Micro-CT imaging reveals<i> Mekk3 </i>heterozygosity prevents cerebral cavernous malformations in <i>Ccm2</i>-deficient mice

Mutations in CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation in humans. Mouse models of CCM disease have been established by deleting Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. However, the full value of these animal models is limited by the lack of an accurate and quantitative method to assess lesion burden and progression. In the present study we have established a refined and detailed contrast enhanced X-ray micro-CT method to measure CCM lesion burden in mouse brains. As this study utilized a voxel dimension of 9.5μm (leading to a minimum feature size of approximately 25μm), it is therefore sufficient to measure CCM lesion volume and number globally and accurately, and provide high-resolution 3-D mapping of CCM lesions in mouse brains. Using this method, we found loss of Ccm1 or Ccm2 in neonatal endothelium confers CCM lesions in the mouse hindbrain with similar total volume and number. This quantitative approach also demonstrated a rescue of CCM lesions with simultaneous deletion of one allele of Mekk3. This method would enhance the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases

Overview of micro-CT imaging procedure.

<p>Mouse brains were dissected and fixed as described. Following the fixation, only the hindbrain was stained with OsO₄ as a contrast staining. Osmium stained hindbrain was then scanned using micro-CT that produces series of radiographs. These radiographs were reconstructed and AVIZO software was used to produce 3D image and quantify lesions in the hindbrain.</p

Three-dimensional images of mouse brain with CCM lesions.

<p><b>A)</b> Macroscopic images of CCM lesions in the hindbrains of <i>Ccm1</i>^<i>iECKO</i> mice. <b>B-G)</b> 3-D visualization of CCM lesions at various levels and orientations in the hindbrain. CCM lesions were visualized with the reference to overall hindbrain anatomy (<b>B</b>), from the back (<b>C</b>) and above (<b>D</b>) of hindbrain, and at different depth (<b>E-G</b>) inside hindbrain (red refers to CCM lesions).</p

<i>Mekk3</i> heterozygous deletion rescues CCM lesions in <i>Ccm2i</i>^<i>ECKO</i> mice.

<p>CCM lesions were qualitatively analyzed by 3-D visualization. Representative Micro-CT scans (<b>A</b>) and 3-D projections (<b>C</b>) of <i>Ccm2i</i>^<i>ECKO</i> mouse and rescued mice with <i>Mekk3</i> heterozygous deletion (<i>Ccm2</i>^<i>iECKO</i><i>; Mekk3</i>^<i>het</i>) (<b>B</b> and <b>D</b>). Red refers to CCM lesions. <b>E</b>) Quantitative comparison of CCM lesions volume in <i>Ccm2i</i>^<i>ECKO</i> (n = 5 from 4 litters) and <i>Ccm2i</i>^<i>ECKO</i>; <i>Mekk3het</i> (n = 8 from 4 litters) mouse.</p

Qualitative and quantitative comparison of CCM lesions in <i>Ccm1i</i>^<i>ECKO</i> and <i>Ccm2i</i>^<i>ECKO</i> mouse models using micro-CT.

<p><b>A-H)</b> 2-D graphs and rendered 3-D images of CCM lesions in <i>Ccm1i</i>^<i>ECKO</i> (<b>A-C</b>), <i>Ccm2i</i>^<i>ECKO</i> (<b>D-F</b>) and littermate control (<i>Ccm1</i>^<i>fl/fl</i> and <i>Ccm2</i>^<i>fl/fl</i>) (<b>G-H</b>) mice (red refers to CCM lesions). <b>I-J</b>) Quantitative comparison of CCM lesions volume (<b>I</b>) and number per brain (<b>J</b>) in <i>Ccm1i</i>^<i>ECKO</i> (n = 9 from 4 litters) and <i>Ccm2i</i>^<i>ECKO</i> mice (n = 5 from 4 litters).</p

Optimization of micro-CT imaging of CCM lesions in the neonatal mouse hindbrain.

<p>Different micro-CT settings were tested to identify optimal images by which to detect CCM lesions. Initially, three different settings were tested: <b>A)</b> 450 projections x 2 seconds (1 hour scanning time), <b>B)</b> 450 projections x 10 seconds (2 hours) and <b>C)</b> 1800 projections x 4 seconds (5 hours). Furthermore, to determine the optimal beam voltage for highest attenuation of osmium, three different energy levels were tested: <b>D)</b> 50keV, <b>E)</b> 65keV and <b>F)</b> 78keV. <b>G)</b> X-ray form factor graph of osmium and the distribution of chosen energy level in the spectrum. <b>H-I)</b> Reconstructed images which detected CCM lesions (green arrows) (<b>H</b> and <b>I</b>) and distinguished blood-filled lesions (red arrows) (<b>I</b>).</p