Bu çalışma hibe anlaşması n ° 201.024 ve n ° 202.213 (Avrupa İnme Network) kapsamında Avrupa Topluluğu Yedinci Çerçeve Programı (FP7/2007-2013) tarafından finanse edildi. Ek finansman Bundesministerium für Bildung und Forschung (Stroke Araştırma Berlin Merkezi) ve Deutsche Forschungsgemeinschaft (Exzellenzcluster NEUROCURE) tarafından verildi. Yazarlar animasyon üretmek için çalışma ve Elke Ludwig (Charité Video Servisleri) Mareike Thielke (Deneysel Nöroloji Dep., Charite Berlin) sırasında destek için teşekkür etmek istiyorum.

Yüksek kaliteli ve tekrarlanabilirlik garanti etmek için, Standart Çalışma Prosedürleri (SOP) kullanmanızı tavsiye ederiz. Bu video, laboratuvarda özel olarak geliştirilen ve kullanılan yayınlanan SOP uygulanır . 1 1. Orta Serebral Arter Tıkanıklığı <ol><li> Fareler, veteriner personel ile istişarede uygun bir anestezi rejimi ile anestezi . (Örnek: indüksiyon 1.5 - 1.0 ile% 2 İsofluranın ve bakım - 2 / 3 N2O ve bir buharlaştırıcı kullanarak 1 / 3 O2 İsofluranın% 1.5). <ol><li> Farelerin Vücut sıcaklığı 36,5 ° C ± 0.5 ° C ısıtma plakası ile ameliyat sırasında korunur. Fare rektal sıcaklık göre ısıtan bir geri besleme kontrollü bir ısıtma yastığı, şiddetle tavsiye edilir. </li><li> Deri ve kürk çevredeki uygun bir ajan (örneğin% 70 etil alkol) ile dezenfekte edin ve daha sonra kurulayın. </li></ol></li><li> Bir orta hat boyun insizyonu yapılır ve yumuşak dokuların dışında çekti. </li><li> Sol karotis arter (LCCA) dikkatle çevresindeki sinirler (vagus siniri zarar vermeden) ve ligatür 6.0/7.0 dize kullanılarak yapılır disseke edilir. 5.0 dize de kullanılabilir. </li><li> Sol eksternal karotis arter (Leça) sonra ayrılır ve ikinci bir düğüm yapılır. </li><li> Sonra, sol internal karotid arter (Lica) izole edilir ve bir düğüm bir filament 6.0 ile hazırlanmıştır. </li><li> Sol internal karotid arter (Lica) ve sol pterigopalatin arter (LPA) iyi bir görüş aldıktan sonra, hem de arterlerde mikrovasküler klip kullanarak kırpılır. </li><li> Leca ve Lica çatallanmasıdır önce küçük bir delik LCCA kesilir. Silikon sertleştirici karışımı (aşağıya bakınız) ile kaplı 8.0 naylondan yapılmış bir monofilament klibi durana kadar sonra, Lica tanıtıldı. Dikkat oksipital arter girmek için ödenecek. (Şekil 1) </li><li> Filament Willis daire LMCA kökeni kapamak için Lica takılı iken kırpılır arterler açıldı. </li><li> Lica üçüncü düğüm filament konumunu düzeltmek için kapalıdır. </li><li> Fareler hacim ikmal olarak subkutan 0,5 mL salin alırsınız. Ağrı kesici, Lidokain jel topikal yara uygulanır. </li><li> Reperfüzyon amaçlanan ise, fareler için 30 kalır ısıtılmış bir kafes içinde 90 dakika oklüzyon, yara küçük bir dikiş klip ile kapalı olabilir. Daha sonra, ikinci bir anestezi yapılır, ICA üçüncü düğüm anlık açılan ve filament geri. </li><li> Kalan dikişler kısaltılmış ve deri cerrahi sütür ile uyarlanmıştır. </li><li> Tüm hayvanlar, yukarıda açıklandığı gibi ikinci bir birim ikmal alırsınız. </li><li> Hayvanlar vücut ısısını kontrol etmek için iki saat boyunca ısıtmalı bir kafese konur. <ol><li> Hayvanların rahatsızlık belirtileri için ameliyat sonrası günlük olarak kontrol edilmelidir. Fareler bazı cerrahi sonrası kilo kaybı gösterebilir. Onlar yeme teşvik etmek için zemin üzerine yerleştirilen bir petri-çanak püresi gıda alırsınız. Yemek yedi gün boyunca günlük olarak değiştirilir. </li></ol></li></ol> 2. Şam Operasyonu <ol><li> Sahte işlemler için filament LMCA kapamak için yerleştirilir ve anında reperfüzyon (1.8) izin hemen geri çekilir. Ikinci bir anestezi de dahil olmak üzere daha sonraki çalışma serebral iskemi (1.14 1.9) gören hayvanlar üzerinde yapılan aynıdır. </li></ol> 3. Filament hazırlanması <ol><li> Filament Sterilite düşünülmelidir. Sterilize filament yanı sıra, uygun bir işleme ekipman kullanımı daha sonra steril bir cerrahi için bir ön koşuldur. Filament Dezenfeksiyon, zor, birçok ortak sterilizasyon yöntemleri filament kalite kötüleştirebilir. Ancak, radyasyon gibi yöntemlerle, UV ışık veya γ ışınları, veya kimyasal sterilizasyon örneğin, örneğin, etilen oksit gibi yüksek reaktif gazlar ile uygulanabilir. </li><li> 8.0 naylon filament mikroskop altında 11 mm uzunluğa bölünür </li><li> Filament ucu Xantopren M Mukoza ve aktivatör NF Optosil sertleştirici karışımı ile 8 mm uzunluğu boyunca tamamen ve eşit şekilde kaplanmış olmalıdır </li></ol> 4. Temsilcisi Sonuçlar Kan akımı kısıtlaması süresine bağlı olarak, farklı motor ve davranışsal açıkları sonucu,. Her ikisi de 30 ve 60 dk serebral iskemi, sonra hayvanların çoğu durumda yanal itme azalmış direnç göstermek ve hareketlilik içinde rahatsızlık nedeniyle çizerek. Daha hafif lezyonlar ön li bir fleksör pozisyon olarak tezahürmbs. Bu kolayca gözlemlenebilir işaretler operasyonun başarısı için temel bir puan olarak kullanılabilir. 2 Morfolojik lezyon histoloji veya Manyetik Rezonans Görüntüleme (MRI) kullanılarak değerlendirilebilir. Orta serebral arter tıkanıklığı Altmış dakika, 30 dakika iskemi striatum sınırlı esas nöronal hücre ölümüne neden ise doku pannecrosis striatum ve neokorteks de dahil olmak üzere bir alan üretir. 3 (Şekil 2) infarkt hacmi açısından, biz bekliyoruz bir standart sapma bir dizi operasyon% 30 daha düşüktür. 60 dakika sonra% 20 - Ölüm 30 iskemi ve 10 dakika sonra yaklaşık% 5 ile kapama süresi bağlıdır. Diğer minimal invaziv olasılık başarısını doğrudan bir kontrolü sağlayan işlem sırasında lazer Doppler flowmetre (LDF) kullanımı. Bir bireysel hayvan, 10 azalma - preocclusion değerleri% 20 fokal serebral iskemi başarılı indüksiyon açıkça gösterir 4 Ancak, LDF kişiler arası karşılaştırmalar yöntemi olarak kullanılır olması değil., LDF sadece kan nicel değişiklikler (yüzde olarak) ölçmek. Beri. küçük ve sınırlı bir doku örneği hacmi içinde akar. Bu kan akımını azaltma mekansal boyutları hakkında bilgi vermez. 5 Yürüme analizi 6,7, Rotarod 8, kutup testi 9,10, yapışkan kaldırma testi 11,12, merdiven testi 13,14, 15,16 merdiven basamak testi ve Morris su labirenti de dahil olmak üzere, inme sonrası davranışsal yönlerini değerlendirmek için çeşitli testler vardır 17. Tüm bu testler, MCAo için maruz kalan farelerde kontrol hayvanlarına göre daha az başarılı bir şekilde gerçekleştirin. <img src="/files/ftp_upload/2423/2423fig1.jpg" alt="Şekil 1" /> Şekil 1 fare beyin (arka plan tasvir) besleyen damar mimari Programı. Farklı suşlar değişkenlik gösterir, örneğin oksipital arter bazen internal karotid arter bırakır. <img src="/files/ftp_upload/2423/2423fig2.jpg" alt="Şekil 2" /> Şekil 2. Tipik lezyon boyutları proksimal MCAo modelinde reperfüzyon farklı bir zaman geçtikten sonra şematik gösterimi. Ortasında, fonksiyonel aktivite ve MCAo sonra serebral kan akımı tipik seyri tasvir edilmektedir. (MCAo: Orta serebral arter tıkanıklığı, LDF: Lazer Doppler akım ölçümü)

