In vivo MR-angiography for the assessment of aortic aneurysms in an experimental mouse model on a clinical MRI scanner: Comparison with high-frequency ultrasound and histology.

BACKGROUND: MR-angiography currently represents one of the clinical reference-standards for the assessment of aortic-dimensions. For experimental research in mice, dedicated preclinical high-field MRI scanners are used in most studies. This type of MRI scanner is not available in most institutions. The aim of this study was to evaluate the potential of MR-angiography performed on a clinical MR scanner for the assessment of aortic aneurysms in an experimental mouse model, compared to a preclinical high-resolution ultrasound imaging system and histopathology. METHODS: All in vivo MR imaging was performed with a clinical 3T MRI system (Philips Achieva) equipped with a clinical gradient system in combination with a single-loop surface-coil (47 mm). All MR sequences were based on clinically used sequences. For ultrasound, a dedicated preclinical high-resolution system (30 MHz linear transducer, Vevo770, VisualSonics) was used. All imaging was performed with an ApoE knockout mouse-model for aortic aneurysms. Histopathology was performed as reference-standard at all stages of aneurysm development. RESULTS: MR-angiography on a clinical 3T system enabled the clear visualization of the aortic lumen and aneurysmal dilation at different stages of aneurysm development. A close correlation (R2 = 0.98; p < 0.001) with histological area measurements was found. Additionally, a good agreement between MR and ultrasound area measurements in systole (R2 = 0.91; p < 0.001) and diastole (R2 = 0.94; p < 0.001) were measured. Regarding interobserver reproducibility, MRI measurements yielded a smaller 95% confidence interval and a closer interreader correlation compared to ultrasound measurements (-0.37-0.46; R2 = 0.97 vs. -0.78-0.88; R2 = 0.87). CONCLUSION: This study demonstrates that MR-angiography, performed on a clinical 3T MR scanner, enables the reliable detection and quantification of the aortic dilatation at different stages of aneurysm development in an experimental mouse model.

A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice.

Objective. Glyoxalase-1 is an enzyme detoxifying methylglyoxal (MG). MG is a potent precursor of advanced glycation endproducts which are regarded to be a key player in micro- and macrovascular damage. Yet, the role of Glo1 in atherosclerosis remains unclear. In this study, the effect of Glo1 on mouse metabolism and atherosclerosis is evaluated. Methods. Glo1 knockdown mice were fed a high fat or a standard diet for 10 weeks. Body weight and composition were investigated by Echo MRI. The PhenoMaster system was used to measure the energy expenditure. To evaluate the impact of Glo1 on atherosclerosis, Glo1(KD) mice were crossed with ApoE-knockout mice and fed a high fat diet for 14 weeks. Results. Glo1 activity was significantly reduced in heart, liver, and kidney lysates derived from Glo1(KD) mice. Yet, there was no increase in methylglyoxal-derived AGEs in all organs analyzed. The Glo1 knockdown did not affect body weight or body composition. Metabolic studies via indirect calorimetry did not show significant effects on energy expenditure. Glo1(KD) mice crossed to ApoE(-/-) mice did not show enhanced formation of atherosclerosis. Conclusion. A Glo1 knockdown does not have major short term effects on the energy expenditure or the formation of atherosclerotic plaques.

Assessment of shear stress related parameters in the carotid bifurcation using mouse-specific FSI simulations.

The ApoE(-)(/)(-) mouse is a common small animal model to study atherosclerosis, an inflammatory disease of the large and medium sized arteries such as the carotid artery. It is generally accepted that the wall shear stress, induced by the blood flow, plays a key role in the onset of this disease. Wall shear stress, however, is difficult to derive from direct in vivo measurements, particularly in mice. In this study, we integrated in vivo imaging (micro-Computed Tomography-µCT and ultrasound) and fluid-structure interaction (FSI) modeling for the mouse-specific assessment of carotid hemodynamics and wall shear stress. Results were provided for 8 carotid bifurcations of 4 ApoE(-)(/)(-) mice. We demonstrated that accounting for the carotid elasticity leads to more realistic flow waveforms over the complete domain of the model due to volume buffering capacity in systole. The 8 simulated cases showed fairly consistent spatial distribution maps of time-averaged wall shear stress (TAWSS) and relative residence time (RRT). Zones with reduced TAWSS and elevated RRT, potential indicators of atherosclerosis-prone regions, were located mainly at the outer sinus of the external carotid artery. In contrast to human carotid hemodynamics, no flow recirculation could be observed in the carotid bifurcation region.

