Bu çalışma Amerikan Kalp Derneği Büyükşehir Güneydoğu Ortaklık (09GRNT2060421), Amerikan Tıp Derneği, ve Florida Klinik ve Translational Bilim Enstitüsü Üniversitesi bağışları ile desteklenmiştir. Adam Mekke bir NIH / NINDS, NRSA predoctoral fellow (F30 NS-060335) &#39;dir. Robert Regenhardt Hipertansiyon Florida Multidisipliner Eğitim Programı Üniversitesi (T32 HL-083810) den predoctoral burs desteğini aldı.

Bu protokol Florida Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu (IACUC) tarafından onaylanmış ve uygun olduğunu &quot;Bakım Rehberi ve Laboratuvar Hayvanlarının Kullanımı&quot; (sekizinci baskı, Ulusal Bilimler Akademisi, 2011) oldu. Malzemeler <ol><li> Hayvanlar: Ameliyat sırasında 250-300 g Sekiz haftalık, erkek, Sprague Dawley sıçan (Charles River Çiftlikler, Wilmington, ABD MA). </li><li> Anestezi <ol class="lalpha"><li> İnhalasyon anestezi sistemi (VetEquip A.Ş., Pleasanton, CA, USA) </li><li> Izofluran anestezi (Baxter İlaç sanayii, Deerfield, IL, ABD) </li></ol></li><li> Stereotaksik sistemi (David Kopf Instruments, Tujunga, CA, USA) <ol class="lalpha"><li> Küçük hayvan stereotaksik sistemi </li><li> Sıçanlarda Non-rüptürü kulak çubukları </li><li> Sıçanlar için gaz anestezi kafa tutucu </li></ol></li><li> Sıcaklık ayarı <ol class="lalpha"><li> BAT-12 mikroprob termometre (DünyaHassas Instruments, Inc, Sarasota, FL, ABD) </li><li> T / POMPA, TP600 Termal battaniye (Gaymar Industries, Inc, Orchard Park, NY, ABD) </li></ol></li><li> Cerrahi aletler <ol class="lalpha"><li> Metzenbaum makas, iris forseps, bulldog klemp Retraktörler, 26 ayar eğimli iğne, Bovie koter kiti (Dünya Precision Instruments, Inc, Sarasota, FL, USA) 10 ul şırınga </li><li> Mikromotor matkap (Stoelting, Wood Dale, IL, ABD) </li><li> 0,8 mm yuvarlak matkap frez (Roboz Cerrahi Instrument Co, Inc, Gaithersburg, MD, USA) </li><li> STORZ Bonn kardioraphie Forseps (Bausch ve Lomb, Inc, Rochester, NY, ABD) </li></ol></li><li> Cerrahi Malzemeleri <ol class="lalpha"><li> 3,0 naylon sütür (Oasis, Mettawa, IL, ABD) </li><li> Pamuklu çubuklar, Puralube göz merhemi (Fisher Scientific, Pittsburg, PA, USA) </li><li> Cerrahi noktaları ve şeritler (Medtronic Xomed, Inc, Jacksonville, FL, ABD) </li><li> Elektrikli saç kesme makineleri (Oster, Providence, RI, ABD) </li></ol></li&gt; <li> Kimyasallar <ol class="lalpha"><li> Endotelin-1 (Amerikan Peptid, Sunnyvale, CA, USA) </li><li> Klorheksidin% 2 (Agrilabs, Joseph, MO, ABD) </li></ol></li><li> Görselleştirme Ekipmanları <ol class="lalpha"><li> Cerrahi mikroskop (Seiler Instrument ve Üretim; St Louis, MO, USA) </li><li> Sony Handycam HDR-SR12 (Sony, Minato, Tokyo, Japonya) </li><li> Fiber Optik aydınlatıcı (TechniQuip Corp, Livermore, CA, USA) </li></ol></li><li> Damar çapı ölçümü <ol class="lalpha"><li> VLC media player (Paris, Fransa) </li><li> Image J yazılımı (ImageJ 1.42q yazılım, ABD Ulusal Sağlık Enstitüleri, Bethesda, MA, ABD) </li></ol></li></ol> 1. Ön cerrahi Adımlar <ol><li> Ameliyat