**Biomechanic and Hemodynamic Perspectives in Abdominal Aortic Aneurysm Rupture Risk Assessment**

Nikolaos Kontopodis, Konstantinos Tzirakis, Emmanouil Tavlas, Stella Lioudaki and Christos Ioannou

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76121

#### **Abstract**

Abdominal aortic aneurysms (AAAs) pose a significant source of mortality for the elderly, especially if they go on undetected and ultimately rupture. Therefore, elective repair of these lesions is recommended in order to avoid risk of rupture which is associated with high mortality. Currently, the risk of rupture and thus the indication to intervene is evaluated based on the size of the AAA as determined by its maximum diameter. Since AAAs actually present original geometric configurations and unique hemodynamic and biomechanic conditions, it is expected that other variables may affect rupture risk as well. This is the reason why the maximum diameter criterion has often been proven inaccurate. The biomechanical approach considers rupture as a material failure where the stresses exerted on the wall outweigh its strength. Therefore, rupture depends on the pointwise comparison of the stress and strength for every point of the aneurysmal surface. Moreover, AAAs hemodynamics play an essential role in AAAs natural history, progression and rupture. This chapter summarizes advances in AAAs rupture risk estimation beyond the "one size fits all" maximum diameter criterion.-

**Keywords:** abdominal aortic aneurysm, rupture risk, wall stress, shear stress, wall strength, biomechanics, hemodynamics, intraluminal thrombus, rupture potential index-

#### **1. Introduction**

Abdominal aortic aneurysms (AAAs) are balloon like dilatations of the abdominal aorta with a- diameter exceeding 50% of the diameter of the normal vessel [1, 2]. These are lesions affecting-

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mostly elderly male patients and have been related to smoking and family history [1, 2]. Patients- with AAA are at risk of rupture which is the most devastating complication of this condition and- is accompanied by a striking overall mortality of approximately 80% [3, 4]. Therefore, elective- repair of AAAs is being performed to avoid the former scenario which of course, similar to any- interventional therapy, is not without its own risks. Specifically, surgical treatment of AAAs is followed by a 3–4% periprocedural mortality which is reported to be as low as 1% in centers of excellence but is significantly increased and can reach up to 10% in case of compromised patients [5–8].- Endovascular modalities have significantly reduced operational risks but again carry a significant- risk for renal morbidity, continuous need for surveillance with CT imaging with the associated- exposure to radiation and a considerable risk for late complications and need for re-interventions- in the long run [9, 10]. Therefore, the need for elective repair has to be cautiously balanced against- the risk of rupture in order to determine optimal therapeutic management in a patient-specific- basis. Currently, the maximum diameter criterion is being used as the sole predictor of rupture- risk and the critical determinant of the need for intervention [1, 2]. Large randomized control trials- have defined appropriate thresholds for repair which are 55mm of diameter for male and 52mm- for female patients [11–14]. Nevertheless, this criterion is not always accurate and may frequently- lead to therapeutic failures in the management of these patients. Specifically, in a contemporary- systematic review, rupture rates for small AAAs, under the threshold for surgical repair, have- been reported to reach 1.61 ruptures per 100 person-years [15]. Furthermore, in a more recent- report, Laine etal. examining a large cohort of ruptured AAAs indicated that a remarkable 5.6%- of men and 11.5% of women presented a maximum diameter under 55 and 52mm, respectively,- which are the thresholds for intervention according to the European guidelines [16].-

#### **2. The maximum diameter criterion**

Actually, the physical principle behind the maximum diameter criterion is the Law of Laplace which states that the stress exerted on the wall of a pipe is proportional to its radius. Admittedly, this law is valid for cylindrical or spherical shapes with rigid, thin walls [17, 18]. None of these perquisites is valid in the living arterial system and therefore the presumption that maximum diameter can be used as an index to estimate wall stress exerted in the vessel wall is an oversimplification. Specifically, the arterial wall is distensible and not rigid, it has a variable thickness and more importantly AAAs present unique 3D geometric configurations which are original to each patient, presenting myriads of shapes and variable major and minor wall curvatures, not at all resembling simple geometrical shapes [19]. Therefore, relevant tools have subsequently been developed in order to stimulate biomechanical conditions inside AAAs and through computational modeling, calculate the stresses exerted on the arterial wall [20]. This progressively led to the next step of AAAs rupture risk estimation.-

#### **3. Wall stress**

#### **3.1. General**

Stress is a measure of the loading sustained per unit area of the arterial wall, due to systemic pressurization and blood flow [21]. Pressure-induced, in-plane wall stress is orders of magnitudegreater than flow-induced shear stress and is considered the main force that contributes to- arterial wall pressurization and the driving force leading to rupture [21]. Peak wall stress- (PWS) is the maximum value of stress throughout the surface under evaluation, in other words- the maximum stress exerted on the aneurysmal wall during systolic pressurization [22].-

Stress acting on the aneurysm sac is estimated through finite element analysis (FEA) which is a numerical method to solve the differential equations of physics [23]. According to this process, any continuous quantity such as wall stress can be approximated by a discrete model composed of a set of simple continuous functions. In other words, in the case of AAAs where the complex geometry precludes a mathematical expression of the behavior of the whole system, one can divide this into a finite number of elements and then study the behavior in a single element or sub-region level. Since these elements have a small size and a simple geometric configuration, the description of their behavior is straightforward. Subsequently, the whole system can be resembled through the description of the behavior of all the elements taken together, since these collectively approximate the shape of the system [23].-

In order to perform FEA, information regarding the boundary conditions, the material's constitutive law (stress-strain relationship) and its geometric configuration are required. Then the 3D geometry is loaded with a fixed or patient-specific value of systemic pressure and the mathematical problem is solved taking into account the equations of mechanical equilibrium and conservation of momentum [24].-

Another approach is to apply a non-uniform pressure taking into account the pattern of pressure changes and the wall motion during the cardiac cycle. This is called fluid structure interaction (FSI) and provides a more realistic pressure distribution along the AAA luminal surface. Despite being more physiologically sound, such an approach needs increased computational complexity and thus it has not yet been determined if the benefit regarding the accuracy of the results justify the additional burden of complex calculations [25, 26]. Additionally, due to the lack of subject specific wall material properties, its superior accuracy remains a universal question.-

Regarding the index geometry, initial studies considered simple representations of AAA- shapes which mostly resembled standard geometrical shapes, rather than the complex configuration of real AAAs. Stringfellow etal. as early as 1987 used simple 2D geometries and- indicated that aortic size was important in determining wall stress which was also dependent- upon aneurysm wall thickness. Maximum longitudinal wall stress was located at the site of- aneurysm's maximum diameter [27]. Mower etal. suggested that doubling the diameter of the- 2D AAA model resulted in a proportional increase in wall stresses, while the same result was- observed in case the wall thickness was reduced in half [28]. Inzoli etal. studied the influence- of intraluminal thrombus (ILT) in the wall stress, indicating that this may reduce maximum- stress values by up to 30% [29]. Others indicated a significant effect of AAA shape to magnitude and distribution of stress [19]. Actually, the influence of other geometric variables such as- vessel asymmetry was found to be similarly important to that of maximum diameter, indicating that similar sized AAAs may in fact present significant differences in wall stresses [30].-

With the rapid progression of imaging techniques and computational modeling, the reconstruction of patient-specific rather than idealized anatomies became feasible. Various techniques and- softwares were developed in order to post-process medical images and reconstruct individualanatomies, from simple axial 2D CT images to complex patient-specific AAA models. The process of AAA 3D reconstruction and estimation of wall stresses is displayed in **Figure 1**.-

#### **3.2. PWS and rupture risk**

Fillinger etal. were the first to indicate that PWS was significantly higher in AAAs that needed- emergent repair (ruptured and symptomatic) compared to those that were electively repaired,- while no significant differences in maximum diameter or blood pressure were found [31]. In- a subsequent study, these authors recorded AAAs progression over time and indicated that- baseline PWS was significantly higher in cases that went on to develop symptoms and require- urgent treatment compared to those that did not. Despite that baseline diameter was also significantly different between these groups, PWS was far more accurate in predicting adverse outcomes [32]. Other authors confirmed the findings that ruptured AAAs present a significantly- higher PWS compared to intact cases [31–44]. These data are summarized in **Figures 2** and **3**.-

#### **3.3. PWS and rapid growth**

Apart from rupture risk, there are data in the literature to suggest that high PWS may be related to a rapid AAA expansion, as well. Speelman etal. studied 69 paired CTs of AAAs and found that a relatively low AAA wall stress was associated with a lower aneurysm growth rate [45]. The same authors in a subsequent study suggested that AAA growth may be driven rather by ILT accumulation and not PWS.-Specifically, in the group of AAAs with rapid growth, a greater ILT volume was recordedalong with a lower PWS.-Of course, ILT has been found to reduce stresses exerted on the aneurysmal wall which is the reason why many suggest a biomechanical cushioning effect of this structure, which is discussedlater [46]. The contradicted data of the two abovementioned studies could be explained by the fact that in the first, the authors did not take into account the presence of ILT during PWS estimation. Others have demonstrated that concentrations of high stresses in the region of the aneurysm shoulder may result in a rapid growth rate. Specifically, baseline AAA shoulder stress was higher in patients with fast growth compared to those with slow and presented a strong and significant correlation with growth rate, whereas AAA diameter did not display any significant effect [47].

**Figure 1.** The process of biomechanical analysis is displayed. From 2D CT images, with manual or automated segmentation, 3D AAA models are reconstructed. Then a mesh is constructed and finite element analysis is performed. The final map of wall stress distribution is finally obtained.-

Biomechanic and Hemodynamic Perspectives in Abdominal Aortic Aneurysm Rupture Risk Assessment 7 http://dx.doi.org/10.5772/intechopen.76121

**Figure 2.** Metanalysis of the studies examining PWS in ruptured and intact AAAs. A consistent finding is that ruptured cases present significantly higher values of PWS compared to elective cases. The high heterogeneity between studies is due to differences in methodology (differences in assumptions for FEA, loading of the AAA model with patientspecific or standard values of pressure, inclusion of ILT in the final model, etc.). In many of the studies there were significant differences in maximum diameter between ruptured and intact AAAs which could have confounded results. This metanalysis has been performed by the authors for the purposes of this chapter only and has not been published elsewhere.-


**Figure 3.** Metanalysis of the same outcome as in **Figure 2**. Only studies which performed matching for maximum diameter or in which differences were not significant are included. The results are similar with the overall comparison, so PWS seems superior to maximum diameter in differentiating ruptured from intact AAAs. This metanalysis has been performed by the authors for the purposes of this chapter only and has not been published elsewhere.-

**Figure 4.** Lateral, posterior and anterior views of the stress distribution in two AAAs are displayed. Case in the first row presented a rapid growth rate while that in the second a much slower one. The difference in stress distribution can be observed. In the rapidly growing AAAhigh stresses are concentrated in the posterior wall, while in the case with relatively stable size, this is more uniform. According to Metaxa etal. [48] higher posterior wall stress may foretell a potential for rapid expansion.-

Shang etal. in a contemporary study also indicated that there is a strong and statistical significant correlation between PWS and AAA growth rate. This is a particularly important finding since rapid growth has been shown to foretell a high rupture risk. Therefore, a high baseline PWS could identify lesions in risk for such adverse outcomes [48]. Moreover, Metaxa etal. divided their patient cohort into fast and slow growth rate subgroups and observed a significant variability in the distribution of stresses along the AAA surface: the fast growth rate group presented significantly higher wall stresses in the posterior portion of the AAA sac compared to the slow growth rate group [49]. Interestingly, they did not record any significant differences in the PWS between those groups. A representative example is presented in **Figure 4**. Finally, Martufi etal. studied a cohort of AAAs taking into account the baseline and a follow-up CT scan and quantified regional growth by dividing the two 3D AAA models in 100 cross sections and registering each section of the initial phase with the corresponding one from the final state. They indicated that for the aortic wall not covered with ILT, the local growth rate was strongly related with the local values of wall stress. The high stress sensitivity of non-dilated aortic walls suggests that wall stress could initiate AAA formation and expansion [50].-

#### **4. Wall strength**

According to the biomechanical approach, rupture of AAAs follows the basic principles of failure applyingin any given material. Therefore, material failure occurs when the mechanical stress exerted on that material surpasses its strength. Accordingly, rupture depends on the pinpoint comparison of the wall stress and strength for every point throughout the aneurysmal surface. Therefore, and taking into account that a significant regional variation of mechanical properties and strength of the AAAs' wall has been shown, a means to quantify the local arterial wall strength non-invasively and provide a map of its distribution similar to that of wall stress was required in order to provide a sound biomechanical rupture risk estimation [51]. Vande Geest etal. in a landmark study that they published in 2006 recorded several demographic and morphometric information of AAA cases and identified significant predictors of wall strength values by relating those to the tensile testing of surgically procured AAA wall specimens. Using this methodology, a four-parameter statistical model was developed, in which the significant predictors that were included were sex, family history, ILT thickness and normalized transverse diameter. Demonstrative application of the model resulted in an original, complex distribution of wall strength over the aneurysmal surface [52].-

$$\begin{aligned} \text{STRENGTH} &= 71.9 - 37.9 \times (\text{ILT1}/2 - 0.81) - 15.6 \times (\text{NORD} - 2.46) \\ &- 21.3 \times \text{HST} + 19.3 \times \text{SEX} \end{aligned} \tag{1}$$

