Measurement of enhanced vasa vasorum density in a porcine carotid model using photon counting detector CT.

Purpose: The onset of atherosclerosis is preceded by changes in blood perfusion within the arterial wall due to localized proliferation of the vasa vasorum. The purpose of this study was to quantify these changes in spatial density of the vasa vasorum using a research whole-body photon-counting detector CT (PCD-CT) scanner and a porcine model. Approach: Vasa vasorum angiogenesis was stimulated in the left carotid artery wall of anesthetized pigs ( n = 5 ) while the right carotid served as a control. After a 6-week recovery period, the animals were scanned on the PCD-CT prior to and after injection of iodinated contrast. Annular regions of interest were used to measure wall enhancement in the injured and control arteries. The exact Wilcoxon-signed rank test was used to determine whether a significant difference in contrast enhancement existed between the injured and control arterial walls. Results: The greatest arterial wall enhancement was observed following contrast recirculation. The wall enhancement measurements made over these time points revealed that the enhancement was greater in the injured artery for 13/16 scanned arterial regions. Using an exact Wilcoxon-signed rank test, a significantly increased enhancement ratio was found in injured arteries compared with control arteries ( p = 0.013 ). Vasa vasorum angiogenesis was confirmed in micro-CT scans of excised arteries. Conclusions: Whole-body PCD-CT scanners can be used to detect and quantify the increased perfusion occurring within the porcine carotid arterial wall resulting from an increased density of vasa vasorum.

Single-Dose Neoadjuvant AKT Pathway Inhibitor Reduces Growth of Hepatocellular Carcinoma after Laser Thermal Ablation in Small-Animal Model.

BackgroundLocal recurrence following thermal ablation of hepatocellular carcinoma (HCC) larger than 2-3 cm remains a challenging clinical problem. Prior studies suggest that phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR)-dependent protein kinase B (AKT) signaling mediates HCC cell survival caused by moderate heat stress in vitro, but these findings need in vivo validation.PurposeTo test the hypothesis that neoadjuvant inhibition of PI3K/mTOR/AKT signaling reduces HCC tumor growth in vivo after laser ablation and to evaluate the effects of moderate heat stress on molecular signaling and cellular function in HCC cells in vitro.Materials and MethodsHCC tumor-bearing mice were randomized to neoadjuvant PI3K/mTOR inhibitor (BEZ235) or control groups followed by an intentional partial laser ablation or sham ablation; there were at least nine mice per group. Postablation tumor growth was monitored up to 7 days. Tumor volumes were compared for drug or ablation groups by using two-way analysis of variance. N1S1 HCC cells pretreated with BEZ235 or control and subjected to moderate heat stress (45°C for 10 minutes) or control (37°C for 10 minutes) were analyzed by using mass spectrometry. Protein interaction networks were derived from protein expression analysis software, and cellular function activation state (Z-score) and fold-change in AKT phosphorylation were calculated.ResultsThere was a 37%-75% reduction in HCC tumor volume by day 7 after ablation in the BEZ235 plus ablation group (713 mm3 ± 417) compared with vehicle plus sham (1559 mm3 ± 552), vehicle plus ablation (1041 mm3 ± 591), and BEZ235 plus sham (1108 mm3 ± 523) groups (P < .001, P = .04, and P = .005, respectively). PI3K/mTOR inhibition prevented moderate heat stress-induced AKT signaling (Z-score, -0.2; P < .001) and isoform-specific AKT phosphorylation compared with the vehicle plus heat stress group. PI3K/mTOR inhibition prevented moderate heat stress-induced global effects on HCC molecular signaling and cellular function, including decreased cell survival, growth, and proliferation (Z-score, -0.3 to -3.2; P < .001) and increased apoptosis and cell death (Z-score, 0.4-1.1; P < .001).ConclusionModerate heat stress induces PI3K/mTOR/AKT-dependent global effects on hepatocellular carcinoma (HCC) cell survival, function, and death. Neoadjuvant PI3K/mTOR/AKT inhibition reduces postablation HCC tumor growth.© RSNA, 2019Online supplemental material is available for this article.See also the editorial by White in this issue.

