SN ve MDD NIH K08 NS083740 ve Nöroloji UCLA Bölümü&#39;nden destek aldı. AJG Dr. Miriam ve Sheldon G. Adelson Tıbbi Araştırma Vakfı ve Larry L. Hillblom Vakfı tarafından destek onaylar. KLN minnetle Amerikan Kalp Derneği 14BFSC17760005 ASA-Bugher İnme Merkezi&#39;nden destek kabul eder. ILL, EGS ve STC NIH R01 NS071481 tarafından desteklendi. JDH NIH K08 NS083740 destek kabul eder.

Bu protokolde hayvanların kullanımı Kaliforniya Los Angeles Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi tarafından onaylanan prosedürlere uygun olarak yapılmıştır. Not: Hedef fare nüfusu belirleyerek başlayın. Önceki çalışmalarda, sadece erkek doğal tipte C57 / BL6 fareleri, ancak çeşitli transgenik ya nakavt fareleri de kullanılabilir, kullanılmıştır. stereotaktik koordinatlar C57 / BL6 anatomi dayalı olduğunu unutmayın. Her kullanıcı başlangıçta beyaz cevher inme lokalizasyonu doğrulamak tavsiye edilir. 1. Beyaz Matter İnme İndüksiyon - medial Açılı Yaklaşım <ol><li> Distal çapı 15-25 um, 11 arasında olacak şekilde 0.5 mm kapiler tüp kullanılarak çekilen cam pipet hazırlanmasıyla başlar. </li><li> steril% 0.9 normal tuzlu su içinde 27.4 mg / ml (130 uM) de - L-NIO bir steril 10 ul alikosu ((1) -iminoethyl-L-ornitin HCI N- (5)) hazırlamak. </li><li> çekti cam pipet Ön doldurmakcam pipet sabitlenmesiyle, L-NIO (2-5 | il), küçük bir hacme sahip bir vakum hattına bağlı boruya. tezgah üstünde pipet düz Lay ve L-Nio çözeltisi içine çekti ucunu. <ol><li> Pipet 0.5 mm&#39;lik bölümünün en az 2 mm dolana kadar vakum uygulanır. Vakumu kapatın ve pipet çekin. Adım 1.12 kadar bir kenara koyun. </li></ol></li><li> Indüksiyon odasında fare yerleştirin ve standart% 32 izofluran kullanarak fare anestezi neden bir buharlaştırıcı akan (5 L / dk 5 L / dk oksijen ve 0,5 L / dk ile inhale N2) 1 dakika boyunca ya da derin anesteziye kadar. stereotaktik mikroskop ile donatılmış bir stereotaktik aparatı fareyi aktarın. Bir buharlaştırıcı akan% 32 izofluran kullanarak bakım anestezi sağlamak ve bir burun konisi (2 L / dk 5 L / dk oksijen ve 0.5 L / dak N 2 ile inhale). Bir ayak tutam anestezi derinliğini kontrol edin. </li><li> 36 ° enjeksiyon kolunu ayarlayın. </li><li> Affixa düşük hacimli basınçlı enjeksiyon sistemi uzak ucuna cam pipet tutucu çekti ve stereotaktik kurulum enjeksiyon koluna takın. </li><li> Coat iki gözün üstünde vazelin ve yer suni gözyaşı merhemi ile anestezi hayvanın bıyık. başının üzerinde bir 5-10 cm açıklığı olan hayvanın başının üzerinde steril örtüyü koyarak steril cerrahi alanında hazırlayın. kafatası örten kürk tıraş tarafından aspetic cerrahi yüzey hazırlayın. alternatif betadin ve% 70 alkol bezlerden ile kafa derisi temizleyin. </li><li> kafatası yüzeyi ortaya çıkarmak için steril ince makas ile 1,5 cm orta hat kafa derisi kesi olun. Steril bir pamuklu çubukla kafatası kurutun ve 1-3X büyütme stereotaktik mikroskop kullanılarak, steril bir mikro nokta aracını kullanarak herhangi bir üstte periost doku kaldırmak. </li><li> ince bir nokta kalem kullanarak bir referans