Andrew J. Vercnocke

Complementary vascular and matrix regulatory pathways underlie the beneficial mechanism of action of sorafenib in liver fibrosis.

Paracrine signaling between hepatic stellate cells (HSCs) and liver endothelial cells (LECs) modulates fibrogenesis, angiogenesis, and portal hypertension. However, mechanisms regulating these processes are not fully defined. Sorafenib is a receptor tyrosine kinase inhibitor that blocks growth factor signaling in tumor cells but also displays important and not yet fully characterized effects on liver nonparenchymal cells including HSCs and LECs. The aim of this study was to test the hypothesis that sorafenib influences paracrine signaling between HSCs and LECs and thereby regulates matrix and vascular changes associated with chronic liver injury. Complementary magnetic resonance elastography, micro–computed tomography, and histochemical analyses indicate that sorafenib attenuates the changes in both matrix and vascular compartments that occur in response to bile duct ligation–induced liver injury in rats. Cell biology studies demonstrate that sorafenib markedly reduces cell–cell apposition and junctional complexes, thus reducing the proximity typically observed between these sinusoidal barrier cells. At the molecular level, sorafenib down‐regulates angiopoietin‐1 and fibronectin, both released by HSCs in a manner dependent on the transcription factor Kruppel‐like factor 6 , suggesting that this pathway underlies both matrix and vascular changes associated with chronic liver disease. Conclusion: Collectively, the results of this study demonstrate that sorafenib inhibits both matrix restructuring and vascular remodeling that accompany chronic liver diseases and characterize cell and molecular mechanisms underlying this effect. These data may help to refine future therapies for advanced gastrointestinal and liver diseases characterized by abundant fibrosis and neovascularization. (HEPATOLOGY 2011;)

Anatomic Changes in Schlemm's Canal and Collector Channels in Normal and Primary Open-Angle Glaucoma Eyes Using Low and High Perfusion Pressures

PURPOSE To examine the anatomy of Schlemms canal (SC) and collector channels (CCs) in normal human and primary open-angle glaucoma (POAG) eyes under low and high perfusion pressure. METHODS In normal (n = 3) and POAG (n = 3) eye pairs, one eye was perfused at 10 mm Hg while the fellow eye was perfused at 20 mm Hg for 2 hours. Eyes were perfusion fixed at like pressures, dissected into quadrants, embedded in Epon Araldite, and scanned by three-dimensional micro-computed tomography (3D micro-CT). Schlemms canal volume, CC orifice area, diameter, and number were measured using ANALYZE software. RESULTS Normal eyes showed a larger SC volume (3.3-fold) and CC orifice area (9962.8 vs. 8825.2 μm(2)) and a similar CC diameter (34.3 ± 17.8 vs. 32.7 ± 13.0 μm) at 10 mm Hg compared to 20 mm Hg. In POAG eyes, SC volume (2.0-fold), CC orifice area (8049.2 μm(2)-6468.4 μm(2)), and CC diameter (36.2 ± 19.1 vs. 29.0 ± 13.8 μm) were increased in 10 mm Hg compared to 20 mm Hg perfusion pressures. Partial and total CC occlusions were present in normal and POAG eyes, with a 3.7-fold increase in total occlusions in POAG eyes compared to normal eyes at 20 mm Hg. Visualization of CCs increased by 24% in normal and by 21% in POAG eyes at 20 mm Hg compared to 10 mm Hg. Schlemms canal volume, CC area, and CC diameter were decreased in POAG eyes compared to normal eyes at like pressures. CONCLUSIONS Compensatory mechanisms for transient and short periods of increased pressure appear to be diminished in POAG eyes. Variable response to pressure change in SC and CCs may be a contributing factor to outflow facility change in POAG eyes.

Intrinsic microvasculature of the sciatic nerve in the rat.

Microvasculature associated with the sciatic nerve was examined using high‐resolution micro‐CT scanning in one group of rats and surgical exploration in another. The results indicate that blood supply to the sciatic nerve is an “open‐ended” system in which the vessels run longitudinally within the epineurium and connect with external vasculature primarily at junction points. Although the range of vasculature found extended down to 4–5 μ, only a few isolated vessels of this size were found, with no capillary “mesh” as such, possibly because of the close proximity of the intrinsic vessel to nerve fibers within the epineurium. While the study did not include direct measurements of flow or nerve function, the “open‐ended” pattern of vasculature found has important implications regarding the relationship between the two. Specifically, the nerve is less vulnerable to a severe or complete disruption in blood supply than it would be under a close‐ended system such as that of the heart or brain, where a severe disruption can occur with the obstruction of only a single vessel. Indeed, the pattern of vasculature found, subject to further study of vasculature at the capillary level, suggests that flow within the intrinsic vessels may be in either direction, depending on circumstances, somewhat like flow within the circle of Willis in the cerebral circulation.