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H., Garner, J. P., Wurbel, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+standardization:+cure+or+cause+of+poor+reproducibility+in+animal+experiments.">Environmental standardization: cure or cause of poor reproducibility in animal experiments.</a> Nat Methods. 6, 257-261 (2009).</li><li>Liesz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spectrum+of+systemic+immune+alterations+after+murine+focal+ischemia:+immunodepression+versus+immunomodulation.">The spectrum of systemic immune alterations after murine focal ischemia: immunodepression versus immunomodulation.</a> Stroke. 40, 2849-2858 (2009).</li><li>Meisel, C., Schwab, J. 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>

Landesamt für Gesundheit und Soziales, Berlin, Almanya tarafından belirlenen kurallara ve yönetmeliklere uygun olarak hayvanlar üzerinde deneyler yapıldı.

Geçici, proksimal MCA oklüzyonu 18,19 modeli hastalarda iskemik inme en yaygın türlerinden biri taklit sundu. Değişken reperfüzyon kez 20 modeli büyük geçici iskemik atak (TIA) kadar hasar farklı sınıflarda iskemik yarımkürenin büyük parçalar da dahil olmak üzere infarktlar. Bu vuruş sonra araştırmacı farklı patofizyolojik mekanizmalar çalışma sağlar. 20,21 Cerrahi kısa bir süre içinde gerçekleştirilir ve yüksek tekrarlanabilir lezyonlar olabilir. Bununla birlikte, bu karışıklıklar tam bir kontrolü gerektirir. Operasyon tekniği 22 küçük farklılıklar infarkt farklı etkileri hesaba olabilir. 23,24 Ayrıca, serebral vasküler anatomi sapmaların nedeniyle, farklı fare suşları farklı bir sonuç göstermektedir. 25,26 Vücut sıcaklık, nörolojik hasar hipotermi daha ciddi eksiklikler küçük lezyonların 27 ve hipertermi önde gelen etkiler. Buna göre 28, sıcaklık kontrolü ve bakımı, bu modelde son derece alakalı. Buna ek olarak, kan basıncı ve kan gazları 29 sonuç önemli karışıklıklar ve izlenecektir. 30,31 hızlı ve minimal invaziv yöntemler kullanmak (non-invaziv uygun kan basıncı ölçümü ve kolay erişilebilir bir kan toplama sitelerine) tavsiye edilir. Anestezik seçim İsofluranın örneğin bazı nöroprotektif etkileri ve / veya vazodilatörler olması gibi, aynı zamanda son derece önemlidir. Sonuç olarak 32, anestezi maruz kalma, mümkün ve standart olarak kısa olmalıdır . 15 dakika daha uzun ameliyatı geçirmiş hayvanlar dahil değildir. Cerrahi sitesi Tıraş microabrasions ve inflamasyon üretir ve saç parçaları bültenleri. Bu da iltihabı artırabilir ve lokal enfeksiyon, inme patofizyolojisinde etkisi olabilir teşvik olabilir. Konut koşulları, özellikle zenginleştirme kullanımı, inme sonucu etkileyebilir ve standardize edilmiş ve araştırma raporları açıklanan olmalıdır. 6,33,34 tekrarlanabilirlik çevresel zenginleştirme ve etkisi kullanılması tartışma konusudur. 35 Bir başka önemli karıştırıcı özellikle iskemi (36) uzun zamanlar sonra enfeksiyon indüklenen inme riski, hangi ek morbidite ve artmış mortalite açar. 37,38 enfeksiyonları 5 gün yaklaşık 3 semptomatik hale geldikçe, bu tür modeller takip uzun vadeli önemli sonuçları vardır. Inme, standardizasyon ve kalite kontrolü için yeni tedavi stratejilerinin geliştirilmesi için ilgili üretmek için preklinik translasyonel inme araştırmalarında son derece önemlidir 39 &quot;İyi Laboratuar Uygulamaları&quot; 40,41 zorunlu kılmaktadır. <ol><li> kullanılan hayvanların uygun ve detaylı bir tanımı; </li><li> örneklem büyüklüğü beklenen etki büyüklüğü hesaplanması ve raporlanması; </li><li> içerme ve dışlama kriterleri, çalışma öncesi; </li><li> gruplar halinde tahsisi randomizasyon; </li><li> araştırmacılara göre dağılımı gizlenmesi; </li><li> analizin dışında hayvanların raporlama; </li><li> sonucu kör değerlendirmesi; </li><li> ilgi ve çalışma fonları potansiyel çatışmalar raporlama. </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>
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modeling stroke in mice - middle cerebral artery occlusion with the filament model

