="Abbildung 1"/> Abbildung 1. Vertretung von Schlaganfallpatienten vor Ort. AB, gebeizt TTC ganze Gehirn Bilder, 24 h nach der MCA-Ligation. Weiß-Bereich stellt die Infarkt. <img src="/files/ftp_upload/3039/3039fig2.jpg" alt="Abbildung 2"/> Abbildung 2. Repräsentative Ergebnisse. (A&gt; G), TTC gefärbt 1mm Hirnschnitten, 24 h nach MCAL. Scheiben sind von vorne nach hinten ausgerichtet. Weiß-Bereich stellt die Infarkt.

<ol>
	<li>Britton, M., Rafols, J., Alousi, S., Dunbar, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+middle+cerebral+artery+occlusion+on+central+nervous+system+apoptotic+events+in+normal+and+diabetic+rats.">The effects of middle cerebral artery occlusion on central nervous system apoptotic events in normal and diabetic rats.</a> Int J Exp Diabesity Res. 4, 13-20 (2003).</li><li>Ciceri, P., Rabuffetti, M., Monopoli, A., Nicosia, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+leukotrienes+in+a+model+of+focal+cerebral+ischaemia+in+the+rat.">Production of leukotrienes in a model of focal cerebral ischaemia in the rat.</a> Br J Pharmacol. 133, 1323-1329 (2001).</li><li>Jin, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+transplantation+of+human+neural+precursor+cells+improves+outcome+from+focal+cerebral+ischemia+in+aged+rats.">Delayed transplantation of human neural precursor cells improves outcome from focal cerebral ischemia in aged rats.</a> Aging Cell. 9, 1076-1083 (2010).</li><li>McCaig, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+GADD34+expression+after+focal+cerebral+ischaemia.">Evolution of GADD34 expression after focal cerebral ischaemia.</a> Brain Res. 1034, 51-61 (2005).</li><li>Shirotani, T., Shima, K., Chigasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+studies+of+extracellular+metabolites+in+the+striatum+after+distal+middle+cerebral+artery+occlusion+in+stroke-prone+spontaneously+hypertensive+rats.">In vivo studies of extracellular metabolites in the striatum after distal middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats.</a> Stroke. 26, 878-884 (1995).</li><li>Wu, Y. P., Tan, C. K., Ling, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Fos-like+immunoreactivity+in+the+brain+and+spinal+cord+of+rats+following+middle+cerebral+artery+occlusion.">Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion.</a> Exp Brain Res. 115, 129-136 (1997).</li><li>Tamura, A., Graham, D. I., McCulloch, J., Teasdale, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+cerebral+ischaemia+in+the+rat:+1.+Description+of+technique+and+early+neuropathological+consequences+following+middle+cerebral+artery+occlusion.">Focal cerebral ischaemia in the rat: 1. Description of technique and early neuropathological consequences following middle cerebral artery occlusion.</a> J Cereb Blood Flow Metab. 1, 53-60 (1981).</li><li>Bederson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+middle+cerebral+artery+occlusion:+evaluation+of+the+model+and+development+of+a+neurologic+examination.">Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination.</a> Stroke. 17, 472-476 (1986).</li><li>Carswell, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+gender+influences+on+sensitivity+to+focal+cerebral+ischemia+in+the+stroke-prone+spontaneously+hypertensive+rat.">Genetic and gender influences on sensitivity to focal cerebral ischemia in the stroke-prone spontaneously hypertensive rat.</a> Hypertension. 33, 681-685 (1999).</li><li>Mary, V., Wahl, F., Uzan, A., Stutzmann, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enoxaparin+in+experimental+stroke:+neuroprotection+and+therapeutic+window+of+opportunity.">Enoxaparin in experimental stroke: neuroprotection and therapeutic window of opportunity.</a> Stroke. 32, 993-999 (2001).</li><li>Menzies, S. A., Hoff, J. T., Betz, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Middle+cerebral+artery+occlusion+in+rats:+a+neurological+and+pathological+evaluation+of+a+reproducible+model.">Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model.</a> Neurosurgery. 31, 100-107 (1992).</li><li>Iaci, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+growth+factor+2+promotes+functional+recovery+with+treatment+initiated+up+to+7+days+after+permanent+focal+ischemic+stroke.">Glial growth factor 2 promotes functional recovery with treatment initiated up to 7 days after permanent focal ischemic stroke.