Fig. 9.1
AngII infusion in mice induces both thoracic and abdominal aortic aneurysms. Male mice were infused with either saline or AngII (1000 ng/kg/min) for 4 weeks. Thoracic and abdominal aortas dissected from mice infused with saline (a and c). Thoracic and abdominal aortas dissected from mice infused with AngII (b and d). Asterisks (*) indicate aneurysmal area. Light green color (a and b) is 2 % agarose gel containing dye that is introduced into the aortic lumen to maintain patency and improve visualization
9.2 Selection of Mice and Infusion Rate of AngII
The initial study reported that AngII infusion at a rate of 500 ng/kg/min induced AAAs in male LDL receptor −/− mice at the age of 5 months [6]. Our subsequent study infused 1000 ng/kg/min to female apoE −/− aged 6 months fed with a normal laboratory diet [7]. Later studies have demonstrated pronounced male gender preference in hypercholesterolemic mice [11, 12]. Studies have also demonstrated that AngII infusion induces AAAs in normocholesterolemic C57BL/6 mice, but the incidence is 5–10 times lower than hypercholesterolemic mice [16, 24–26]. In contrast, fat-enriched diet or genetic obesity promotes AngII-induced AAAs in C57BL/6 mice without pronounced hypercholesterolemia [15]. The basis for hypercholesterolemia promoting AAAs is attributable to increased apoB-containing lipoproteins [16]. Although aging is a critical risk factor for human AAAs, there has been no systematic study demonstrating changes in incidence or severity of AngII-induced AAAs with increasing age. Most studies have been performed on mice that are ~8–12 weeks old.
Mouse selection for the study of AngII-induced AAAs can vary depending on the hypothesis of the experimental study. For example, if administration of a drug or manipulation of a gene of interest is expected to reduce AngII-induced AAAs, it is preferable to have a robust presence of the disease in the placebo or vehicle group. This would be achieved using hypercholesterolemic male mice infused with 1000 mg/kg/min or higher of AngII. If the experimental outcome is expected to promote AAAs, the presence of AAAs can be reduced in controls by reducing AngII infusion rate and/or using normocholesterolemic mice.
In contrast to profound contributions of hypercholesterolemia and male gender to AngII-induced AAAs, AngII-induced TAAs are not associated with increases in plasma cholesterol concentrations or gender differences [20]. Therefore, if AngII-induced TAAs are the focus of a proposed experiment, normocholesterolemic mice of either gender can be used. As with AngII-induced AAAs, an infusion rate of 500 ng/kg/min or less is preferable if augmentation of AngII-induced TAAs is expected, whereas an infusion rate of 1000 ng/kg/min is desirable if the outcome is attenuation of AngII-induced TAAs.
9.3 Determination of AngII Infusion Rate and Selection of Pump Models
Development of a protocol to permit consistent infusion rates of AngII is a critical aspect of experimental design. AngII mass to be infused is calculated after determination of infusion rate and study duration [27]. Infusion is performed using an Alzet (DURECT Corporation) osmotic pump model selected from those listed in Table 9.1. Selection of a pump model for a specific experiment is based on infusion rate, study duration, and mouse body weight. Although most reported studies use an AngII infusion rate of 1000 ng/kg/min for 28 days, infusion rates and study duration can vary depending on experimental purpose. Insights into selecting AngII rates are provided in Sect. 9.2. If early changes prior to overt aortic aneurysm formation are the focus of an investigation, infusions of AngII for less than 10 days will suffice [9]. If advanced stages of AAAs are the focus of a study, longer infusion intervals can be performed by sequential replacement of pumps [18, 28]. In addition to infusion duration, an important determinant to the selection of Alzet pump used is mouse body weight (Table 9.1).
Table 9.1
Alzet osmotic pump models commonly used in mice
Pump model | 1007D | 2001 | 1002 | 2002 | 1004 | 2004 | 2006 |
|---|---|---|---|---|---|---|---|
Infusion duration (days) | 7 | 7 | 14 | 14 | 28 | 28 | 42 |
Mouse weight (g) | >10 | >20 | >10 | >20 | >10 | >20 | >20 |
Different pump models have various priming intervals to initiate peptide delivery. For example, model 2004 pumps initiate extrusion 40 h after filling, when submersed in saline at 37 °C or implanted subcutaneously. In our standard protocol, we implant pumps after incubation in saline at 37 °C for 12–24 h on the basis that mice have 16–28 h to recover from the surgery prior to AngII delivery. However, if prolonged infusion of AngII (more than 4 or 6 weeks) is needed, this requires replacement with a second pump that should be primed to immediately deliver AngII on implantation. In this case, a filled model 2004 must be immersed in saline and incubated at 37 °C for at least 40 h before the implantation surgery.