İnme, ölüm ve yetişkin sakatlık, özellikle çok gelişmiş ülkelerde en sık rastlanan nedenleri arasında yer almaktadır. Ancak, bugüne kadar tedavi seçenekleri çok sınırlı. Yeni terapötik yaklaşımlara ihtiyacı karşılamak için, deneysel inme araştırma, sık sık fokal serebral iskemi kemirgen modelleri kullanmaktadır. Çoğu araştırmacılar, fareler ya da sıçanlar orta serebral arter (MCA) kalıcı veya geçici oklüzyon kullanın. Intraluminal sütür tekniği (Filament veya dikiş modeli olarak da adlandırılır) ile orta serebral arter (MCA) proksimal oklüzyon muhtemelen deneysel inme araştırmalarında en sık kullanılan model. Intraluminal MCAO model nispeten non-invaziv bir şekilde MCA topraklarının tekrarlanabilir geçici veya kalıcı iskemi inducing avantajı sunmaktadır. Intraluminal yaklaşımlar tüm topraklarının bu arter kan akışını keser. Filament oklüzyon böylece tutuklamalar, bazal ganglia kaynağı lenticulo-yivler arterler, proksimal akışı. Filament tekrarlanabilir lezyonlar korteks ve striatum MCA sonuçları tıkanıklığı ve kalıcı veya geçici olabilir. Buna karşılık, distal inducing modelleri (lenticulo yivler arterlerin dallanma) MCA oklüzyonu tipik striatum ve yedek neokorteks öncelikle dahil. Buna ek olarak bu modellerin kraniektomi gerektirir. Bu makale gösterilen model, bir silikon kaplı filament karotis artere tanıtıldı ve orta serebral arter kökenli bloklar Willis, Çember içine iç karotis arteri boyunca gelişmiş. Hastalarda, orta serebral arter oklüzyonlar iskemik inme en sık görülen nedenleri arasında yer almaktadır. Reperfüzyon sırasında noktası bağlı olarak iskemik lezyonların şiddeti değişen derecelerde olabilir, bu modelde değişik iskemik aralıklarla serbestçe seçilmiş olabilir bu yana. Kan akışının kısmen tromboembolik insanlarda pıhtı kendiliğinden veya tedavi (tPA) Parçalama işleminden sonra restorasyon modelleri en azından kapatılmasıy filament kaldırılması Reperfüzyon. Bu video temel tekniğinin yanı sıra, bu modelin prediktif değeri sınırı en büyük tuzaklar ve karışıklıklar sunacak.

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

İpliksi Orta serebral arter oklüzyonu farelerde iskemik inme eğitimi için ortak bir model.

Fare Modelleme İnme - Filament Model Orta Serebral Arter Tıkanıklığı

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

91.4K Görüntüleme.  Center for Stroke Research Berlin, Charité Universitätsmedizin. İpliksi Orta serebral arter oklüzyonu farelerde iskemik inme eğitimi için ortak bir model.

Video: 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.

Orta Serebral Arter Tıkanıklığı Fare Modeli

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 ...

Tıp

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.

Fare lazer Doppler flowmetre Rehberlik İskemik İnme intraluminal Orta Serebral Arter Tıkanıklığı (MCAO) Model

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.

Fare Daimi Orta Serebral Arter ligasyon Of Uygulama

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.

Rat Lazer Doppler Flowmetri Rehberlik ile İskemik İnme için Endotelin-1 Bağlı Orta Serebral Arter Tıkanıklığı Modeli

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.

Endotelin-1 Bağlı Orta Serebral Arter Tıkanıklığı sırasında Orta Serebral Arter görselleştirin bir Kranial Pencere kullanmak

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.

Intraluminar Orta Serebral Arter Tıkanıklığı ve Sıçan Strok Model Serebral Perfüzyon İzleme için Optimize Sistemi

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.

Farelerde Modelleme İnme: Distal Orta Serebral Arter Daimi Pıhtılaşma

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.

Sıçanlar Homolog Kan pıhtısı ile İskemik İnme emboli Orta Serebral Arter Tıkanıklığı (MCAO)

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.

Farelerde Tekrarlanan Hipoksik Önkoşullanma ve Geçici Orta Serebral Arter Tıkanıklığı ardından Nörovasküler Koruma Kantitasyonu

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.

Yaşlı Sıçanlarda Ortak Karotis Arter Tıkanıklığı ile Kalıcı Distal Orta Serebral Sahne Kortikal İskemi Sürekli Engelli Eğitim için

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