</a> Neuropharmacology. 59, 640-649 (2010).</li><li>Richard, M. J. P., Khan, B. V. C. B. J., Saleh, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+mechanisms+by+which+lipoic+acid+confers+protection+during+the+early+stages+of+cerebral+ischemia:+A+possible+role+for+calcium.">Cellular mechanisms by which lipoic acid confers protection during the early stages of cerebral ischemia: A possible role for calcium.</a> Neuroscience Research. , (2011).</li><li>Heiss, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+derangement+of+periinfarct+viable+tissue+in+ischemic+stroke.">Progressive derangement of periinfarct viable tissue in ischemic stroke.</a> J Cereb Blood Flow Metab. 12, 193-203 (1992).</li><li>Nedergaard, M., Gjedde, A., Diemer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+ischemia+of+the+rat+brain:+autoradiographic+determination+of+cerebral+glucose+utilization,+glucose+content,+and+blood+flow.">Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow.</a> J Cereb Blood Flow Metab. 6, 414-424 (1986).</li><li>Nowicki, J. P., Assumel-Lurdin, C., Duverger, D., MacKenzie, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+evolution+of+regional+energy+metabolism+following+focal+cerebral+ischemia+in+the+rat.">Temporal evolution of regional energy metabolism following focal cerebral ischemia in the rat.</a> J Cereb Blood Flow Metab. 8, 462-473 (1988).</li><li>Butcher, S. P., Bullock, R., Graham, D. I., McCulloch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+amino+acid+release+and+neuropathologic+outcome+in+rat+brain+following+middle+cerebral+artery+occlusion.">Correlation between amino acid release and neuropathologic outcome in rat brain following middle cerebral artery occlusion.</a> Stroke. 21, 1727-1733 (1990).</li><li>Arlicot, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+remote+microglial+activation+in+rodent+models+of+focal+ischaemia+using+the+TSPO+radioligand+CLINDE.">Detection and quantification of remote microglial activation in rodent models of focal ischaemia using the TSPO radioligand CLINDE.</a> Eur J Nucl Med Mol Imaging. 37, 2371-2380 (2010).</li><li>Moyanova, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+for+type+4+metabotropic+glutamate+receptors+against+ischemic+brain+damage.">Protective role for type 4 metabotropic glutamate receptors against ischemic brain damage.</a> J Cereb Blood Flow Metab. , (2010).</li><li>Ortolano, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+imaging+of+new+targets+for+pharmacological+intervention+in+stroke:+real-time+tracking+of+T-cells+in+the+ischaemic+brain.">Advances in imaging of new targets for pharmacological intervention in stroke: real-time tracking of T-cells in the ischaemic brain.</a> Br J Pharmacol. 159, 808-811 (2010).</li><li>O'Neill, M. J., A, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rodent+models+of+focal+cerebral+ischemia.">Rodent models of focal cerebral ischemia.</a> Current Protocols in Neuroscience. , 9.6.1-9.6.32 (2000).</li><li>Lin, T. N., He, Y. Y., Wu, G., Khan, M., Hsu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+brain+edema+on+infarct+volume+in+a+focal+cerebral+ischemia+model+in+rats.">Effect of brain edema on infarct volume in a focal cerebral ischemia model in rats.</a> Stroke. 24, 117-121 (1993).</li><li>Filiano, A. J., Tucholski, J., Dolan, P. J., Colak, G., Johnson, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transglutaminase+2+protects+against+ischemic+stroke.">Transglutaminase 2 protects against ischemic stroke.</a> Neurobiol Dis. 39, 334-343 (2010).</li></ol>

Die permanente MCA Ligationsmethode gibt gut reproduzierbare Infarktvolumen und erhöhte postoperative Überlebensrate im Vergleich zu anderen Methoden zur Verfügung. Die Leichtigkeit und der kurzen Dauer (~ 30 min) des Verfahrens machen es noch praktischer. Die Methode ist weit verbreitet in Mäusen und Ratten verwendet. Diese Technik erfordert einen invasiven Chirurgie unter einem Stereomikroskop. Deshalb ist Erfahrung im Betrieb unter einem Mikroskop und Perfektionierung einer erfolgreic...