noktası olarak Bregma işaretleyin. </li><li> steril ince stip cerrahi matkap ucu kullanarak 2 mm ellipitical kraniotomi Matkap0; bregma posterior başlayan ve öne sadece orta hat sol uzanan. kemik parçaları çıkarın ve serebral korteks görüntülenmiştir böylece yumuşak doku örten. </li><li> aralıklı steril serum fizyolojik damla uygulayarak cerrahi alan ve nemli kortikal yüzey tutun. </li><li> Stereotaktik aparatın enjektör koluna çekti cam pipet yapıştırın. Bregma ile pipet distal ucunu hizalayın ve stereotaktik koordinatlar sıfır. </li><li> İlk ön / arka (A / P) pipet ilerlemek ve / Tablo 1&#39;de verilmiştir koordinatları (M / L) yanal medial. </li><li> kortikal yüzeye pipet ilerlemek ve dorsal / ventral (D / V) ölçümü sıfır. </li><li> Yavaş yavaş ilk D / V Tablo 1&#39;de koordinat ulaşana kadar beyin içine pipet geçmektedir. </li><li> beyne L-NIO 100 nl enjekte ve reflü önlemek için 5 dakika bekleyin, 20 msn bakliyat için 20 psi ayarlanmış bir düşük hacimli basınçlı enjeksiyon sistemi kullanarakPipet parça yukarı. <ol><li> Stereotaktik mikroskop mercek ve 3X bir büyütme kalibre reticle kullanın. </li><li> Buna uygun olarak, koordinat her biri için çekilen cam pipet (100 NL karşılık gelen 0.5 mm çapında bir pipet 0.5 mm uzunluğunda), 0.100 mm&#39;lik toplam 3 yerinden. Bir el çantası kullanarak, ölçmek ve her kullanılan büyütme ve ölçekler bağlı olarak değişir kurmak beri standardize. </li><li> hava-sıvı menisküs bir sagital görünümü vardır, böylece her enjeksiyon sırasında doğru hacim ölçümü için, yan mikroskopla açılı pipet yaklaşım. Menisküs pipet iç ve dış duvarın hem aynı odak düzlemi görünmelidir. </li></ol></li><li> Yavaşça pipet çekilme ve adımları tekrarlayın 1.13-1.16.3 Tablo 1&#39;de verilen koordinatlar ikinci ve üçüncü sette de. </li><li> Son enjeksiyondan sonra, pipet kaldırmak ve kraniyotomi sitesini dolduracak kadar kemik mumu yerleştirin. birKafa derisi yara kenarlarını pproximate dermal yapıştırıcı ile bağlanır. </li><li> Kafa derisi kesi ile ilişkili yerel ağrıyı önlemek için önlemek için steril bir 30 G iğne kullanılarak yara marjları içine% 0.5 marcaine 0.1 ml enjekte edilir. </li><li> 5 gün boyunca içme suyu konut ve arz post-operatif antibiyotik hayvan (0.48 mg / ml trimetoprim-sulfametoksazol veya 0.5 mg / ml Levofloksasin) döndürür. </li></ol> 2. Beyaz Matter İnme İndüksiyon - Posterior Açılı Yaklaşım <ol><li> posterior anterior odaklı 45 dereceye stereotaktik kurulum enjeksiyon kolunu ayarlamak dışında, medial açılı yaklaşım protokolünde olduğu gibi adımları 1.1-1.12 gerçekleştirin. </li><li> İlk A / P pipet ilerlemek ve M / L Tablo 2&#39;de verilmiştir koordinatları. </li><li> Komple kalan adımları yanal açılı yaklaşım protokolde olduğu gibi 1,14-1,20. </li></ol> 3. Retrograd Sinir Etiketleme <ol><li> 10 steril hazırlanması% 0.9 normal serum fizyolojik içinde 54.8 mg / ml L-NIO ul alikosu. </li><li> % 0.9 normal serum fizyolojik içinde% 20 Fluororuby (veya% 20, biyotinlenmiş dekstran amin ya da% 2 Fluorogold) steril bir kısım hazırlayın. </li><li> 