These authors also suggested a new biomechanical index to estimate rupture risk which was the Rupture Potential Index (RPI). This integrated information about wall stress and strength and was basically the *stress:strength* ratio for any given point of the aneurysm wall. This ranged from 0 (low stress exerted in aneurysms with high wall strength) to 1 (high stress exerted in AAAswith low wall strength). **Figure 5** illustrates color maps for the distribution of wall stress, wall strength and RPI in a patient-specific AAA model. Subsequently, the same Biomechanic and Hemodynamic Perspectives in Abdominal Aortic Aneurysm Rupture Risk Assessment 9 http://dx.doi.org/10.5772/intechopen.76121

**Figure 5.** A patient-specific AAA model is presented where distribution of stress, strength and RPI can be seen. It can be observed that a weak region (decreased strength) at the site of maximum diameter results in a comparatively high RPI value, while at the same site a low stress value had been recorded. The implementation of strength in biomechanical calculations with the introduction of RPI seems superior than using wall stress alone.-

authors compared between a small cohort of ruptured and non-ruptured AAAs indicating that RPI was superior in differentiating these groups than PWS alone. Due to small sample size, statistical significance was not reached. Other studies that included this marker in the biomechanical estimation of AAAs rupture risk consistently showed that RPI could improve risk prediction. Gasser etal. examined a diameter-matched cohort of 18 intact and 16 ruptured AAAs and indicated that both PWS and RPI were significantly higher in the former group of patients. Similar results were obtained when cases were matched for maximum diameter and blood pressure values. Overall, these authors suggested that RPI reinforces PWS as a biomechanical rupture risk index [39]. In a larger population including 203 intact and 40 ruptured AAAs, the same authors indicated that both PWS and RPI were significantly different between groups and that a linear relation existed between PWS and maximum diameter, while an exponential one fitted the relation between RPI and maximum diameter [41]. Erhart etal. analyzed CTA data from 13 asymptomatic AAAs experiencing rupture at a later stage who had imaging during the time of rupture as well. FEA was performed to calculate PWS and RPI and identify location of those values in the pre-rupture state. A statistical comparison was performed between the pre-rupture state and that at the time of rupture. Moreover, this group was compared with a 23-patient diameter-matched asymptomatic AAA control group that underwent elective surgery. The AAAs that subsequently went on to rupture displayed significantly higher values of RPI at the pre-rupture state compared with the diametermatched group of asymptomatic AAAs, while the differences of PWS were not significant. Regarding in-group comparisons between the AAAs at the pre-rupture state and at the time of rupture, again RPI displayed significant differences, while PWS alone did not [44]. Overall, according to published data, RPI seems to advance rupture risk estimation and provide a more accurate biomechanical prediction compared to PWS alone. Studies examining RPI are summarized in **Table 1**.-


**Table 1.** Studies that examine RPI during biomechanical analysis are presented, along with absolute values and statistical significance of the differences between intact and ruptured cases and main authors' conclusions.-

#### **5. Equivalent diameters**

Despite the fact that the abovementioned data provide consistent evidence of the superiority of stress and stress/strength calculation over the maximum diameter criterion for the evaluation of AAAs rupture risk, clinical applicability of these findings remain limited. A possible explanation could be the complexity of the process along with the requirement of sophisticated software, increased computational time and specially trained personnel. Indeed, computational modeling and mathematical algorithms that may be required in order to perform biomechanical calculations are often puzzling and confusing to clinical doctors. In order to deal with this problem and translate biomechanical indices into a more relevant clinical variable, the concept of "equivalent diameters" has been recently introduced. According to this approach, the PWS and RPI values are determined from a reference population of intact AAAs and these are plotted against the maximum diameter to obtain a graphical representation of Biomechanic and Hemodynamic Perspectives in Abdominal Aortic Aneurysm Rupture Risk Assessment 11 http://dx.doi.org/10.5772/intechopen.76121

**Figure 6.** A graphical representation of the concept of equivalent diameters is presented. According to that, the equivalent diameter is determined based on the PWS or RPI value of a given AAA which is related to the diameter of the average AAA with similar PWS or RPI values. For example, it can be seen from these figures that two AAAs with the same maximum diameter, may present large differences in their equivalent diameters depending on biomechanical analysis.-

their relationship. Subsequently, the values of PWS and RPI for any given AAA are related to those of an average AAA and the diameter of the latter is nominated "equivalent diameter".-

For example, a 45mm AAA could correspond to a stress equivalent 65mm AAA, if a higher PWS or RPI is calculated. The concept of equivalent diameters relates results of biomechanical analysis to currently accepted diameter thresholds being determined from large clinical AAA trials, and hence manifests a sound clinical interpretation of biomechanical results [41]. The number of studies that have used this concept remains limited at the moment, but a consistent finding of larger equivalent diameters in ruptured compared with intact AAAs even when diameter matching was performed has been consistently reported [41, 43, 44]. As already mentioned, the relation between the maximum diameter and PWS is a linear one while that between diameter and RPI is exponential. This is because while stress is expected to increase as a function of diameter, in the case of RPI, strength has been shown to decrease as a result of an increased diameter too. Therefore, in that instance, a larger aneurysm size results in both higher stress values and lower strength values which are displayed in the exponential form of the relation between the RPI and the maximum diameter. A graphical representation of this concept is presented in **Figure 6**.-

#### **6. Wall shear stress**

The wall shear stress (WSS) is the tangential force acting on the arterial wall due to blood flow.- This traditionally had been considered to play a negligible role in AAAs expansion and progression to rupture for several reasons. Specifically, not only AAAs almost universally contain- ILT which acts as an impediment between the blood flow and the endothelial layer of the arterial wall, but also it has been suggested that AAAs mostly lack a proper intimal layer that wouldbe affected by shear stress [20]. More importantly, the flow-induced shear stress acting on the- AAA wall is orders of magnitude smaller than the in-plane pressure-induced wall stress, which- until recently was believed to be the only force that could impair structural integrity of the wall- leading to rupture. Specifically physiological values of wall stress is about 104 orders higher- than WSS (wall stress is measured in 104 Pa, whereas WSS in Pa) [53].-

Nevertheless, lately there have been data in the literature, to indicate a role of WSS in the natural history of AAAs. The main variable that seems to be related to WSS is the accumulation of ILT.-Specifically, it has been suggested that ILT deposition has a significant negative relation with WSS.-In other words thrombus tends to accumulate in regions where WSS is minimal. WSS typically ranges from 1.5 to 4-Pa-[53]. Tzirakis etal. used longitudinal data for AAA patients and related initial hemodynamic parameters with subsequent ILT accumulation during follow-up, using an original technique that divided AAA surface into patches, in order to achieve registration between the initial and the final state. They indicated that a low local WSS was related with later ILT formation, with a value <0.5-Pa be indicative of a higher probability for thrombus deposition [54]. Representative AAA cases are presented in **Figure 7**. Similarly, Arzani etal. examined the relationship between changes in ILT and hemodynamic indices at mid-aneurysm cross section and suggested that thrombus growth mainly occurred in regions where WSS displayed values between 0.2 and 0.3-Pa-[55]. To provide an answer to the obvious contradiction that intracranial saccular aneurysms, despite presenting low WSS, almost never exhibit thrombus accumulation, Gasser etal. suggested that initial platelet activation inside a proximal recirculation zone, such as the aneurysm neck, where relatively high-shear stresses act long enough to activate platelets, must precede their convection toward the wall at the distal portion of the sac, in order to initiate the cascade that ultimately results in ILT deposition [56]. Moreover, the rate of ILT accumulation has been reported to be similar to that of AAA expansion, while AAAs with thrombus exhibited a significantly faster enlargement compared to those without, with the former group presenting lower values of WSS.-These

**Figure 7.** Initial hemodynamics (Time Average Wall Shear Stress-TAWSS, Oscillatory Shear Index-OSI, Relative Residence Time-RRT) and thrombus deposition thickness at follow-up for two cases. Adapted with permission from Tzirakis etal., [54].-

findings imply a causal relation between low WSS and rapid AAA growth which could be mediated by the accumulation of ILT [57]. Finally, a recent study indicated that WSS independently predicted the growth of AAA volume and these investigators suggested that since aneurysmal wall lacks endothelial cells, blood flow properties could only indirectly influence AAA growth through stimulation of the biochemical environment within the ILT [58].-

In fact, ILT has been suggested to play an active role in AAAs' natural history. Most researchers- believe that it has a negative effect through its proteolytic activity and promotion of inflammation. ILT thickness has been associated with vascular smooth muscle cell apoptosis and elastin- degradation, while it is positively associated with the concentration of proteolytic enzymes in- the underlying wall [59]. Moreover, segments of the AAA sac under a thick layer of ILT have- been recorded to be hypoxic and present significantly more neovascularization compared to- those covered by no or minimum ILT.-More importantly, regions of thicker ILT presented a- decreased wall strength, which could make them more susceptible to rupture [60]. Additionally,- there are longitudinal and computational AAA studies that also suggest a negative effect of ILT- in AAAs progression. Speelman etal. recorded a higher growth rate in AAAs containing larger- amounts of ILT despite the fact that those presented significantly lower values of PWS [46]. In- a contemporary study which recorded regional growth of AAAs, it had been demonstrated- that the local growth was positively related to local values of wall stress only in cases where- ILT was absent. On the other hand, in the presence of ILT, local growth was dependent on local- ILT thickness but not wall stress [50]. Therefore, these data may imply that ILT plays a more- imminent role in AAAs progression than wall stress. Additionally, it has been suggested that- larger ILT deposition may be related to AAA expansion, rupture and even with cardiovascular- events [61–63]. On the other hand, it should also be mentioned that biomechanical analysis has- demonstrated a cushioning effect of thrombus which acts as a buffer reducing stresses exerted- on the wall. Many studies have examined this effect recording a reduction in PWS values up- to 30% [64]. Therefore, there is wide consensus that ILT should be included in computational- simulations in order to have a realistic and accurate estimation of stress magnitude and distribution. Additionally, while there is general agreement that ILT plays an active role in AAAs- progression, not being an "innocent bystander" its exact role is still debatable, but most evidence points to a negative overall effect of ILT.-All in all, taking into account the definitive role- of ILT in AAAs progression and its well established relation with the shear stresses and the- overall hemodynamic environment inside the aneurysm sac, a significant impact of hemodynamic forces in the AAAs' natural history has started to become evident.-

### **7. Clinical implications**

All the abovementioned indices and diagnostic methods point toward developing a predictive model that will be able to estimate AAAs rupture risk in an individualized, patient-specific basis. This would allow identification of patients with small AAAs presenting a higher than average rupture risk, thus being suitable for prompt elective repair at a lower diameter, but also those with larger aneurysms and low rupture potential who would benefit from conservative treatment. Subsequently, optimization of patients' management with the selection of the most appropriately suited treatment (i.e. conservative or interventional/surgical) for each patient would reduce rupture rates of AAAs at the same time obviating unnecessary procedural risks of patients that do not actually need to undergo surgical intervention. A new promising tool that will probably receive much attention in the near future and will have an upgraded role in AAAs' diagnostics is ultrasonography which is a cheap and readily available bedside imaging modality which has recently been used to estimate biomechanical variables of AAAs with promising results [65].-

#### **8. Limitations**

Despite the fact that biomechanical analysis seems to have advanced rupture risk prediction which is a consistent finding of all relevant studies, this approach is not without limitations. Specifically, the stresses and strains which are obtained are dependent on several model assumptions taken into account during FEA.-For example inclusion or not of the ILT, consideration of the arterial wall as isotropic or anisotropic, linear or non-linear material properties, consideration of the pre-stress state as well as accuracy of the 3D reconstruction, meshing and number of finite elements used, all can have a great influence on calculated values. As a consequence, interpretation of results in many studies can be difficult since these are often not comparable. Differences in PWS due to different model assumptions can be up to 210% in extreme cases. Overall, in order for comparisons between individual reports to be valid, information about preconditions and model assumptions should be provided [26]. Moreover the need for special software and/or highly trained special personnel to make these complex calculations along with the fact that data are not directly comparable with information from randomized trials which have taken into account the maximum diameter criterion alone limit applicability of biomechanical analysis in the every-day clinical practice.-

#### **9. Conclusion**

Despite the fact that currently therapeutic management of AAAs is based on the maximum diameter criterion, there is evidence that this can often be inaccurate. New methods have been developed in order to advance rupture risk estimation. Biomechanical indices of wall stress and rupture potential index have been consistently shown to be superior to maximum diameter in this regard. The concept of equivalent diameters may provide a comprehensive means to translate results of biomechanical analysis into a simple clinical index which may be appropriate for use in a clinical setting. An important role of hemodynamic conditions which can have a significant effect on AAAs progression, mainly through its relation with ILT accumulation, has recently started to become evident as well.-