<table border="1"><tbody><tr><th> NAMEN </th><th> UNTERNEHMEN UND Katalognummer </th></tr><tr><td> A. Solutions and Chemicals </td><td></td></tr><tr><td> Povidon-Jod-Lösung, Surgical Scrub </td><td> Aplicare, 82-209 </td></tr><tr><td> 70% Ethanol </td><td> Koptec, V1001 </td></tr><tr><td> 2,3,5 Triphenyltetrazoliumchlorid (TTC) </td><td> Sigma, T8877 </td></tr><tr><td> 4% Paraformaldehyd </td><td> Electron Microscopy Sciences, 159SP </td></tr><tr><td> Phosphate Buffered Saline, pH: 7,4 </td><td> Auf und sterilisiert im Labor. </td></tr><tr><td> Mineralöl </td><td> PML Microbiologicals, R6570 </td></tr><tr><td> Isofluoran </td><td> Der Butler Company, 029405 </td></tr><tr><td> Buprenorphin (0.3mg/ml) </td><td> Hospira, NDC 0409-2012-32 </td></tr><tr><td> Künstliche Tränen </td><td> Der Butler Company, 007312 </td></tr><tr><td> Recovery-Gel </td><td> Klare H 2 O, 72-01-5022 </td></tr><tr><td> B. chirurgische Materialien und Geräte </td><td></td></tr><tr><td> C57/BL6 Maus, 10-12 Wochen alt </td><td> Die Kolonie wurde in der Universitätsstadt Vivarium gezüchtet </td></tr><tr><td> 100% Sauerstoff-Tank </td><td> Airgas </td></tr><tr><td> Anästhetika Verdampfer und Durchflussmesser </td><td> Surgivet, Modell 100 </td></tr><tr><td> Heizkissen mit Rektalsonde </td><td> Feine Science Tools, TR200 </td></tr><tr><td> Präpariermikroskop </td><td> Zeiss, Stemi 2000 </td></tr><tr><td> Heizplatte </td><td> Petco Services Ltd 0307-013 </td></tr><tr><td> Small Vessel Cauterizer Set </td><td> Feine Science Tools, 18000-00 </td></tr><tr><td> Mini Schlauchpumpen </td><td> Harvard Apparatus, MPII </td></tr><tr><td> 1ml Spritze </td><td> BD, 329650 </td></tr><tr><td> 18G Nadel </td><td> Becton Dickinson, 305196 </td></tr><tr><td> Chirurgische 5,0 Nylonnaht </td><td> Ethilon, 698H </td></tr><tr><td> Sectioning blockieren </td><td> Kent Scientific, 1mm </td></tr><tr><td> Single Edge Razor Blades </td><td> Electron Microscopy Sciences, 71962 </td></tr><tr><td> Etikettenband </td><td> Scienceware, Bel-art Produkte F13463-2075 </td></tr><tr><td> Gazetupfer </td><td> Kendall cherheit 2252, Johnson und Johnson 6415 </td></tr><tr><td> Cotton Tip Applikatoren </td><td> Puritan 806-WCL </td></tr><tr><td> C. Chirurgie Werkzeuge </td><td></td></tr><tr><td> Chirurgische Frühling Schere </td><td> Feine Science Tools 15003-08 </td></tr><tr><td> Microdissecting Pinzette abgewinkelt </td><td> Feine Science Tools 5 / 45 </td></tr><tr><td> Microdissecting Pinzette gebogen </td><td> Feine Science Tools 5 Ti </td></tr><tr><td> Feine gerade Schere </td><td> Feine Science Tools 14568-12 </td></tr><tr><td> Gebogene Knochen Rongeur </td><td> Feine Science Tools 16021-14 </td></tr><tr><td> Hartman Hemostat Curved </td><td> Feine Science Tools 13003-10 </td></tr></tbody></table> Tabelle 1. Die Liste der Materialien, Werkzeuge und Chemikalien für die MCA Ligation Verfahren verwendet. A. Solutions and Chemicals, B. chirurgische Materialien und Equipment, C. Surgery Tools.

