Name: induction and micro-ct imaging of cerebral cavernous malformations in mouse model
Uploaded: 2017-09-04T15:00:00.000+00:00
Duration: 5 min 12 s
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&#160;to generate litters with Cdh5-CreErt2; Ccm2fl/fl&#160;(Ccm2iECKO) pups and littermate controls (Ccm2fl/fl). Cdh5-CreErt2 and Ccm2fl/fl animals have been previously described19.</li>
	<li>Dissolve 4HT in 100% ethanol and store at -80 &#176;C in aliquots of 30 &#181;L (4HT concentration: 10 mg/mL). On the day of use, dilute aliquoted 4HT in corn oil (0.5 mg/mL).</li>
	<li>Intragastrically inject 50 &#181;L of 4HT (0.5 mg/mL) to neonatal pups at P1 to induce experimental CCM lesions using an insulin syringe.</li>
</ol>
2. Sample Preparation for Micro-CT Scans
<ol>
	<li>Euthanize neonatal pups at P8 via carbon dioxide asphyxiation.</li>
	<li>Perform intra-cardiac perfusion by inserting a 29-gauge needle with 3 mL of 2% paraformaldehyde in PBS in a 10 mL syringe into the ventricle of the mouse heart. 
	CAUTION: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>Detach the head from the body using a scissors. Remove all the skin off the head using a scissors and peel off the skull using forceps to dissect the whole brain.</li>
	<li>Take images of the brain with stereomicroscope at 7.82x magnification, 1x gain, 0.6 gamma, and 20 ms exposure time.</li>
	<li>Fix the dissected brains with 4% paraformaldehyde in PBS solution overnight. 
	CAUTION: Paraformaldehyde is toxic; wear appropriate protection.</li>
	<li>On the following day, detach hindbrains using a forceps and wash with PBS solution.</li>
	<li>Incubate the hindbrains in Lugol&#39;s iodine solution for 48 h.</li>
	<li>Following the incubation, briefly air dry the hindbrains to remove excess Lugol&#39;s iodine solution.</li>
	<li>Pack the Lugol&#39;s iodine stained hindbrains in 0.65 mL microcentrifuge tubes and seal completely with plastic paraffin film to avoid tissue shrinkage (Figure 2A).</li>
	<li>Place microcentrifuge tubes in 5 mL plastic tubes with sponges to prevent them from moving during scan (Figure 2B).</li>
</ol>
3. Micro-CT Scan of CCM in the Mouse Brain
<ol>
	<li>Vertically mount the hindbrain packed in a tube on an aluminum holder in the micro-CT system.</li>
	<li>Set the scanning parameters to 540 projections and 2 s exposure time with source conditions of 50 kV and 10 W to acquire the tomographic datasets (image resolution 9.5 &#956;m/pixel).</li>
	<li>Radiographs 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 (&#34;TXM&#34; file).</li>
</ol>
4. Analysis of Micro-CT Datasets
<ol>
	<li>Open the reconstructed image file (&#34;TXM&#34; file) in the 3-D analysis software and visualize the brain using the &#34;volume rendering&#34; function.</li>
	<li>Transform the hindbrain to the desired orientation using the &#34;bounding box&#34; and &#34;clipping plane&#34; functions.</li>
	<li>Resample the transformed image in extended mode and preserve the voxel size.</li>
	<li>Create &#34;edit new label field&#34; on the transformed image (4.3) and highlight only the hindbrain using greyscale intensity.</li>
	<li>Add the highlighted areas and run &#34;label analysis&#34; to obtain measurements of the whole hindbrain (i.e., total volume and area). Export measurements in an Excel file.</li>
	<li>Visualize the hindbrain using the &#34;isosurface rendering&#34; function.</li>
	<li>Create another &#34;edit new label field&#34; on the transformed image and highlight areas with lesions using greyscale intensity.</li>
	<li>Add the highlighted areas and run &#34;label&#34; analysis to obtain measurements of the lesions within the hindbrain (i.e., total volume and area). Export measurements in an Excel file.</li>
	<li>Visualize the lesions using the &#34;generate surface&#34; and &#34;surface view&#34; functions.</li>
	<li>Snapshot the images of the hindbrain at desired orientations or generate a movie for 3-D visualization of the lesions.</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>

The authors have nothing to disclose.

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&#039;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 ...

