Yazarlar imkanları ve Sydney mikroskobu ve Microanalysis araştırma tesisi (AMMRF) ve Avustralya Merkezi bilimsel ve teknik yardım mikroskobu &amp; Microanalysis (ACMM) Sydney Üniversitesi için kabul edersiniz. Bu çalışmalar Avustralya Ulusal Sağlık tarafından desteklenen edildi ve tıbbi araştırma Konseyi (NHMRC) projesi 161558 ve APP1124011 (XZ) hibe.

tüm hayvan etik ve protokolleri Sydney yerel sağlık ilçe hayvan refahı Komitesi ve kurumsal hayvan bakım ve kullanım Komitesi (IACUC) Tianjin Tıp Üniversitesi tarafından onaylanmıştır. Tüm deneyler yüzüncü Enstitüsü, University of Sydney ve Tianjin Tıp Üniversitesi yönergeleri/düzenlemeler altında yapılmıştır 1. Fare modellerinde serebral derin malformasyon indüksiyon <ol><li>çapraz Cdh5-CreErt2; Ccm2 fl/fl çöp ile oluşturmak için Ccm2 fl/fl ile fareler Cdh5-CreErt2; Ccm2 fl/fl (Ccm2 iECKO) pups ve littermate denetimleri (Ccm2 fl/fl). Cdh5-CreErt2 ve Ccm2 fl/fl hayvan-si olmak be önceden açıklanan 19.</li>
	<li>4HT % 100 etanol içinde olur ve mağaza içinde 30 µL aliquots-80 ° C'de (4HT konsantrasyonu: 10 mg/mL). Kullanım günü, mısır yağı (0,5 mg/mL) bölünmemeli 4HT sulandırmak.</li>
	<li>İntragastrically 4HT 50 µL enjekte (0,5 mg/mL) için bir insülin Ģırınga kullanarak deneysel CCM lezyonlar ikna etmek için P1, Yenidoğan pups.</li>
</ol> 2. Örnek mikro-CT inceden inceye gözden geçirmek için hazırlık <ol><li>karbon dioksit Havasızlıktan Boğulma yoluyla P8, Yenidoğan pups ötenazi.</li>
	<li>29'lik iğne %2 paraformaldehyde 3 mL ile PBS içinde bir 10 mL şırınga fare Kalp karıncığı ekleyerek intra kardiyak perfüzyon gerçekleştirmek. 
	Dikkat: Paraformaldehyde toksik; uygun koruma giymek.</li>
	<li>Bir makas kullanarak gövdesini başından bağlantısını kesin. Bir makas kullanarak kafa derisini kaldırmak ve forseps kullanarak tüm beynini incelememize kafatası soyma.</li>
	<li>Al stereomicroscope ile beyin görüntüleri 7.82 x büyütme, 1 x kazanç, 0.6 gama ve 20 ms pozlama süresi,.</li>
	<li>Tamir disseke beyin ile %4 paraformaldehyde PBS çözümde gecede. 
	Dikkat: Paraformaldehyde toksik; uygun koruma giymek.</li>
	<li>Ertesi gün hindbrains bir forseps kullanarak bağlantısını kesin ve yıkama PBS çözüm.</li>
	<li>Hindbrains içinde Lugol kuluçkaya &#39; s iyot çözüm için 48 h.</li>
	<li>Kuluçka kısa bir süre hava kuru aşırı Lugol kaldırmak için hindbrains &#39; s iyot çözüm.</li>