#### **Conflict of interest-**

None to declare.-

### **Author details**

Nikolaos-Kontopodis<sup>1</sup> \*, Konstantinos-Tzirakis<sup>2</sup> , Emmanouil-Tavlas<sup>1</sup> , Stella-Lioudaki<sup>1</sup> and Christos-Ioannou<sup>1</sup>

\*Address all correspondence to: kontopodisn@yahoo.gr-

1-Vascular Surgery Unit, Department of Cardiothoracic and Vascular Surgery, University of Crete, Medical School, Heraklion, Greece-

2-Institute of Applied Mathematics, Foundation for Research and Technology-Hellas,- Heraklion, Greece-

#### **References**


## **Experimental Models in Abdominal Aortic Aneurysm**

#### Zerrin Pulathan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.79393

#### **Abstract**

Abdominal aortic aneurysm (AAA) is a potentially fatal disease and survival rate is very low when rupture occurs. Experimental models related with abdominal aortic aneurysm are performed on intact and ruptured aneurysm (RAAA) models. By using AAA models; complex mechanisms of aneurysm formation, aneurysm progression, chance of rupture, preventative and treating methods are researched. Most commonly used methods for creating aneurysm are utilization of transgenic or knockout animals; intra/extraluminal pharmacologic treatments such as elastase, calcium chloride or angiotensin II; hyperlipidemic diet application and surgical interventions such as xenograft, stenosis or graft. Pathogenesis of aneurysm is predominantly examined on rodents whereas studies aimed at development of treatment modalities such as surgical or endovascular interventions are predominantly performed on large animals like rabbit, porcine or dog. Experimental studies modeling aneurysm rupture (RAAA) simulate shock (total hypoperfusion) occurred due to rupture and ischemia/reperfusion (I/R) occurred due to surgical treatment; without creating aneurysm. In this model, end organ or distal organ injuries and methods for reducing these injuries or their hemodynamic effects are investigated by creating shock +I/R.

**Keywords:** abdominal aortic aneurysm, ruptured abdominal aortic aneurysm, experimental models, elastase, rat

#### **1. Introduction**

Abdominal aortic aneurysm is a degenerative disease characterized by structural degeneration and progressive dilatation in aorta wall. Progressive increase in arterial diameter results with rupture which is a life-threatening condition. Only 50% of cases with ruptured aneurysm can reach to hospital and 30–50% of these patients dies in hospital. It is an important health problem which has been seen in 5–9% of men and 1–1.3% of women aged 65. It is 10th cause

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of death in developed countries and its incidence increases as population ages. However, complex and multifactorial pathophysiology of AAA has not been thoroughly understood [1].

Many animal models have been developed for understanding pathophysiology of AAAs and developing treatment models. It has firstly been incidentally observed by Ponseti IV etal. in 1952 that medial necrosis, dissection and aneurysm formation occurred after a special diet. Abdominal aortic aneurysm has been created with different techniques in many different models, its pathophysiology and drugs for preventing aneurysm formation have been investigated [2].

Among these models, most effective models developed for learning disease progression are elastase-based models performed on small animals. Large animal models have been required for endovascular or current surgical treatment methods, many surgical models like saccular or aortic patch have been developed. Most commonly used models among many different AAA animal models, difference between models and their applications will be explained in this chapter [3].

#### **2. Pathogenesis of abdominal aortic aneurysm**

In this section, pathogenesis of aneurysm will be briefly reviewed for clarify the development mechanism of experimental models of abdominal aortic aneurysm (AAA).

#### **2.1. Building stones of aortic wall**

#### *2.1.1. Elastin and collagen*

Two major building stones of aortic wall are elastin and collagen. Elastin is a structural protein produced by fibroblasts. Collagen is a solid, insoluble and fibrous protein which is majorly produced by fibroblasts and also produced by cells like chondroblast and osteoblast. Elastin is the major lifting structure against aneurysm development whereas collagen is the safety barrier which is resistant to high pressure and which provides protection against rupture after aneurysm is occurred. Degeneration of elastin and collagen results with aneurysm and rupture. Some studies revealed that these two proteins are reduced in intima and media layers of aneurysmatic aortas [4].

Presence of various mechanisms in development of AAAs which has been known as progressive dilatation of aortic wall has been well-recognized. Most important one of all factors causing degeneration in aortic wall is altered homeostasis between matrix synthesis and degradation due to inflammation. Adventitial and medial inflammatory cell infiltration, elastin fragmentation and degeneration, medial attenuation are observed during aneurysm development [5].

Collagen synthesis in the media and adventitia layers (especially type I and III) increases in favor of repair during first stages of aneurysm formation, then it becomes excessively degraded like other extracellular matrix macromolecules such as elastin during late stage and causes aortic rupture. Inflammatory cells like polymorphonuclear neutrophils, T cells, macrophages, mast cells, NK cells are present in all layers of aneurysm wall and intraluminal thrombus. These cells secrete various humoral-inflammatory factors like cytokines, chemokines, leukotrienes, reactive oxygen species (ROS) and immunoglobulins. Inflammatory cells enter to aortic intima and media layers through vasa vasorum vessels. Neovascularization and decreased number of smooth muscle cells in medial layer are typical features of aneurysm. Intraluminal thrombus causes functional hypoxia in luminal intima and media layers, therefore neovascularization and inflammation increase. Also, inflammatory cells in thrombus secrete active proteases like matrix metalloproteinase (MMP-9) and urokinase-type plasminogen activator (u-PA). Therefore, AAAs occur as a complex pathology consisted of many cellular and humoral mechanisms like inflammatory cells, various enzymes and complement system [6].

#### *2.1.2. Cellular and molecular mechanisms*

#### *2.1.2.1. Proteases*

Elastase is a group of serine endopeptidases which catalyzes degradation of elastin and other proteins to simpler molecules; breaks polypeptide chains in the bonds including carbonyl group of amino acids; and is secreted from neutrophils and macrophages. α-1 antitrypsin is a protease inhibitor and it suppresses elastase activity and protects the tissue from inflammation. Elastase levels have been found to be high in ruptured aneurysms whereas α-1 antitrypsin levels have been found to be low [7].

Matrix metalloproteinases (MMPs) are homolog peptidases which contain zinc in their active region; and can degrade extracellular matrix and basal membrane components. They are enzymes playing role in physiological processes like tissue regeneration, morphogenesis and wound healing.

Tissue inhibitor of metalloproteases (TIMPs) are antiprotease enzymes. MMPs secretion degrades structural proteins of aortic wall. Impaired balance between MMPs and TIMPs plays an important role in development of acute and chronic cardiovascular diseases. Important MMPs which play an important role in development of AAA are MMP-1, -2, -3, -9, -12 and -13. There have been inadequate evidence for other MMPs; however, it has been stated that MMP-3 plays a very important role for AAA [8]. Elastin degradation and extracellular matrix loss in addition to destruction of smooth muscle cells via MMPs cause media layer thinning and aortic dilatation. Especially in enlarged aneurysms, intraluminal thrombus along with local inflammation and proteolysis occurs. Local hemodynamic forces and weakened vessel wall increases aneurysm enlargement. If wall stress exceeds tensile strength; rupture occurs. Inflammation, matrix remodeling and neovascularization reduce tensile strength [9].

Others are serine proteases, tissue plasminogen activators (t-PA, u-PA), plasmin, neutrophile elastase, cysteine protease (cathepsin D, L, K and S); also cysteine and serine proteases have been shown in all AAAs. Concentrations of dipeptidyl peptidase which is a lysosomal cysteine protease, is found normal at aneurysm wall or abundant in stenotic arterial walls when neutrophile elastase and other proteases are activated [6, 10].

#### *2.1.2.2. Phospholipids*

Phospholipids play an important role in cell membrane structure; also they have been known as very important inflammatory mediators. 5-lipoxygenase (5-LO) and leukotriene C4 synthase levels have been found high in human AAA tissues [11]. Association between AAA and cyclooxygenase (COX) and its sub-component prostaglandin E2 (PGE2) has been demonstrated, indometacin which is a non-selective COX inhibitor has been shown as preventing AAA created with elastase in rat [12]. PGE2 has been shown to activate IL-6 secretion of macrophages in studies performed on human aortic tissue or aortic smooth muscle cells [13].

#### *2.1.2.3. Inflammatory cells-*

Most commonly seen inflammatory cells among many inflammatory cells identified in AAA tissue are macrophages and it has been known that they play an active role in aneurysm formation by many macrophage-mediated inflammatory responses [11, 14]. T and B lymphocytes have also identified in AAA tissues and functional insufficiency of CD25+ T regulator cells in AAAs- patients has been reported [15]. Neutrophils have been identified in both human AAAs and- animal aneurysm models, also L-selectin which is an adhesion molecule has been shown as an important mediator in AAA formation created with elastase in rats. Neutrophil depletion in mice with aortic perfusion of elastase led to attenuation of AAAs [16]. In addition, mast cells have also been identified in human AAA tissues and animal models. These cells secrete many proteases- and inflammatory mediators which play role in inflammation and immunity. It has been shown- that mast cell insufficiency in rat and mouse models decreases aneurysm formation [17].

#### *2.1.2.4. Complement system-*

Complement activation is an immune response started with classic antigen–antibody reaction, lectin pathway or alternative C3 hydrolysis pathway. Factor B is the most important component of alternative pathway whereas C4 is the most important component of both classic and alternative pathway. Factor B insufficiency decreases the development of AAA created by elastase in rats [18].

#### *2.1.2.5. Cytokines and chemokines-*

Cytokines regulate expressions of matrix metalloproteases (MMPs), serine proteases and cathepsin.

Wıthout a doubt, tumor necrosis factor (TNF)-α has a very important place among many cytokines and chemokines which are related with inflammatory response. Increased plasma and tissue TNF-α levels have been found in AAA patients. Genetic or pharmacological (with infliximab) TNF-α inhibition has been shown to decrease calcium chloride-induced AAA formation in rats [19].

Another cytokine which plays an important role in inflammatory process is transforming growth factor (TGF)-β. This cytokine acts as a protector from inflammation and cell death. It has been shown that systemic blockage of TGF-β activity causes smooth muscle cell death, elastin degradation and vascular inflammation in AAAs created with angiotensin II in hypercholesterolemic rats with genetic tendency [20, 21].

#### *2.1.2.6. MicroRNAs*

MicroRNAs are small and single-stranded RNA molecules which directs genes and complex pathophysiologic events in many diseases; and a few of them have been known to contribute AAA development. It was found that miR-29b which is one of 3 miR-29s (miR-29a, miR-29b and miR-29c) of MicroRNA family is increased in AAA tissues [22]. Another mediator responsible for smooth muscle cell proliferation and apoptosis in aneurysm tissue is miR-21; and its overexpression prevents aneurysm formation whereas its inhibition increases [23].

#### *2.1.2.7. Gender-dependent mediators*

Male gender is an important risk factor AAAs in humans. In pharmacological aneurysm creation models of animals, it was observed that aneurysm expansion is more in males and protection from aneurysm disappeared when female aortas are transplanted to males whereas aneurysm diameter reduces when estradiol is given to male rats [24].

#### **2.2. Hemodynamic effects-**

Hemodynamic forces defines kinetic energy applied on arteries and veins by blood flow. Vascular endothelial and smooth muscle cells are constantly exposed to dynamic effect of blood flow during blood circulation. Three important hemodynamic components play role in AAA pathogenesis.


It is well-known that AAA pathophysiology involves many factors as biological, biochemical and biomechanical processes. Although biochemical and biological factors are well-defined in AAA, role of biomechanical factors in AAA pathology is still poorly understood.

Altered flow types (turbulence etc.) may contribute aneurysm development by injuring arterial endothelium and increasing progression of arterial wall degeneration. Flow oscillation areas and areas with extreme shear stress are correlated with atherosclerosis development in aorta. Flow types in AAA have been demonstrated as smooth and laminar or irregular and turbulent; however, effects of wall shear stress on aneurysm is still poorly known. Geometry of the aneurysm sac and surrounding vasculature (including existence, size and symmetry of branches arising near the aneurysm) as well as position of the aneurysm sac relative to parent vessel affect intraaneurysmal flow [25, 26].

Coarctation increases hemodynamic stress on the aortic wall and alters flow dynamics. In some studies, it was shown that hemodynamic stress facilitates AAA predisposition and flow alterations significantly affect arterial lumen diameter. Also, poststenotic dilatation was detected at the area of oscillatory shear stress distal to the cast in some studies [27].