27.4 mg / ml L-Nio ve% 10 Fluororuby nihai konsantrasyonları için 1: Birlikte: 1 seyreltin. </li><li> adımlarda 1,3-1,23 olarak yukarıdaki vuruş protokolü gerçekleştirin. </li><li> Daha önce 8 açıklandığı gibi perfüzyon fiksasyon, cryosectioning ve mikroskopi ile doku bölümünde doğal floresan tracer gözünüzde canlandırın. </li></ol> İmmünofloresan 4. Doku İşleme <ol><li> 3 saat dan inme sonrası 14 gün arasında değişen uygun bir inme sonrası aralıklarla, izofluran doz aşımı ya da yerel IACUC üzerinden fareler prosedür onaylı euthanize. </li><li> açılı makas kullanarak göğüs boşluğu açın ve sol ventrikül içine bir 23 G kelebek iğne takın. </li><li> perfüzyon sıvısı için bir çıkış izini izin ince makas kullanarak sağ atrium küçük bir kesim yerleştirin. </li><li> Transkardiyal oda sıcaklığında, 10 ml / dk&#39;lık bir oranda soğuk% 4 paraformaldehit 30-40 ml ve ardından soğutulmuş fosfat tamponlu tuz 30-40 ml serpmek. </li><li> fare başını kesmek ve posterior kafatası açmak için steril makas kullanarak beyin kaldırmak ve sonra yavaşça 24 saat süreyle soğuk% 4 PFA halinde beyin, bir spatula ile örten kafatası kaldırmak yerleştirin ve daha sonra 48 saat süreyle PBS içinde% 30 sakaroz transfer . </li><li> Bir kriyostat kullanarak devre mikron kayan kesitler hazırlamak ve daha önce 6-8 tarif edildiği gibi, antikor işlemi gerçekleştirir. Bu çalışmada aşağıdaki antikorların kullanımı: tavşan anti-sinir lifi 200 (1: 500 seyreltme); tavşan anti-vimentin (1: 500); keçi anti-GFAP (1: 500); tavşan anti-IBA-1 (1: 1000). </li></ol> Protein veya RNA Analizi 5. Doku İşleme <ol><li> 3 saat dan inme sonrası 14 gün arasında değişen uygun bir inme sonrası aralıklarla, izofluran doz aşımı yoluyla euthanize ya da yerel IACUC prosedürü onayladı. </li><li> başını kesmekFare posteriora kafatası açmak için steril makas kullanarak beyin kaldırmak ve daha sonra bir spatula ile hafifçe örten kafatası kaldırmak ve. </li><li> Koku ampul ve optik sinirleri koparmaya beynin ön kısmında steril 4 mm spatula yerleştirin. Yavaşça calvarium üzerinden beyin kaldırın ve (1x Hank Dengeli Tuz Çözeltisi, 25 mM HEPES-KOH, pH 7.4, 35 mM glukoz, 4 mM sodyum bikarbonat, ve 0.01 mg / ml cyclohexamide) buz soğukluğunda tahlil tamponuna içine koyun. </li><li> Bir beyin blok ve steril yeni Jilet kullanarak, soğuk diseksiyon tampon içine inme ve yeri içeren 2-3 mm döşeme hazırlamak. </li><li> bir mikroskop altında enjekte yarımkürede beyaz cevher altta yatan motor korteksin tespit. uzun inme sonrası aralıklarla, bölge görsel fokal nekroz ve miyelin solukluk tarafından tespit edilebilir. (Daha önce inme sonrası aralıklarla,% 10 1 ul ile karıştırılmış L-NIO enjeksiyonu ise Hızlı Yeşil inme görsel kimlik izin verebilir: Not <strong&gt; Şekil 4A). </li><li> Bir mikroskop rehberliğinde ve taze bir neşter kullanılarak altında, dikkatle Hızlı Yeşil etiketleme ya da doku kaybı ya tarafından belirlenen inme içeren beyaz cevher bölgeyi incelemek. istediğiniz gibi örten korteks ve altta yatan striatum çıkarın. </li></ol>