Production of dairy cows fed distillers dried grains with solubles in low- and high-forage diets.

The objective of the study was to investigate the effects of dietary forage and distillers dried grains with solubles (DDGS) concentration on the performance of lactating dairy cows. Twelve Holstein cows were blocked by parity and milk production and assigned to replicated 4 × 4 Latin squares with a 2 × 2 factorial arrangement of treatments. Diets were formulated to contain low forage [LF; 17% forage neutral detergent fiber (NDF)] or high forage (HF; 24.5% forage NDF) and DDGS at 0 or 18% of diet dry matter. The forage portion of the diet consisted of 80% corn silage and 20% alfalfa hay (dry matter basis). A portion of the ground corn and all of the expeller soybean meal and extruded soybeans from 0% DDGS diets were replaced with DDGS to formulate 18% DDGS diets. Overall, we found no interactions of forage × DDGS concentrations for any of the production measures. We observed no effect of diet on dry matter intake. Milk yield was greater when cows were fed LF diets compared with HF diets (43.3 vs. 41.5 kg/d). Milk fat concentration (3.03 vs. 3.38%) was lower for cows fed LF diets compared with HF diets, whereas protein concentration (3.11 vs. 2.98%) and yield (1.34 vs. 1.24 kg/d) were greater for cows fed LF diets compared with HF diets. Yields of fat, total solids, energy-corrected milk, and feed efficiency were not affected by diets. Cows partitioned equally for milk, maintenance, and body reserves. Replacing starch from ground corn and protein from soybean feeds with DDGS at either 17 or 24.5% of forage NDF concentration in the diet was cost-effective and did not affect the production performance of lactating dairy cows.

Heat Stress and Thermal Ablation Induce Local Expression of Nerve Growth Factor Inducible (VGF) in Hepatocytes and Hepatocellular Carcinoma: Preclinical and Clinical Studies.

The purposes of this study were to test the hypothesis that heat stress and hepatic thermal ablation induce nerve growth factor inducible (VGF) and to determine intrahepatic versus systemic VGF expression induced by thermal ablation in vivo and in patients. Hepatocytes and HCC cells were subjected to moderate (45°C) or physiologic (37°C) heat stress for 10 min and assessed for VGF expression at 0-72 h post-heat stress (n ≥ 3 experiments). Orthotopic N1S1 HCC-bearing rats were randomized to sham or laser thermal ablation (3 W × 90 s), and liver/serum was harvested at 0-7 days postablation for analysis of VGF expression (n ≥ 6 per group). Serum was collected from patients undergoing thermal ablation for HCC (n = 16) at baseline, 3-6, and 18-24 h postablation and analyzed for VGF expression. Data were analyzed using ordinary or repeated-measures one-way analysis of variance and post hoc pairwise comparison with Dunnett's test. Moderate heat stress induced time-dependent VGF mRNA (3- to 15-fold; p < 0.04) and protein expression and secretion (3.1- to 3.3-fold; p < 0.05). Thermal ablation induced VGF expression at the hepatic ablation margin at 1 and 3 days postablation but not remote from the ablation zone or distant intrahepatic lobe. There was no detectable serum VGF following hepatic thermal ablation in rats and no increase in serum VGF following HCC thermal ablation in patients at 3-6 and 18-24 h postablation compared to baseline (0.71- and 0.63-fold; p = 0.27 and p = 0.16, respectively). Moderate heat stress induces expression and secretion of VGF in HCC cells and hepatocytes in vitro, and thermal ablation induces local intrahepatic but not distant intrahepatic or systemic VGF expression in vivo.

Heat Stress and Hepatic Laser Thermal Ablation Induce Hepatocellular Carcinoma Growth: Role of PI3K/mTOR/AKT Signaling.