öncesinde, sıçanlar su ve kemirgen chow ücretsiz erişim ile 00:12 aydınlık / karanlık döngüsü altında yer alır. </li><li> Anestezi indüksiyon odasında% 100 O 2 gaz karışımı içinde izofluran% 4 ile indüklenir. </li><li> Karga<html> baş n elektrikli saç makasları ile traş edilir. </li><li> Sıçan sıcaklık kontrollü bir çalışma yüzeyi (termal paket) ve baş gaz anestetik yüz maskesi yerleştirilmesi ile başlayarak stereotaksik alet yerleştirilir yatan emici bir ped üzerinde bir yüzüstü konur. </li><li> Sonraki, kulak çubukları yerleştirilir ve sıkılır. </li><li> Prosedür anestezi sırasında% 100 O 2 gaz karışımı içinde izofluran% 2 ile korunur. </li><li> Oftalmoloji yağ merhemi hem gözlere uygulanır ve göz kapaklarının cerrahi prosedür sırasında göz kuruma önlemek için kapatılır. </li><li> Rektal sıcaklık probu ± 0.5 ° C sıcaklıkta 37 sabit bir hayvansal çekirdek sıcaklığı korumak için eklenir </li><li> Stereotaksik cihazı sıkıca tutulan anestezi sıçan baş ile cerrahi alan klorheksidin ve tuzlu üç kez dönüşümlü olarak temizlenir. </li></ol> 2. Ön kranial Pencere Hazırlama ontent &quot;&gt; Önce bir kraniyal pencere oluşturmak için, sıçan diğer gerekli donanım implantasyonu herhangi deneyler için hazırlanmalı ve gerekli cerrahi almalıdır. Bu protokolü için, biz daha önce endotelin-1 (ET-için bir kılavuz kanül implante oylandı 1) indüklenen MCAO başlıklı bir arkadaşı yayında gösterilen &quot;Rat Lazer Doppler Flowmetri Rehberlik ile İskemik İnme için Endotelin-1 Bağlı Orta Serebral Arter Tıkanıklığı Modeli.&quot; 3. Kranial Pencere Oluşturma Bir kılavuz kanül ve deneyler için gerekli olan ekipmanların yerleştirilmesinden sonra, bir kranyal pencere doğrudan bir vuruş prosedürü sırasında orta serebral arterin proksimal kısımları görselleştirmek için oluşturulur. <ol><li> İlk olarak, makas medial başlangıç ​​ve yanal olarak çalışan temporal kas örten cilt kesilirken için kullanılır. </li><li> Temporal kas elektrokoter kullanılarak örtülmüş ve sonra visua ila 3,0 naylon sütür kullanılarak geri çekilir<html> temporal kemiğin skuamöz kısmı lize. </li><li> Bu temporal kemik dışına yansıtır gibi bir yaklaşık 3-4 mm kare yörüngesine kaudal ve elmacık sürecinin temel üstün temporal kemiğin skuamöz kısmı çizilir. </li><li> Bir matkap kademeli olarak temporal kemik serbest kemik anahat parça kesmek için kullanılmaktadır. Bakım bu dura veya serebral kortekse zarar mümkün olduğundan matkap çok fazla baskı uygulamaktan kaçının için alınmalıdır. </li><li> Steril serum fizyolojik ile sık durular cerrahi alanın görselleştirme geliştirmek ve kafatası aşırı ısınmayı önlemek için yapılmaktadır. </li><li> Gevşek bir köşeden başlayarak, temoporal kemik parça özenle dura ile ilişkili herhangi bir gemi gözyaşı değil emin yaparken ince sıçan dişli forseps kullanılarak kaldırılır. </li><li> Dura değişmeden kalır ve enkaz steril serum fizyolojik kullanılarak uzak durulanır. </li></ol> 4. Serebral Arter Konstriksiyon kaydedilmesi gerçek zamanlı, ET-1 ile