<ol>
	<li>Go, A. S., 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=Heart+disease+and+stroke+statistics--2014+update:+a+report+from+the+American+Heart+Association.">Heart disease and stroke statistics--2014 update: a report from the American Heart Association.</a> Circulation. 129, 28-292 (2014).</li><li>Saini, M., 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=Silent+stroke:+not+listened+to+rather+than+silent.">Silent stroke: not listened to rather than silent.</a> Stroke. 43, 3102-3104 (2012).</li><li>Koton, S., 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=Burden+and+outcome+of+prevalent+ischemic+brain+disease+in+a+national+acute+stroke+registry.">Burden and outcome of prevalent ischemic brain disease in a national acute stroke registry.</a> Stroke. 44, 3293-3297 (2013).</li><li>Jiwa, N. S., Garrard, P., Hainsworth, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+vascular+dementia+and+vascular+cognitive+impairment:+a+systematic+review.">Experimental models of vascular dementia and vascular cognitive impairment: a systematic review.</a> J Neurochem. 115, 814-828 (2010).</li><li>Sozmen, E. G., Hinman, J. D., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+that+matter:+white+matter+stroke+models.">Models that matter: white matter stroke models.</a> Neurotherapeutics. 9, 349-358 (2012).</li><li>Sozmen, E. G., Kolekar, A., Havton, L. A., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+white+matter+stroke+model+in+the+mouse:+axonal+damage,+progenitor+responses+and+MRI+correlates.">A white matter stroke model in the mouse: axonal damage, progenitor responses and MRI correlates.</a> J Neurosci Methods. 180, 261-272 (2009).</li><li>Rosenzweig, S., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+exacerbation+of+white+matter+stroke+outcomes:+a+role+for+oxidative+damage+and+inflammatory+mediators.">Age-dependent exacerbation of white matter stroke outcomes: a role for oxidative damage and inflammatory mediators.</a> Stroke. 44, 2579-2586 (2013).</li><li>Hinman, J. D., Rasband, M. N., Carmichael, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+the+axon+initial+segment+after+focal+cortical+and+white+matter+stroke.">Remodeling of the axon initial segment after focal cortical and white matter stroke.</a> Stroke. 44, 182-189 (2013).</li><li>McCall, T. B., Feelisch, M., Palmer, R. M., Moncada, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+N-iminoethyl-L-ornithine+as+an+irreversible+inhibitor+of+nitric+oxide+synthase+in+phagocytic+cells.">Identification of N-iminoethyl-L-ornithine as an irreversible inhibitor of nitric oxide synthase in phagocytic cells.</a> Brit j pharmacol. 102, 234-238 (1991).</li><li>Gadea, A., Aguirre, A., Haydar, T. F., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin-1+regulates+oligodendrocyte+development.">Endothelin-1 regulates oligodendrocyte development.</a> J Neurosci. 29, 10047-10062 (2009).</li><li>Dean, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+(pulling)+of+needles+for+gene+delivery+by+microinjection.">Preparation (pulling) of needles for gene delivery by microinjection.</a> CSH prot. , (2006).</li><li>Hughes, P. M., 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=Focal+lesions+in+the+rat+central+nervous+system+induced+by+endothelin-1.">Focal lesions in the rat central nervous system induced by endothelin-1.</a> J. Neuropathol. Exp. Neurol. 62, 1276-1286 (2003).</li><li>Whitehead, S. N., Hachinski, V. C., Cechetto, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+a+rat+model+of+cerebral+ischemia+and+beta-amyloid+toxicity:+inflammatory+responses.">Interaction between a rat model of cerebral ischemia and beta-amyloid toxicity: inflammatory responses.</a> Stroke. 36, 107-112 (2005).</li><li>Frost, S. B., Barbay, S., Mumert, M. L., Stowe, A. M., Nudo, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+animal+model+of+capsular+infarct:+endothelin-1+injections+in+the+rat.">An animal model of capsular infarct: endothelin-1 injections in the rat.</a> Behav Brain Res. 169, 206-211 (2006).</li><li>Horie, N., 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=Mouse+model+of+focal+cerebral+ischemia+using+endothelin-1.">Mouse model of focal cerebral ischemia using endothelin-1.</a> J Neurosci Methods. 173, 286-290 (2008).</li><li>Wang, M., 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=Cognitive+deficits+and+delayed+neuronal+loss+in+a+mouse+model+of+multiple+microinfarcts.">Cognitive deficits and delayed neuronal loss in a mouse model of multiple microinfarcts.</a> Neuroscience. 32, 17948-17960 (2012).</li><li>Shih, A. Y., 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=The+smallest+stroke:+occlusion+of+one+penetrating+vessel+leads+to+infarction+and+a+cognitive+deficit.">The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.</a> Nat Neurosci. 16, 55-63 (2013).</li><li>Jung, K. J., 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=The+role+of+endothelin+receptor+A+during+myelination+of+developing+oligodendrocytes.">The role of endothelin receptor A during myelination of developing oligodendrocytes.</a> J Korean Med Sci. 26, 92-99 (2011).</li></ol>