Purpose To determine if heat stress and hepatic laser thermal ablation induce hepatocellular carcinoma (HCC) growth and to identify growth factors induced by heat stress. Materials and Methods Non-heat-stressed HCC cells were cocultured with HCC cells or hepatocytes that were heat stressed at 37°C (physiologic), 45°C (moderate), or 50°C (severe) for 10 minutes and proliferation monitored with bioluminescence imaging for up to 6 days after heat stress (three experiments). Rats bearing orthotopic N1S1 HCC were randomly assigned to undergo immediate sham or laser thermal (3 W for 60 or 90 seconds; hereafter, 3W×60s and 3W×90s, respectively) ablation of the median (local) or left (distant) hepatic lobe, and tumor growth was monitored with magnetic resonance imaging for up to 18 days after ablation (six or more rats per group). Experiments were repeated with rats randomly assigned to receive either the adjuvant phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor (NVP-BEZ235) or the vehicle control. Heat-stressed HCC cells and hepatocytes were analyzed by using microarray or quantitative real-time polymerase chain reaction analysis for growth factor expression (three or more experiments). Groups were compared by using one- or two-way analysis of variance, and post hoc pairwise comparison was performed with the Dunnett test. Results There were more non-heat-stressed HCC cells when cells were cocultured with cells subjected to moderate but not physiologic or severe heat stress (P < .001 for both). Local intrahepatic N1S1 tumors were larger at day 18 in the 3W×60s (mean, 3102 mm3 ± 463 [standard error]; P = .004) and 3W×90s (mean, 3538 mm3 ± 667; P < .001) groups than in the sham group (mean, 1363 mm3 ± 361) but not in distant intrahepatic tumors (P = .31). Adjuvant BEZ235 resulted in smaller N1S1 tumors in the BEZ235 and laser thermal ablation group than in the vehicle control and laser thermal ablation group (mean, 1731 mm3 ± 1457 vs 3844 mm3 ± 2400, P < .001). Moderate heat stress induced expression of growth factors in HCC cells and hepatocytes, including heparin-binding growth factor, fibroblast growth factor 21, and nerve growth factor (range, 2.9-66.9-fold; P < .05). Conclusion Moderate heat stress and laser thermal ablation induce hepatocellular carcinoma growth, which is prevented with adjuvant PI3K/mTOR/protein kinase B inhibition.

Heat stress induced, ligand-independent MET and EGFR signalling in hepatocellular carcinoma.

PURPOSE: The aims of the present study were 2-fold: first, to test the hypothesis that heat stress induces MET and EGFR signalling in hepatocellular carcinoma (HCC) cells and inhibition of this signalling decreases HCC clonogenic survival; and second, to identify signalling pathways associated with heat stress induced MET signalling. MATERIALS AND METHODS: MET+ and EGFR+ HCC cells were pre-treated with inhibitors to MET, EGFR, PI3K/mTOR or vehicle and subjected to heat stress or control ± HGF or EGF growth factors and assessed by colony formation assay, Western blotting and/or quantitative mass spectrometry. IACUC approved partial laser thermal or sham ablation was performed on orthotopic N1S1 and AS30D HCC tumours and liver/tumour assessed for phospho-MET and phospho-EGFR immunostaining. RESULTS: Heat-stress induced rapid MET and EGFR phosphorylation that is distinct from HGF or EGF in HCC cells and thermal ablation induced MET but not EGFR phosphorylation at the HCC tumour ablation margin. Inhibition of the MET and EGFR blocked both heat stress and growth factor induced MET and EGFR phosphorylation and inhibition of MET decreased HCC clonogenic survival following heat stress. Pathway analysis of quantitative phosphoproteomic data identified downstream pathways associated with heat stress induced MET signalling including AKT, ERK, Stat3 and JNK. However, inhibition of heat stress induced MET signalling did not block AKT signalling. CONCLUSIONS: Heat-stress induced MET and EGFR signalling is distinct from growth factor mediated signalling in HCC cells and MET inhibition enhances heat stress induced HCC cell killing via a PI3K/AKT/mTOR-independent mechanism.