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Research

JoVE Journal

Medicine

10.9K Views.  Centenary Institute. Dit protocol toont de inductie van cerebrale cavernous malformatie ziekte in een muismodel en gebruik contrast enhanced micro computertomografie voor het meten van de laesie Last. Deze methode verbetert de waarde van gevestigde Muismodellen te bestuderen van de moleculaire basis en mogelijke therapieën voor cerebrale cavernous misvorming en andere cerebrovasculaire ziekten.

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

Contrast Enhanced Vessel Imaging using MicroCT

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between different soft tissues and body fluids is inherently poor in micro-CT images 1. Under these circumstances, visualization of blood vessels becomes a nearly impossible task. To overcome this and to improve the visualization of blood vessels exogenous contrast agents can be used. Herein, we present a methodology for visualizing the vascular network in a rodent model. By using a long-acting aqueous colloidal polydisperse iodinated blood-pool contrast agent, eXIA 160XL, we optimized image acquisition parameters and volume-rendering techniques for finding blood vessels in live animals. Our findings suggest that, to achieve a superior contrast between bone and soft tissue from vessel, multiple-frames (at least 5-8/ frames per view), and 360-720 views (for a full 360&deg; rotation) acquisitions were mandatory. We have also demonstrated the use of a two-dimensional transfer function (where voxel color and opacity was assigned in proportion to CT value and gradient magnitude), in visualizing the anatomy and highlighting the structure of interest, the blood vessel network. This promising work lays a foundation for the qualitative and quantitative assessment of anti-angiogenesis preclinical studies using transgenic or xenograft tumor-bearing mice.

Contrast enhanced small animal vessel imaging by microCT is a rapid, cost-effective and high-throughput technique for serial in situ examination for tumor development, for analyzing the network of blood vessels that nourish them, and for following the response of tumors to preclinical therapeutic intervention(s).

Microscopic computed tomography (microCT) offers high-resolution volumetric imaging of the anatomy of living small animals. However, the contrast between ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

Neuroscience

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that requires manual estimation. Here, we describe a simple, straightforward method for quantifying lesions and locating electrodes in the brain that is less laborious and yields more detailed results. Whole brains are stained with osmium tetroxide, embedded in resin, and imaged with a micro-CT scanner. The scans result in 3D digital volumes of the brains with resolutions and virtual section thicknesses dependent on the sample size (12&#8211;15 and 5&#8211;6 &#181;m&#160;per voxel for rat and zebra finch brains, respectively). Surface and deep lesions can be characterized, and single tetrodes, tetrode arrays, electrolytic lesions, and silicon probes can also be localized. Free and proprietary software allows experimenters to examine the sample volume from any plane and segment the volume manually or automatically. Because this method generates whole brain volume, lesions and electrodes can be quantified to a much higher degree than in current methods, which will help standardize comparisons within and across studies.

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

Lesion and electrode location verification are traditionally done via histological examination of stained brain slices, a time-consuming procedure that ...

Simultaneous Imaging of Microglial Dynamics and Neuronal Activity in Awake Mice

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in the brain sense various biological conditions in the periphery and transmit the signals to neurons. Microglia, immune cells in the brain, are involved in synaptic development and plasticity. Therefore, the contribution of microglia to neural circuit construction in response to the internal state of the body should be tested critically by intravital imaging of the relationship between microglial dynamics and neuronal activity.
Here, we describe a technique for the simultaneous imaging of microglial dynamics and neuronal activity in awake mice. Adeno-associated virus encoding R-CaMP, a gene-encoded calcium indicator of red fluorescence protein, was injected into layer 2/3 of the primary visual cortex in CX3CR1-EGFP transgenic mice expressing EGFP in microglia. After viral injection, a cranial window was installed onto the brain surface of the injected region. In vivo two-photon imaging in awake mice 4 weeks after the surgery demonstrated that neural activity and microglial dynamics could be recorded simultaneously at the sub-second temporal resolution. This technique can uncover the coordination between microglial dynamics and neuronal activity, with the former responding to peripheral immunological states and the latter encoding the internal brain states.

Here, we describe a protocol combining adeno-associated virus injection with cranial window implantation for simultaneous imaging of microglial dynamics and neuronal activity in awake mice.

Since brain functions are under the continuous influence of the signals derived from peripheral tissues, it is critical to elucidate how glial cells in ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

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.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

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.