indüklenen MCAO geçiyor bir sıçan görüntüleri yakalamak için nasıl göstermek için çadır &quot;&gt; Bu protokol için kullanılır. <ol><li> Vazoaktif bileşiklerin uygulamasından önce, bir temel video en az 1 dakika süreyle kaydedilmelidir. Iğne önceki ET-1, enjeksiyon ile beyin parankima içine indirilir fakat edildikten sonra, ET-1 indüklenmiş MCAO için, taban kayıt yapılır. </li><li> Şırınga pompası enjeksiyon için başladı ve 1 saat süre ile ya da arzu edilen son nokta kadar kaydedilir. İğne odak düzlemi bozuklukları önlemek için video kaydı sırasında yerinde bırakılır. </li><li> Sıçan bu prosedürü izleyerek onaylanmış protokole göre derin anestezi ve ötenazi olmalıdır. </li></ol> 5. Görüntü Analizi Gemi çapı görüntülenmiştir MCA herhangi bir kısmı için tespit edilebilir. Bir örnek olarak, ET-1 enjeksiyon öncesi ve sonrası zaman noktalarında damar çapını ölçmek için MCA bir dal kullanır. Stivideo ll kare VLC media player (VideoLAN) kullanarak 1 dakika aralıklarla yakalanır. <ol><li> VLC player yüklü ve açılır. </li><li> Araçları ve daha sonra tercihlerini seçerek ayar altında &quot;All&quot; olarak değiştirildi &quot;Ayarları göster.&quot; </li><li> &quot;Video menüsü&quot; sol kenar çubuğundaki genişletilir ve &quot;çıkış modülleri&quot; sonra genişletilir. &quot;Sahne filtreleri&quot; sahnesi video filtresi menüyü getirmek için seçilir. </li><li> &quot;Sahne&quot; dosya adı öneki için kutunun içine yazılır. </li><li> Bir dizin yolu öneki tanımlanır. Hareketsiz kareleri kaydedilir yerdir. </li><li> Bir kayıt oranının karesi yakalama istenilen oranı esas alınarak seçilir. Bir video saniyede 29 kare, sonra da &quot;1749&quot; (29 kare / sn x 60 sn / dk) yakalandı Eğer hala her 1 dakikada çerçeveye kaydetmek kutusuna girilmelidir. Tüm değişiklikler sonra kaydedilir. </li><li> İstediğiniz video sonra otomatik olarak her 1 dakikada bir kez yine kare kaydetmek için VLC player açıldı. </li><li> Damar çapı daha sonra bu kullanılarak ölçülürgörüntüler. Birincisi, ImageJ yazılım (NIH) açıldı. </li><li> Sonra ölçmek olacağı hala çerçeveleri ImageJ açıldı. </li><li> &quot;Analiz&quot; menüsünü seçerek, &quot;ölçümler set&quot; sonra açılır ve tüm kutuları işaretlemeyin vardır. </li><li> Sonraki, düz bir çizgi aracı seçilir. </li><li> Klavye kısayolu &quot;Ctrl +&quot; gerektiği gibi yakınlaştırmak için kullanılır ve bir çizgi damar çapı ölçmek için geminin yoluna dik yerleştirilir. </li><li> Son olarak, damar uzunluk damar uzunluğu elde etmek için &quot;önlemi&quot; (CTRL + M) sonra &quot;analiz&quot; menü seçimi ile elde edilir. </li><li> Bu işlem, en azından üç kez tekrarlandı ve yine de her bir kare cinsinden ölçülen her bir ölçülü bir kap için ortalaması alınmıştır. </li><li> Çoklu karşılaştırmalar sıçan kullanılarak yapılabilir, böylece her bir zaman noktasında damar çapı baz damar çapı ile normalize edilir. Bunu yapmak için, her bir kabın% taban çapı hesaplamak için x% 100 formülüne akım çapı / taban çapı kullanır. </li></ol>