	<li>Lugol paketi &#39; s iyot lekeli hindbrains 0.65 mL microcentrifuge tüpler ve mühür ile doku büzülme ( Şekil 2 A) önlemek için tamamen plastik parafin film.</li>
	<li>5 mL plastik tüpler ( Şekil 2 B) tarama sırasında hareket etmesini engellemek için sünger ile yer microcentrifuge tüpler.</li>
</ol> 3. Mikro-CT inceden inceye gözden geçirmek, CCM fare beyin <ol><li>dikey olarak mikro-CT sistemdeki bir tüp bir alüminyum kutusunda paketlenmiş arka beyin mount.</li>
	<li>540 projeksiyonlar ve 2 s çekim hızı 50 kaynak şartlar ile tarama parametrelerini ayarlamak kV ve 10 W tomografik veri kümeleri (görüntü çözünürlüğü 9.5 mikron/piksel) elde etmek için.</li>
	<li>Üzerinden tarama ilerlemenin yeniden otomatik olarak mikro-CT sistem tarafından sağlanan donanım tabanlı projeksiyon imar yazılım tarafından 16-bit Aksiyel dilimler görüntü dizi üreten (&#34; TXM &#34; dosya).</li>
</ol> 4. Mikro-CT veri analizi <ol><li>resimle dosyasını açın (&#34; TXM &#34; dosyası) 3 boyutlu analiz yazılımı ve beyin kullanarak görselleştirmek &#34; toplu oluşturma &#34; işlev.</li>
	<li>Dönüştürmek istediğiniz yönlendirme kullanarak arka beyin &#34; sınırlayıcı kutu &#34; ve &#34; kırpma uçak &#34; fonksiyonları.</li>
	<li>Genişletilmiş Mod dönüştürülmüş görüntüyü yeniden örnekle ve Voksel boyutu korumak.</li>
	<li>Oluşturma &#34; yeni etiket alanını düzenleyin &#34; dönüştürülmüş görüntü (4.3) üzerinde ve sadece gri tonlama yoğunluğu kullanarak arka beyin vurgulayın.</li>
	<li>Vurgulanan alanlar ekleyin ve çalıştırın &#34; etiket analysis &#34; bütün arka beyin (Yani, Toplam hacim ve alan) ölçümleri elde etmek için. Ölçümler bir Excel dosyasındaki dışa.</li>
	<li>Arka beyin kullanarak görselleştirmek &#34; isosurface işleme &#34; işlev.</li>
	<li>Başka bir hesap oluşturun &#34; yeni etiket alanını düzenleyin &#34; gri tonlama yoğunluğu kullanarak lezyonlar ile dönüştürülmüş görüntü ve vurgulamak alanlarda.</li>
	<li>Vurgulanan alanlar ekleyin ve çalıştırın &#34; etiket &#34; lezyonları (Yani, Toplam hacim ve alan) arka beyin içinde ölçümleri elde etmek için analiz. Ölçümler bir Excel dosyasındaki dışa.</li>
	<li>Lezyonlar kullanarak görselleştirmek &#34; yüzey oluşturmak &#34; ve &#34; yüzey görünümü &#34; fonksiyonları.</li>
	<li>İstenen yönler, arka beyin görüntüleri anlık görüntü görüntü görüntüsünü veya lezyonlar, 3-D görüntüleme için bir film üretmek.</li>
</ol>