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

Occlusion of Distal Middle Artery to Develop an Ischemic Stroke Mouse Model

Induction of Acute Ischemic Stroke in Mice Using the Distal Middle Artery Occlusion Technique

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic stroke patients are eligible to receive intravascular thrombolysis or mechanical thrombectomy therapy within the optimal time window. Among those stroke survivors, around two-thirds suffer neurological dysfunctions over an extended period. Establishing a stable and repeatable experimental ischemic stroke model is extremely significant for further investigating the pathophysiological mechanisms and developing effective therapeutic strategies for ischemic stroke. The middle cerebral artery (MCA) represents the predominant location of ischemic stroke in humans, with the MCA occlusion serving as the frequently employed model of focal cerebral ischemia. In this protocol, we describe the methodology of establishing the distal MCA occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6 mice. Since the occlusion site is located at the cortical branch of MCA, this model generates a moderate infarcted lesion restricted to the cortex. Neurological behavioral and histopathological characterization have demonstrated visible motor dysfunction, neuron degeneration, and pronounced activation of microglia and astrocytes in this model. Thus, this dMCAO mouse model provides a valuable tool for investigating the ischemiastroke and worth of popularization.

Author Spotlight: Establishing a Reliable Distal MCA Occlusion Model in Mice for Stroke Research

Here, we present a protocol to establish a distal middle cerebral artery occlusion (dMCAO) model through transcranial electrocoagulation in C57BL/6J mice and evaluate the subsequent neurological behavior and histopathological features.

Ischemic stroke remains the predominant cause of mortality and functional impairment among the adult populations globally. Only a minority of ischemic ...

Dolichoectasia: Vascular Dementia and Alzheimer&#39;s Disease Models

A Visual Approach for Inducing Dolichoectasia in Mice to Model Large Vessel-Mediated Cerebrovascular Dysfunction

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to enter and expel excessive amino acids or toxins from the brain. To model how the BBB can be compromised in diseases like vascular dementia (VaD) or Alzheimer's disease (AD), researchers developed novel methods to model vessel dilatation. A compromised BBB in these disease states can be detrimental and result in the dysregulation of the BBB leading to untoward and pathological consequences impacting brain function. We were able to modify an existing technique that enabled us to inject directly into the Cisterna magna (CM) to induce dilatation of blood vessels using elastase, and disrupt the tight junctions (TJ) of the BBB. With this method, we were able to see various metrics of success over previous techniques, including consistent blood vessel dilatation, reduced mortality or improved recovery, and improving the fill/opacifying agent, a silicone rubber compound, delivery for labeling blood vessels for dilatation analysis. This modified minimally invasive method has had promising results, with a 19%-32% increase in sustained dilatation of large blood vessels in mice from 2 weeks to 3 months post-injection. This improvement contrasts with previous studies, which showed increased dilatation only at the 2 week mark. Additional data suggests sustained expansion even after 9.5 months. This increase was confirmed by comparing the diameter of blood vessels of the elastase and the vehicle-injected group. Overall, this technique is valuable for studying pathological disorders that affect the central nervous system (CNS) using animal models.

Author Spotlight: Modeling Vascular Contributions to Alzheimer's Disease in Transgenic Mice

We demonstrate chemically inducing large blood vessel dilatation in mice as a model for investigating cerebrovascular dysfunction, which can be used for vascular dementia and Alzheimer's disease modeling. We also demonstrate visualizing the vasculature by injecting silicone rubber compound and providing clear visual guidance for measuring changes in blood vessel size.

The blood-brain (BBB) is a crucial system that regulates selective brain circulation with the periphery, as an example, allowing necessary nutrients to ...

Cre-LoxP Mediated Induction of Cerebral Cavernous Malformations in a Mouse Model

Source: Choi, J. P., et al. <a href="https://app.jove.com/v/56476">Induction and micro-CT imaging of cerebral cavernous malformations in mouse model.</a> J. Vis. Exp. (2017)
In this video, a genetically modified mouse pup carrying the Ccm2 gene flanked by loxP sites and Cre recombinase fused to an estrogen receptor is used to induce cerebral cavernous malformations (CCMs). The administration of 4-hydroxytamoxifen activates Cre in endothelial cells, leading to the deletion of Ccm2 and the formation of CCMs in the brain.

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

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