Reproducibility of Global and Local Reconstruction of Three-Dimensional Micro-Computed Tomography of Iliac Crest Biopsies

Variation in computed tomography (CT) image grayscale and spatial geometry due to specimen orientation, magnification, voxel size, differences in X-ray photon energy and limited field-of-view during the scan, were evaluated in repeated micro-CT scans of iliac crest biopsies and test phantoms. Using the micro-CT scanner on beamline X2B at the Brookhaven National Laboratorys National Synchrotron Light Source, 3-D micro-CT images were generated. They consisted of up to 1024 X 24002, 4-mum cubic voxels, each with 16-bit gray-scale. We also reconstructed the images at 16-, 32-, and 48-mum voxel resolution. Scan data were reconstructed from the complete profiles using filtered back-projection and from truncated profiles using profile-extension and with a Local reconstruction algorithm. Three biopsies and one bonelike test phantom were each rescanned at three different times at annual intervals. For the full-data-set reconstructions, the reproducibility of the estimates of mineral content of bone at mean bone opacity value, was plusmn28.8 mg/cm3 , i.e., 2.56%, in a 4-mum cubic voxel at the 95% confidence level. The reproducibility decreased with increased voxel size. The interscan difference in imaged bone volume ranged from 0.86 plusmn 0.64% at 4-mum voxel resolution, and 2.64 plusmn 2.48% at 48 mum.

Anatomy of hepatic arteriolo-portal venular shunts evaluated by 3D micro-CT imaging

The liver differs from other organs in that two vascular systems deliver its blood - the hepatic artery and the portal vein. However, how the two systems interact is not fully understood. We therefore studied the microvascular geometry of rat liver hepatic artery and portal vein injected with the contrast polymer Microfil(®). Intact isolated rat livers were imaged by micro-CT and anatomic evidence for hepatic arteriolo-portal venular shunts occurring between hepatic artery and portal vein branches was found. Simulations were performed to rule out the possibility of the observed shunts being artifacts resulting from image blurring. In addition, in the case of specimens where only the portal vein was injected, only the portal vein was opacified, whereas in hepatic artery injections, both the hepatic artery and portal vein were opacified. We conclude that mixing of the hepatic artery and portal vein blood can occur proximal to the sinusoidal level, and that the hepatic arteriolo-portal venular shunts may function as a one-way valve-like mechanism, allowing flow only from the hepatic artery to the portal vein (and not the other way around).

Arterial wall perfusion measured with photon counting spectral x-ray CT

Early atherosclerosis changes perfusion of the arterial wall due to localized proliferation of the vasa vasorum. When contrast agent passes through the artery, some enters the vasa vasorum and increases radiopacity of the arterial wall. Technical challenges to detecting changes in vasa vasorum density include the thin arterial wall, partial volume averaging at the arterial lumen/wall interface and calcification within the wall. We used a photon-counting spectral CT scanner to study carotid arteries of anesthetized pigs and micro-CT of these arteries to quantify vasa vasorum density. The left carotid artery wall was injected with autologous blood to stimulate vasa vasorum angiogenesis. The scans were performed at 25-120 keV; the tube-current-time product was 550 mAs. A 60 mL bolus of iodine contrast agent was injected into the femoral vein at 5mL/s. Two seconds post injection, an axial scan was acquired at every 3 s over 60 s (i.e., 20 time points). Each time point acquired 28 contiguous transaxial slices with reconstructed voxels 0.16 x 0.16 x 1 mm3. Regions-of-interest in the outer 2/3 of the arterial wall and in the middle 2/3 of the lumen were drawn and their enhancements plotted versus time. Lumenal CT values peaked several seconds after injection and then returned towards baseline. Arterial wall CT values peaked concurrent to the lumen. The peak arterial wall enhancement in the left carotid arterial wall correlated with increased vasa vasorum density observed in micro-CT images of the isolated arteries.

A Three-Dimensional Micro–Computed Tomographic Study of the Intraosseous Lunate Vasculature: Implications for Surgical Intervention and the Development of Avascular Necrosis

Background: The purpose of this study was to use micro–computed tomography to demonstrate the intraosseous vascularity of the lunate within a three-dimensional orientation to identify areas of greatest perfusion and define vascular “safe zones” for surgical intervention. Methods: Fourteen upper extremities were injected with a lead-based contrast agent. The lunates were harvested and scanned using a micro–computed tomography scanner. The intraosseous vascularity was incorporated into a three-dimensional image. Vessel number, diameter, distribution, and pattern were evaluated and analyzed. Vascularity of all specimens was projected onto one representative lunate to identity areas of higher and lower vascularity. Results: Twelve specimens had nutrient vessels entering the bone from volar and dorsal; two specimens had no dorsal vessels. The intraosseous vascularity could be classified according to the Y, I, and X patterns described by Gelberman et al. Average number and diameter of vessels were 2.3 and 118.1 &mgr;m, respectively, for volar; and 1.4 and 135.8 &mgr;m, respectively, for dorsal. The long axis of the lunate showed the highest vascularity on both axial and lateral views. Lower vascularity was observed in the dorsoradial and volar-ulnar quadrants on the axial view, and in the proximal part on the lateral view. Lunate shape was not associated with an increase or decrease in nutrient vessels or vascular pattern. Conclusions: Vascular safe zones were identified, allowing for potentially safer surgical interventions to the lunate. Volar approaches to the lunate may result in localized ischemia in a subset of patients with absent dorsal nutrient vessels. This study may help to better define patients at risk for Kienböck disease.