<ol>
	<li>Fischer, A., Zalvide, J., Faurobert, E., Albiges-Rizo, C., Tournier-Lasserve, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+cavernous+malformations:+from+CCM+genes+to+endothelial+cell+homeostasis.">Cerebral cavernous malformations: from CCM genes to endothelial cell homeostasis.</a> Trends Mol Med. 19 (5), 302-308 (2013).</li><li>Liquori, C. L., 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=Mutations+in+a+gene+encoding+a+novel+protein+containing+a+phosphotyrosine-binding+domain+cause+type+2+cerebral+cavernous+malformations.">Mutations in a gene encoding a novel protein containing a phosphotyrosine-binding domain cause type 2 cerebral cavernous malformations.</a> Am J Hum Genet. 73 (6), 1459-1464 (2003).</li><li>Laberge-le Couteulx, 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=Truncating+mutations+in+CCM1,+encoding+KRIT1,+cause+hereditary+cavernous+angiomas.">Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas.</a> Nat Genet. 23 (2), 189-193 (1999).</li><li>Sahoo, T., 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=Mutations+in+the+gene+encoding+KRIT1,+a+Krev-1/rap1a+binding+protein,+cause+cerebral+cavernous+malformations+(CCM1).">Mutations in the gene encoding KRIT1, a Krev-1/rap1a binding protein, cause cerebral cavernous malformations (CCM1).</a> Hum Mol Genet. 8 (12), 2325-2333 (1999).</li><li>Denier, C., 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=Mutations+within+the+MGC4607+gene+cause+cerebral+cavernous+malformations.">Mutations within the MGC4607 gene cause cerebral cavernous malformations.</a> Am J Hum Genet. 74 (2), 326-337 (2004).</li><li>Bergametti, F., 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=Mutations+within+the+programmed+cell+death+10+gene+cause+cerebral+cavernous+malformations.">Mutations within the programmed cell death 10 gene cause cerebral cavernous malformations.</a> Am J Hum Genet. 76 (1), 42-51 (2005).</li><li>McDonald, D. A., 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=A+novel+mouse+model+of+cerebral+cavernous+malformations+based+on+the+two-hit+mutation+hypothesis+recapitulates+the+human+disease.">A novel mouse model of cerebral cavernous malformations based on the two-hit mutation hypothesis recapitulates the human disease.</a> Hum Mol Genet. 20 (2), 211-222 (2011).</li><li>Boulday, G., 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=Developmental+timing+of+CCM2+loss+influences+cerebral+cavernous+malformations+in+mice.">Developmental timing of CCM2 loss influences cerebral cavernous malformations in mice.</a> J Exp Med. 208 (9), 1835-1847 (2011).</li><li>Chan, A. C., 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=Mutations+in+2+distinct+genetic+pathways+result+in+cerebral+cavernous+malformations+in+mice.">Mutations in 2 distinct genetic pathways result in cerebral cavernous malformations in mice.</a> J Clin Invest. 121 (5), 1871-1881 (2011).</li><li>Zheng, X., 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=Cerebral+cavernous+malformations+arise+independent+of+the+heart+of+glass+receptor.">Cerebral cavernous malformations arise independent of the heart of glass receptor.</a> Stroke. 45 (5), 1505-1509 (2014).</li><li>McDonald, D. A., 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=Fasudil+decreases+lesion+burden+in+a+murine+model+of+cerebral+cavernous+malformation+disease.">Fasudil decreases lesion burden in a murine model of cerebral cavernous malformation disease.</a> Stroke. 43 (2), 571-574 (2012).</li><li>Maddaluno, L., 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=EndMT+contributes+to+the+onset+and+progression+of+cerebral+cavernous+malformations.">EndMT contributes to the onset and progression of cerebral cavernous malformations.</a> Nature. 498 (7455), 492-496 (2013).</li><li>Gibson, C. C., 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=Strategy+for+identifying+repurposed+drugs+for+the+treatment+of+cerebral+cavernous+malformation.">Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.</a> Circulation. 131 (3), 289-299 (2015).</li><li>Choi, J. 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=Micro-CT+Imaging+Reveals+Mekk3+Heterozygosity+Prevents+Cerebral+Cavernous+Malformations+in+Ccm2-Deficient+Mice.">Micro-CT Imaging Reveals Mekk3 Heterozygosity Prevents Cerebral Cavernous Malformations in Ccm2-Deficient Mice.</a> PLoS One. 11 (8), 0160833 (2016).</li><li>Zhou, Z., 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=Cerebral+cavernous+malformations+arise+from+endothelial+gain+of+MEKK3-KLF2/4+signalling.">Cerebral cavernous malformations arise from endothelial gain of MEKK3-KLF2/4 signalling.</a> Nature. 532 (7597), 122-126 (2016).</li><li>Metscher, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroCT+for+comparative+morphology:+simple+staining+methods+allow+high-contrast+3D+imaging+of+diverse+non-mineralized+animal+tissues.">MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.</a> BMC Physiol. 9, 11 (2009).</li><li>Johnson, J. T., 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=Virtual+histology+of+transgenic+mouse+embryos+for+high-throughput+phenotyping.">Virtual histology of transgenic mouse embryos for high-throughput phenotyping.</a> PLoS Genet. 2 (4), 61 (2006).</li><li>Anderson, R., Maga, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Procedure+for+Rapid+Imaging+of+Adult+Mouse+Brains+with+MicroCT+Using+Iodine-Based+Contrast.">A Novel Procedure for Rapid Imaging of Adult Mouse Brains with MicroCT Using Iodine-Based Contrast.</a> PLoS One. 10 (11), 0142974 (2015).</li><li>Zheng, X., 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=Dynamic+regulation+of+the+cerebral+cavernous+malformation+pathway+controls+vascular+stability+and+growth.">Dynamic regulation of the cerebral cavernous malformation pathway controls vascular stability and growth.</a> Dev Cell. 23 (2), 342-355 (2012).</li></ol>