the application of permanent middle cerebral artery ligation in the mouse

Fokaler zerebraler Ischämie ist eine der häufigsten Form des Schlaganfalls bei Patienten gesehen. Durch die klinische Bedeutung hat es eine längere Anstrengung, um geeignete Tiermodelle zu entwickeln, um die Ereignisse, die während der ischämischen Insult entfalten Studie. Diese Techniken umfassen vorübergehend oder bleibend, fokale oder globale Ischämie Modelle mit vielen verschiedenen Tiermodellen, die häufigsten sind Nagetiere. Die permanente MCA Ligationsmethode die auch als pMCAo ist in der Literatur wird weitgehend als fokale Ischämie-Modell in Nagetieren 1-6 verwendeten. Diese Methode wurde ursprünglich für die Ratten durch Tamura et al. im Jahr 1981 sieben. In diesem Protokoll eine Kraniotomie wurde verwendet, um Zugriff auf die MCA und der proximalen Regionen durch Elektrokoagulation waren verschlossen. Die Infarkte betreffen meist kortikale und manchmal striatalen Regionen, je nach Lage der Okklusion. Diese Technik ist inzwischen gut etabliert und wird in vielen Labors 8-13. Der frühe Einsatz dieser Technik führte zur Definition und Beschreibung von &quot;Infarktkern&quot; und &quot;Penumbra&quot; 14-16, und es wird oft benutzt, um potenzielle neuroprotektive Verbindungen 10, 12, 13, 17 zu bewerten. Obwohl die anfänglichen Studien an Ratten durchgeführt wurden, hat permanent MCA Ligation erfolgreich an Mäusen mit leichten Modifikationen 18-20 verwendet. Dieses Modell liefert reproduzierbare Infarkte und eine erhöhte post-Überlebensraten. Etwa 80% der ischämischen Schlaganfälle bei Menschen in der MCA Bereich 21 passieren und somit ist dies ein sehr wichtiges Modell für Schlaganfall-Studien. Derzeit gibt es einen Mangel an wirksamen Therapien zur Verfügung Schlaganfall-Patienten, und somit gibt es einen Bedarf für gute Modelle, um potenzielle pharmakologische Verbindungen testen und zu evaluieren physiologische Ergebnisse. Diese Methode kann auch für die intrazelluläre Hypoxie Reaktionsmechanismen in vivo verwendet werden. Hier präsentieren wir die MCA Ligation Operation in einem C57/BL6 Maus. Wir beschreiben die präoperative Vorbereitung, MCA Ligation Chirurgie und 2,3,5 Triphenyltetrazoliumchlorid (TTC)-Färbung für die Quantifizierung der Infarktgröße Bände.

Watch this Scientific Journal Video about The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse at JoVE.com

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

A. cerebri media (MCA) Ligation ist eine Technik, fokaler zerebraler Ischämie im Tiermodell zu studieren. Bei dieser Methode ist die mittlere Hirnarterie durch Kraniotomie freigelegt und ligiert durch Kauterisation. Diese Methode liefert gut reproduzierbare Infarktvolumen und erhöhte postoperative Überlebensrate im Vergleich zu anderen Methoden zur Verfügung.

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 ...

the-application-permanent-middle-cerebral-artery-ligation

Research

JoVE Journal

Medicine

19.5K Aufrufe.  University of Rochester. A. cerebri media (MCA) Ligation ist eine Technik, fokaler zerebraler Ischämie im Tiermodell zu studieren. Bei dieser Methode ist die mittlere Hirnarterie durch Kraniotomie freigelegt und ligiert durch Kauterisation. Diese Methode liefert gut reproduzierbare Infarktvolumen und erhöhte postoperative Überlebensrate im Vergleich zu anderen Methoden zur Verfügung.

Serie: The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

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 ...

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 ...

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) 1. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex 2, is the most common site of human stroke 3, and ischemia within its territory can result in extensive dysfunction or death 1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.
Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

We describe a highly reproducible method for the permanent occlusion of a rodent major cerebral blood vessel. This technique can be accomplished with very little peripheral damage, minimal blood loss, a high rate of long-term survival, and consistent infarct volume commensurate with the human clinical population.