Practical assessment of the quantification of atherosclerotic lesions in apoE⁻/⁻ mice.

Genetic manipulations have enabled the mouse to be widely used as an animal model for investigating the mechanisms of human atherosclerotic disease. However, there is no standard method for quantifying atherosclerotic lesions among different laboratories. The present study introduces a thorough and precise quantitative assessment of atherosclerotic lesions in mice. In the present study, 6­week­old apoE­/­ mice were fed either a chow diet or a high­fat diet (HFD) for 12 weeks. Plasma lipid levels were measured every four weeks. Aortic atherosclerotic lesions were quantified and analyzed using an image analysis system. The aortic tree was isolated and stained with Oil Red O to measure the gross lesion area. The heart was removed and divided into sequential cross sections, which were then assessed for microscopic intimal lesions in the aortic root as follows: (1) Elastic van Gieson staining was performed to determine the area of the atherosclerotic lesion; (2) cross sections were stained with hematoxylin and eosin for histological analysis; and (3) cross sections were stained with Oil Red O and immunohistochemical staining for quantitative analysis of the cellular components within the lesions. ApoE­/­ mice fed with either the chow diet or HFD developed severe atherosclerosis in the aortic root, however, there were few lesions in the remainder of the aortic tree. Compared with the control group, the HFD apoE­/­ mice had increased plasma lipid levels and increases in the gross lesion area in the aortic tree, the microscopic lesion area in the aortic root and the number of macrophages, vascular smooth muscle cells and neutral lipids present within the lesions. HFD feeding in the apoE­/­ mice accelerated the development of atherosclerosis. The quantitative method described in the present study may be used to assist in future investigations of atherosclerosis in mice.

In vivo assessment of intraplaque and endothelial fibrin in ApoE(-/-) mice by molecular MRI.

OBJECTIVE: Molecular magnetic resonance imaging (MRI) has emerged as a promising non-invasive modality to characterize atherosclerotic vessel wall changes on a morphological and molecular level. Intraplaque and endothelial fibrin has recently been recognized to play an important role in the progression of atherosclerosis. This study aimed to investigate the feasibility of intraplaque and endothelial fibrin detection using a fibrin-targeted contrast-agent, FTCA (EPIX Pharmaceuticals, Lexington, MA), in a mouse model of atherosclerosis. METHODS: Male apolipoproteinE-knockout mice (ApoE(-/-)) were fed a high fat diet (HFD) for one to three months. MRI of the brachiocephalic artery was performed prior to and 90 min after the administration of FTCA (n=8 per group). Contrast to noise ratios (CNR) and longitudinal relaxation rates (R1) of plaques were determined and compared to ex vivo fibrin density measurements on immunohistological sections stained with a fibrin-specific antibody and gadolinium concentrations measured by inductively coupled mass spectroscopy (ICP-MS). RESULTS: Molecular MRI after FTCA administration demonstrated a significant increase (p<0.05) in contrast agent uptake in brachiocephalic artery plaques. In vivo CNR measurements were in good agreement with ex vivo fibrin density measurements on immunohistochemistry (y=2.4x+11.3, R(2)=0.82) and ICP-MS (y=0.95x+7.1, R(2)=0.70). Late stage atherosclerotic plaques displayed the strongest increase in CNR, R1, ex vivo fibrin staining and gadolinium concentration (p<0.05). CONCLUSION: This study demonstrated the feasibility of intraplaque and endothelial fibrin imaging using FTCA. Direct in vivo fibrin detection and quantification could be useful for characterization and staging of coronary and carotid atherosclerotic lesions, which may aid diagnosis and intervention.