Yazarlar ifşa gerek yok.

CCM bireyler1ilâ % 0,5 etkiler ortak bir vasküler malformasyon var. CCM durum sporadik veya ailesel formunda oluşabilir. CCM lezyonlar beklenmedik biçimde kontur ve diğer nörolojik sonuçları neden rüptürü gibi hasta prognoz genellikle belli değildir. Şu anda, tek tedavi seçeneği cerrahi olarak yüksek riskli tarafından eşlik eder lezyonları kaldırmaktır.
İnsan CCM koşulları son zamanlarda çoğaltılamaz içinde hayvan modelleri7...

CCM ince duvarlı, insan nüfusunun%10.5'yaygınlığı ile vasküler malformasyon beyin büyümüş. CCM nedeniyle işlev kaybı mutasyonlar birinde üç genleri dominant bozukluk olarak miras: CCM1 (aka KRIT1), CCM2ve CCM3 ( PDCD10olarak da bilinir)2,3,4 ,5,6. Bu genlerin tek bir karmaşık sinyal mevcut.
Çeşitli modelleri modeli insan CCM hastalık ve akış aşağı yolları CCM7,8,9,10için sorumludur CCM genlerin anlamak için geliştirilmiştir. En güçlü modeli yeni doğan yavrular8,10 Ccm genlerinde tamoxifen indüklenebilir Cdh5-CreERT2 , P1 ile herhangi bir koşullu olarak silmektir. Bu pups ileriye P6 beyin lezyonlarının CCM geliştirmek ve arama mekanizmaları için önceden klinik çalışmalar ve CCM hastalıkları tedavisinde terapötik ajanlar için ideal bir model olacağı tahmin ediliyor.
CCM lezyon yük fare beynindeki ölçülen öncelikle Histoloji tabanlı yöntemler tarafından son derece zaman alıcı bir yaklaşım ve araştırmacı tabi10,11,12önyargı. MRI tabanlı yöntemleri CCM lezyon yük yetişkin fare modeli9,13' te değerlendirmek için kullanılmaktadır. Ancak, son derece uzmanlaşmış küçük hayvan MRI enstrüman ve gece için birkaç saat uzun inceden inceye gözden geçirmek zaman CCM lezyonlar belirlenmesi için tatmin edici bir çözüme ulaşmak için gereklidir. Ayrıca, MRI CCM lezyonlar yenidoğan farelerde algılamak için kullanılıp kullanılamayacağı değil bildirilmiştir ve çözünürlük hassasiyeti sınırlayabilir.
Son zamanlarda bir mikro-CT teknik resim ve CCM lezyon14,15çözümlemek için geliştirdik. Bu yüksek çözünürlüklü, zaman ve maliyet-etkin yöntem önemli ölçüde mekanik ve tedavi edici çalışmalar CCM hastalığı modelinde değerini artırdı. Yumuşak doku ve fare embriyo16,17mikro-CT görüntüleme için kontrast arttırıcı, Bütün Dağı boyama yöntemleri kullanılmaktadır. Daha önce biz bir boyama Osmiyum tabanlı mikro-CT görüntüleme beyin14CCM lezyonların için kontrastı Artır için kullandık. Bu gazetede bir daha az toksik, yıkıcı olmayan-kullandık ve maliyet-etkin reaktifi tabanlı bir iyot Lugol'ın çözümü, mikro-CT görüntüleme için kontrastı artırmak için. İyot beyin diffüz ve kan18için yüksek bir yakınlık vardır.
Detaylı iletişim kuralı burada bir yenidoğan fare modeli ile birlikte görüntüleme lezyonlarının CCM indüksiyon ve CCM lezyonlar kontrast tabanlı mikro tomografi ile analizi için sunulan Bu mikro-CT tabanlı yöntem CCM lezyon birimin nicel küresel ölçüm sağlar, doğru sayı ve 3-b farenin beyin lezyonlarının CCM konumunu tanımlar ve büyük ölçüde maliyeti azaltır ve bu hayvanların fenotip için gerekli süre.

