Name: induction and micro-ct imaging of cerebral cavernous malformations in mouse model
Uploaded: 2017-09-04T15:00:00
Description: Watch this Scientific Journal Video about Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model at JoVE.com

作者承认悉尼大学显微镜 &amp; 微量分析研究设施 (AMMRF) 和澳大利亚显微镜 &amp; 微微量分析中心 (ACMM) 的设施和科学和技术援助。这些研究得到了澳大利亚国家卫生和医学研究理事会 (澳洲) 项目赠款161558和 APP1124011 的支持。

所有动物伦理和议定书均经悉尼地方卫生区动物福利委员会和天津医科大学动物保育与使用委员会 (IACUC) 批准。所有实验都是根据悉尼大学和天津医科大学百年研究所的指导方针/条例进行的, 1。小鼠脑内海绵状畸形的诱导模型 <ol> <li> 跨 Cdh5-CreErt2;Ccm2 fl/fl 与 Ccm2 fl/fl 和 #160 的小鼠; 用 Cdh5-CreErt2 生成凋落物;Ccm2 fl/fl 和 ...

<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>

CCM 是一种常见的血管畸形, 影响多达0.5% 的个人1。CCM 可以以零星或家族形式出现。患者的预后往往不清楚, 因为 CCM 病变可能破裂意外导致中风和其他神经后果。目前, 唯一的治疗方法是手术切除病灶, 伴随着高风险。
人类 CCM 条件最近被复制在动物模型7,8,9,10。?...

CCM 是薄壁, 扩张血管畸形在大脑中的流行率高达 0.5%, 在人口1。CCM 可以遗传为显性障碍, 由于功能突变的三基因之一: CCM1 (aka Krit1), CCM2, 和CCM3 (也称为PDCD10)2,3,4 ,5,6。这些基因存在于单一信号复合体中。
各种模型已经开发出模型人类 ccm 疾病和了解的 ccm 基因的下游途径, 负责 ccm7,8,9,10。最健壮的模型是有条件地删除任何一个Ccm基因与他莫昔芬诱导的Cdh5-CreERT2在 P1 在新生幼崽8,10。这些幼崽从 P6 发展到大脑中的 ccm 病变, 并有望成为临床前研究的理想模型, 以寻找治疗 ccm 疾病的机制和治疗剂。
在小鼠大脑中, CCM 病变的负担主要是用 histology-based 方法测量的, 这种方法非常耗时, 并受调查者的偏见10,11,12。采用 MRI 方法对成人小鼠模型9,13中的 CCM 损伤负荷进行评估。然而, 一个高度专业化的小动物磁共振成像仪和长时间扫描-一夜之间需要达到一个满意的分辨率, 以确定 CCM 病变。还有, MRI 是否可用于检测新生小鼠的 CCM 病变尚未报告, 分辨率可能会限制灵敏度。
我们最近开发了一种显微 CT 技术来图像和分析 CCM 病变14,15。这种高分辨率、时间和 cost-effective 的方法极大地提高了 CCM 疾病模型在机械和治疗研究中的价值。对比增强, 全贴装染色方法已用于软组织和小鼠胚胎的显微 CT 成像16,17。以前, 我们使用了 osmium-based 染色增强了在脑部14中 CCM 病变的微 CT 成像的对比度。本文采用了一种无毒、无损、低成本的试剂--碘基卢戈溶液, 增强了微 CT 成像的对比度。碘可以弥漫整个大脑和具有高亲和力的血液18。
详细的协议是在这里提出的诱导 ccm 病变的新生儿小鼠模型, 连同成像和分析的 ccm 病变的 contrast-based 微 CT。该方法对 ccm 病变体积进行了定量的全球测量, 准确地确定了小鼠脑内 ccm 病变的数目和3维位置, 大大降低了这些动物表型的成本和时间。

<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

突变在CCM1 (aka KRIT1), CCM2,或CCM3 (aka PDCD10)基因导致大脑海绵状畸形 (CCM) 在人类。在产后动物中, 用他莫昔芬引起的ccm基因缺失, 建立了小鼠 ccm 疾病模型。这些小鼠模型为研究 CCM 疾病的分子机制和治疗方法提供了宝贵的工具。准确和定量的方法来评估病变的负担和进展是必不可少的充分利用这些动物模型的价值。在这里, 我们演示了 ccm 疾病的诱导在一个小鼠模型和使用对比增强 X 射线显微计算机断层扫描 (微 CT) 方法测量 ccm 病变负荷的小鼠脑。在产后日 1 (P1), 我们使用 4-hydroxytamoxifen (4HT) 激活重组活动从 Cdh5-CreErt2 转基因, 以切割 floxed 等位基因的Ccm2。P8 对小鼠脑内 CCM 病变进行分析。在微 CT, 碘基卢戈的解决方案被用来提高对比度的脑组织。我们优化了扫描参数, 并利用了9.5 µm 的体素维数, 这导致了大约25µm 的最小特征尺寸。该分辨率足以全面准确地测量 ccm 病变体积和数量, 为小鼠脑内的 ccm 病变提供高质量的3维图谱。该方法提高了已建立的小鼠模型的价值, 研究了 CCM 和其他脑血管疾病的分子基础和潜在的疗法。

单注射4HT 在 P1 是足以诱发 CCM 病变的小脑。卢戈的碘对比微 CT 充分检测 CCM 病变, 并可以量化其体积和数量。利用优化的微 CT, 我们在Ccm2iECKO 小鼠的 hindbrains 中对 CCM 病变进行了成像。扫描的 X 射线图像重建, 以产生3维图像的鼠标的大脑, 这使得可视化整个病变的脑实质在不同的深度和方向, 和评估的结构和3的位置的病变在大脑中。重要的是, 这个图像数据?...

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

本协议演示了在小鼠模型中诱导脑海绵状畸形的疾病, 并使用对比增强微计算机断层扫描来测量病变的负担。该方法提高了建立的小鼠模型的价值, 以研究脑海绵状畸形和其他血管性疾病的分子基础和潜在的疗法。

小鼠脑海绵体畸形的诱导及显微 CT 成像

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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