Visualization of Complex Flow Patterns in Angiotensin II-Induced Dissecting Murine Abdominal Aortic Aneurysms with High Frequency Ultrasound

Abdominal aortic aneurysm (AAA) rupture is a common cause of mortality in the United States. Current treatments are only employed once the risk of rupture outweighs the risks associated with surgery. Murine models have been developed to characterize AAA pathogenesis in the hope that new treatments will be developed. For this study, angiotensin II (AngII) was infused subcutaneously into apolipoprotein E-deficient (ApoE-/-) mice using an osmotic mini-pump over 28 days. ApoE-/- mice (16-week-old, 3 females, 2 males) were imaged using a VisualSonics Vevo 2100 high frequency ultrasound before pump implantation and 3, 7, 14, 21, and 27 days following implantation. Images were acquired in the transverse and longitudinal planes from the suprarenal region of the aorta. Blood pressure measurements were taken using a tail-cuff system (CODA, Kent Scientific). Three mice (1 female, 2 male) developed aneurysms within the first 14 days of infusion. Pre-study abdominal aortas had a diastolic diameter of 0.84±0.09 mm and a systolic diameter of 0.96±0.08 mm. By day 21, AAAs had a diastolic diameter of 1.51±0.59 mm and a systolic diameter of 1.56±0.59 mm. Initially, mice had a systolic blood pressure of 111.94±6.53 mmHg and a diastolic pressure of 82.38±5.13 mmHg. These pressures steadily elevated but eventually began to plateau. By day 27, systolic pressure had risen to 154.92±11.43 mmHg and diastolic pressure to 115.77±10.25 mmHg. Color Doppler images revealed complex, recirculating flow within the aneurysms, a phenomenon which could affect vessel remodeling. In conclusion, this study utilized in vivo sonographic methods to characterize AAA development

In Vivo Flow Measurements of Murine Renal Arteries and Veins with High Frequency Ultrasound

The number of glomeruli in the kidneys has been shown to have an effect on the decline in renal function over time (Brenner, Garcia, Anderson 1988). Furthermore, flow in the renal arteries and veins may depend on the number of glomeruli in the kidney. Consistent in vivo measurements of volumetric flow in the renal arteries and veins are difficult to obtain. Thus, the purpose of this study was to develop non-invasive imaging techniques capable of estimating arterial and venous flow to kidneys. A high-frequency small animal ultrasound system was chosen based upon its excellent spatial and temporal resolution when imaging mice (Vevo 2100, VisualSonics, Inc.). Velocity profiles of the renal arteries and veins in C57BL/6 male mice (n=4) were measured. Motion, color Doppler, and pulsed wave Doppler data were acquired and used to determine renal diameter, maximum velocity, mean velocity, and volumetric flow for both kidneys. For the renal artery the average volumetric flow was 33.31±7.16 mm3/s and for the renal vein it was 30.23±4.58 mm3/s. The next step will be imaging the same animals multiple times to ensure that these measurements are consistent over prolonged periods of time. Then data will be collected from different breeds of mice to conclude whether or not differences in glomeruli number affect renal flow. Measurement of volumetric flow in the renal arteries and veins can lead to important insights into how the glomeruli density in kidneys relates to renal flow and function

Murine Ultrasound-Guided Transabdominal Para-Aortic Injections of Self-Assembling Type I Collagen Oligomers

Abdominal aortic aneurysms (AAAs) represent a potentially life-threatening condition that predominantly affects the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Cell- or pharmaceutical-based therapeutics designed to prevent AAA expansion are currently being evaluated with these animal models, but more minimally invasive strategies for delivery could improve their clinical translation. The purpose of this study was to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. Here we show the success and reliability of a para-aortic, ultrasound-guided technique for injecting quickly-polymerizing collagen oligomer solutions into mice to form a collagen-fibril matrix at body temperature. A commonly used infrarenal mouse AAA model was used to determine the target location of these collagen injections. Ultrasound-guided, closed-abdominal injections supported consistent delivery of collagen to the area surrounding the infrarenal abdominal aorta halfway between the right renal artery and aortic trifurcation into the iliac and tail arteries. This minimally invasive approach yielded outcomes similar to open-abdominal injections into the same region. Histological analysis on tissue removed on day 14 post-operatively showed minimal in vivo degradation of the self-assembled fibrillar collagen and the majority of implants experienced minimal inflammation and cell invasion, further confirming this material's potential as a method for delivering therapeutics. Finally, we showed that the typical length and position of this infrarenal AAA model was statistically similar to the length and targeted location of the injected collagen, increasing its feasibility as a localized therapeutic delivery vehicle. Future preclinical and clinical studies are needed to determine if specific therapeutics incorporated into the self-assembling type I collagen matrix described here can be delivered near the aorta and locally limit AAA expansion.