<table >
	
		
		
		
		
	
	<tbody>
		<tr >
			<td >4-hydroxy tamoxifen</td>
			<td >Sigma-Aldrich</td>
			<td >H6278</td>
			<td >To activate Cdh5-CreErt2</td>
		</tr>
		<tr >
			<td >Corn oil</td>
			<td >Sigma-Aldrich</td>
			<td >C8267-500ML</td>
			<td >To dilute 4-hydroxy tamoxifen</td>
		</tr>
		<tr >
			<td >Stereomicroscope</td>
			<td >Leica</td>
			<td >M205FA</td>
			<td >To take macroscopic images</td>
		</tr>
		<tr >
			<td >Lugol&#39;s Iodine solution</td>
			<td >Sigma-Aldrich</td>
			<td >L6146</td>
			<td >To stain samples for contrast micro-CT</td>
		</tr>
		<tr >
			<td >Plastic paraffin film</td>
			<td >Parafilm</td>
			<td >PM992</td>
			<td >To package samples</td>
		</tr>
		<tr >
			<td >Micro-CT</td>
			<td >Xradia</td>
			<td >MicroXCT-400</td>
			<td >Micro-CT</td>
		</tr>
		<tr >
			<td >3D rendering software</td>
			<td >FEI Visualization Science group</td>
			<td >Avizo 3D image processing software</td>
			<td >To analyse micro-CT scans</td>
		</tr>
	</tbody>
</table>

induction and micro-ct imaging of cerebral cavernous malformations in mouse model

Mutasyonların CCM1 (aka KRIT1), CCM2, veya CCM3 (aka PDCD10) gene neden serebral derin malformasyon (CCM) insanlarda. Fare modelleri CCM hastalığının indüklenen tamoxifen silme postnatal hayvanlarda Ccm genlerin tarafından kurulmuştur. Bu fare modelleri moleküler mekanizması ve CCM hastalığı için tedavi yaklaşımları araştırmak için çok değerli çözümler sağlar. Lezyon yük ve ilerlemeyi değerlendirmek için doğru ve nicel bir yöntem bu hayvan modellerin tam değeri koşum için esastır. Burada, biz bir fare modeli hastalığında CCM indüksiyon göstermek ve x-ışını mikro Bilgisayarlı Tomografi (mikro-CT) yöntemi ölçü CCM lezyon yük fare beynindeki için geliştirilmiş kontrast kullanın. Doğum sonrası gün 1 (P1), Ccm2floxed alleli ayırmak için Cdh5-CreErt2 transgene Cre recombinase aktivitesinden etkinleştirmek için 4-hydroxytamoxifen (4HT) kullanılır. Fare beyin lezyonlarının CCM P8 analiz edildi. Mikro-CT için iyot dayalı Lugol'ın çözüm beyin dokusunda kontrast geliştirmek için kullanıldı. Biz tarama parametreleri en iyi duruma getirilmiş ve 9,5 µm, voxel boyutunun yaklaşık 25 µm minimum özellik boyutu için yol faydalanır. Bu çözünürlük CCM lezyon birimi ve numarasını genel olarak ve doğru bir şekilde ölçmek ve fare beynindeki CCM lezyonların yüksek kaliteli 3-b eşleme sağlamak yeterli olur. Bu yöntem moleküler temeli ve potansiyel tedaviler CCM ve diğer serebrovasküler hastalıklar için çalışmaya kurulan fare modelleri değerini artırır.