Accurate assessment of carotid artery stenosis in atherosclerotic mice using accelerated high-resolution 3D magnetic resonance angiography.

OBJECT: High-resolution magnetic resonance angiography (MRA) enables non-invasive detection and longitudinal monitoring of atherosclerosis in mouse models of human disease. However, MRA is hampered by long acquisition times putting high demands on the physiological stability of the animal. Therefore, we evaluated the feasibility of accelerated MRA using the parallel imaging technique SENSE with regard to both lesion detection and quantification. MATERIALS AND METHODS: MRA acquisitions of supra-aortic vessels were performed in ApoE (-/-) mice that have been shown to develop atherosclerotic plaques. Findings obtained from accelerated data sets were compared to fully sampled reference data sets and histology. RESULTS: Our results revealed only minor differences in detecting vascular lesions for data collections accelerated by factors of up to 3.3 using a four-element coil array. For vessels with a mean lumen diameter of 500 µm, morphometry of stenotic lesions revealed no substantial deviations from reference (fully sampled) data for all investigated acceleration factors. For the highest acceleration factor of 3.3, an average deviation of the degree of stenosis of 4.9 ± 3.6% was found. Common carotid stenoses assessed by in vivo MRA displayed a good correlation with histological analyses (slope of linear regression = 0.97, R (2) = 0.98). CONCLUSION: According to the results of this work, we have demonstrated the feasibility and accuracy of accelerated high-resolution 3D ToF MRA in mice suitable for detailed depiction of mouse supra-aortic vessels and amenable to non-invasive quantification of small atherosclerotic lesions.

Pharmacological inhibition of C-C chemokine receptor 2 decreases macrophage infiltration in the aortic root of the human C-C chemokine receptor 2/apolipoprotein E-/- mouse: magnetic resonance imaging assessment.

UNLABELLED: Purpose- This study assessed the pharmacological effect of a novel selective C-C chemokine receptor (CCR) 2 antagonist (GSK1344386B) on monocyte/macrophage infiltration into atherosclerotic plaque using magnetic resonance imaging (MRI) in an atherosclerotic mouse model. METHODS AND RESULTS: Apolipoprotein E(-/-) mice expressing human CCR2 were fed a Western diet (vehicle group) or a Western diet plus10 mg/kg per day of GSK1344386B (GSK1344386B group). After the baseline MRI, mice were implanted with osmotic pumps containing angiotensin II, 1000 ng/kg per minute, to accelerate lesion formation. After five weeks of angiotensin II administration, mice received ultrasmall superparamagnetic iron oxide, an MRI contrast agent for the assessment of monocyte/macrophage infiltration to the plaque, and underwent imaging. After imaging, mice were euthanized, and the heart and aorta were harvested for ex vivo MRI and histopathological examination. After 5 weeks of dietary dosing, there were no significant differences between groups in body or liver weight or plasma cholesterol concentrations. An in vivo MRI reflected a decrease in ultrasmall superparamagnetic iron oxide contrast agent uptake in the aortic arch of the GSK1344386B group (P<0.05). An ex vivo MRI of the aortic root also reflected decreased ultrasmall superparamagnetic iron oxide uptake in the GSK1344386B group and was verified by absolute iron analysis (P<0.05). Although there was no difference in aortic root lesion area between groups, there was a 30% reduction in macrophage area observed in the GSK1344386B group (P<0.05). CONCLUSIONS: An MRI was used to noninvasively assess the decreased macrophage content in the atherosclerotic plaque after selective CCR2 inhibition.

In vivo assessment of blood flow patterns in abdominal aorta of mice with MRI: implications for AAA localization.