Subkortikal inme örneksiz bir dizi sıçan 12-14 ve fare 6,15 iç kapsüle endotelin-1 fokal enjeksiyonlar subkortikal beyaz madde ve striatum içeren tarif edilmiştir. Küçük odak vuruşları daha yeni modeller kolesterol mikroemboli karotis arter 16 enjeksiyon ve tek delici arteriole 17 Fototrombotik tıkanıklığı kullanmışlardır. Bu modellerin her biri avantaj ve dezavantajları 5 hem de sahiptir. Şu anda açıklanan model aksonal anormallikler ve zara...

Stroke affecting the subcortical white matter is a common clinical entity, accounting for up to 25% of clinical strokes annually in the US 1. Ischemic damage to white matter also occurs silently at a significantly higher rate and contributes to the development of vascular dementia 2,3. Presently, patients with this form of cerebral ischemia have few, if any treatment choices. Despite the clinical importance of this disease, few clinically relevant animal models exist 4,5. 
The goal of this protocol is to produce a focal ischemic lesion within the murine white matter. This murine model of human disease allows the specific study of axonal injury response to stroke and how the cellular elements of white matter, namely oligodendrocytes and astrocytes along with axons, respond to and repair after stroke.
Previous reports have described a model of subcortical white matter stroke using endothelin-1 (ET-1) 6 that is similar to the one described here. Several key changes to the experimental protocol have been made thereby the potential uses of this model have expanded 7,8. This protocol provides a reliable and modifiable strategy to produce a focal stroke within mouse brain white matter. 
The major advantages of this model are the use of a chemical endothelial nitric oxide synthase (eNOS) inhibitor N(5)-(1)-iminoethyl-L-ornithine HCl (L-Nio) 9 with no known paracrine effects on cellular elements of white matter which had been a complication of models using endothelin-1 10. In addition, the stereotactic targeting of white matter in the mouse allows the use of any variety of transgenic or knockout strains, greatly expanding the available tools to determine the effect of stroke on brain white matter. Here, two variations on this technique are described and demonstrate some of the additional variations that can be utilized to enhance the understanding of axonal and white matter damage and repair after stroke.

<table><tbody><tr><td>L-N5-(1-Iminoethyl)ornithine, Dihydrochloride</td><td>Calbiochem</td><td>400600-20MG</td><td></td></tr><tr><td>Isoflurane</td><td>Phoenix Pharmaceutical, Inc.</td><td>NDC 57319-559-06</td><td></td></tr><tr><td>Capillary tubes</td><td>World Precision Instruments</td><td>50-821-807</td><td></td></tr><tr><td>Picospritzer</td><td>Parker Instrumentation</td><td>Picospritzer II</td><td></td></tr><tr><td>Stereotactic setup</td><td>Kent Scientific</td><td>KSC51725</td><td></td></tr><tr><td>Pipette puller</td><td>KOPF</td><td>Model 720</td><td></td></tr><tr><td>Stereomicroscope SZ51</td><td>Olympus</td><td>88-124</td><td></td></tr><tr><td>Fine scissors</td><td>Fine Scientific Tools</td><td>14084-08</td><td></td></tr><tr><td>Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8547</td><td></td></tr><tr><td>Curved Forceps</td><td>Harvard Apparatus</td><td>PY2 72-8598</td><td></td></tr><tr><td>Blunt dissection tool</td><td>Fine Scientific Tools</td><td>10066-15</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>8220-1/28</td><td></td></tr><tr><td>Drill bits</td><td>Fine Scientific Tools</td><td>19007-05</td><td></td></tr><tr><td>Vetbond</td><td>3M</td><td>1469SB&amp;nbsp;</td><td></td></tr><tr><td>Marcaine</td><td>HOSPIRA</td><td>NDC 0409-1610-50</td><td></td></tr><tr><td>Trimethoprim-Sulfamethaxole</td><td>STI Pharmacy</td><td>NDC 54879-007-16</td><td></td></tr><tr><td>Fluororuby</td><td>Fluorochrome Inc</td><td>30 mg</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>O4042-500</td><td></td></tr><tr><td>Sucrose</td><td>Fisher</td><td>BP220-10</td><td></td></tr><tr><td>Cryostat</td><td>Leica CM3050 S</td><td>14047033518</td><td></td></tr><tr><td>Glass slides</td><td>Fisher</td><td>12-544-7</td><td></td></tr><tr><td>Fast Green&amp;nbsp;</td><td>Sigma</td><td>F7252-5G</td><td></td></tr><tr><td>Dissection microscope</td><td>Nikon</td><td>SMZ1500</td><td></td></tr><tr><td>23 G butterfly needle</td><td>Fisher</td><td>14-840-35</td><td></td></tr><tr><td>10x Hank&#039;s Balanced Salt Solution</td><td>Life Technologies</td><td>14065056</td><td></td></tr><tr><td>1 M HEPES-KOH, pH 7.4</td><td>Affymetrix</td><td>16924</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G8270</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Cyclohexamide</td><td>Sigma</td><td>01810</td><td></td></tr></tbody></table>