#### **3. Intact aneurysm models**

Various experimental models have been used for creating abdominal aneurysm. Pharmacological methods, xenograft, large animal models are a few of them. Most commonly used pharmacological methods are methods like intraluminal elastase, periaortic calcium chloride application, systemic angiotensin II infusions. Application of these models in rodents will be explained in detail in a different chapter. It is briefly presented in this part.-

#### **3.1. Elastase model**

Elastase is a member of serine proteases. Its endoluminal infusion alters the normal structure of tunica media by causing elastic fiber destruction. It stimulates receptors which are activated by- protease in the smooth muscle cells on the aortic wall; therefore inhibits Ca2+ inflow required for- vascular contraction. This inhibition of smooth muscle contractions causes aortic dilatation [28].

As the response to acute elastase damage, elastic lamellae become fragmented by the leucocytes invading media layer; then formation of intraluminal thrombus (ILT) starts due to endothelial injury. This process promotes aneurysm growth by causing release of endogenous proteases activated by fibrinolytic system, activation of MMP-2, secretion of urokinase and leucocyte elastase.-

This model can be utilized on animal species like rat, mice, hamster and rabbit (**Figure 1**). It can also be applied on larger species like dogs; however, intra-aortic perfusion, aortic balloon angioplasty and simultaneous collagenase infusion may be performed for avoiding problems caused by high dose of elastase infusion [28, 29].

Waiting period is approximately 7–14 days after the elastase infusion in rodents. An increase over 300% in aortic diameter in first week has been observed in this model. If rupture is not occurred in first week, stabilization is occurred at 2–3weeks due to mesenchymal cells colonized into intraluminal thrombus (ILT) and fibrosis.-

Generally, aneurysm development is completed in this time period, also histopathologic changes- on aortic wall at the level of cellular and inflammatory levels are arised along with mechanic- dilatation occurred in first week. Relaparotomy is performed at the end of these durations, aortic- measurements are performed and aorta tissue is excised for histopathologic examinations [30].

#### **3.2. Calcium chloride model**

Local CaCl<sup>2</sup> is applied on adventitial layer without direct intervention to abdominal aorta in this model which has firstly been designed for developing aneurysm on rabbit carotid artery. Therefore, it is technically easier than intraluminal elastase infusion model. In this model which has especially been used in rats, a CaCl<sup>2</sup> -impregnated gauze is directly applied

**Figure 1.** Intraluminal elastase infusion in rat abdominal aorta.

to infrarenal aorta. Calcium ions have high degree of affinity to elastin. After application, ionized calcium intracellularly turns into calcium phosphate (CaPO<sup>4</sup> ) due to alkaline phosphatase activity from vascular smooth muscle cells (vSMC) and this compound precipitates as hydroxyapatite crystals in elastin fibers, causing their mechanical degradation. In some models, this process is accelerated with applying local phosphate-buffered saline (PBS) after CaCI2 application. It has also been known that human aneurysm wall calcification is caused by these CaPO4 crystals. Aortic dilatation in CaCI<sup>2</sup> model increases with time and it is caused by progressive infiltration of mast cells and T lymphocyte to adventitia layer. Vascular smooth muscle cells disappear due to calcification and fragmentation in elastic fibers and they are replaced by neutrophils [27]. Most important aneurysm formation mechanisms of CaCI<sup>2</sup> application are medial degeneration and leucocyte infiltration. Endoluminal and intramural thrombus are not seen in these type of aneurysms, also rupture chance is very low. Although it is mostly used in mice, it can also be applied on rats and pigs [31].

#### **3.3. Elastase and calcium chloride combined model**

It has been developed by Tanaka. In this model, CaCI<sup>2</sup> application is performed around the aorta while intraluminal elastase infusion through femoral artery is being given. CaCI<sup>2</sup> impregnated gauze is applied around aorta along with 30 U elastase infusion into aorta (**Figure 2**). Total duration is 20 minutes. Elastase infusion duration is decreased from 120 to 20 minutes. In this model, it has been reported that no atherosclerosis and intraluminal thrombus occurs and it can easily be performed [28].

**Figure 2.** Intraluminal elastase infusion and adventitial CaCI<sup>2</sup> application.

#### **3.4. Angiotensin II (AngII) model**

In this model, intravascular infusion and aortic exploration are not performed. It is the most easily performed aneurysm model on mice. It has been firstly described by Daughtery at 2000. It is only used on mice and it is the most frequently used method on this species.-

Angiotensin II is a potent vasoconstrictor octapeptide. It is produced from angiotensin I after the removal of two amino acids at the C-terminal by angiotensin converting enzyme. It maintains blood pressure and body fluid/sodium balance by causing construction of blood vessels.-

It has been shown that continuous infusion of angiotensin II causes vascular remodeling; especially causes atherosclerosis in transgenic or knockout animals; therefore it is an important model for researching AAA development and preventative mechanisms [32, 33].

#### **3.5. Spontaneously mutated and transgenic mice models of AAA**

Genetically determined types are Blotchy, Lox (lysyl oxidase) deficiency, MMP-3 or TIMP-1(tissue inhibitor of matrix metalloproteinase) deficiencies, LDL receptor −/−, ApoE −/−, eNOS −/−,- C57BL/6 and "transgenic mice overexpressing renin and angiotensin". Food supplements, different feeding methods or some drugs increase rupture risk of aneurysm. For example, betaaminoproprionitrile, sweet pea, diethylstilbestrol, monoamine oxidase inhibitors and hydralazine are some of them; and they increase rupture risk by reacting collagen in media layer without causing any hemodynamic effect. On the contrary, propranolol and reserpine decrease rupture- risk by their hemodynamic effect; propranolol also decreases rupture risk by causing connective- tissue with increasing cross-linkage of elastin.

Blotchy Mouse is the mutant with impaired intestinal copper absorption due to X chromosome mutation. It is one of the species with spontaneous aneurysm development. Copper is co-factor of lysyl oxidase (Lox). Lox plays a role in vascular growth and extracellular matrix (ECM) production. Elastin and collagen productions are impaired in Lox-deficient mice, neutrophil infiltration in tunica adventitia occurs and spontaneous aneurysm formation is seen in male rats in 3weeks. Elastic fiber fragmentation and disintegration of smooth muscle cell layers of aortic wall are seen. Although saccular or fusiform aneurysm formations occur through whole aorta in these mice; thoracic aorta is most commonly involved. However; it has not been primarily preferred model for experimental aneurysm studies because it usually results in spontaneous thoracic aorta rupture [34].

Spontaneous aneurysm development on both abdominal and thoracic aortas is seen in MMP-3 and TIMP-1 deficient mice. The importance of MMPs for AAA formation was- further investigated by Eskandari et al. They demonstrated a protective role for tissue inhibitor of metalloproteinase (TIMP)-1 on elastase induced AAA in mice. Compared with- wild type mice, TIMP-1 deficient mice developed larger AAAs after AAA induction with- elastase [35].

Although LDL receptor and ApoE deficient mice are more commonly used in atherosclerosis studies, suprarenal AAAs may occur when they are fed with high-fat diet for 6 months. Adventitia thickening along with media layer injury in these animals prevent rupture. These aneurysms are very similar to atherosclerotic aneurysms due to presence of elastin degradation medial electrolysis, vascular dilatation and necrotic core. Duration of aneurysm development can be shortened with pharmacologic methods like intraluminal elastase, periaortic CaCI<sup>2</sup> application or subcutaneous AngII infusion in these mice.

It has been demonstrated that suprarenal AAA occurred with a rate of 25% in ApoE+ eNOS deficient mice which had been fed with high-fat diet for 4–6months. These aneurysms are characterized with perimedial thrombotic and fibrous material accumulation [31, 33, 34].

Chronic hypertension was created in transgenic mice by cross-mating with human renin or human angiotensin genes, then occurrence of aortic rupture was observed when they were fed with water containing 1% sodium chloride. Aortic aneurysm was predominantly occurred in aortic arch or juxtarenal segments in these mice [36].

Role of MMPs in AAA formation in genetically altered mice have been defined. It was shown that after CaCl<sup>2</sup> -mediated aortic injury was created; aneurysm did not occur in MMP2 −/− and MMP9 −/− knockout mice whereas aortic dilatation was decreased in MMP12 −/− mice when compared with wild-types. Studies indicating regulation of matrix metalloproteinases by TIMPs have been conducted [31, 36].

#### **3.6. Large animal models**

Aneurysm models performed on large animals have been predominantly developed for preclinic research of surgical or endovascular treatment methods. Most important of these methods are Elastase model, xenograft model, graft models (patch, pouch, interposition grafts), coarctation model and balloon dilatation model.

#### *3.6.1. Elastase model*

This model which has been mostly used on rodents has also been used on large animals. Aneurysm occurs due to destruction of medial elastic lamellae. Intraaortic elastase infusion may be applied by reaching aorta through femoral artery without laparotomy, alone or with applying methods like balloon angioplasty, collagenase infusion, CaCI<sup>2</sup> application. Aorta aneurysm may be created in swine and dog aortas with this method by creating intimal hyperplasia, medial elastic fiber rupture and matrix degeneration. It is the most similar method to human aneurysm without tendency towards rupture [37].

Complications like livedo, lower limb paraplegia, neurologic bladder and rectal prolapse may occur in this method. Changes in aortic wall and aneurysmatic dilatation, calcification and blood flow are followed with weekly ultrasonographic examination under sedation or anesthesia. After 2 weeks, it is possible to transformation of dilatation to an aneurysm (>50%). Experiment is terminated at the end of third week; then aortic exploration and necessary examinations are performed. It has been reported that chance of rupture is increased in monitoring period which exceeds 4 weeks.

#### *3.6.2. Xenograft model*

It is also called "decellularized aortic xenograft model". In this model, aorta implantation between- two different species is performed. This model is based on vascular smooth muscle cell suppression- and extracellular matrix immunogenicity between species. In this model, roles of immune system and extracellular matrix proteins in aneurysm development can be researched and pharmacological or immunologic mechanisms preventing aneurysm development can be examined [38].

#### *3.6.2.1. Experimental application*

A 1cm infrarenal aorta segment of Guinea pig (300–350g) is excised after ligating collateral branches with median laparotomy under general anesthesia; then it is decellularized in sodium dodecyl sulphate (SDS 1%, Sigma, St-Louis, USA) at 37°C for 18hours. After this- procedure, it is washed with Triton X-100 solution (Sigma) and process is completed after- washing four times in 24hours with 0.1% phosphate buffered-saline (PBS) solution. Xenograft- prepared with this method is transplanted to Lewis rats in orthotopic position by using 10/0 sutures with microsurgery method [39]. Decellularized aortic xenograft triggers immune reactions without an acute fatal rejection. All cells on the distal part of aorta are removed during donor graft preparation; however, collagen and elastin network of extracellular matrix is preserved. Degraded guinea pig extracellular matrix becomes infiltrated by intimal monocytes- and T-lymphocytes, luminal thrombus along with aortic dilatation starts. AAA occurs due to reaction between species in extracellular matrix after 14 days, xenograft destruction may result with aortic rupture. Doubling time of aortic diameter is short as 10 days in this model [31, 40].

#### *3.6.3. Graft models*

In these models, aneurysm is created by performing biologic or prosthetic graft interposition to abdominal aorta. Tubular graft or patch application may be performed. Most frequently used biologic grafts are peritoneum, bovine pericardium, fascia of rectus muscle, jejunum whereas prosthetic materials are dacron or polytetrafluoroethylene (PTFE) grafts. Most common graft application methods are patch model, pouch (saccular aneurysm model), graft interposition and coarctation models.

#### *3.6.3.1. Patch graft model*

In this experimental model developed on large animals like swine and dog, developing new endovascular devices for AAA repair by creating aneurysm similar to human anatomy became possible. It is an easily applicable method. Most commonly used one of this method is the an elliptic patch application. Patch materials used in this method which is also named as anterior patch model are materials such as prosthetic grafts, venous grafts (iliac vein or jugular vein), rectus fascia, jejunum treated with glutaraldehyde and gastric serosa.

In this procedure, abdominal aorta is explored between renal artery and iliac bifurcation by performing median laparotomy under general anesthesia. One each silicon loops are placed on renal arteries and above of aortic bifurcation. Aortic segment is occluded with silicon loops after systemic heparin injection (200 U/kg). Inferior mesenteric artery and lumbar arteries are temporary closed with mini hemoclips. If juxtarenal aneurysm is created, renal arteries are also temporary clamped; then segments of 2–3 mm from edge of incision are longitudinally excised by performing 5–10 cm aortotomy. Patch graft is sutured to aortotomy incision with 5/0 prolene suture by using continuous technique. Aorta clamps are opened with well-known air removal- techniques and circulation is restored. Incisions are closed. Aneurysm formation is generally- seen in first 1months. Rupture rate varies according to graft types and length of aneurysmatic- segment. It has been reported that segments whose length is more than 6 cm have a rupture rate of 70%. Lowest rupture rate has been seen in iliac vein patches (0%) whereas highest rupture rates have been seen in jejunum patches (100% in 42 hours), jejunum patches treated with glutaraldehyde (66% at 11days) and peritoneal patches (50%, 2weeks). For preparing peritoneal patches, peritoneal part is isolated and resected with blunt dissection and it is shaped as an ovoid-shaped patch whose length is 5–10 cm and width is 2–3 cm. A double-layered peritoneal patch is created after folding the free end on itself. It is kept in saline solution for 30 minutes before use, and it is anastomosed with continuous suture like other grafts [41].