Measuring arterial wall perfusion using photon-counting computed tomography (CT): improving CT number accuracy of artery wall using image deconvolution.

Changes in arterial wall perfusion mark the onset of atherosclerosis. A characteristic change is the increased spatial density of vasa vasorum (VV), the microvessels in the arterial walls. Measuring this increased VV (IVV) density using contrast-enhanced computed tomography (CT) has had limited success due to blooming effects from contrast media. If the system point-spread function (PSF) is known, then the blooming effect can be modeled as a convolution between the true signal and the PSF. We report the application of image deconvolution to improve the CT number accuracy in the arterial wall of a phantom and in a porcine model of IVV density, both scanned using a whole-body research photon-counting CT scanner. A 3D-printed carotid phantom filled with three concentrations of iodinated contrast material was scanned to assess blooming and its effect on wall CT number accuracy. The results showed a reduction in blooming effects following image deconvolution, and, consequently, a better delineation between lumen and wall was achieved. Results from the animal experiment showed improved CT number difference between the carotid with IVV density and the normal carotid artery after deconvolution, enabling the detection of VV proliferation, which may serve as an early indicator of atherosclerosis.

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 × 0.16 × 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.

Heat Stress-Induced PI3K/mTORC2-Dependent AKT Signaling Is a Central Mediator of Hepatocellular Carcinoma Survival to Thermal Ablation Induced Heat Stress.

Thermal ablative therapies are important treatment options in the multidisciplinary care of patients with hepatocellular carcinoma (HCC), but lesions larger than 2-3 cm are plagued with high local recurrence rates and overall survival of these patients remains poor. Currently no adjuvant therapies exist to prevent local HCC recurrence in patients undergoing thermal ablation. The molecular mechanisms mediating HCC resistance to thermal ablation induced heat stress and local recurrence remain unclear. Here we demonstrate that the HCC cells with a poor prognostic hepatic stem cell subtype (Subtype HS) are more resistant to heat stress than HCC cells with a better prognostic hepatocyte subtype (Subtype HC). Moreover, sublethal heat stress rapidly induces phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dependent-protein kinase B (AKT) survival signaling in HCC cells in vitro and at the tumor ablation margin in vivo. Conversely, inhibition of PI3K/mTOR complex 2 (mTORC2)-dependent AKT phosphorylation or direct inhibition of AKT function both enhance HCC cell killing and decrease HCC cell survival to sublethal heat stress in both poor and better prognostic HCC subtypes while mTOR complex 1 (mTORC1)-inhibition has no impact. Finally, we showed that AKT isoforms 1, 2 and 3 are differentially upregulated in primary human HCCs and that overexpression of AKT correlates with worse tumor biology and pathologic features (AKT3) and prognosis (AKT1). Together these findings define a novel molecular mechanism whereby heat stress induces PI3K/mTORC2-dependent AKT survival signaling in HCC cells and provide a mechanistic rationale for adjuvant AKT inhibition in combination with thermal ablation as a strategy to enhance HCC cell killing and prevent local recurrence, particularly at the ablation margin.

Myocardial Perfusion: Characteristics of Distal Intramyocardial Arteriolar Trees.

A combination of experimental, theoretical, and imaging methodologies is used to examine the hierarchical structure and function of intramyocardial arteriolar trees in porcine hearts to provide a window onto a region of myocardial microvasculature which has been difficult to fully explore so far. A total of 66 microvascular trees from 6 isolated myocardial specimens were analyzed, with a cumulative number of 2438 arteriolar branches greater than or equal to 40 µm lumen diameter. The distribution of flow rates within each tree was derived from an assumed power law relationship for that tree between the diameter of vessel segments and flow rates that are consistent with that power law and subject to conservation of mass along hierarchical structure of the tree. The results indicate that the power law index increases at levels of arteriolar vasculature closer to the capillary level, consistent with a concomitant decrease in shear stress acting on endothelial tissue. These results resolve a long standing predicament which could not be resolved previously because of lack of data about the 3D, interconnected, arterioles. In the context of myocardial perfusion, the results indicate that the coefficient of variation of flow rate in pre-capillary distal arterioles is high, suggesting that heterogeneity of flow rate in these arterioles is not entirely random but may be due at least in part to active control.