Three Dimensional Quantification of Angiotensin II-Induced Murine Abdominal Aortic Aneurysms Using High Frequency Ultrasound

Abdominal aortic aneurysms (AAAs), a localized dilation of the vessel wall of 50% or more above normal, claims approximately 14,000 U.S. lives yearly due to aortic rupture. This commonly asymptomatic disease can only be treated by endovascular stent grafts or invasive surgery, usually after the AAA diameter reaches 5 cm. Because these treatment methods carry serious risk, stem cell therapy is being explored in order to provide a low risk option for managing smaller AAAs. To determine if stem cell therapy, once administered, could stabilize or reduce AAA growth, baseline 3D ultrasound measurements in a control group were first needed. High frequency ultrasound was used on apolipoprotein E-deficient (apoE-/-) mice given angiotensin II (AngII) from subcutaneously implanted osmotic mini pumps. This mouse model developed dissecting AAAs, containing a false and true lumen, which were clearly visualized and quantified using 3D ultrasound imaging. With this ultrasound technique, we found that aneurysm diameter, total volume, and false lumen volume all increased steadily over a period of 28 days once AAAs formed. These data suggest our noninvasive, 3D ultrasound technique can be used to monitor the progression of aneurysms that may be delayed once stem cell therapy is administered

Nonlinear Optical Microscopy of Murine Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is a cardiovascular disease characterized by dilation and weakening of the vessel wall. AAA rupture is responsible for approximately 14,000 deaths annually in the United States [1]. Nonlinear optical (NLO) microscopy presents new possibilities for analyzing AAA tissue samples from murine models. Common NLO techniques are two-photon excitation fluorescence (TPEF), which detects the intrinsic autofluorescent properties of elastin, and second-harmonic generation (SHG), which is specific for collagen fibrils. Elastin and collagen, two major extracellular matrix components, help the aortic wall withstand internal pressure. Murine AAAs were created through 1) subcutaneous continuous systemic infusion of angiotensin II (AngII) in hyperlipidemic apolipoprotein E-deficient mice and 2) by intraluminal infusion of elastase (low 0.5 U/ml and high 25 U/ml concentrations) into the infrarenal aorta of rats [2]. We imaged aneurysmal and control tissue using TPEF and SHG and compared the resulting images to sections stained with standard elastin and collagen markers. TPEF images revealed disorganized elastin sheets and SHG images indicated collagen turnover after aneurysm formation. We quantified the relative degree of elastin degradation and collagen content in the aortic media within a user-defined area on sections stained with Verhoeff-van Gieson (VVG) or Masson’s trichrome (MTC), as well as on TPEF and SHG images. Our analysis with VVG-stained sections shows that elastin content in AAA tissue is significantly decreased by 64% in AngII models (P=0.02), by 34% in low concentration elastase models (P=0.07), and by 99% in high concentration elastase models (P=0.03), relative to control aortic tissue

Murine Ultrasound-Guided Transabdominal Para-Aortic Injections of Type I Collagen

Abdominal aortic aneurysms (AAAs) are a potentially life-threatening disease that predominantly affect the infrarenal aorta. Several preclinical murine models that mimic the human condition have been developed and are now widely used to investigate AAA pathogenesis. Therapeutics designed to prevent AAA expansion are currently being evaluated using these animal models, but improvements in minimally invasive strategies for delivery could help in the translation of these therapeutics into the clinic. The purpose of this study is to investigate the use of self-assembling type I collagen oligomers as an injectable therapeutic delivery vehicle in mice. With the aid of noninvasive high frequency ultrasound, we describe the accuracy and reliability of an image-guided technique for murine para-aortic injections of collagen oligomers that quickly polymerize to form a collagen-fibril matrix at body temperature. Ultrasound-guided closed-abdominal injections successfully delivered collagen to the infrarenal abdominal aorta halfway between the right renal artery and aortic bifurcation into the iliac arteries. This minimally invasive approach proved to be similar to injections into the same region after opening the abdominal cavity. Finally, we observed minimal in vivo degradation of the self-assembled collagen over 14 days, further confirming this material’s potential as a method for delivering therapeutics. Future preclinical and clinical studies will be needed to determine if therapies incorporated into the self-assembling type I collagen matrix can effectively limit AAA expansion