#### *3.6.3.2. Saccular aneurysm model-*

It is an another aneurysm model which has been firstly used by Perini. Biologic or prosthetic- material used in this model is cut into a material sized 3X6cm and its both sides are sutured after- folding it on itself. It becomes a sac sized approximately 3X3 cm; then opening of the sac is anastomosed to an aortotomy sized 3 cm which is created on the anterior part of aorta. Result is a saccular aneurysm. Bovine pericardium is most frequently used material. Venous graft materials or- prosthetic materials may also be used; however, largest dilatation is acquired with biomaterials.-

Swines whose weight are approximately 20 kg are used for applying this model. Similar to anterior patch model which uses median laparotomy, aorta between renal arteries and bifurcation is explored. A 3cm segment is chosen for aneurysm. After administrating IV heparin (with- a dose of 100 UI/kg), proximal and distal aorta are clamped and 3 cm longitudinal aortotomy is

performed on the chosen area; then previously prepared saccular bovine pericardium is anastomosed to this area with continuous technique using 6/0 polypropylene. Result is a saccular- aneurysm. Retroperitoneum and abdomen are appropriately closed. Stabilization is provided a few weeks after surgery, mortality and morbidity of this procedure are low. Continuity of- aneurysm is followed with Doppler ultrasonography with intervals of 15 days, aorta diameter increases by more than 50% and it is most frequently used for evaluating endovascular methods.- Terminal branches of aorta and lumbar plexus are preserved, partial thrombus formation is seen in lumen. Endovascular graft applications may easily be performed on aneurysm formation created with this model, it is a good model for endoleak researches due to patency of side branches. Most important disadvantages are lack of characteristic features of human aneurysm like atherosclerosis, medial degeneration, medial or adventitial lymphocyte infiltration. Complications- like renal failure, intestinal perforation, sepsis, iliac artery thrombus may be seen in this model. Bovine pericardium is cheaper than synthetic materials because it can be acquired easily [42].

Most frequently used animal in this model due to anatomic and hematologic (coagulation and fibrinolytic system) similarity to humans is swine. In addition, it is an easily manipulable model. Lipid metabolism, lipoprotein profile, thrombocyte aggregation/thrombus formation and fibrin deposits after intimal injury, histologic structure of neointimal are also very similar to human. Disadvantages are rapid growth of animal, its low tolerance to anesthesia, high cost and possible paralysis due to medullary ischemia. Pericardium used in this model is treated with glutaraldehyde for reducing antigenicity and increasing resistance to degeneration. Monitoring is performed with Doppler ultrasonography in this model, other imaging modalities like angiography is not required.-

#### *3.6.3.3. Interposition grafts*

In this model, various types of grafts are interposed to infrarenal area of aorta. Graft whose diameter is twofold of abdominal aorta is replaced to aorta after spinal artery are ligated. Pigs are generally used and endovascular approaches can be used after two weeks. In this model, biologic materials (bovine jugular vein treated with glutaraldehyde), fusiform-shaped dacron grafts or PTFE grafts dilated with balloon are used. For creating aneurysm, a 8mm PTFE graft is dilated with balloon until its final diameter reaches 30mm. Graft which becomes fusiform-shaped is anastomosedas it is placed between renal arteries and trifurcation. Caudal paraplegia may occur due to ligated spinal arteries. Two lumbar arteries are re-implanted through posterior of aorta with Carrel patch technique in endoleak researches. Endovascular repair can be done 2 weeks after surgery. Type II endoleak researches can be done after placing intraluminal pressure transducer into sac during surgery [43].

#### *3.6.4. Stenosing cuff-*

Aneurysm development can be maintained due hemodynamic effect by creating stenosis at the infrarenal area of aorta. Stenosis below renal arteries can be created by nylon tape or plastic cuff whose width are generally 5mm. Dilatation of aortic wall and aneurysm formation are seen due to turbulent flow after stenosis. This model is generally used together with intraluminal elastase infusion and balloon angioplasty.

 After performing median laparotomy, aortic exploration and entrance right above aortic trifurcation; balloon plasty and elastase infusion are performed. Amount of administered elastase when a pig weighting approximately 30kg is 10ml; and stenosing cuff is placed below renal arteries by performing balloon dilatation after infusion. Presence of palpable thrill on aorta is the indicator of adequate stenosis. Parameters like "pulsatility index" which provides quantitative measurement of degree of stenosis may also be used [44].

Increase in aortic diameter is expected over 50%. Most important advantage of this model is preservation of lumbar arteries. Disadvantages are requirement of laparotomy, occurrence of retroperitoneal fibrosis and aneurysm extension limited at proximal [2]. Turbulent flow in this model provides appropriate hemodynamic effect for damaging intercellular matrix after protective barriers like tunica intima and lamina elastic interna are weakened by elastase and effect of balloon; rather than creating aneurysm alone [45].

#### *3.6.5. Balloon dilatation*

Balloon dilatation alone cannot produce enough aneurysmatic dilatation in large animal models. Therefore, it is always used with elastase or collagenase infusion or sometimes both of them. Infrarenal stenosing cuff is also occasionally used with them. High pressure balloons with width of 10–12 cm and length of 4 cm are used. Angioplasty balloons produced for peripheral arteries may be used for this purpose. Applications can be performed with or without stenting. It is percutaneously performed and whole side branches of aorta are preserved. It results with moderate degree of dilatation [2].

#### **4. Ruptured abdominal aortic aneurysm (RAAA) model**

Clinical condition occurred in aneurysm rupture is modeled in this experimental model without- creating a real aneurysm. In this model which has been firstly described by Thomas Lindsay at- 1995, first aneurysm rupture by creating shock, ischemic stage of surgical treatment by placing- aortic clamp then revascularization and reperfusion processes after removing clamp are modeled. Lindsay identified factors like degree of pulmonary injury, ideal clamping area and duration by measuring "lung permeability index" and "neutrophil sequestration" levels; he reported- that highest damage had been observed on rats with created lower torso ischemia due to 1 hour of shock + supramesenteric clamp. This model has also been used by various investigators [46, 47].

In experimental RAAA model, hemorrhagic shock studies evaluating hemodynamic effects of all kinds of drugs, molecules or resuscitation fluids as well as ischemia/reperfusion researches evaluating their effects on remote or end organ injuries can be conducted [48].

Most important features which differ RAAA model from other aortic ischemia/reperfusion (I/R) studies or hypovolemic shock studies are initial shock creation and placement of aortic clamps on both supramesenteric level and aortic bifurcation level. Total body hypoperfusion due to initial hypovolemic shock, lower torso ischemia with aortic clamp and then reperfusion are done by creating both shock and I/R [49–51].

Therefore an effect stronger than both of them alone is acquired. Besides, most important cause of high mortality in RAAA is comorbidity of these two important pathology [52].

Application: Most commonly used animals are rats; however, large animals may also be used. Right carotid artery for measuring mean arterial pressure (MAP) and jugular vein for venous access are cannulated with cut-down method in anesthetized rats with spontaneous respiration (No 22 cannula) (**Figure 3**).

Heart rate, MAP, rectal temperature and respiratory rate are monitored. Saline infusion with a rate of 3 ml/kg/h is given during whole experiment period for preventing insensible losses. Rectal temperature is kept at 36.5°C by using heat lamp. After stabilization is acquired, shock is created as MAP is set to 50 mmHg for 60 minutes by drawing blood in plastic injector containing standard heparin; aneurysm rupture is simulated and withdrawn blood is kept in room temperature (**Figure 4**).

Blood which will be drawn is calculated as not exceeding 30% of total blood volume. Lower torso ischemia is created at the end of 1 hour by clamping abdominal aorta with microvascular clamps on superior mesenteric level and iliac bifurcation level after performing median laparotomy and systemic heparinization (250 U/kg). Half of the withdrawn blood is slowly reinfused through venous line; therefore surgical x-clamp and resuscitation are simulated (**Figure 5**).

**Figure 3.** Cannulations of jugular vein and carotid artery and monitorization.-

**Figure 4.** Drawing blood from carotid artery to create shock.

At the end of ischemic period which is 60 minutes long, all of remaining withdrawn blood is re-infused right before opening clamp and the subject is left to reperfusion for 120 minutes after removing clamps and closing abdomen. During reperfusion period, MAP is kept at approximately 100mmHg and fluid replacement is performed if necessary. Hemodynamic values are recorded in every 10 minutes (**Figure 6**). All given fluids are recorded and most commonly used fluid is Ringer lactate. At the end of the period, rats are sacrificed by drawing blood method; then necessary blood and tissue samples are collected.

Experiment can be modified with different ways.-

**Figure 5.** Aortic clamps on superior mesenteric and iliac bifurcation levels in rat aorta.

**Figure 6.** Mean arterial blood pressure during the experiment.

#### **5. Conclusion**

Until today, many animal experimental models have been developed for investigating development mechanisms, factor affecting expansion and treatment methods of AAA which has been- a very common disease with high mortality in community. It is obvious that as the technology advances, larger number of studies which are more sophisticated will be needed for both better- understanding etiopathogenesis and developing less invasive methods for treatment.

#### **Conflict of interest-**

Author declares that there is no conflict of interests regarding the publication of this paper.-

#### **Author details**

#### Zerrin Pulathan

Address all correspondence to: zerrin.pulathan@gmail.com

Faculty of Medicine, Karadeniz Technical University, Trabzon, Turkey-

#### **References**


## **Experiment and Animal Models of AAA**

#### Karel Houdek

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78988

#### Abstract

Introduction: The incidence of abdominal aortic aneurysms has been increasing throughout the world. The etiology and pathophysiology of this disease are very complicated and complex and include biomechanical aspects as well as biological processes. The effect of these mechanisms is the degradation of the aortic wall, which leads to its dilation and rupture. The possibilities for studying such complex pathophysiology in humans are very limited. That is why we use various mathematical models and a number of different animal models of aneurysm. Methods: A summary of the basic characteristics, findings and examples of using the most widely used animal models of abdominal aortic aneurysm. Information has been obtained from our own experience with laboratory animals and from studies published and available on the Pubmed Internet database. The following search terms were used: aneurysm, aorta, animal model and experiment. Conclusion: Animal models of aortic aneurysms are a usable and useful tool in the study of AAA etiopathogenesis. They also serve as a means to find novel therapeutic pathways. Each model, like any animal species, is different and has its own limitations, advantages and disadvantages, which we should always consider during their use and while interpreting the results.

Keywords: experiment, aneurysm, aorta, animal, model

#### 1. Introduction

#### 1.1. Introduction

Infrarenal aortic aneurysm is a disease, which puts patients at risk primarily due to its long, asymptomatic course, often resulting in abrupt pain caused by rupture as the first sign of the disease [1]. Aneurysmal rupture often has a fatal outcome. Infrarenal aortic aneurysm is not a single group of diseases. The etiology is different in patients with congenital connective tissue

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

disorders in Marfan syndrome and Ehlers-Danlos syndrome [2], different in infectious aneurysms with bacterial agents clearly confirmed by culture [3] and different in aneurysms classified as degenerative [4], which represent the most common ones. These are diseases with etiology that has not been completely elucidated. The pathophysiology of aneurysmal development is a very complex process with complicated interrelated and interconnected physical and biological mechanisms that lead to the degradation of the molecular and cellular structure of the vessel wall [4]. It is exactly this complicated and not yet fully elucidated etiopathogenesis that makes aneurysms the subject of continued interest across scientific disciplines. One of the options to study the individual processes at different levels are animal models and experimental animal research. Animal models, unlike aneurysm samples obtained from surgery or autopsy, are used to study the individual mechanisms from the early stages of aneurysmal development. Studies in humans are conducted to examine the changes at an advanced stage.

Experimental work with animals requires strict adherence to the rules, careful planning and a lot of effort. The advantage is the possibility to see the individual processes and mechanisms in the context of the whole body, including all interactions.

#### 1.2. Materials and methods

This chapter gives a summary of basic characteristics of most commonly used animal models of aortic aneurysms. By giving few examples of each model, it also points out the advantages and disadvantages and their practical application. The authors gain the information from published studies that are available on the Pubmed Internet database. For searching in the database, the words experiment, aneurysm, aorta, animal and model were used. Only those papers were read and accepted, if the full text was written in English. Another source of knowledge presented in this chapter is a long-time experience and practice with laboratory animals of different species in various models and studies on the authors' place of work. Due to the nature of this chapter and many variables, no statistical analysis is presented.

### 2. Experimental work and models of AAA

#### 2.1. General conditions for working with laboratory animals

The current issues of experimental work with animals are subject to European and global conventions on the protection of animal rights, which may be further regulated and specified by national legislations. Several fundamental rules apply in this field. In general, there have been attempts to reduce the total number of animals used for experimental purposes. The interests of researchers may be in conflict with those of animal rights defenders and a reasonable compromise should be sought. The conditions in which the animals are kept, how they are treated during transportation and throughout the experiments, including the killing and subsequent handling of the remains, have been constantly improved. The basic principles and rules of working with experimental animals, which are valid still today, were defined by William Russell and Rex Burch in the mid-twentieth century in the book "The Principles of Human Experimental Technique" [5]. They can be summarized in three points or rules known as "3R"—Replacement, Reduction, Refinement [6].