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).

Changes in CT angiographic opacification of porcine coronary artery wall with patchy altered flow in vasa vasorum.

To evaluate the potential of whole-body CT to detect localized areas of decreased or increased vascularity in coronary arterial walls. We used both microsphere embolization of coronary artery vasa vasorum to generate small areas of hypoperfusion and surrounding hyperperfusion of the arterial wall and diet-induced hypercholesterolemia. As a stimulus for localized angiogenesis, such as occurs in early plaque formation in the coronary arterial wall, microspheres were injected selectively into the LAD coronary artery lumens of anesthetized pigs. Fourteen pigs (acute) then had a segment of their LAD harvested during injection of contrast medium and snap-frozen for subsequent cryo-static micro-CT. An additional thirteen pigs (chronic) were allowed to recover, fed a high cholesterol diet and 3 months later were again anesthetized and a segment of the LAD artery harvested and scanned. The spatial distribution of the contrast agent within the arterial wall was measured in contiguous micro-CT images at right angles to the lumen axis with the area of wall in each cross-sectional image being approximately (0.1 mm)(3) in size. In the acute animals there were no localized areas of increased contrast around the hypoperfused embolized perfusion territories in the arterial wall, but in the chronic animals the hypoperfused areas were surrounded by increased contrast. These results suggest that CT might be able to detect localized regions of increased vascularity in the arterial wall as an indicator of early atherosclerotic stimulation of vasa vasorum proliferation.

AS30D model of hepatocellular carcinoma: tumorigenicity and preliminary characterization by imaging, histopathology, and immunohistochemistry.

PURPOSE: This study was designed to determine the tumorigenicity of the AS30D HCC cell line following orthotopic injection into rat liver and preliminarily characterize the tumor model by both magnetic resonance imaging (MRI) and ultrasound (US) as well as histopathology and immunohistochemistry. MATERIALS: AS30D cell line in vitro proliferation was assessed by using MTT assay. Female rats (N = 5) underwent injection of the AS30D cell line into one site in the liver. Rats subsequently underwent MR imaging at days 7 and 14 to assess tumor establishment and volume. One rat underwent US of the liver at day 7. Rats were euthanized at day 7 or 14 and livers were subjected to gross, histopathologic (H&E), and immunohistochemical (CD31) analysis to assess for tumor growth and neovascularization. RESULTS: AS30D cell line demonstrated an in vitro doubling time of 33.2 ± 5.3 h. MR imaging demonstrated hyperintense T2-weighted and hypointense T1-weighted lesions with tumor induction in five of five and three of three sites at days 7 and 14, respectively. The mean (SD) tumor volume was 126.1 ± 36.2 mm(3) at day 7 (N = 5). US of the liver demonstrated a well-circumscribed, hypoechoic mass and comparison of tumor dimensions agreed well with MRI. Analysis of H&E- and CD31-stained sections demonstrated moderate-high grade epithelial tumors with minimal tumor necrosis and evidence of diffuse intratumoral and peritumoral neovascularization by day 7. CONCLUSIONS: AS30D HCC cell line is tumorigenic following orthotopic injection into rat liver and can be used to generate an early vascularizing, slower-growing rat HCC tumor model.

Molecular bioluminescence imaging as a noninvasive tool for monitoring tumor growth and therapeutic response to MRI-guided laser ablation in a rat model of hepatocellular carcinoma.