Permanent Cerebral Gefäßverschluss<em&gt; Über</em&gt; Double Blattschraube und Durchtrennung

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow ...

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 ...

Permanent Ligation of the Left Anterior Descending Coronary Artery in Mice: A Model of Post-myocardial Infarction Remodelling and Heart Failure

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It is characterized by fluid retention, shortness of breath, and fatigue, in particular on exertion. Heart failure is a growing public health problem, the leading cause of hospitalization, and a major cause of mortality. Ischemic heart disease is the main cause of heart failure.
Ventricular remodelling refers to changes in structure, size, and shape of the left ventricle. This architectural remodelling of the left ventricle is induced by injury (e.g., myocardial infarction), by pressure overload (e.g., systemic arterial hypertension or aortic stenosis), or by volume overload. Since ventricular remodelling affects wall stress, it has a profound impact on cardiac function and on the development of heart failure. A model of permanent ligation of the left anterior descending coronary artery in mice is used to investigate ventricular remodelling and cardiac function post-myocardial infarction. This model is fundamentally different in terms of objectives and pathophysiological relevance compared to the model of transient ligation of the left anterior descending coronary artery. In this latter model of ischemia/reperfusion injury, the initial extent of the infarct may be modulated by factors that affect myocardial salvage following reperfusion. In contrast, the infarct area at 24 hr after permanent ligation of the left anterior descending coronary artery is fixed. Cardiac function in this model will be affected by 1) the process of infarct expansion, infarct healing, and scar formation; and 2) the concomitant development of left ventricular dilatation, cardiac hypertrophy, and ventricular remodelling.
Besides the model of permanent ligation of the left anterior descending coronary artery, the technique of invasive hemodynamic measurements in mice is presented in detail.

Heart failure is the leading cause of hospitalization and a major cause of mortality. A model of permanent ligation of the left anterior descending coronary artery in mice is applied to investigate ventricular remodelling and cardiac dysfunction post-myocardial infarction. The technique of invasive hemodynamic measurements in mice is presented.

Permanent Ligation des linken vorderen absteigenden Koronararterie bei Mäusen: Ein Modell der Post-Myokardinfarkt Umbau und Herzinsuffizienz

Heart failure is a syndrome in which the heart fails to pump blood at a rate commensurate with cellular oxygen requirements at rest or during stress. It ...

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, ...

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 ...

Middle Cerebral Artery Occlusion Allowing Reperfusion via Common Carotid Artery Repair in Mice

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue plasminogen activator (tPA) as an approved drug treatment to target ischemic strokes. Current research in the field of ischemic stroke focuses on better understanding the pathophysiology of stroke, to develop and investigate novel pharmaceutical targets. Reliable experimental stroke models are crucial for the progression of potential treatments. The middle cerebral artery occlusion (MCAO) model is clinically relevant and the most frequently used surgical model of ischemic stroke in rodents. However, the outcomes of this model, such as lesion volume, are associated with high levels of variability, particularly in mice. The alternative MCAO model described here allows the reperfusion of the common carotid artery (CCA) and the increased perfusion of the middle cerebral artery (MCA) territory, using a tissue pad with fibrinogen-based sealant to repair the vessel, and the improved welfare of the mice by avoiding external carotid artery (ECA) ligation. This reduces the reliance on the Circle of Willis, which is known to be highly anatomically variable in mice. Representative data show that using this alternative surgical approach decreases the variability in lesion volumes between the traditional MCAO approach and the alternative approach described here.

Intraluminal filament occlusion of the middle cerebral artery is the most frequently used in vivo model of experimental stroke in rodents. An alternative surgical approach to allow common carotid artery repair is performed here, which allows the reperfusion of the common carotid artery and a full reperfusion to the middle cerebral artery territory.

Mittlere zerebrale Arterie Okklusion so dass Reperfusion über gemeinsame Halsschlagader Reparatur bei Mäusen

The ischemic stroke is a major cause of adult long-term disability and death worldwide. The current treatments available are limited, with only tissue ...