Replacement: an effort to find other, alternative methods of conducting research without the use of laboratory animals. When considering the initiation of research, we should first ask and answer the question of whether it is possible to obtain the result without using laboratory animals. The current level of knowledge allows the use of a variety of mathematical or computer models. Cell or tissue cultures alone can often be used to verify hypotheses. If this is not possible, we should always try to use animals from lower evolutionary groups. If work with animals is a part of teaching programs, it can often be replaced by video recordings.

Reduction: an effort to reduce the total number of laboratory animals used. This rule is closely related to the previous one. The already mentioned use of nonanimal models and cell and tissue cultures should include the careful planning of experimental work so that we do not duplicate experiments that have already been carried out unnecessarily or do not verify hypotheses that have already been adequately verified. The total number of laboratory animals used can be reduced by appropriate selection of the animal species, choice of appropriate sex and age. Careful consultation with the statistician (appropriately chosen model, number of animals in each group, length and ways of monitoring) should be an integral part of the planning.

Refinement: includes measures to improve the living conditions and the environment of laboratory animals. Working with laboratory animals requires the possession of authorizations that can be obtained based on professional education and experience. The Federation of European laboratories animal science associations (FELASA) determines four categories of authorization (A-D) according to the level of education and length of practice. Correct or wrong animal handling can significantly affect the results of the experiment. Any handling of animals, including transportation and environmental changes, is stressful for animals. In addition to stress, transportation also poses the risk of the transmission of infections not only to the animal but also to the transporter, and it is therefore necessary to choose suitable transport boxes (air conditioning, protection). Acclimatization to the new environment is always necessary between transportation and the beginning of the experiment. The acclimatization time varies according to the type of animal chosen and also serves to normalize changes caused by stress during transportation (weight loss, change in heart rate). The environment in which the animal is kept (box size, number of animals in the box, temperature, humidity, observation of circadian rhythm, appropriate feeding) and how the animal is treated is very important. Smaller laboratory animals are less expensive, and handling them is not so physically demanding and does not require much space. On the other hand, greater size of the animals, such as a rabbit or a pig, may be an advantage when handling organs and tissues. Any painful handling, investigations, and procedures should be performed under anesthesia, and suitable analgesia should be provided, including in the postoperative period. The method of anesthesia should be selected according to the type of animal chosen and plays an important role in the successful completion of the experiment and achievement of the necessary results.

The anesthesiologist should be sufficiently experienced and knowledgeable about the specific differences of the chosen animal species.

Strict adherence to the established rules and standardized conditions is an inherent part of any experimental work so that the results of the work are reproducible, repeatable and the statistical analysis is valid.

At present, multiple animal species are used in animal experiments. The same is true for experimental works related to aneurysms. Wild-type (WT) animals, whose genome is not modified, can be used for each animal species. Interindividual differences, for example, in enzymatic activity are a certain disadvantage when studying such populations [7]. This is one of the reasons why genetically modified strains of animals are often used in studies in which a population of similar or virtually identical animals is being studied [7]. Another advantage of using modified strains is a specific modification that allows for the monitoring of, for example, the involvement of a particular enzyme and its activity in the studied process. Especially in mice, a large variety of different genetically modified strains are available. With a properly chosen animal, we are able to model very specific situations. A properly chosen animal type and methodology can significantly influence the results of the work in both positive and negative terms, as documented below in the text. When choosing an animal model, it is necessary to answer the question of whether it will be possible to compare the model with the real situation in human medicine and to what extent the conditions studied will be similar (enzymatic equipment etc.) or different from the reality.

#### 2.2. Animal models of AAA

Animal aneurysm models help clarify the complex etiopathogenesis, can be used to develop new treatment methods or to improve endovascular and surgical procedures. The first animal aneurysm models were published in the 1960s, and many other methods and models have been developed since then and have been variously upgraded and improved [8–11]. In principle, the methods of inducing an aneurysm in animals can be divided into those using different chemicals and those using physical laws and their various combinations. Papers that are presenting research with different models of aneurysm and different animal species are summarized in Table 1.

#### 2.2.1. Elastase model

Perhaps the most important changes that can be observed in the aneurysm wall in humans are degeneration of extracellular matrix—degradation of elastin in the presence of matrix metalloproteinases 1, 2, 3 and 9 (MMPs) and the inflammatory infiltration. The first attempts to develop an experimental aneurysm used proteolytic enzymes to cause the degradation of elastin fibers. Wills et al. [8] used porcine aortic tissue to demonstrate the effect of exogenous elastase in the development of degenerative changes of the extracellular matrix. He confirmed the results and observations attained by Anidjar et al. [21]. Anidjar repeatedly demonstrated the possibility of establishing an aortic aneurysm model in rats by applying porcine pancreatic elastase (PPE). Anidjar's model represents the basis for a various PPE model modifications. In


Table 1. Animals and models of experimental aneurysm used in presented studies.

this model, a segment of infrarenal aorta is perfused with a PPE solution through a directly inserted tube or needle. The authors and models can differ in the concentration of PPE, method of perfusion (pump, single or repeated applications, or application with increased pressure), duration of perfusion and the laboratory animal [24–26, 28, 52]. Anidjar perfused a 1 cm long segment of aorta of rat with a porcine elastase solution. Other proteases (papain, trypsin, and collagenase) can lead to the development of an aneurysm as well. Carsten et al. [23] studied several batches of elastase and confirmed the need for inflammatory infiltration with activated macrophages to achieve the necessary extracellular matrix degradation and aneurysmal development in rats. PPE model was widely used to study the pathophysiology and possible treatment options of AAA. For this purpose, genetically modified mice were used [12] and many anti-inflammatory acting drugs and agents were studied (TIMPs, doxycycline, indomethacin) [13–16, 21]. Periadventitial application of elastase in mice may cause similar changes and lead to development of AAA as well [17]. Nie et al. [30] induced an aortic aneurysm using PPE in the New Zealand White Rabbit within 14 day. Despite mild differences in the method of perfusion, similar conclusions were made by Bi et al. [31] and Kobayashi et al. [32]. Both used higher pressure for the perfusion. In elastase-induced models small animals are commonly used. In large animals, such as different species of pigs or in dogs, the results are not so unambiguous. Marinov et al. [29] observed elastin fiber destruction, inflammatory infiltration, a change in wall thickness and changes in smooth muscle cells, and even calcium depositions after aortic perfusion with PPE in Yucatan miniature swine, but he did not observe the development of aneurysmal dilation after 3 weeks. Strindberg et al. [27] wanted to use the elastase-induced aneurysm model in a dog for control and development of stent grafts. He compared the changes while using different elastase concentrations, different perfusion times, and the combined use of elastase with collagenase and/or an inflated intraluminal balloon catheter. By extending the perfusion time to 2 h and using elastase alone or in combination with collagenase, aortic dilation of 65.6 ˜ 20.8% was present, which was not enough for his need. Degradation of elastin fibers, a reduced number of smooth muscle cells and an intimal thickening were present during the examination of the aorta samples. Many modifications of the elastase-induced aneurysm model employ differently genetically modified and specified animal clones [18–20].

#### 2.2.2. Calcium chloride model (CaCl2)

Inflammatory infiltration is another significant contributor to the development of aneurysms. This reaction can also be induced by an external insult to the adventitia. The use of calcium chloride to induce an aneurysm was first described in the carotid arteries of rabbits [9] more or less as a secondary observation. However, the histological structure of these aneurysms somewhat differs from findings in human aneurysms. In both cases, we can see changes in the media with wall thickening and inflammatory reaction, but in carotid artery aneurysms in rabbits, the wall thickening is more pronounced with marked intimal hyperplasia and marked calcification of elastin fibers in the media. For the abdominal aorta, this methodology and experience was described by Freestone et al. [33]. He studied the effects of different concentrations of calcium chloride and sodium chloride solutions applied to the surface of the infrarenal aorta for 15 min. He also examined the possibility of influencing the effect of calcium chloride by added sodium thioglycolate and a high-cholesterol diet. Histological changes (intimal hyperplasia, media injury, calcification of the media) increased with the increasing calcium chloride concentrations. The leading symptom was infiltration of the media and adventitia by macrophages and increased activity of MMP2 and 9. Aneurysms developed at a concentration of 0.25 mol/L. High cholesterol and/or thioglycolate levels did not significantly affect the development of aneurysms. The effect of sodium chloride has not been demonstrated as well. Chiou et al. [11] provided a similar comparable study but he used mice as a laboratory animal. Calcification of the vascular wall is a common denominator of a number of vascular diseases. We can find calcification in the aortic and aneurysm walls. Basalyga et al. [35] used the application of calcium salts at various concentrations in unmodified and genetically engineered mice to verify the association of elastin degradation caused by the action of MMP and the resulting calcification. Watanabe et al. [34] used genetically engineered mouse clones with calcium chloride-induced aneurysm for studying the role of phospholipase A2 (PLA2) and inflammation in the pathogenesis of AAA. Other study confirmed a protective effect of PLA2 inhibitor. Using the calcium chloride-induced aneurysm model in mice, Gacchina et al. [36] referred the role of vascular smooth muscle cells (VSMC) for the AAA growth.

#### 2.2.3. Angiotensin II model

Like previous aneurysm models, another model that uses the effect of angiotensin II has several common characteristics with human aneurysms. It is an association with hyperlipidemia, wall remodeling, inflammation and thrombosis and also a higher incidence in males [53]. The model is more animal-specific and uses apolipoprotein E deficient mice (ApoE ˜/˜). Daugherty et al. [54] examined the effect of Angiotensin II on the development of atherosclerosis in relation to hyperlipidemia. He administered angiotensin II to ApoE ˜/˜ clones of mice for 1 month using a minipump. In addition to the development of atherosclerotic changes, both by the action of higher blood pressure and independent of elevated blood pressure on the basis of activation of the monocyte-macrophage system and oxidative stress, Daugherty observed development of aneurysm as a secondary effect. This phenomenon was not dependent on blood pressure or lipid levels or their distribution in the blood. The mice thus treated were found to have a number of macrophages and lymphocytes, that is, inflammatory infiltration in the external elastic lamina and adventitial hypertrophy. In contrast to human aneurysm, the effects of angiotensin II result in dilation and development of aneurysm in the suprarenal segment [10]. This is explained by a higher proportion of fat cells in the adventitia region in the suprarenal segment of the aorta. Dissection and rupture have been reported to occur more frequently [38, 39]. In animals, rupture of the media occurs with thrombus formation and further stimulation and activation of macrophages with elastin disintegration and matrix remodeling. The described changes and the rate of aortic dilation are not the same in identical animals even under the same experimental conditions [10, 37]. Based on these differences, four subtypes of angiotensin II-induced aneurysm models can be distinguished. This heterogeneity can also be observed when comparing samples from different levels of aneurysm in one animal [40]. In this model, further growth of the aneurysm occurs for several weeks after the last angiotensin infusion [39]. Another animal that was used as an angiotensin-induced aneurysm model was the zebrafish. This is primarily due to similar vasculogenesis with humans [42]. This model was used primarily to investigate the effects of smoking tobacco.

#### 2.2.4. Combined and other newer models

Very often, experimental aneurysm models combining the effects of calcium chloride and pancreatic porcine elastase are used. These models are often associated with rats. As an example, we can mention the Tanaka group [47], who achieved aneurysmatic dilation in almost 93% of animals by using the combined approach, but only in 25% and 0% of animals when using PPE alone or CaCl2 alone, respectively. Even histological changes copied this trend: less elastin, more pronounced infiltration by inflammatory cells, and higher activity of cytokines and MMPs 2 and 9 were recorded in the group combining the effects of PPE and calcium chloride. Morimoto et al. [48] used this combined model in rats to study the effects of free oxygen radicals. Molacek et al. [49] compared different AAA animal models in pigs. He compared the PPE model, stenosing cuff model, Dacron patch model and their combinations. He observed best results in combination of PPE model with hemodynamical changes caused by a stenosing cuff placed around the subrenal aorta (p < 0.0156) and the same group used this knowledge to influence the growth of experimentally created aneurysm in rats and pigs with atorvastatin [50]. They observed no thrombus, lipid deposition, media necrosis, intramural hematoma, dissection, or rupture in this combined model. Figures 1–3 show the combination of placed stenosing cuff and PPE infusion and the aortic dilatation after 4 weeks in pig. Figures 4 and 5 are images from ultrasound, showing dilatation of porcine infrarenal aorta after 2 weeks.

Figure 1. Porcine infrarenal aorta with stenosing cuff day 0. Black arrow—stenosing cuff; yellow arrow—infrarenal aorta; blue arrow—aortic bifurcation; red arrow—inferior caval vein.