OBJECTIVES: The objective of this study was to quantitatively compare tumor imaging by magnetic resonance imaging (MRI) and molecular bioluminescence imaging (BLI) and test the feasibility of monitoring the effect of MRI-guided laser ablation on tumor viability by 2-dimensional BLI and 3-dimensional diffuse luminescence tomography (3D DLIT) in an orthotopic rat model of hepatocellular carcinoma. MATERIALS AND METHODS: This study was approved by the animal care committee. Rats underwent injection of N1S1 cells stably transfected with an empty vector (n = 3) or a heat shock element luciferase reporter (HSE-luc; n = 4) into the liver. All rats underwent MRI to assess tumor establishment and volume and 2-dimensional BLI to assess tumor luminescence at day 7 with subsequent MRI and 2D BLI and 3D DLIT in select animals at days 14 and 21. Magnetic resonance imaging-guided laser ablation of the tumor was performed with preablation and postablation 2D BLI and/or 3D DLIT (n = 2). The tumors underwent histopathologic analysis to assess tumor viability. RESULTS: The MRI scans demonstrated hyperintense T2-weighted lesions at 3 of 3 and 4 of 4 sites in the empty vector and HSE-luc rats, respectively. Two-dimensional BLI quantitation demonstrated 23.0-fold higher radiance in the HSE-luc group compared with the empty vector group at day 7 (P < 0.01) and a significant correlation with tumor volume by MRI (r = 0.86; P < 0.03). Tumor dimensions by 3D DLIT and MRI demonstrated good agreement. Three-dimensional DLIT quantitation demonstrated better agreement with the percentage of nonviable tumor by histopathology than did 2D BLI quantitation after the MRI-guided laser ablation. CONCLUSIONS: Bioluminescence imaging is feasible as a noninvasive, quantitative tool for monitoring tumor growth and therapeutic response to thermal ablation in a rat model of hepatocellular carcinoma.

IVUS detection of vasa vasorum blood flow distribution in coronary artery vessel wall.

There is an increased body of evidence to suggest that the vasa vasorum play a major role in the progression and complications of vulnerable plaque leading to acute coronary syndrome. We propose that detecting changes in the flow in the vascular wall by intravascular ultrasound signals can quantify the presence of vasa vasorum. The results obtained in a porcine model of atherosclerosis suggest that intravascular ultrasound-based estimates of blood flow in the arterial wall can be used in vivo in a clinical research setting to establish the density of vasa vasorum as an indicator of plaque vulnerability.

Development and preliminary testing of a translational model of hepatocellular carcinoma for MR imaging and interventional oncologic investigations.

PURPOSE: To develop a translational rat hepatocellular carcinoma (HCC) disease model for magnetic resonance (MR) imaging and image-guided interventional oncologic investigations. MATERIALS AND METHODS: Male rats underwent sham control surgery (n = 6), selective bile duct ligation (SBDL; n = 4), or common bile duct ligation (CBDL; n = 6), with procedure optimization in four rats and N1S1 hepatoma cell injection into two or three sites in the livers of 12 rats. All rats subsequently underwent MR imaging to assess tumor establishment and volume. Mesenteric angiography and percutaneous MR-guided laser ablation of the liver were performed in a subgroup of animals (n = 4). Animal weight and liver test results were monitored. After harvesting, the livers were subjected to gross and microscopic analysis. Tumor volume and laboratory parameters were assessed between ligation groups. RESULTS: MR imaging demonstrated hyperintense T2 and hypointense T1 lesions with tumor induction in five of 10 (50.0%), seven of eight (87.5%), and 12 of 12 (100%) sites in the control, SBDL, and CBDL groups, respectively. Tumor volumes differed significantly by group (P < .02). Mesenteric angiography demonstrated an enhancing tumor stain. Clinical and laboratory assessment revealed a significant decrease in weight (P = .01) and albumin level (P < .01) and an increase in total bilirubin level (P = .02) in CBDL rats but not SBDL rats (P = 1.0). Histologic examination showed high-grade HCCs with local and vascular invasion within the context of early fibrosis in CBDL and SBDL rats. MR-guided laser ablation generated a 1-2-cm ablation zone with histologic findings consistent with reversible and irreversible injury. CONCLUSIONS: A biologically relevant rat HCC disease model has been developed for MR imaging and preliminary interventional oncologic applications.

Fetuin-A expression in early venous stenosis formation in a porcine model of hemodialysis graft failure.