Abdominal aortic aneurysms (AAA) localize in the infrarenal aorta in humans, while they are found in the suprarenal aorta in mouse models. It has been shown previously that humans experience a reversal of flow during early diastole in the infrarenal aorta during each cardiac cycle. This flow reversal causes oscillatory wall shear stress (OWSS) to be present in the infrarenal aorta of humans. OWSS has been linked to a variety of proatherogenic and proinflammatory factors. The presence of reverse flow in the mouse aorta is unknown. In this study we investigated blood flow in mice, using phase-contrast magnetic resonance (PCMR) imaging. We measured blood flow in the suprarenal and infrarenal abdominal aorta of 18 wild-type C57BL/6J mice and 15 apolipoprotein E (apoE)-/- mice. Although OWSS was not directly evaluated, results indicate that, unlike humans, there is no reversal of flow in the infrarenal aorta of wild-type or apoE-/- mice. Distensibility of the mouse aortic wall in both the suprarenal and infrarenal segments is higher than reported values for the human aorta. We conclude that normal mice do not experience the reverse flow in the infrarenal aorta that is observed in humans.

Assessment of macrophage infiltration in a murine model of abdominal aortic aneurysm.

PURPOSE: To evaluate the use of an ultrasmall superparamagnetic iron oxide (USPIO) contrast agent as a marker for the detection of macrophage in a preclinical abdominal aortic aneurysm animal (AAA) model. MATERIALS AND METHODS: Osmotic pumps were implanted subcutaneously in apoE(-/-) mice for continuous infusion of Angiotensin II (Ang-II). Weekly bright-blood gradient echo scans were performed on the suprarenal abdominal aorta to evaluate aneurysm development. Once an AAA was detected, animals were administered 1000 mumol/kg of the USPIO contrast agent ferumoxtran-10 (Combidex) followed by in vivo scanning 24 h post-USPIO administration. After in vivo imaging, aortas were harvested for ex vivo imaging, histology, iron quantification, and gene expression analysis. RESULTS: Reduced signal intensity was evident in the post-USPIO transverse images of the abdominal aorta. The areas of reduced signal were primarily along the aneurysm shoulder and outer perianeurysm areas and corresponded to regions of macrophage infiltration and colocalized USPIO determination by means of histological staining. The absolute iron content measured significantly correlated to the area of signal reduction in the ex vivo images (r = 0.9; P < 0.01). In the AAA tissue, the macrophage-driven cytokine gene expression was up-regulated along with a matrix metalloproteinase known to mediate extracellular matrix breakdown in this disease model. CONCLUSION: These results demonstrate the feasibility of using an USPIO contrast agent as a surrogate for detecting the acute inflammatory process involved in the development of abdominal aneurysms.

Imminent cardiac risk assessment via optical intravascular biochemical analysis.

Heart disease is by far the biggest killer in the United States, and type II diabetes, which affects 8% of the U.S. population, is on the rise. In many cases, the acute coronary syndrome and/or sudden cardiac death occurs without warning. Atherosclerosis has known behavioral, genetic and dietary risk factors. However, our laboratory studies with animal models and human post-mortem tissue using FT-IR microspectroscopy reveal the chemical microstructure within arteries and in the arterial walls themselves. These include spectra obtained from the aortas of ApoE-/- knockout mice on sucrose and normal diets showing lipid deposition in the former case. Also pre-aneurysm chemical images of knockout mouse aorta walls, and spectra of plaque excised from a living human patient are shown for comparison. In keeping with the theme of the SPEC 2008 conference 'Spectroscopic Diagnosis of Disease...' this paper describes the background and potential value of a new catheter-based system to provide in vivo biochemical analysis of plaque in human coronary arteries. We report the following: (1) results of FT-IR microspectroscopy on animal models of vascular disease to illustrate the localized chemical distinctions between pathological and normal tissue, (2) current diagnostic techniques used for risk assessment of patients with potential unstable coronary syndromes, and (3) the advantages and limitations of each of these techniques illustrated with patent care histories, related in the first person, by the physician coauthors. Note that the physician comments clarify the contribution of each diagnostic technique to imminent cardiac risk assessment in a clinical setting, leading to the appreciation of what localized intravascular chemical analysis can contribute as an add-on diagnostic tool. The quality of medical imaging has improved dramatically since the turn of the century. Among clinical non-invasive diagnostic tools, laboratory tests of body fluids, EKG, and physical examination are still the first line of defense. However, with the fidelity of 64-slice CT imaging, this technique has recently become an option when the patient presents with symptoms of reduced arterial flow. Single photon emission computerized tomography (SPECT) treadmill exercise testing is a standard non-invasive test for decreased perfusion of heart muscle, but is time consuming and not suited for emergent evaluation. Once the invasive clinical option of catherization is chosen, this provides the opportunity for intravascular ultrasound (IVUS) imaging. As the probe is pulled through the artery, the diameter at different parts is measurable, and monochrome contrast in the constricted area reveals the presence of tissue with a different ultrasonic response. Also, via an optical catheter with a fiber-optic conductor, the possibly of spectroscopic analysis of arterial walls is now a reality. In this case, the optical transducer is coupled to a near-infrared spectrometer. Revealing the arterial chemical health means that plaque vulnerability and imminent risk could be assessed by the physician. The classical emergency use of catherization involves a contrast agent and dynamic X-ray imaging to locate the constriction, determine its severity, and possibly perform angioplasty, and stent placement.