Figure 2. Infusion of clamped infrarenal aorta with porcine pancreatic elastase day 0.

Figure 3. Dilatation of porcine infrarenal aorta. Combined model—stenosing cuff + PPE. Day 28. Black arrow—stenosing cuff; yellow arrow—dilated infrarenal aorta; blue arrow—aortic bifurcation; red arrow—inferior caval vein.

Figure 4. Ultrasound image of dilated porcine infrarenal aorta. Translumbal approach. Transverse view. Day 14. Combined model—stenosing cuff + PPE. Yellow arrow—dilated infrarenal aorta; red arrow—inferior caval vein.

Another models that combine the use of PPE, CaCl2 or Angiotensin II in mice, rats, rabbits or pigs were used to explain the effects of various statins and other drugs [41, 55–60].

The possibilities of using stem cells to influence the growth and rupture of aneurysms have been increasingly studied in recent years. This topic is studied by many authors and no consensus has been achieved as to the optimal experimental model or laboratory animal. Mesenchymal stem cells (MSCs) have been used in studies to treat a number of cardiovascular diseases, such as critical limb ischemia, cerebral ischemia or myocardial infarction. It is believed that mesenchymal stem cells (MSCs) could help to inhibit degenerative changes in the AAA wall and promote its regeneration. Turnbull et al. [51] attempted to demonstrate the uptake and the presence of stem cells in the aortic wall after insult. She used an experimental pig model, where she combined physical (balloon dilation) and chemical (the effect of PPE and collagenase) methods, and administered stem cells to the pigs. Her methods have led to the

Figure 5. Ultrasound image of dilated porcine infrarenal aorta. Translumbal approach. Longitudinal view. Day 14. Combined model—stenosing cuff + PPE. Black arrow—stenosing cuff; yellow arrow—dilated infrarenal aorta; blue arrow—aortic bifurcation; red arrow—inferior caval vein.

development of aneurysms with characteristics close to human ones, such as expression of MMP2 and 9. By proving the presence of stem cells in the affected aortic wall, she verified her hypothesis and provided the basis for further research. Regeneration of the damaged aortic wall largely depends on the capabilities and presence of VSMC. Schneider et al. [46] was able to improve the regeneration of the aortic wall and thereby influence the progression of aortic dilation in the negative sense using mesenchymal stem cells with a wide differentiation capacity. The effect of MSC was greater than that of VSMC alone. In his work, he induced aneurysms in rats by implanting an aortic graft from guinea pigs. Before the implantation, the xenografts were perfused with a solution containing VSMC or MSC or with a cell-free solution in a control group. The development of aneurysms occurred 14 days after. Grafts colonized by MSC showed significantly less dilation after 1 and 4 weeks compared to those colonized by VSMC and to the controls, where further dilation occurred (p = 0.006). The presence of MSC led to a reduction in inflammatory cell infiltration, a decrease in activity of MMPs, increased TIMP-1 activity, and triggered regeneration of the damaged aortic wall.

#### 3. Discussion

Experimental studies have an irreplaceable role in a research of etiopathogenesis and possible treatment options of AAA. Experimental works with animals and aneurysm models, in contrast to human aneurysms, allow us to monitor the development of aneurysms over time and take samples for analysis at any time during the development. Exploitation of experimental animal models provides, beyond the research of etiopathogenesis, a wide range of possibilities for studying therapeutic interventions, influencing growth or preventing aneurysmal development and rupture. Pharmacotherapy used in experimental models is strongly influencing the initial changes and triggers, and in some models, even a pretreatment is used. To better understand the etiopathogenesis of infrarenal aortic aneurysm, especially how to prevent the growth and rupture, comprehensive studies are needed. Triggers and initial steps leading to the development of aneurysms in animals under experimental conditions are known. Studies with animal AAA models have promising results, but if they are repeated in humans, the results are inferior. The models are representing "acute" aneurysms. Aneurysms in humans are growing slowly usually with degenerative changes. Degenerative aneurysms usually develop in humans over many years. For animal models, this time is significantly shorter, ranging from days to weeks. There are differences not only between animal AAA model and AAA in human, but also various changes in the results, if a different animal species or different AAA model is used. Table 2 summarizes the advantages and disadvantages of each model. Not all animal aneurysm models are capable of achieving sustained growth and dilation, and ruptures of already existing aneurysms cannot be observed in all models. Specifically, no ruptures were observed in models with calcium chloride alone. The presence of thrombus in the aneurysm is common in the human aorta, but thrombus formation does not occur in most animal models. Common for majority of animal AAA models is the degradation of extracellular matrix and elastin fibers, increased MMP activity and inflammatory infiltration of aortic wall.

The angiotensin II model is, to a certain extent, very specific not only due to the choice of animal (apolipoprotein deficient mouse clone), but in contrast to other models, the aortic dilation occurs predilectively, in the suprarenal region, and more than other models encounters dissection and rupture, and the development of dilation may be less predictable.

The use of PPE alone to induce aneurysm model is effective in small animals (mouse, rat), can be used in large animals (rabbit, pig, dog) as well, but in large animals, this model is less effective. With respect to the proven and dominant changes in the wall of such aneurysms (inflammatory infiltration, degradation of elastin fibers, increased MMP activity), which are more or less consistent with the changes that can be observed in human aneurysms, such model can be considered to be appropriate. It has been used extensively to study possible


Table 2. Advantages and disadvantages of different models of experimental aneurysm.

prophylactic and therapeutic methods and to explore the individual pathogenetic mechanisms of aneurysmal development. PPE can also be used to study isolated aortic tissue.

Small animal—mice is commonly used for the calcium chloride-induced aneurysm model as well. Changes and characteristics are comparable to human. It is most often used in the infrarenal region; the aneurysmatic wall contains calcifications with inflammatory cellular infiltration. Oxidative stress, degradation of elastin fibers and changes in SMC play role in this model. In addition, the mechanisms involved in the induction of aneurysms in this model appear to be involved in the pathogenesis of aneurysm in humans, for example, sPLA2 and plasminogen. Unlike human aneurysms, no rupture, intraluminal thrombus or atherosclerotic changes other than calcification have been observed in this model. Studies have confirmed that this model can be used in both WT animals as well as in genetically modified animals. This aneurysm model is perhaps more often used in combination with other techniques of aneurysm modeling in different animal species.

Most of the models described herein were used in more than one animal species. The advantage of larger laboratory animals, such as pigs or rabbits, is their size and hence the size of the aorta, which improves tissue handling. On the other hand, the size itself may also be a disadvantage in terms of spatial capacity and handling of the animal itself. The pig has an anatomy and physiology generally similar to humans, which is undoubtedly important for interpreting the results and possible use in human medicine. If we select a mouse as a laboratory animal, we have the option of choosing wild species or a variety of genetically modified strains. Lower financial burden is certainly a great advantage of small laboratory animals. In any case, adequate methods of application and administration of pharmaceutical doses should be observed for the selected laboratory animal and aneurysm model. We have mentioned contrast between animal models and the real human aneurysm.

Examples were included for all the abovementioned animal models of AAA, where the possibilities of positive pharmacological effects on aneurysm growth and potential rupture were studied. The effects of drugs should be first verified in laboratory animals or in tissue culture and afterwards in a clinical trial.

#### 4. Conclusion

Animal models of AAA are still essential in searching for novel treatment options. Successful aneurysm induction depends on the choice of the right laboratory animal in each method. In general, small laboratory animals are preferred in experimental studies. Small animals are cheaper, handling with them is easier and they require less space. This enables to design trials with more individuals. There are different genetically modified mouse clone available on the market and that makes mouse a widely used laboratory animal. Regarding current experiences, no universal animal AAA model can be recommended. The aim of the study, advantages and disadvantages of each model should be taken into consideration when preparing the design of a new study. The most commonly observed features of various animal models and human aneurysms are the presence of cellular inflammatory infiltration in the aortic wall, degradation of the elastin fiber network, increased activity of MMP2 and 9, and a lower number of smooth muscle cells, but many differences and contrasts are observed as well. Because of these contrasts, each observation and result of animal study have to be confirmed in clinical study before they can be implanted into daily medical practice. Unfortunately, ideal model similar to human's AAA remains undeveloped.

#### Acknowledgements

Financial support: AZV Grant No. 15-32727, Czech Republic.

Charles University Research Fund (Progress Q39), Czech Republic.

#### Conflict of interest

None of the authors are aware of facts that will represent conflict of interest.

#### Acronyms and abbreviations


#### Author details

Karel Houdek

Address all correspondence to: houdekkarel7@gmail.com

University Hospital and Faculty of Medicine in Pilsen, Charles University, Pilsen, Czech Republic

#### References


**Preoperative Planning and Dilemas** 

**Chapter 4**

**Provisional chapter**

**Planning and Sizing with OsiriX/Horos**

**Planning and Sizing with OsiriX/Horos**

DOI: 10.5772/intechopen.78018

It is known that endovascular aneurysm repair (EVAR) requires a precise deployment of the graft and so the anatomical and morphological characteristic study of the aorta and its branches is mandatory. The increase of endovascular surgeons' interest on tomography image edition through software is marked specially when the increasing frequency of these procedures and its complexity have impelled surgeons to face additional and successive risk to occupational radiation exposure. Thus, a meticulous study of the angio-CT during EVAR preparation allows the reduction of unnecessary radiation exposure, as it also reduces consecutive image acquisition and contrast use (that may be related to renal overload in susceptible patients). Although some studies propose effective strategies to optimize the procedure, they rely on the use of additional specific and advanced equipment, available only in major centers. As an alternative, a simpler technique through image manipulation on the software OsiriX/Horos, aiming to reduce both exposures, is presented.

Over the last decades, since the first published results by Juan Parodi in 1991 [1], endovascular aneurysm repair (EVAR) became the vascular surgeon's most preferential technique to treat aortic aneurysms due to its benefit of early clinical and surgical outcomes with good long-term durability. EVAR has progressively replaced open surgical repair (OSR), especially in the infrarenal territory, representing currently over half of the surgeries for abdominal aneurysms [2, 3]. The development of new modern devices (with features that can adapt to different morphologic presentations of this aortic disease, which in the past were considered as not eligible for EVAR), like low-profile delivery systems, comformability and flexibility, has required some new aptitudes beyond endovascular skills for this type of repair, directly

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Giovani José Dal Poggetto Molinari

Giovani José Dal Poggetto Molinari

http://dx.doi.org/10.5772/intechopen.78018

**Abstract**

**1. Introduction**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Keywords:** EVAR, angio-CT, planning, sizing

## **Planning and Sizing with OsiriX/Horos**

Giovani José Dal Poggetto Molinari

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.78018

#### **Abstract**

It is known that endovascular aneurysm repair (EVAR) requires a precise deployment of the graft and so the anatomical and morphological characteristic study of the aorta and its branches is mandatory. The increase of endovascular surgeons' interest on tomography image edition through software is marked specially when the increasing frequency of these procedures and its complexity have impelled surgeons to face additional and successive risk to occupational radiation exposure. Thus, a meticulous study of the angio-CT during EVAR preparation allows the reduction of unnecessary radiation exposure, as it also reduces consecutive image acquisition and contrast use (that may be related to renal overload in susceptible patients). Although some studies propose effective strategies to- optimize the procedure, they rely on the use of additional specific and advanced equipment, available only in major centers. As an alternative, a simpler technique through image manipulation on the software OsiriX/Horos, aiming to reduce both exposures, is presented.

**Keywords:** EVAR, angio-CT, planning, sizing

#### **1. Introduction**

Over the last decades, since the first published results by Juan Parodi in 1991 [1], endovascular aneurysm repair (EVAR) became the vascular surgeon's most preferential technique to treat aortic aneurysms due to its benefit of early clinical and surgical outcomes with good long-term durability. EVAR has progressively replaced open surgical repair (OSR), especially in the infrarenal territory, representing currently over half of the surgeries for abdominal aneurysms [2, 3]. The development of new modern devices (with features that can adapt to different morphologic presentations of this aortic disease, which in the past were considered as not eligible for EVAR), like low-profile delivery systems, comformability and flexibility, has required some new aptitudes beyond endovascular skills for this type of repair, directly

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

related to specific technical knowledge of each brand's endograft and their usage facing each patient's anatomy.

Consequently, the image study in the pre-operatory time took the uttermost importance in order to ensure the adequate selection of patient candidates for EVAR, the decision to endograft type and size, and additional details for postoperatory follow-up. Different from OSR, EVAR relies on knowing the patient's anatomy well enough to choose the appropriate device preoperatively [4]. Adequate planning is an essential and indispensable step for technical success, in order to promote appropriate adjustment of the graft to the vessel wall and consequently aneurysm sac exclusion with blood flow reorientation [5, 6]. The implications of an inefficient planning can be seen immediately—or after the procedure—by endoleaks' formation or late aneurysm sac growth [6]. Even an underestimation of 2 millimeters in the vessel size can result in fixation and sealing failure, creating endoleaks, migration, and secondary interventions needs (including OSR conversion of late aortic rupture). Moreover, this step warrants the foreknowledge of additional surgical strategies for EVAR viability, like angioplasties, bypass or conduits, and hypogastric occlusion [4].

