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

The authors acknowledge the facilities and the scientific and technical assistance of the Sydney Microscopy &amp; Microanalysis Research Facility (AMMRF) and the Australian Centre for Microscopy &amp; Microanalysis (ACMM) at the University of Sydney. These studies were supported by Australian National Health and Medical Research Council (NHMRC) project grant 161558 and APP1124011 (XZ).

All animal ethics and protocols were approved by The Sydney Local Health District Animal Welfare Committee and Institutional Animal Care and Use Committee (IACUC) of Tianjin Medical University. All experiments were conducted under the guidelines/regulations of Centenary Institute, University of Sydney and Tianjin Medical University
1. Induction of Cerebral Cavernous Malformations in Mouse Models
<ol>
	<li>Cross Cdh5-CreErt2; Ccm2fl/fl mice with Ccm2fl/fl</e...

<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 is a common vascular malformation that affects up to 0.5% of individuals1. CCM can occur in sporadic or familial form. Patient prognosis is often unclear as CCM lesions can rupture unexpectedly to cause stroke and other neurological consequences. Currently, the only treatment option is to surgically remove lesions, which are accompanied by high risk.
Human CCM conditions have recently been reproduced in animal models7,<sup class="x...

CCM are&#160;thin walled, dilated vascular malformations in the brain with prevalence of up to 0.5% in the human population1. CCM can be inherited as a dominant disorder due to loss-of-function mutations in one of three genes: CCM1 (aka Krit1), CCM2, and CCM3 (also called PDCD10)2,3,4,5,6. These genes are present in a single signaling complex.
Various models have been developed to model human CCM disease and to understand the downstream pathways of CCM genes that are responsible for CCM7,8,9,10. The most robust model is to conditionally delete any one of the Ccm genes with tamoxifen-inducible Cdh5-CreERT2 at P1 in newborn pups8,10. These pups develop CCM lesions in the brain from P6 onward and are expected to be an ideal model for pre-clinical studies in search for mechanisms and therapeutic agents in treating CCM diseases.
CCM lesion burden in mouse brain has been measured primarily by histology-based methods, an approach that is extremely time-consuming and subject to investigator bias10,11,12. MRI based methods have been used to assess CCM lesion burden in the adult mouse model9,13. However, a highly specialized small animal MRI instrument and long scan-time of several hours to overnight is required to achieve a satisfactory resolution to identifying CCM lesions. Also, whether MRI can be used to detect CCM lesions in neonatal mice has not been reported and resolution may limit sensitivity.
We have recently developed a micro-CT technique to image and analyze CCM lesion14,15. This high-resolution, time and cost-effective method dramatically boosted the value of the CCM disease model in mechanistic and therapeutic studies. Contrast-enhancing, whole mount staining methods have been used for micro-CT imaging of soft tissues and mouse embryos16,17. Previously, we have used an osmium-based staining to enhance contrast for micro-CT imaging of CCM lesions in brain14. In this paper, we used a less toxic, non-destructive, and cost-efficient reagent, an iodine based Lugol&#39;s solution, to enhance contrast for micro-CT imaging. Iodine can diffuse throughout the brain and has a high affinity for blood18.
The detailed protocol is presented here for the induction of CCM lesions in a neonatal mouse model along with the imaging and analysis of CCM lesions with a contrast-based micro-CT. This micro-CT based method provides quantitative global measurement of CCM lesion volume, accurately identifies the number and 3-D location of CCM lesions in the mouse brain, and greatly reduces the cost and time required to phenotype these animals.

<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

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 been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

A single injection of 4HT at P1 was sufficient to induce CCM lesions in the cerebellum. Lugol&#39;s iodine contrasted micro-CT sufficiently detected CCM lesions and could quantify its volume and number. Utilizing the optimized micro-CT, we imaged CCM lesions in the hindbrains of Ccm2iECKO mice. Scanned X-ray images were reconstructed to produce 3-D images of the mouse brain, which allowed visualization of entire lesions in the brain parenchyma at different depths and o...

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

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

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 ...

The overall goal of this procedure is to induce cerebral cavernous malformations in neonatal mice, and measure the disease burden using micro-CT. This method can help answer key questions in cerebral vascular disease field, such as cerebral cavernous malformations. The main advantage of this technique is that cerebral cavernous malformation disease burden in neonatal mouse models can be determined accurately and efficiently compared to existing methods.Visual demonstration of this method is critical, as induction of cerebral cavernous malformation and micro-CT analyzing steps are difficult to learn, because it requires experience and use of sophisticated software. Begin by dissolving 4-HT in 100%ethanol to a concentration of 10 milligrams per milliliter. Prepare 30 microliter alaquads, and then store at minus 80 degrees Celsius.On the day of use, dilute the alaquad at 4-HT in corn oil to 0.5 milligrams per milliliter. Then use an insulin syringe to introgastricallly inject 50 microliters of 4-HT into P1 neonatal pups to induce experimental CCM lesions. Begin sample preparation by filling a 10 milliliter syringe equipped with a 29 gauge needle with three milliliters of 2%paraformaldehyde and PBS.Then perform intracardiac profusion on euthenized P8 pup by inserting the needle into the ventricle of the mouse heart, and slowly injecting the full volume of paraformaldehyde. After using scissors to detach the head, remove the skin from the head and peel off the skull using forceps to dissect the whole brain into 4%paraformaldehyde and PBS. Post fix the brains overnight at 4-HT degrees Celsius.The following day, detach the hind brains using forceps, and rinse with PBS solution. Then incubate the hind brains in Lugol's iodine solution, for 48 hours. Following the incubation, briefly air dry the hind brains to remove excess Lugol's iodine solution.Then pack the Lugol's iodine stained hind brains in 0.65 microliter microcentrifuge tubes, and seal completely with plastic paraffin film to avoid tissue shrinkage. Place the microcentrifuge tubes in five milliliter plastic tubes with sponges to prevent them from moving during the scan. Vertically mount the tube containing a hind brain on an aluminum holder in the micro-CT system.Set the scanning parameters. Use 540 projections and a two second exposure time with source conditions of 50 kilovolts and 10 Watts to acquire the tomographic datasets. Radio graphs from the scan are reconstructed automatically by hardware-based projection reconstruction software, supplied by the micro-CT system, producing an image series of 16-bit axial slices.Lugol's iodine contrasted micro-CT sufficiently detected CCM lesions. Scanned X-ray images were reconstructed to produced 3D images of the mouse brain, which allowed visualization of entire lesions in the brain parenchyma, at different depths and orientations, and assessment of the structure in 3D location of lesions in the brain. This table shows an example of quantitative output of the labeled individual lesions.After its development, this technique paved way for researchers in the field of vascular disease to explore mechanism and therapeutic options for cerebral cavernous malformations in mouse models. After watching this video, you should have a good understanding of how to induce CCM lesion in mouse models, and how to acquire and analyze CCM lesion burden with micro-CT technique.

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