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

Çıkar çatışması ilan etti.

Bu küçük değişiklikler 4, 5 ile birçok deneylerin ihtiyaçlarını karşılamak için değiştirilebilir gibi Özetle, bu kraniyal pencere hazırlama tekniği çok yönlüdür. Örneğin, başarılı bir kraniyal pencere (Mekke AP 2009 ve 2011) ile görselleştirilmiş bir serebral arter doğrudan odaklanmak için lazer Doppler flowmetre kullanarak belirli MCA dallarında serebral kan akımı takip ettik. Buna ek olarak, dura kesik ile benzer bir hazırlama vivo vasküler reaktivite banyo i...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> İnhalasyon anestezi sistemi </td><td> VetEquip Inc, Pleasanton, CA, USA </td><td> 901806 </td><td></td></tr><tr><td> İzofluran anestezik </td><td> Baxter İlaç sanayii, Deerfield, IL, ABD </td><td> 1001936060 </td><td></td></tr><tr><td> Küçük hayvan stereotaksik sistemi </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 900 </td><td></td></tr><tr><td> Sigara rüptürü kulak çubukları, sıçan </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 957 </td><td></td></tr><tr><td> Sıçan gaz anestezi kafa tutucu </td><td> David Kopf Instruments, Tujunga, CA, USA </td><td> 1929 </td><td></td></tr><tr><td> BAT-12 mikroprob termometre </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> BAT-12 </td><td&gt; </td></tr><tr><td> T / POMPA, Termal Battaniye </td><td> Gaymar Industries, Inc, Orchard Park, NY, ABD </td><td> T / POMPA, TP600 </td><td></td></tr><tr><td> Metzenbaum Makas </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> 501254 </td><td></td></tr><tr><td> Iris forseps </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> 15915 </td><td></td></tr><tr><td> Bulldog kelepçe retraktörler </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> 14119-G </td><td></td></tr><tr><td> Şırınga 10 ul 26-gaugue </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> SGE010RNS </td><td></td></tr><tr><td> Bovie, yüksek sıcaklık koter kiti </td><td> Dünya Precision Instruments, Inc, Sarasota, FL, ABD </td><td> 500392 </td><td></td></tr><tr><td> Sıçan diş 0.12 forseps </td><td> Stotz </td><td> E1811 </td><td></td></tr><tr><td> Mikromotor matkap </td><td> Stoelting,Wood Dale, IL, ABD </td><td> 51449 </td><td></td></tr><tr><td> 0,8 mm yuvarlak matkap bur </td><td> Roboz Cerrahi Instrument Co, Inc, Gaithersburg, MD, USA </td><td> RS-6280C-1 </td><td></td></tr><tr><td> STORZ Bonn sütür forseps </td><td> Bausch and Lomb, Inc, Rochester, NY, ABD </td><td></td><td></td></tr><tr><td> Naylon Dikiş, büyüklüğü 3.0 </td><td> Oasis, Mettawa, IL, ABD </td><td> MV-663 </td><td></td></tr><tr><td> Pamuklu çubuklar </td><td> Fisher Scientific, Pittsburg, PA, USA </td><td> 22-029-488 </td><td></td></tr><tr><td> Puralube göz merhemi </td><td> Fisher Scientific, Pittsburg, PA, USA </td><td> NC0138063 </td><td></td></tr><tr><td> Elektrikli saç kesme makineleri </td><td> Oster, Providence, RI, ABD </td><td> 78005-301 </td><td></td></tr><tr><td> ET-1 PBS içinde 80 μ M konsantrasyonda seyreltildi </td><td> Amerikan Peptid, Sunnyvale, CA, USA </td><td> 88-1-10A </td><td></td></tr><tr><td> Klorheksidin,% 2 </td&gt; <td> Agrilabs, St Joseph, MO, USA </td><td> 1040, Rev 6-06, NAC No: 10580322 </td><td></td></tr><tr><td> Cerrahi mikroskop </td><td> Seiler Instrument ve İmalat, St Louis, MO, USA </td><td> Evrim XR6 </td><td></td></tr><tr><td> Sony Handycam </td><td> Sony, Minato, Tokyo, Japonya </td><td> HDR-SR12 </td><td></td></tr><tr><td> Fiber optik aydınlatma </td><td> TechniQuip Corp, Livermore, CA, USA </td><td> FO1-150 </td><td></td></tr><tr><td> VLC Media Player </td><td> (Paris, Fransa) </td><td></td><td></td></tr><tr><td> Image J yazılımı </td><td> ABD Sağlık, Bethesda, National Institutes of MA, USA </td><td></td><td></td></tr></tbody></table>