In vivo serial assessment of aortic aneurysm formation in apolipoprotein E-deficient mice via MRI.

BACKGROUND: Hyperlipidimic mice administered angiotensin II have been used for the study of abdominal aortic aneurysms (AAAs). The purpose of this study was to examine the use of MRI for studying AAA development and for examining the effects of pharmacological intervention on AAA development in the apolipoprotein E-deficient mouse. METHODS AND RESULTS: Suprarenal aortic aneurysms were generated in apolipoprotein E-deficient mice administered angiotensin II (1000 ng/kg per min) for up to 28 days. In vivo MRI was performed serially (once weekly) to assess AAA development and rupture. Comparison of AAA size as measured by in vivo and ex vivo MRI resulted in excellent agreement (r=0.96, P<0.0001). In addition, MRI correlated with histology-derived AAA area assessment (in vivo versus histology: r=0.84, P<0.0001; ex vivo versus histology: r=0.89, P<0.0001). In a separate study, angiotensin II-administered apolipoprotein E-deficient mice were treated with doxycycline (broad-based matrix metalloproteinase inhibitor; 30 mg/kg per day for 28 days). MRI was able to noninvasively assess a reduced rate of AAA development (46% versus 71%, P<0.05), a decreased AAA area (2.56 versus 4.02 mm(2), P<0.01), and decreased incidence of rupture (43% versus 100%) in treated versus control animals. Inhibition of aorta matrix metalloproteinase 2/9 activity was observed in the treated animals. CONCLUSIONS: These results demonstrate the use of MRI to noninvasively and temporally assess AAA development on pharmacological intervention in this preclinical cardiovascular disease model.

Alpha2beta1 integrin and development of atherosclerosis in a mouse model: assessment of risk.

OBJECTIVE: The alpha2beta1 integrin serves as a collagen or collagen/laminin receptor on many cell types, including endothelial cells and platelets. Many studies indicate that the alpha2beta1 integrin is a critical mediator of platelet adhesion to collagen. Epidemiologic studies suggest a direct correlation between the genetically determined platelet surface density of the alpha2beta1 integrin and the risk of thrombotic diseases, such as myocardial infarction and stroke, in the young, which are well-established complications of atherosclerosis. We have now used the alpha2beta1 integrin-deficient mouse to evaluate the contributions of the alpha2beta1 integrin to the development of atherosclerosis. METHODS AND RESULTS: We generated wild-type (alpha2+/+) or alpha2beta1 integrin-deficient (alpha2-/-) mice that were also deficient in the apolipoprotein E (ApoE) gene (ApoE-/-) and compared atherosclerotic lesion development in alpha2+/+ ApoE-/- and alpha2-/- ApoE-/- mice that were fed a high-fat, cholesterol-containing diet for 6 or 15 weeks. Total lesional area did not differ significantly between the alpha2-null animals and the wild-type animals at either 6 or 15 weeks. CONCLUSIONS: Our results suggest that risk for arterial thrombotic disease associated with high-level alpha2beta1 integrin expression is not attributable to enhanced development of atherosclerosis per se but may rather be a consequence of thrombotic complications at the plaques.