PURPOSE: Because fetuin-A is a cytokine with multifunctional effects on vascular smooth muscle cells and fibroblasts, the authors examined the course of its expression in early venous stenosis formation in a porcine model of chronic renal insufficiency with polytetrafluoroethylene (PTFE) arteriovenous (AV) hemodialysis grafts. MATERIALS AND METHODS: Pigs had chronic renal insufficiency created by complete embolization of the left kidney and partial embolization of the right kidney. Twenty-eight days later, PTFE AV grafts were placed from the carotid artery to the ipsilateral jugular vein, and the animals were euthanized 3 days (n = 4), 7 days (n = 4), or 14 days (n = 4) later. Expression of fetuin-A was determined by Western blot analysis of the venous stenosis, control veins, and plasma. Immunohistochemical analysis of the venous stenosis and control vein was performed. Blood urea nitrogen (BUN) and creatinine were measured before embolization and at the time of graft placement. RESULTS: The mean BUN and creatinine levels at graft placement were significantly higher than before embolization (P < .05). Severe venous neointimal hyperplasia occurred by day 14 and was characterized by primarily alpha-smooth muscle actin-positive cells. By day 14, fetuin-A levels had increased significantly (P < .05) at the venous stenosis compared with control veins and in the serum compared with measurements before embolization. CONCLUSIONS: Significantly increased expression of fetuin-A was observed in early venous stenosis by day 14 and in serum compared with baseline measurements. Understanding the role of fetuin-A in venous neointimal hyperplasia could help in improving outcomes in patients undergoing hemodialysis.

The rat femoral arteriovenous fistula model: increased expression of matrix metalloproteinase-2 and -9 at the venous stenosis.

PURPOSE: To determine whether a femoral arteriovenous (AV) fistula model in a rat was feasible and whether there is increased expression of matrix metalloproteinase (MMP)-2 and -9 and the tissue inhibitors of MMPs (TIMPs) at the venous stenosis of the fistula. MATERIALS AND METHODS: Fifteen male Sprague-Harley rats weighing 353 g +/- 26 underwent creation of an AV fistula between the left femoral artery and ipsilateral femoral vein, with the contralateral femoral vessels serving as controls. The animals were euthanized at day 14 (n = 5) and day 28 (n = 10) after fistula creation. Zymography and Western blot analysis for TIMP-1 and TIMP-2 were performed at the venous stenosis and in control vessels. Hematoxylin and eosin, Verhoeff-van Gieson, Masson trichrome, and alpha-smooth muscle staining were performed at the stenosis and in controls at day 28 in four animals. The intima/media ratio was determined at day 28. RESULTS: By day 14, pro-MMP-2 measurements were 8.13 +/- 1.06 at the venous stenosis and 4.1 +/- 1.33 in controls (P < .05). By day 28, they had increased to 18.95 +/- 4.8 at the stenosis and 12.11 +/- 4.84 in controls (P < .05). By day 14, active MMP-2 measurements were 7.38 +/- 1.25 at the stenosis and 2.31 +/- 1.04 in controls (P < .05). By day 28, they had increased to 12.12 +/- 3.45 at the stenosis and 9.26 +/- 3.97 in controls (P < .05). By day 28, pro-MMP-9 measurements were 11.77 +/- 4.71 at the stenosis and 7.78 +/- 3.49 in controls (P < .05), with no difference at day 14. There was no difference in expression of TIMP-1 and TIMP-2. The average intima/media ratio of the stenosis increased by 28% versus controls, and the neointima was composed of primarily alpha-smooth muscle actin-positive cells. CONCLUSIONS: A rat femoral AV fistula model was created with venous stenosis formation characterized by thickened neointima composed of alpha-smooth muscle actin-positive cells compared with controls. At the venous stenosis, there was increased expression of pro-MMP-2 and active MMP-2 by days 14 and 28, with significantly increased expression of pro-MMP-9 by day 28.

In vivo vibroacoustography of large peripheral arteries.