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

Kranyal pencere oluşturulması 1-3 beyin kortikal yüzey üzerinde doğrudan görüntüleme yapılarının sağlayan bir yöntem. Bu teknik, sıçan beyin örten çok noktadan yapılabilir, ancak, en kolay şekilde kolayca ulaşılabilir ya da ön parietal kemik üzerinde bir kraniektomi oluşturarak gerçekleştirilir. En sık, biz proksimal MCA bitişik beyin parankimi içine endotelin-1 enjeksiyonu ile meydana orta serebral arter damar çapı değişiklikleri ölçmek için iskemik inme endotelin-1 orta serebral arter oklüzyon modeli ile birlikte bu tekniği kullanmış 4, 5. Endotelin -1 indüklenen MCAO sırasında MCA proksimalinden görselleştirmek için, biz sıçan kafatası (Şekil 1) lateralinde temporal kemik yoluyla kraniyal pencere oluşturmak için bir teknik kullanır. Serebral arterlerin dura bozulmamış veya dura kazıma ve RETRA ile ya görsellenebilircted. En sık, biz beyin parankimi içine vazokonstriktör peptid teslimini içerir endotelin-1 ile indüklenen MCAO yana görselleştirme sırasında dura olduğu gibi bırakabilirsiniz. Bu ilaç teslim görüntülenmiştir damarları üzerinde doğrudan dura kesilirken ihtiyaç atlar. Bu protokol de bu yöntem ile ilgili potansiyel tuzaklar birçok önlemek için ne gibi, adım adım moda serebral arterlerin görselleştirmek için bir kraniyal pencere oluşturmak nasıl anlatacağız.

Hala çekilen video gösterisi alınan görüntülerin bundan sonra serebral arter çapı değişikliği ET-1 enjeksiyonu kolaylıkla bu kraniyal pencere tekniği (Şekil 2) kullanılarak takdir edilebilir. ET-1 enjeksiyonu birkaç dakika içinde, damar daralması ile başlayacak. Sonunda gemiler görselleştirmek için zor olacaktır ve beyin dokusu soluk olacak. Yaklaşık 20 dakika sonra ET-1 etkileri azalacak ve gemilerin giderek yaklaşık 45 dakika sonra bazal çap dönen, dilate başlayacaktır. ...

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

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

Bu makalede, orta serebral arterin proksimal bölümlerini görüntülemek için temporal kraniektomi kullanılarak kranyal pencereden sıçan serebral arterlerin görüntülenmesi için bir yöntem açıklanır (<strong&gt; Şekil 1</strong&gt;). Bu çok yönlü yöntem serebral arter reaktivitesi ölçmek için ilaç dağıtım çeşitli teknikleri ile kombine edilebilir<em&gt; In vivo</em&gt;.

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 ...

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

Research

JoVE Journal

Medicine

14.8K Görüntüleme.  University of Florida. Bu makalede, orta serebral arterin proksimal bölümlerini görüntülemek için temporal kraniektomi kullanılarak kranyal pencereden sıçan serebral arterlerin görüntülenmesi için bir yöntem açıklanır ( Şekil 1). Bu çok yönlü yöntem serebral arter reaktivitesi ölçmek için ilaç dağıtım çeşitli teknikleri ile kombine edilebilir In vivo.

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

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

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

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

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 ...

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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 ...

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 ...

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 ...

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, ...

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

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

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

Farelerde Orta Serebral Arter Tıkanıklığı ve Reperfüzyon Kaynaklı İnme Cerrahi Yaklaşım

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

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

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

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

P10 Sıçanlarda Yenidoğan İnme Geçici Orta Serebral Arter Tıkanıklığı Modeli

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