Development of Non-Invasive In Vivo Ultrasound Imaging Techniques for Elastase-Induced Experimental Abdominal Aortic Aneurysms

Abdominal aortic aneurysms (AAAs) are pathological dilations of the aorta which are associated with significant morbidity and mortality. The underlying mechanisms that cause this inflammatory disease are not fully understood and thus, are currently under investigation. In the hopes of preventing disease progression, rodent models that mimic the human condition have been developed to provide insight into the pathogenesis of AAAs. In this study, porcine pancreatic elastase (0.44 U; Sigma-Aldrich) was infused into the infrarenal aortas of male, Sprague Dawley rats to induce aneurysms. To perform the surgery, temporary ligatures were placed around proximal and distal sections of the abdominal aorta and a catheter was inserted into the vessel through an aortotomy slightly above the trifurcation to infuse elastase (30 minutes). Rats were imaged using a VisualSonics Vevo 2100 high-frequency ultrasound prior to and following surgery on days 3, 7, 14, 21, and 28. Of the 8 rats used in this study, 4 survived for 28 days and developed aneurysms. Pre-surgery abdominal aortas had a systolic diameter of 1.16 ± 0.19 mm and a diastolic diameter of 0.99 ± 0.17 mm. By day 14, AAAs had a systolic diameter of 2.32 ± 0.63 mm and a diastolic diameter of 2.25 ± 0.64 mm. Our efforts using this rat model will benefit future mesenchymal stem cell work aimed at preventing aneurysm formation by modulating the immune response. In conclusion, this study utilized high-frequency ultrasound to characterize an elastase-induced rat AAA model and will help increase our understanding of aneurysm pathogenesis

Morphological and Biomechanical Differences in the Elastase and AngII apoE−/− Rodent Models of Abdominal Aortic Aneurysms

An abdominal aortic aneurysm (AAA) is a potentially fatal cardiovascular disease with multifactorial development and progression. Two preclinical models of the disease (elastase perfusion and angiotensin II infusion in apolipoprotein-E-deficient animals) have been developed to study the disease during its initiation and progression. To date, most studies have used ex vivo methods to examine disease characteristics such as expanded aortic diameter or analytic methods to look at circulating biomarkers. Herein, we provide evidence from in vivo ultrasound studies of the temporal changes occurring in biomechanical parameters and macromolecules of the aortic wall in each model. We present findings from 28-day studies in elastase-perfused rats and AngII apoE−/− mice. While each model develops AAAs specific to their induction method, they both share characteristics with human aneurysms, such as marked changes in vessel strain and blood flow velocity. Histology and nonlinear microscopy confirmed that both elastin and collagen, both important extracellular matrix molecules, are similarly affected in their levels and spatial distribution. Future studies could make use of the differences between these models in order to investigate mechanisms of disease progression or evaluate potential AAA treatments

Imaging of Small Animal Peripheral Artery Disease Models: Recent Advancements and Translational Potential

Peripheral artery disease (PAD) is a broad disorder encompassing multiple forms of arterial disease outside of the heart. As such, PAD development is a multifactorial process with a variety of manifestations. For example, aneurysms are pathological expansions of an artery that can lead to rupture, while ischemic atherosclerosis reduces blood flow, increasing the risk of claudication, poor wound healing, limb amputation, and stroke. Current PAD treatment is often ineffective or associated with serious risks, largely because these disorders are commonly undiagnosed or misdiagnosed. Active areas of research are focused on detecting and characterizing deleterious arterial changes at early stages using non-invasive imaging strategies, such as ultrasound, as well as emerging technologies like photoacoustic imaging. Earlier disease detection and characterization could improve interventional strategies, leading to better prognosis in PAD patients. While rodents are being used to investigate PAD pathophysiology, imaging of these animal models has been underutilized. This review focuses on structural and molecular information and disease progression revealed by recent imaging efforts of aortic, cerebral, and peripheral vascular disease models in mice, rats, and rabbits. Effective translation to humans involves better understanding of underlying PAD pathophysiology to develop novel therapeutics and apply non-invasive imaging techniques in the clinic