OBJECTIVE: Vibroacoustography allows imaging of objects on the basis of their acoustic signal emitted during low-frequency (kHz) vibrations produced by 2 intersecting ultrasound beams at slightly different frequencies. This study tested the feasibility of using vibroacoustography to distinguish between normal and calcified femoral arteries in a pig model. MATERIALS AND METHODS: Thirteen normal porcine femoral arteries, 7 with experimentally induced arterial calcifications, and 1 control artery injected with saline only were scanned in vivo. Images were obtained at 45 kHz using a 3 MHz confocal transducer. The acoustic emission signal was detected with a hydrophone placed on the animal's limb. Images were reconstructed on the basis of the amplitude of the acoustic emission signal. Vessel patency, vessel dimensions, and the extent of calcified plaques were confirmed in vivo by angiography and conventional ultrasound. Excised arteries were reexamined with vibroacoustography, X-ray radiography, and histology. RESULTS: In vivo, vibroacoustography produced high-resolution, speckle-free images with a high level of anatomic detail. Measurements of femoral artery diameter were similar by vibroacoustography and conventional ultrasound (mean difference +/- SD, 0.1 +/- 0.4 mm). Calcified plaque area measured by different methods was comparable (vibroacoustography, in vivo: 1.0 +/- 0.9 cm; vibroacoustography in vitro: 1.1 +/- 0.6 cm2; X-ray radiography: 0.9 +/- 0.6 cm2). The reproducibility of measurements was high. Sensitivity and specificity for detecting calcifications were 100% and 86%, respectively, and positive and negative predictive values were 77% and 100%, respectively. CONCLUSIONS: Vibroacoustography provides accurate and reproducible measurements of femoral arteries and vascular calcifications in living animals.

Reproducibility of measurements of regional myocardial blood flow in a model of coronary artery disease: Comparison of H215O and 13NH3 PET techniques.

UNLABELLED: PET absolute myocardial blood flow (MBF) with H(2)15O and 13NH3 are widely used in clinical and research settings. However, their reproducibility with a 16-myocardial segment model has not been examined in chronic coronary artery disease (CAD). We examined the short-term reproducibility of PET H(2)15O MBF and PET 13NH3 MBF in an animal model of chronic CAD. METHODS: Twelve swine (mean weight +/- SD, 38 +/- 5 kg) underwent percutaneous placement of a copper stent in the mid circumflex coronary artery, resulting in an intense inflammatory fibrotic reaction with luminal stenosis at 4 wk. Each animal underwent repeated resting MBF measurements by PET H(2)15O and PET 13NH3. Attenuation-corrected images were analyzed using commercial software to yield absolute MBF (mL/min/g) in 16 myocardial segments. MBF was also normalized to the rate.pressure product (RPP). RESULTS: By Bland-Altman reproducibility plots, the mean difference was 0.01 +/- 0.18 mL/min/g and 0.01 +/- 0.11 mL/min/g, with confidence limits of +/-0.36 and +/-0.22 mL/min/g for uncorrected regional PET H(2)15O MBF and for uncorrected regional PET 13NH3 MBF, respectively. The repeatability coefficient ranged from 0.09 to 0.43 mL/min/g for H(2)15O and from 0.09 to 0.18 mL/min/g for 13NH3 regional MBF. RPP correction did not improve reproducibility for either PET H(2)15O or PET 13NH3 MBF. The mean difference in PET H(2)15O MBF was 0.03 +/- 0.14 mL/min/g and 0.02 +/- 0.19 mL/min/g for infarcted and remote regions, respectively, and in PET 13NH3 MBF was 0.03 +/- 0.11 mL/min/g and 0.00 +/- 0.09 mL/min/g for infarcted and remote regions, respectively. CONCLUSION: PET H(2)15O and PET 13NH3 resting MBF showed excellent reproducibility in a closed-chest animal model of chronic CAD. Resting PET 13NH3 MBF was more reproducible than resting PET H(2)15O MBF. A high level of reproducibility was maintained in areas of lower flow with infarction for both isotopes.