4HT P1, tek bir enjeksiyon beyincik lezyonlarının CCM ikna etmek için yeterli idi. Lugol'ın tezat iyot mikro-CT yeterince CCM lezyonlar tespit ve kendi birim ve sayı ölçmek. En iyi duruma getirilmiş mikro-CT kullanmak, biz hindbrains Ccm2iECKO farelerin lezyonlarının CCM görüntüsü. Taranan X-ray görüntüleri görselleştirme tüm lezyonların beyin parankimi farklı derinliklerde ve yönelimleri izin fare beyin 3 boyutlu görüntüleri ve değerlendirm...

Watch this Scientific Journal Video about Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model at JoVE.com

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Bu iletişim kuralı bir fare modeli hastalığında serebral derin malformasyon indüksiyon gösterir ve kullanır kontrast mikro bilgisayarlı tomografi lezyon yük ölçmek için geliştirilmiş. Bu yöntem moleküler temelini ve serebral derin malformasyon için potansiyel tedaviler ve diğer serebrovasküler hastalıklar çalışmaya kurulan fare modelleri değerini artırır.

İndüksiyon ve fare modeli serebral derin malformasyon mikro-CT görüntüleme

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

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10.8K Görüntüleme.  Centenary Institute. Bu iletişim kuralı bir fare modeli hastalığında serebral derin malformasyon indüksiyon gösterir ve kullanır kontrast mikro bilgisayarlı tomografi lezyon yük ölçmek için geliştirilmiş. Bu yöntem moleküler temelini ve serebral derin malformasyon için potansiyel tedaviler ve diğer serebrovasküler hastalıklar çalışmaya kurulan fare modelleri değerini artırır.

Video: Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse 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 ...

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Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Biyoparlaklık ve Micro CT Görüntüleme Tümör Metastazlari ve osteolitik Lezyonlar İzleme

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Oral Skuamöz Hücre Karsinomu Ortotopik Fare Modeli Tümör Hücre Invasion Multi-foton Görüntüleme

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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 ...

Non-invasive Optical Measurement of Cerebral Metabolism and Hemodynamics in Infants

Perinatal brain injury remains a significant cause of infant mortality and morbidity, but there is not yet an effective bedside tool that can accurately screen for brain injury, monitor injury evolution, or assess response to therapy. The energy used by neurons is derived largely from tissue oxidative metabolism, and neural hyperactivity and cell death are reflected by corresponding changes in cerebral oxygen metabolism (CMRO2). Thus, measures of CMRO2 are reflective of neuronal viability and provide critical diagnostic information, making CMRO2 an ideal target for bedside measurement of brain health.
Brain-imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) yield measures of cerebral glucose and oxygen metabolism, but these techniques require the administration of radionucleotides, so they are used in only the most acute cases.
Continuous-wave near-infrared spectroscopy (CWNIRS) provides non-invasive and non-ionizing radiation measures of hemoglobin oxygen saturation (SO2) as a surrogate for cerebral oxygen consumption. However, SO2 is less than ideal as a surrogate for cerebral oxygen metabolism as it is influenced by both oxygen delivery and consumption. Furthermore, measurements of SO2 are not sensitive enough to detect brain injury hours after the insult 1,2, because oxygen consumption and delivery reach equilibrium after acute transients 3. We investigated the possibility of using more sophisticated NIRS optical methods to quantify cerebral oxygen metabolism at the bedside in healthy and brain-injured newborns. More specifically, we combined the frequency-domain NIRS (FDNIRS) measure of SO2 with the diffuse correlation spectroscopy (DCS) measure of blood flow index (CBFi) to yield an index of CMRO2 (CMRO2i) 4,5.
With the combined FDNIRS/DCS system we are able to quantify cerebral metabolism and hemodynamics. This represents an improvement over CWNIRS for detecting brain health, brain development, and response to therapy in neonates. Moreover, this method adheres to all neonatal intensive care unit (NICU) policies on infection control and institutional policies on laser safety. Future work will seek to integrate the two instruments to reduce acquisition time at the bedside and to implement real-time feedback on data quality to reduce the rate of data rejection.

