Christopher C. Hanger

A reinvestigation of murine cranial suture biology: Microcomputed tomography versus histologic technique

Background: Histology remains the standard form to analyze cranial suture in murine models, but this technique provides only limited “snapshots” of the entire suture and requires animal euthanasia with tissue destruction. Because of the bone complex microarchitecture, better methods are required to study the behavior of the cranial suture and its surrounding environment. The authors compared microcomputed tomography and histology as techniques to evaluate murine cranial sutures. Methods: A total of 360 microcomputed tomography images and 160 to 170 histologic sections were processed from a mouse at postnatal days 22 and 45, respectively. After euthanasia, the posterior frontal and sagittal sutures were imaged with a microcomputed tomography system and subsequently processed for histologic analysis. Quantitative analysis of two-dimensional images was performed to determine the percentage of bone in a 1-mm2 sample. Results: Quantitative analysis of the percentage of bone within the sutures showed identical patterns by microcomputed tomography and histology techniques. Both methods demonstrated the posterior frontal suture to have heavier fusion patterns in the anterior and endocranial portions, with variable skip areas of complete patency on the endocranial surface, ectocranial surface, or both at day 45. Conclusions: Cranial suture fusion in the murine model is not an “all-or-none” phenomenon. The posterior frontal suture, previously thought to be completely fused on day 45 by histological analysis, showed variable fusion along the length of the suture by both methods. Quantitative assessment of the percentage of bone within the posterior frontal and sagittal sutures and morphologic assessment of these sutures demonstrated similar findings by both methods. Whereas thorough histologic evaluation of an entire suture would be extremely labor intensive and impractical, these findings help to validate microcomputed tomography as a rapid and reliable method of examining the entire suture in murine models.

Semiautomated skeletonization of the pulmonary arterial tree in micro-CT images

We present a simple and robust approach that utilizes planar images at different angular rotations combined with unfiltered back-projection to locate the central axes of the pulmonary arterial tree. Three-dimensional points are selected interactively by the user. The computer calculates a sub- volume unfiltered back-projection orthogonal to the vector connecting the two points and centered on the first point. Because more x-rays are absorbed at the thickest portion of the vessel, in the unfiltered back-projection, the darkest pixel is assumed to be the center of the vessel. The computer replaces this point with the newly computer-calculated point. A second back-projection is calculated around the original point orthogonal to a vector connecting the newly-calculated first point and user-determined second point. The darkest pixel within the reconstruction is determined. The computer then replaces the second point with the XYZ coordinates of the darkest pixel within this second reconstruction. Following a vector based on a moving average of previously determined 3- dimensional points along the vessels axis, the computer continues this skeletonization process until stopped by the user. The computer estimates the vessel diameter along the set of previously determined points using a method similar to the full width-half max algorithm. On all subsequent vessels, the process works the same way except that at each point, distances between the current point and all previously determined points along different vessels are determined. If the difference is less than the previously estimated diameter, the vessels are assumed to branch. This user/computer interaction continues until the vascular tree has been skeletonized.

X-ray measurement of regional blood flow distribution using radiopaque contrast medium: influence of gravity

X-ray CT measurement of regional blood flow distribution in the lungs is potentially biased because the contrast medium used to track the flow is denser than blood. To evaluate this gravity effect, cross-sectionally uniform boluses (Reno 60, density 1.3) were delivered at the upstream end of a horizontal tube connected to a downstream axisymmetric bifurcation. When the plane of the bifurcation was vertical and actual flow through the two branches was equal, the fraction of contrast medium passing through the downward- directed branch increased with decreasing Reynolds number, increasing length-to-diameter ratio of the horizontal tube, and decreasing bolus volume. In the lungs, Reynolds number decreases and pathway length increases with decreasing vessel diameter. Thus, the results suggest that the spatial resolution of CT flow measurement within the lungs may be limited by density differences between contrast medium and blood.

3D X-ray microtomography applied to structural and mechanical characterization of arterial trees

The authors applied micro-CT imaging to arterial trees in rodent lungs. Morphometric features derived from the images are sensitive to interspecies differences in vascular structure and can reflect the distensibility of arterial walls.

Simple cone beam backprojection reconstruction for robust skeletonization of 3D vascular trees

We describe and demonstrate a semi-automated method for three-dimensional skeletonization of vascular trees in X-ray CT image volumes. The key to the method, which tracks the vessel midline in successive increments, is that estimates of midpoint coordinates at each point along the vessel are obtained from a local reconstruction of the orthogonal vessel cross section at that point. Unlike filtered backprojection reconstruction, which attempts to recover the uniform density of a contrast agent-filled vessel across the entire lumen, simple backprojection reconstruction results in the minimum intensity occurring at the center of gravity of the orthogonal cross section. Our method is shown to provide a robust, semi-automated means for skeletonization of complex vascular trees.

Dispersion of transit times within the pulmonary vasculature from microfocal angiograms

The site and mechanism of the dispersion of blood transit times within the pulmonary vascular bed can be described using x-ray angiography images of bolus passage through the pulmonary vasculature. Time-absorbance curves from the lobar inlet artery and outlet vein, various locations within the arterial and venous trees, and regions of the microvasculature were acquired from the images. The overall dispersion within the lung lobe was determined from the inlet arterial and outlet venous curves by examining the difference in their first and second moments, mean transit time and variance, respectively. Subsequently, the moments at each location within the arterial tree were calculated and compared to those of the lobar inlet artery curve. The transit time variance imparted on the bolus as it traveled through the pulmonary arterial tree upstream from the smallest measured arteries was < 5 percent of the variance attributable to transit through the total lung lobe vascular bed. Similar results were obtained for the venous pathways using reverse-flow conditions. Regional capillary mean transit time and variance were obtained from the measured microvascular residue curves using a mass-balance model. These results suggest that most of the bolus dispersion occurs within the pulmonary capillary bed rather than in large feeding arteries or draining veins.