CT image-based quantification of sub-pixel diameter microparticle accumulations in tissues using á priori biological information

With the increasing use of microspheres and nanoparticles for diagnostic and therapeutic purposes, the need to quantify the spatial distribution and concentration of those particles in a minimally invasive manner, such as by imaging, is required. In the case of CT-imaging, labelling of those particles with elements that have high contrast, and when possible that is specific for that element, is an obvious approach, but this still begs the question as to what extent particles that are smaller than the detector pixel can be quantified over relatively large volumes of tissue. This study is an exploration of three approaches to quantify the spatial distribution and/or size of those microscopic particles by use of; (i) a model of the impact of high contrast opaque particle on the detected x-ray attenuation, (ii) quasi-monochromatic energy CT and (iii) the statistics of random clustering of particles resulting in clusters that are larger than detector pixels and using that information to extrapolate to sub-resolution information about individual particles. To explore the role of particle size relative to detector pixel size we recorded x-ray attenuation in detector pixels smaller than the particle and then retrospectively increased the effective detector pixel size by summing the x-ray signal in contiguous pixels around the particle location.

Micro-CT analysis of sea sponge pore architecture as a model of a cell-populated synthetic tissue scaffold

Sponges consist of a tissue skeleton that provides structure for its elaborate pore system of canals and chambers. Sponges have been noted for their remarkable ability to support cellular life within these pores. For that reason, their structure is of great interest to us since our goal is to create a scaffold that supports cell vitality beyond diffusion depth from the scaffold surface. In the sponge this is achieved by convective transport of nutrients through the pore system. Hence, understanding of the architecture of sea sponges has the potential to aid in the production of better design of porous, cell-populated, synthetic tissue scaffolds. Pore geometry affects depth and distribution of the solute transport needed to sustain the cells lining the pores. To explore this aspect we need accurate 3D measurements of pore architecture and interconnectivity. Three-dimensional micro-CT imaging can be used to characterize the desired microarchitecture labyrinthine pore structure of a sea sponge. The sea sponge was collected, dried, and then rotated in small angular measurements inside the scanner as an x-ray image was obtained at each angle of view. Reconstructed cross-sectional images of the sponge consisted of up to 107 cubic voxels, 20μm on a side. After reconstruction, Analyze 8.1 was used to display and generate measurements of the sponges pores. The pores were subjected to a sequence of morphological erosion and dilation operations, each of which either removed or added one layer of voxels from the outer surface of the segmented pore. Hence, each erosion removed 40μm from the diameter of a pore. Progressive erosions were used to calculate pore volume and to disconnect pores from adjacent pores, thereby identifying connecting throats(s) as well as their diameter. Along with diameter, individual pore volume and surface area were also computed. Results show that the throats were predominately 264±129μm in diameter. Preliminary data show complex pore structures can be analyzed with morphological erosion and dilation image analysis techniques to provide significant quantitative data. Such data has provided information about throat identification and diameter, as well as pore volume and surface area. Ground work has also been laid for computing flow path of least resistance through the pore labyrinth from any point in the labyrinth.

Evaluation of a photon counting Medipix3RX CZT spectral x-ray detector

We assessed the performance of a cadmium zinc telluride (CZT)-based Medipix3RX x-ray detector as a candidate for micro-computed tomography (micro-CT) imaging. This technology was developed at CERN for the Large Hadron Collider. It features an array of 128 by 128, 110 micrometer square pixels, each with eight simultaneous threshold counters, five of which utilize real-time charge summing, significantly reducing the charge sharing between contiguous pixels. Pixel response curves were created by imaging a range of x-ray intensities by varying x-ray tube current and by varying the exposure time with fixed x-ray current. Photon energy-related assessments were made by flooding the detector with the tin foil filtered emission of an I-125 radioisotope brachytherapy seed and sweeping the energy threshold of each of the four charge-summed counters of each pixel in 1 keV steps. Long term stability assessments were made by repeating exposures over the course of one hour. The high properly-functioning pixel yield (99%), long term stability (linear regression of whole-chip response over one hour of acquisitions: y = -0.0038x + 2284; standard deviation: 3.7 counts) and energy resolution (2.5 keV FWHM (single pixel), 3.7 keV FWHM across the full image) make this device suitable for spectral micro-CT. The charge summing performance effectively reduced the measurement corruption caused by charge sharing which, when unaccounted for, shifts the photon energy assignment to lower energies, degrading both count and energy accuracy. Effective charge summing greatly improves the potential for calibrated, energy-specific material decomposition and K edge difference imaging approaches.