Although increases of blood pressure do not contribute to AngII-induced AAAs or TAAs [20, 23, 29], we routinely measure systolic blood pressure at baseline (prior to pump implantation) and during AngII infusion since blood pressure is one parameter that demonstrates effective AngII delivery [30]. Therefore, we recommend measuring blood pressure as a routine procedure before and during AngII infusion. A second validation is measurement of plasma renin concentrations that are decreased during AngII infusion [31].
9.4 Quantification of Aortic Aneurysms
9.4.1 Death Due to Aortic Rupture
Death due to aortic rupture has been a consistent finding during AngII infusion, and it should be qualified in all studies. Analysis of aortic rupture rate or survival curve for the entire study duration is a useful indicator of aneurysm rupture in response to an intervention [28, 32]. Since mice infused with AngII may die of either abdominal or thoracic aortic rupture, it is important to perform a necropsy soon after death to define the location of aortic rupture since pathologies of AngII-induced AAAs and TAAs differ considerably.
9.4.2 Ultrasonography
Ultrasonography is the most convenient and cost-efficient manner of screening for aortic aneurysms in humans [33–35]. In humans, although luminal diameter of 3 cm or above in the infrarenal region is commonly defined as an AAA, an aneurysm is also defined as exceeding the normal luminal diameter of the artery by 50 % [36]. Both sagittal and transverse screens are commonly used, accompanied by color Doppler flow recognition in humans.
Ultrasonography has also been used frequently in mouse aortic aneurysm studies to quantify aortic dilation [28, 37–42]. A major benefit of this method is the ability to sequentially quantify luminal dilation in a live mouse [20, 29, 37]. Any of the VisualSonics models (Vevo 660, 770, or 2100) provide sufficient resolution to determine lumen dimensions of the suprarenal aorta. Although long-axis screening is reported in several studies, accuracy requires demonstrating that lumen diameter is recorded from the midpoint of the vessel to provide maximum diameter. Also, the potential for tortuous regions after AngII infusion can complicate accurate measurement. Hence, it is likely that short-axis screen provides more precise measurements of abdominal aortic dilations in mice.
For these measurements, mice are sedated with isoflurane and their abdominal surface depilated and placed dorsally in a supine position. AngII-induced AAAs are located in the suprarenal region. The left renal artery provides a landmark to ensure consistent screening in the same location. After identifying the left renal artery, two-dimensional images (B mode) can be acquired starting above the right renal artery with Cine loops. Our standard protocol requires acquisition of Cine loops of 100 frames/suprarenal aortic region. The 100 frames are used to determine the maximal luminal diameter of suprarenal aortas. Measurement accuracy of ultrasonographic images depends largely on user skill, knowledge, and experience. It is preferable that two independent, well-trained operators analyze images in a blinded manner. The frequency for ultrasound screening is variable dependent on the experimental design. To monitor the progression of AAAs, suprarenal aortas can be screened at relatively frequent intervals [28, 37].
Ultrasonography has also been used frequently to monitor and determine aortic root and ascending aortic dilations in mouse models [20, 29, 41, 42]. Because of depth of penetration and width of the aortic root and ascending aorta, use of a VisualSonics Vevo 2100 is preferable to acquire these images. There is an enhanced level of technical skill needed to measure TAAs relative to AAAs.
9.4.3 Aortic Tissue Processing
Blood is usually drawn from anesthetized mice through a right ventricular puncture to collect either serum or plasma for biochemical measurements. These measurements typically include concentrations of total cholesterol and cytokines. As noted above, we commonly measure plasma renin concentrations to confirm effective AngII delivery.