We combined frequency-domain near-infrared spectroscopy measures of cerebral hemoglobin oxygenation with diffuse correlation spectroscopy measures of cerebral blood flow index to estimate an index of oxygen metabolism. We tested the utility of this measure as a bedside screening tool to evaluate the health and development of the newborn brain.

Bebeklerde Serebral Metabolizma ve Hemodinamiğin Noninvaziv Optik Ölçüm

Perinatal brain injury remains a significant cause of infant mortality  and morbidity, but there is not yet an effective bedside tool that can  accurately ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Bir Insanlaşmış Fare Modeli epidermal melanin ve Karşı Koruma yanığı Farmakolojik İndüksiyon

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Fare Serebral Hipoksi-iskemi sırasında Eşzamanlı PET / MR Görüntüleme

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

A Battery of Motor Tests in a Neonatal Mouse Model of Cerebral Palsy

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, standardized battery of neonatal mouse motor tests. These tests can validate injury or disease models, determine treatment efficacy and/or assess motor behaviors in new transgenic strains. This paper presents a series of neonatal motor tests to evaluate general motor function, including ambulation, hindlimb foot angle, surface righting, negative geotaxis, front- and hindlimb suspension, grasping reflex, four limb grip strength and cliff aversion. Mice between the ages of post-natal day 2 to 14 can be used. In addition, these tests can be used for a wide range of neurological and neuromuscular pathologies, including cerebral palsy, hypoxic-ischemic encephalopathy, traumatic brain injury, spinal cord injury, neurodegenerative diseases, and neuromuscular disorders. These tests can also be used to determine the effects of pharmacological agents, as well as other types of therapeutic interventions. In this paper, motor deficits were evaluated in a novel neonatal mouse model of cerebral palsy that combines hypoxia, ischemia and inflammation. Forty-eight hours after injury, five tests out of the nine showed significant motor deficits: ambulation, hindlimb angle, hindlimb suspension, four limb grip strength, and grasping reflex. These tests revealed weakness in the hindlimbs, as well as fine motor skills such as grasping, which are similar to the motor deficits seen in human cerebral palsy patients.

Presented is a concise battery of mouse neonatal motor tests. Using these tests, neonatal motor deficits can be demonstrated in a variety of neonatal motor disorders. By having a standardized set of tests, results from different studies can be compared, allowing for better and accurate reporting between groups.

Cerebral Palsy bir Yenidoğan Fare Modeli Motor Testleri A Pil

As the sheer number of transgenic mice strains grow and rodent models of pediatric disease increase, there is an expanding need for a comprehensive, ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Manyetik Rezonans Görüntüleme Kullanarak Deneysel Serebral Sıtma Bir Modelinde Ödem Gelişimi ve Mikrovasküler Patolojinin İn Vivo Takibi

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Quantitative Micro-CT Analysis of Aortopathy in a Mouse Model of &#946;-aminopropionitrile-induced Aortic Aneurysm and Dissection

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of aortic disease exist, in vivo imaging of the vasculature has been limited. In recent years, micro-computerized tomography (micro-CT) has emerged as a preferred modality for imaging both large and small vessels both in vivo and ex vivo. In conjunction with a method of vascular casting, we have successfully used micro-CT to characterize the frequency and distribution of aortic pathology in &#946;-aminopropionitrile-treated C57/Bl6 mice. Technical limitations of this method include variations in the quality of the perfusion introduced by poor animal preparation, the application of proper methodologies for vessel size quantification, and the non-survivability of this procedure. This article details a methodology for the intravascular perfusion of a lead-based radiopaque silicone rubber for the quantitative characterization of aortopathy in a mouse model of aneurysm and dissection. In addition to visualizing aortic pathology, this method may be used for examining other vascular beds in vivo or vascular beds removed post-mortem.

This article describes a detailed methodology of using a radiopaque lead-based silicone rubber to perfuse the murine vasculature for aortic diameter quantification in a mouse model of aortic aneurysm and dissection.

Aortopathy aort anevrizması β-aminopropionitrile-indüklenen ve diseksiyon, fare modeli nicel mikro-CT Analizi

Aortic aneurysm and dissection is associated with significant morbidity and mortality in the population and can be highly lethal. While animal models of ...