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

Summary

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.

Abstract

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's solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 µm, which lead to a minimum feature size of approximately 25 µm. 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.

Introduction

CCM are 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 CCM^7,8,9,10. The most robust model is to conditionally delete any one of the Ccm genes with tamoxifen-inducible Cdh5-CreERT2 at P1 in newborn pups^8,10. 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 bias^10,11,12. MRI based methods have been used to assess CCM lesion burden in the adult mouse model^9,13. 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 lesion^14,15. 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 embryos^16,17. 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'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.

Protocol

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

1. Cross Cdh5-CreErt2; Ccm2^fl/fl mice with Ccm2^fl/fl to generate litters with Cdh5-CreErt2; Ccm2^fl/fl (Ccm2^iECKO) pups and littermate controls (Ccm2^fl/fl). Cdh5-CreErt2 and Ccm2^fl/fl animals have been previously described¹⁹.
2. Dissolve 4HT in 100% ethanol and store at -80 °C in aliquots of 30 µL (4HT concentration: 10 mg/mL). On the day of use, dilute aliquoted 4HT in corn oil (0.5 mg/mL).
3. Intragastrically inject 50 µL of 4HT (0.5 mg/mL) to neonatal pups at P1 to induce experimental CCM lesions using an insulin syringe.

2. Sample Preparation for Micro-CT Scans

1. Euthanize neonatal pups at P8 via carbon dioxide asphyxiation.
2. 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.
3. 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.
4. Take images of the brain with stereomicroscope at 7.82x magnification, 1x gain, 0.6 gamma, and 20 ms exposure time.
5. Fix the dissected brains with 4% paraformaldehyde in PBS solution overnight.
   CAUTION: Paraformaldehyde is toxic; wear appropriate protection.
6. On the following day, detach hindbrains using a forceps and wash with PBS solution.
7. Incubate the hindbrains in Lugol's iodine solution for 48 h.
8. Following the incubation, briefly air dry the hindbrains to remove excess Lugol's iodine solution.
9. Pack the Lugol's iodine stained hindbrains in 0.65 mL microcentrifuge tubes and seal completely with plastic paraffin film to avoid tissue shrinkage (Figure 2A).
10. Place microcentrifuge tubes in 5 mL plastic tubes with sponges to prevent them from moving during scan (Figure 2B).

3. Micro-CT Scan of CCM in the Mouse Brain

1. Vertically mount the hindbrain packed in a tube on an aluminum holder in the micro-CT system.
2. 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 μm/pixel).
3. 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 ("TXM" file).

4. Analysis of Micro-CT Datasets

1. Open the reconstructed image file ("TXM" file) in the 3-D analysis software and visualize the brain using the "volume rendering" function.
2. Transform the hindbrain to the desired orientation using the "bounding box" and "clipping plane" functions.
3. Resample the transformed image in extended mode and preserve the voxel size.
4. Create "edit new label field" on the transformed image (4.3) and highlight only the hindbrain using greyscale intensity.
5. Add the highlighted areas and run "label analysis" to obtain measurements of the whole hindbrain (i.e., total volume and area). Export measurements in an Excel file.
6. Visualize the hindbrain using the "isosurface rendering" function.
7. Create another "edit new label field" on the transformed image and highlight areas with lesions using greyscale intensity.
8. Add the highlighted areas and run "label" analysis to obtain measurements of the lesions within the hindbrain (i.e., total volume and area). Export measurements in an Excel file.
9. Visualize the lesions using the "generate surface" and "surface view" functions.
10. Snapshot the images of the hindbrain at desired orientations or generate a movie for 3-D visualization of the lesions.

Results

A single injection of 4HT at P1 was sufficient to induce CCM lesions in the cerebellum. Lugol'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 Ccm2^iECKO 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...

Discussion

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 models^7,

Materials

Name	Company	Catalog Number	Comments
4-hydroxy tamoxifen	Sigma-Aldrich	H6278	To activate Cdh5-CreErt2
Corn oil	Sigma-Aldrich	C8267-500ML	To dilute 4-hydroxy tamoxifen
Stereomicroscope	Leica	M205FA	To take macroscopic images
Lugol's Iodine solution	Sigma-Aldrich	L6146	To stain samples for contrast micro-CT
Plastic paraffin film	Parafilm	PM992	To package samples
Micro-CT	Xradia	MicroXCT-400	Micro-CT
3D rendering software	FEI Visualization Science group	Avizo 3D image processing software	To analyse micro-CT scans