Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model
September 4th, 2017
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.
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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Overview
This protocol demonstrates the induction of cerebral cavernous malformation disease in neonatal mice and utilizes micro-CT to measure lesion burden. This method enhances the understanding of the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.
Key Study Components
Area of Science
- Neuroscience
- Vascular Biology
- Mouse Models
Background
- Cerebral cavernous malformations (CCMs) are vascular lesions that can lead to significant neurological issues.
- Existing methods for measuring disease burden in mouse models are often inefficient.
- Micro-CT offers a non-invasive way to visualize and quantify lesions.
- Understanding CCMs can provide insights into other cerebrovascular diseases.
Purpose of Study
- To induce cerebral cavernous malformations in neonatal mice.
- To accurately measure the disease burden using micro-CT.
- To improve methodologies for studying cerebrovascular diseases.
Methods Used
- Dissolving 4-HT in ethanol to create a solution for inducing CCMs.
- Preparation of aliquots for storage at -80 degrees Celsius.
- Utilization of contrast-enhanced micro-CT for imaging.
- Visual demonstration of the induction and analysis process.
Main Results
- The method allows for accurate and efficient determination of CCM disease burden.
- Micro-CT provides clear visualization of lesions in the mouse models.
- Induction of CCMs was successfully achieved in neonatal mice.
- This approach can facilitate further research into potential therapies.
Conclusions
- This protocol enhances the value of mouse models for studying CCMs.
- Micro-CT is a powerful tool for assessing cerebrovascular disease burden.
- Future studies can build on this methodology to explore therapeutic options.
Frequently Asked Questions
Executive Industry Relevance
Quantitative micro-CT imaging of cerebral cavernous malformations (CCMs) in genetically engineered mouse models enables precise assessment of lesion burden, supporting mechanistic de-risking and target validation in cerebrovascular disease research. This approach enhances predictive confidence for early-stage therapeutic hypothesis testing and informs portfolio triage by providing robust, reproducible lesion quantification. Integration of high-resolution imaging with genetic disease models positions this workflow as a critical inflection point for translational continuity in vascular biology pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Enables rigorous interrogation of CCM gene function and pathway involvement in disease models.
- Supports biological de-risking by quantifying lesion formation following targeted gene deletion.
- Provides functional target validation through direct measurement of disease-relevant phenotypes.
- Facilitates predictive confidence in selecting or deprioritizing molecular targets for further study.
Screening & Assay Development
- Establishes validated mouse models and imaging protocols for downstream compound screening.
- Delivers standardized, quantitative lesion metrics suitable for assay development and reproducibility assessment.
- Enables scalable, high-throughput evaluation of therapeutic interventions in preclinical settings.
- Supports reliable comparison of experimental conditions through consistent imaging outputs.
Translational & Preclinical Research
- Aligns preclinical models with human disease genetics for translational biomarker development.
- Ensures continuity from genetic discovery to preclinical validation of therapeutic hypotheses.
- Reduces translational risk by providing quantitative endpoints for disease progression and intervention efficacy.
- Strengthens mechanistic understanding of cerebrovascular pathology relevant to clinical translation.
Pipeline & Workflow Integration
This method bridges early discovery and preclinical research by enabling hypothesis-driven genetic manipulation and quantitative phenotyping in mouse models, supporting lead identification and translational biomarker alignment.
- Discovery Biology: Facilitates null hypothesis testing and pathway clarification through genetic lesion induction and quantification.
- Screening: Provides reproducible, quantitative imaging outputs for assay readiness and compound evaluation.
- Analytics: Delivers volumetric and numerical lesion data to support statistical comparison across experimental groups.
- Translational Research: Connects genetic models to disease-relevant endpoints for preclinical continuity.
- Enterprise Reuse: Establishes a reusable imaging and analysis platform for diverse cerebrovascular research programs.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence and reduces mechanistic ambiguity in target validation.
- Operational Value: Standardizes lesion quantification and imaging workflows for reproducibility and scalability.
- Strategic Value: Informs go/no-go decisions and enhances capital efficiency by providing robust preclinical data.
- Portfolio Impact: Supports risk-adjusted prioritization and advancement of vascular disease programs.
Implementation Considerations
- Requires expertise in genetic mouse model handling and micro-CT imaging protocols.
- Demands access to high-resolution micro-CT instrumentation and specialized analysis software.
- Necessitates cross-team standardization of imaging parameters and data interpretation.
- May require adaptation of protocols for different genetic backgrounds or lesion types.
- Dependent on precise sample preparation and imaging conditions to ensure data quality.
Biopharma FAQs
Chapters in this video
0:05
Title
0:57
Induction of Cerebral Cavernous Malformations in Mouse Models
1:38
Sample Preparation for Micro-CT Scans
3:15
Micro-CT Scan of CCM in the Neonatal Mouse Brain
3:57
Results: Rendered 3-D Images Showing CCM Lesions in the Neonatal Mouse
4:34
Conclusion
