Ultra High-Resolution In vivo Computed Tomography Imaging of Mouse Cerebrovasculature Using a Long Circulating Blood Pool Contrast Agent

Abstract

Abnormalities in the cerebrovascular system play a central role in many neurologic diseases. The on-going expansion of rodent models of human cerebrovascular diseases and the need to use these models to understand disease progression and treatment has amplified the need for reproducible non-invasive imaging methods for high-resolution visualization of the complete cerebral vasculature. In this study, we present methods for in vivo high-resolution (19 μm isotropic) computed tomography imaging of complete mouse brain vasculature. This technique enabled 3D visualization of large cerebrovascular networks, including the Circle of Willis. Blood vessels as small as 40 μm were clearly delineated. ACTA2 mutations in humans cause cerebrovascular defects, including abnormally straightened arteries and a moyamoya-like arteriopathy characterized by bilateral narrowing of the internal carotid artery and stenosis of many large arteries. In vivo imaging studies performed in a mouse model of Acta2 mutations demonstrated the utility of this method for studying vascular morphometric changes that are practically impossible to identify using current histological methods. Specifically, the technique demonstrated changes in the width of the Circle of Willis, straightening of cerebral arteries and arterial stenoses. We believe the use of imaging methods described here will contribute substantially to the study of rodent cerebrovasculature.

Figure 1. Effect of contrast agent dose and spatial resolution on visualization of the Circle of Willis.

(A) Vascular CT signal as a function of contrast agent dose and peak voltage. Analysis is performed in the internal carotid artery (ICA) and great cerebral vein of Galen (GVG). The differences in vascular CT attenuation obtained using a contrast agent dose of 1.1 g I/kg and 2.2 g I/kg are statistically significant for ICA and GVG at either peak voltage setting (p < 0.01). Mean background brain tissue CT signal was 51 HU for 1.1 g I/kg and 50 kVp, -87 HU for 1.1 g I/kg and 70 kVp, 73 HU for 2.2 g I/kg and 50 kVp and -20 HU for 2.2 g I/kg and 70 kVp. (B) Coronal thick slab maximum intensity projection images demonstrating the mouse Circle of Willis as a function of contrast agent dose and voxel size. The skull is segmented and subtracted out for better visualization of the mouse Circle of Willis. Scale bar represents 500 μm. (C) CT imaging-derived diameters of major Circle of Willis arteries determined using low-res and high-res scan protocols. Vessel diameter (μm) is expressed as mean ±SD. The differences in the vessel diameter obtained using either of the protocol is not statistically significant (p < 0.05). Analysis was performed on images acquired using a contrast agent dose of 2.2 g I/kg. Literature reported vessel diameters are provided in the final column (^* - ref; ^** -ref39).

Figure 2. Volume-rendered 3D images of the cerebral vasculature in mice.

Panels (A-C): (1) Olfactory artery (OlfA), (2) Anterior communicating artery (AcomA), (3) Anterior cerebral artery (ACA), (4) Middle cerebral artery (MCA), (5) Internal carotid artery (ICA), (6) Posterior communicating artery (PcomA), (7) Posterior cerebral artery (PCA), (8) Superior cerebellar artery (SCA), (9) Basilar artery (BA), (10) Anterior inferior cerebellar artery (AICA), (11) Vertebral artery, (12) Ophthalmic artery, (13) Artery of Percheron, (14) Transverse hippocampal arteries; (15) Azygos pericallosal artery (AzPa), (16) Posterior internal frontal artery; (17) Medial orbitofrontal artery. Panels (D-E): (1) Great cerebral vein of Galen, (2) Longitudinal hippocampal vein, (3) Thalamostriate vein, (4) Transverse sinus, (5) Sigmoid sinus, (6) Long cortical branch. Images (high-res) were acquired using the following parameters: 50 kVp, 1440 projections, 19 μm isotropic voxel; 2.2 gm I/kg contrast agent dose.

Figure 3. Effect of spatial resolution (voxel size) and projection number on vessel visibility.

Representative thick slab maximum intensity projection images showing the effect of these parameters on pontine arteries, transverse hippocampal arteries and Azygos pericallosal artery (AzPA) and branches. Images are acquired at 50 kVp with a contrast agent dose of 2.2 gm I/kg. Scale bar represents 500 μm.

Figure 4. Quantitative analysis of the effect of scan parameters (peak voltage, spatial resolution and projection number) and contrast agent dose on vessel visibility

. Analysis is performed on (A) pontine arteries (PA); (B) transverse hippocampal arteries (THA); and (C) middle cerebral artery branches (MCA branches). (D) Analysis of projection number (360, 720, 1440) is performed on scans acquired at 50 kVp and a contrast agent dose of 2.2 g I/kg. ^* indicates statistically significant difference (p < 0.05).

Figure 5. Coronal volume rendered images for 3D morphometric analysis of cerebral vasculature in mouse model of Acta2 mutations.

Acta2^+/- and Acta2^-/- mice manifests alterations in cerebral vasculature, demonstrated by narrowing of the Circle of Willis (f,j) and straightening of cerebral vessels (d,g) In addition, partial stenotic vessels are seen sporadically in Acta2^+/- and Acta2^-/- mice (e,h) None of the wild type (WT) mice had evidence of similar arterial narrowing (c,a,b).