a versatile murine model of subcortical white matter stroke for the study of axonal degeneration and white matter neurobiology

İnme klinik inme sunumlar% 25&#39;e kadar beyaz madde hesaplarını etkileyen, 5-10 kat daha büyük olabilir fiyatla sessizce gerçekleşir ve vasküler demans gelişmesine önemli ölçüde katkıda bulunur. fokal beyaz cevher inme birkaç model var ve uygun modellerin bu eksikliği inme bu tip sonra yaralanma tepki ve onarımında yer alan nörobiyolojik mekanizmaları anlaşılmasını engelliyordu. diğer subkortikal inme modellerinin ana sınırlama fokal beyaz cevher infarkt kısıtlamak değil ya da öncelikle olmayan fare türlerinde valide edilmiş olmasıdır. Bu beyaz cevher inme nörobiyolojisini incelemek için fare araştırma araçları çok çeşitli uygulama becerisi sınırlar. Burada geri dönüşü olmayan bir eNOS inhibitörü bir lokal enjeksiyonu kullanılarak fare beyaz cevherde fokal inme güvenilir üretimi için bir metodoloji sunuyoruz. Biz de iki benzersiz stereotaktik dahil olmak üzere genel protokol üzerinde çeşitli varyasyonlar mevcutvaryasyonlar, büyük ölçüde bu tekniğin potansiyel uygulamalarını genişletmek retrograd nöronal izleme yanı sıra taze doku etiketleme ve diseksiyonu. Birden fazla yaklaşımlar inme bu ortak ve ilgili yeterli formun nörobiyolojik etkilerini analiz etmek için bu varyasyonlar izin verir.

sunulan modeli kullanarak, beyaz cevher altta yatan ön ayakları sensorimotor korteks güvenilir hedeflenebilir. Tipik olarak insan laküner infarkt görüldüğü gibi kimyasal olarak bağlı kontur modeli, fokal aksonal ve miyelin kaybı, astrositoz ve microgliosis (Şekil 1) üretir. Üç iğne kullanarak, klinik olarak faydalı model ön ayakları, motor görevleri 7 erken yetmezliği ve beyin dokusu deneyimleri küçük ama önemli bir kısmı ile kurula...

Watch this Scientific Journal Video about A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology at JoVE.com

A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Aksonal Dejenerasyon ve Beyaz Cevher Nörobiyoloji Çalışması Çok Yönlü Fare Modeli subkortikal beyaz madde İnme

Stroke affecting white matter accounts for up to 25% of clinical stroke presentations, occurs silently at rates that may be 5-10 fold greater, and ...

a-versatile-murine-model-subcortical-white-matter-stroke-for-study

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

8.0K Görüntüleme.  University of California, Los Angeles. Here we present methodology for the production of a focal stroke in murine white matter by local injection of an irreversible endothelial nitric oxide synthase (eNOS) inhibitor (L-Nio). Presented are two stereotactic variations, retrograde neuronal tracing, and fresh tissue labeling and dissection that expand the potential applications of this technique.

Video: A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

Periventriküler lökomalazi Fare Modelleri

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Nörobilim

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 ...

Tıp

Circumscribed Capsular Infarct Modeling Using a Photothrombotic Technique

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for white matter stroke has hampered research investigations. Here, we describe a novel method for creating a circumscribed capsular infarct that minimizes damage to neighboring gray matter structures. We used pre-surgery neural tracing with adeno-associated virus-green fluorescent protein (AAV-GFP) to identify somatotopic organization of the forelimb area within the internal capsule. The adjustment of light intensity based on different optical properties of gray and white matter contributes to selective destruction of white matter with relative preservation of gray matter. Accurate positioning of optical-neural interface enables destruction of entire forelimb area in the internal capsule, which leads to a marked and persistent motor deficit. Thus, this technique produces highly replicable capsular infarct lesions with a persistent motor deficit. The model will be helpful not only to study white matter stroke (WMS) at the behavioral, circuit, and cellular levels, but also to assess its usefulness for development of new therapeutic and rehabilitative interventions.