For mice infused with AngII for 4 weeks or less, after perfusion with saline to remove blood, aortas are dissected carefully and placed in a fixative solution (e.g., 10 % neutrally buffered formalin) for more than 24 h. For mice infused with AngII for more than 4 weeks, especially those infused with AngII for 12 weeks, it is helpful to excise aortas after pressure perfusion with a fixative solution at ~100 mmHg and injection of 2 % (wt/vol) agarose to maintain aortic patency [18, 28]. Adventitial tissues are then removed carefully to expose the gross structure (outer boundary of the medial layers) of the aorta. Subsequently, aortas are pinned on black wax to acquire aortic images for maximal ex vivo measurements of suprarenal regions (Fig. 9.2). After completion of this measurement, aortas are unpinned and cut open longitudinally (Fig. 9.3a) through the inner curvature and outer curvature with midline cutting through the three major branches (innominate, left common carotid, and left subclavian) of the aortic arch region as described in the literature [43]. Aortas are then pinned to expose intimal surface of the ascending, arch, and descending part of the thoracic aorta on black wax. For TAAs, images are acquired for en face measurements of intimal surface areas (Fig. 9.3b).
Fig. 9.2
Maximal outer width measurement of the suprarenal aorta. Example shown is an aorta from a male LDL receptor −/− mouse infused with AngII 1000 ng/kg/min for 4 weeks. The red line on the abdominal aorta represents maximal outer width of the suprarenal region
Fig. 9.3
En face analysis for measuring thoracic aortic aneurysms. (a). Longitudinal cutting open of aorta. Labeling 1–4 represents the cutting order. 1 represents cutting open the middle of the outer curvature through the innominate artery, 2 through left common carotid artery, and then 3 through left subclavian artery. 4 is cutting open the inner curvature with midline cutting through the entire length of the aorta. (b). Definition of area to determine intimal area of the ascending aortic (yellow box) and aortic arch (ascending aorta in addition to 3 mm from the branch of the subclavian as illustrated by the orange line)
9.4.4 Ex Vivo Imaging
Measurement of the maximal outer width is the most commonly used measurement for AngII-induced AAAs. Measurements can easily be recorded using a digital imaging system. It is preferable to have two independent observers acquire measurements. The measurement of ex vivo diameter can be used to determine AAA incidence. As with the definition of human AAAs, this requires an arbitrary definition of the absolute size or percent increases that determine AAA presence.
A potential issue of ex vivo measurements is that it only provides a two-dimensional measurement of a three-dimensional aorta and it does not provide information on luminal diameters. To overcome this limitation, AAA volume can be measured using the three-dimensional (3D) imaging function of the Vevo imaging system [28, 38]. 3D mode of the Vevo imaging system acquires a series of 2D “slices” that can be assembled into a 3D dataset using a cube view system. This method can provide both outer and inner volumes of each aortic segment analyzed. For this approach, it is optimal to perform fixation in vivo at physiological pressure to maintain the patency of the lumen. With current software configurations, it is time consuming to measure AAA volume.
9.4.5 En Face Imaging
En face analysis of thoracic aortic dilation can be performed on pinned aortas using image analysis. As shown in Fig. 9.3b, this analysis can arbitrarily be defined as ascending aorta and arch regions to determine site-specific effects. The ascending aorta is defined as the intersection of the base of the left cardiac ventricle to the root of innominate artery, and the aortic arch includes the three major aortic branches (innominate, left common carotid, and left subclavian arteries). Ascending aortic dilation is measured by tracing the outline of the intimal surface area in the ascending and arch regions. As with all measurements, we acquire measurements from two independent operators.
9.5 Characterization of Aortic Aneurysmal Pathologies
9.5.1 Serial Sectioning
Visualization and characterization of pathological changes in aneurysmal tissues is optimally performed by histological and immunocytochemical analyses on serial sections of aortas. AAAs have considerable heterogeneity throughout their length. Meaningful analysis of AAA pathologies requires examining the region of lumen expansion which presents in the early stages of AAA formation as a focal transmural media break across all elastin layers. This should include analysis of regions with normal lumen diameters, although these are not the primary areas of disease initiation. Therefore, it is preferable to section the entire length of AngII-induced AAAs, also including a small portion of normal aortic tissues proximal and distal to the AAA region. An example of a protocol for performing this analysis has been described previously [18]. This includes the collection of serial 10 μm sections throughout the AAA. These sections are placed serially on 10 sequential slides. This process provides slides with 9–12 tissue sections that are located at 100 μm intervals. For a 4 mm long AAA with normal aortic tissues, this entails 40 slides with approximately 400 sections.
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