This manuscript describes a modeling technique of capsular infarct. Here we utilized a modified photothrombotic technique with low intensity of light after pre-surgery target mapping. Using this technique, we created a circumscribed capsular infarct model with persistent motor impairment.

Bir FOTOTROMBOTİK Tekniği sınırlı Kapsül Enfarktüs Modelleme

Recent increase in the prevalence rate of white matter stroke demands specific research in the field. However, the lack of a pertinent animal model for ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Yanal Kronik Kranial Pencere Hazırlık sağlar<em&gt; İn Vivo</em&gt; Farelerde Distal Orta Serebral Arter Tıkanıklığı takiben Gözlem

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Pro-enflamatuar Sitokinlerin İntraserebral Enjeksiyonu ile İndüklenen Yaygın Serebral Kortikal Demiyelinasyonun Sıçan Modeli

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Modeling Stroke in Mice: Focal Cortical Lesions by Photothrombosis

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, there remain limited therapeutic options for stroke patients. Therefore, more research is needed for pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Given the inability of in vitro models to reproduce the complexity of the brain, experimental stroke models are essential for the analysis and subsequent evaluation of novel drug targets for these mechanisms. In addition, detailed standardized models for all procedures are urgently needed to overcome the so-called replication crisis. As an effort within the ImmunoStroke research consortium, a standardized photothrombotic mouse model using an intraperitoneal injection of Rose Bengal and the illumination of the intact skull with a 561 nm laser is described. This model allows the performance of stroke in mice with allocation to any cortical region of the brain without invasive surgery; thus, enabling the study of stroke in various areas of the brain. In this video, the surgical methods of stroke induction in the photothrombotic model along with&#160;histological analysis are demonstrated.

Described here is the photothrombotic stroke model, where a stroke is produced through the intact skull by inducing permanent microvascular occlusion using laser illumination after administration of a photosensitive dye.

Farelerde Modelleme İnme: Fototomboz ile Fokal Kortikal Lezyonlar

Stroke is a leading cause of death and acquired adult disability in developed countries. Despite extensive investigation for novel therapeutic strategies, ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Farelerde Modelleme İnme: Dış Karotid Arter yoluyla Geçici Orta Serebral Arter Tıkanıklığı

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

Transkraniyal Orta Serebral Arter Oklüzyon Fareleri Modelinin Kurulması

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Yazar Spotlight: İnme Araştırmalarında Yenilikçi Teknikler ve Gelecekteki Yönelimler 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

Yaşlı Farelerde İnme Sonuçlarını İncelemek için Modifiye Transkraniyal Orta Serebral Arter Oklüzyon Modeli

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

Aksonal Dejenerasyon ve Beyaz Cevher Nörobiyoloji Çalışması Çok Yönlü Fare Modeli subkortikal beyaz madde İnme

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Periventriküler lökomalazi Fare Modelleri

Farelerde Modelleme İnme: Distal Orta Serebral Arter Daimi Pıhtılaşma

Bir FOTOTROMBOTİK Tekniği sınırlı Kapsül Enfarktüs Modelleme

Yanal Kronik Kranial Pencere Hazırlık sağlar

Pro-enflamatuar Sitokinlerin İntraserebral Enjeksiyonu ile İndüklenen Yaygın Serebral Kortikal Demiyelinasyonun Sıçan Modeli

Farelerde Modelleme İnme: Fototomboz ile Fokal Kortikal Lezyonlar

Farelerde Modelleme İnme: Dış Karotid Arter yoluyla Geçici Orta Serebral Arter Tıkanıklığı

Yaşlı Farelerde İnme Sonuçlarını İncelemek için Modifiye Transkraniyal Orta Serebral Arter Oklüzyon Modeli

Yaşlı Farelerde İnme Sonuçlarını İncelemek için Modifiye Transkraniyal Orta Serebral Arter Oklüzyon Modeli

Yaşlı Farelerde İnme Sonuçlarını İncelemek için Modifiye Transkraniyal Orta Serebral Arter Oklüzyon Modeli