Therefore, it is important that the surgeon shows familiarity to all the necessary tools to perform a meticulous analysis of the computed tomography angiography (CTA), an imperative exam for this disease evaluation. Nowadays, the multislice CTA represents one of the most important methods for diagnostics and the assistance of vascular disorders. Its performance is related to modern attributions like better spatial and temporal resolution associated with the characteristic vascular lumen attenuation obtained by intravenous contrast injection [7]. CTA yields thinner tomographic segments that give high-definition properties to superior threedimensional (3D) image reformatting, with the less use of iodinated contrast while captured under faster sweep for image generation [8]. A single intravenous bolus contrast injection can produce slices from thorax, abdomen, and pelvis, with a 0.5–1.5 millimeters thickness. When compared to the conventional angiography, the CTA is less expensive, less invasive, and exposes the patient to lesser radiation doses [9].

Also, technological refinements of these (thinner) slices provides plenty of details that—- associated with software for image manipulation—promotes the study of large anatomical segments (including a complete patient's scan). These tomographic data, known as digital imaging and communications in medicine (DICOM) files, additionally retain information of radiation dose distribution at different levels (such as organs and other structures) that—associated with highly sensitive and precise algorithms of 3D-by-volume rendering—allows a patient's scan to be recreated in these software as interactive models along with their vascular anatomy [10, 11]. A range of data processing of the CTA-DICOM files can be practiced and are as follows: multiplanar reformats, bidimensional (2D) and 3D MPR and 3D MPR curved; minimal and maximal intensity projections, MinIP/MIP; 3D volume and surface rendering; and shaded surface display. Each one of these image formattings has its peculiarity and it is important to identify a specific arterial alteration in other distinct projections, rendered by different techniques [9]. There is no type of image reconstruction that is more effective than another; they all have their own properties and indications, where often it is necessary of more than just one kind to demonstrate properly a disease [7].

The preoperative analysis of the CTA consists of three principal purposes: to determine eligibility for EVAR, to choose the appropriate endograft, and to simulate a plan of intervention. Thereby, decisive information for EVAR execution is extracted—like the morphologic configuration of- aneurysm's neck (tapered, reverse tapered, cylindrical, angulated, the presence of thrombus, etc.); the anatomy of visceral arteries related to the aortic axis (lowest renal artery—LRA); to set- the anatomic areas for proximal and distal sealing and deployment of the graft and its diameters for device sizing; to access the quality of arterial paths (stenosis, tortuosities, vessel wall alterations, etc.); and to apprehend the possible necessity of auxiliary procedures for EVAR completion. These abilities raised the preparatory purpose of EVAR due to the many-times expressive and complex presentation of the arteries in the presence of aneurysmal disease [11, 12].

The most popular software used among vascular surgeons are OsiriX Imaging Software (Pixmeo Labs., Geneva, Switzerland) and the Aquarius iNtuition system (TeraRecon, Inc.) and both are retail versions [13]. Lately, the Horos software (horosproject.com) has been shown as a free downloadable option for OsiriX MD: It has the same interface and functionality, running on a 64-bit platform. Although Horos is a low-cost alternative for EVAR planning, only OsiriX and TeraRecon have FDA and CE Marking. The following techniques, described for image inspection for the EVAR study, can be reproduced in Horos, although to ensure its scientific validity OsiriX MD is recommended (FDA approved).-

### **2. Understanding EVAR morphologic concepts**

Necks are the proximal aortic and distal iliac segments free of aneurysmatic disease. It allows the grafts' adequate apposition and promotes the fabric's sealing to the vessel and stent fixation [6, 13]. By reason of the absence of suture to the artery, the durability and stability of EVAR depend almost exclusively on the stent's radial force and friction to the wall. This is why meticulous studies of aneurysms' necks are decisive.

**a.** The proximal neck:

The proximal neck of an infrarenal aneurysm is defined by the segment from immediately- below the LRA to the beginning of the aortic dilation [14]. It can be analyzed by form, length, diameter, angulation, and related alterations (such as calcifications, ulcers, and thrombus) [13].

Form: Aneurysms' necks can exhibit forms as cylindrical, tapered, and reverse tapered. In cylindrical necks, the diameters' difference between the two extremities is inferior to 15%. Tapered necks display up to 20% of this variation, just as 20% is inversely proportional in reverse-tapered necks [14]. Necks over a 30% change in diameter and reverse tapered are not eligible for EVAR and a revision of treatment strategy should be considered into a more complex network that involves visceral arteries.

Length: The ideal neck's length, according to most of the grafts' instructions for use (IFU), is 15mm. Some devices have active proximal fixation and can adapt to a 10mm neck. Nevertheless, the shorter the neck the greater is the risk of type Ia endoleak. The ideal neck length is longer or equal to 20mm [13, 15, 16].

Diameter: There are clear evidences that cranial progression of the aneurysmal disease can occur independently of the type of care (OSR or EVAR) [6]. Aortic necks over 30–32 mm are likely to be diseased and progress to proximal degeneration. They offer no durability to sealing and evolve in time to type Ia endoleaks and thus to reintervention needs [13, 16]. On the other hand, narrow necks (under 18mm), although less frequent, must be carefully evaluated.- Since the majority of endografts' size starts from 22mm (which confers an above 20% of diameter oversize), there is potential exposure to wall stress, partial graft unfolding with fabric corrugation, and even aortic rupture [14]. Currently, the smallest available main body diameter device is the 20mm Ovation Prime (Endologix Inc., Irvine, CA) [16]. The aortic bifurcation diameter should also be measured. Distal narrow aortic bifurcations (inferior to 20 mm) are not suitable to fit both iliac grafts and may compete for space leading to one of the leg thrombosis by compression exerted by the contralateral branch [17]. This can be avoided by aorticmonoiliac devices (with femoro-femoral bypass and contralateral proximal plug occlusion) or single-piece bifurcated grafts like AFX (Endologix Inc., Irvine, California) [14, 18].

Angulation: The suprarenal aneurysm's angle is defined as the suprarenal axis blood flow and- aneurysm's neck. The infrarenal angle is determined between the aortic neck and the aneurysm axis [19]. It is important to specify these two curvatures once they have implications related to the bare-stent accommodation (i.e., the *free flow*in suprarenal fixations) and correct deployment- of infrarenal stent graft. For the majority of commercially available devices, it is not recommended to exceed an angulation over 60 degrees, except for those specifically designed for- 75–90 degrees angulated necks like Anaconda (Vascutek Terumo Lt., Scotland, UK) [20] and Aorfix (Lombard Medical Inc., Irvine, CA) [21]. There is a consistent risk of irregular deployment if these advices are not observed [13]. Patients with angled necks are more willing to- present other associated morphologic alterations that may define technical challenge for EVAR- execution. Severe angulations can result in endograft's kinking or migration along with a lower-than-the-ideal apposition site at the deployment time. Angled aortas should be pursued by the *functional neck, that is,* the length segment that can adequately suit the graft's sealing and fixation. Its limit is ruled by the internal curvature of these tortuous necks where the extra-stiff- guide wire (e.g., Lunderquist) takes over its trajectory, especially when in short aortic necks. On- the other side, in elongated necks, the curvature can be influenced by the stiffness of the wire,- rectifying it. Under the influence of these wires, the longer the neck, the greater the probability- of readjustment of the aortic axis and angle remodeling. For complex morphologic and severe angle presentations, oversizing the graft above 20% is mandatory [22]: when an endograft is implanted in an angulated aortic neck, it might not land perfectly in line with the vessel but rather angulated, which decreases the "effective" amount of oversizing. [23] Due to the probability of asymmetric deployment of the device (which tends to follow the guide wire path) the *functional neck*assumes a more elliptical shape—being necessary the election of larger main- body diameters to guarantee uniformity of the stent-graft contact to the arterial wall [22].

Thrombus and calcifications: The presence of thrombus or atherosclerotic plaques over 50% or- two-thirds of the neck's circumference prevents ideal proximal sealing, with potential type 1a- endoleak evolution. Thus, EVAR is not recommended for these cases. Furthermore, it can cause atero/thrombo embolic complications by manipulating endovascular instruments (to visceral branches and distal arteries) [13, 14, 24]. On the presence of a heavily calcified neck, the Ovation- Prime (Endologix Inc., Irvine, CA) becomes an alternative due to the polymer properties of filling the gaps between the vessel wall and the graft's fabric, warranting appropriate sealing [16].

#### b. The distal neck:

The distal neck is defined as the bottom site for endograft's anchorage and sealing. It can be analyzed by length, diameter, angulation with the aortic axis, and tortuosity of the access vessels (external iliac and femoral arteries).

Length: It should be longer than 10mm. When given extreme tortuosity, longer lengths are recommended [6]. Thus, the greater the graft's area of contact to the arterial wall at the distal neck, the greater are the friction forces that will restrict stent migration and type 1b endoleaks [25]. Also, the most proximal to the hypogastric ostium is the prosthesis; the greater is the stability untowardly to its migration [26]. Along with the support, iliac fixation is associated with factors related to proximal migration of the endografts [27]. Short iliacs can lead to device unbalance and when a 25 mm iliac total length is not feasible (due to its curtailment), progression of the graft to the external iliac artery must be considered. Alternatively, aortomonoiliac stent grafts with femoro-femoral bypass and common contralateral iliac plug can be performed [28].

Diameter: A common iliac artery above 20mm is considered to be aneurysmatic [13, 28]. Diameters of 20–24mm can be treated with bell-bottom grafts (upon which the bottom-line diameter is enlarged to ensure distal sealing). Bell bottoms must be placed at the nearest level of the hypogastric ostium [28]. The larger graft limbs are of 28 mm, accessible in Ovation iX (Endologix Inc., Irvine, CA) and Endurant II (Medtronic Vascular Inc., Santa Rosa, CA) [29, 30]. However, as in proximal necks that tend to evolve to the degenerative wall progression associated with graft migration risk, this process may also happen in the distal neck [31]. Yet in the presence of aneurysmatic iliacs above the 24 mm diameter, the graft can be anchored in the external iliac artery, along with hypogastric trunk coil embolization to halt aneurysm backflow (type 2 endoleak) [13, 32, 33]. Still, preservation of one of the internal iliacs is advocated when both common iliacs are dilated and it can be reached with the use of branched devices (iliac side-branch device) or parallel/sandwich techniques [14, 34]. Bilateral hypogastric coil embolization comes with the risk of buttock claudication, sexual dysfunction, and, in extreme cases, colonic and medullar ischemia [2]. Staged procedures are justified and considered safe with reasonable morbidity [35, 36].

Angulation to aortic axis: Iliac tortuosity can be rectified with extra support guide wire- or through-and-through technique (femorobraquial), granting uphold for the delivery system progression [13, 37]. Angulations closer than 90 degrees may make device progression- and aneurysm sealing difficult, with risks of stent-graft kinking and thrombosis. It can be- avoided in a crossed-legs deployment design, which, in addition to contralateral cannulation assistance, permits a longer length fixation of the limb and extra-column longitudinal- support [28, 38].

### **3. Recommendations for EVAR graft choice**

There are no current studies that compare the effectiveness of aneurysm exclusion between different endograft companies; some were performed only in observational studies. Moreover, the available data are always relatively obsolete due to constant improvements in technology and design.

**Figure 1.** The inner-to-inner and outer-to-outer measurements.


**Table 1.** Main Endografts available in the market and features descriptions based on IFU and orientations.-

For the augment of EVAR graft-related durability, companies specify normatives presented in instructions for use (IFUs) with precise information regarding device sizing and deployment. They are based under the aneurysm's morphologic parameters and rigorous bench testing [39]. Patients with challenging anatomic presentations may benefit from a specific graft's design, delivery, and deployment attributes. Consequently, this requires a surgeon's better knowledge and experience for device selection [40]. In some cases although if in an attempt to embrace EVAR for patients not eligible for OSR the IFU are neglected it will result in significantly device failure in a mean 2years of follow-up [39]. The most common IFU non-adherence situation is hostile neck anatomies: proximal and distal necks below 10mm, angles over 60 degrees, diameters higher than 28mm, thrombus or calcifications over 50% of the diameter, and conical presentations. These are said to have high aneurysmatic sac growth, early endoleak formation, and greater reintervention needs [41, 42].

An IFU compliance for each company guidance is imperative with regard to graft choice based on diameter measurements. Diameter dimensions can be calculated from intima to intima (inner wall) or adventitia to adventitia (outer wall) (**Figure 1**). When these details are not observed, the impacts on oversizing can be clinically expressive. Stent grafts with innerwall measurement recommendations can be over-dimensioned if a 20% oversizing estimated from adventitia is considered. Inversely, devices with outer-wall assessment can be undersized if estimated from intima. Because measurements are based on a static image of CTA (at any point of the cardiac course), these differences can be more problematic if diameter variations caused by aortic pulsatility seen on ECG-gated CT are considered [43].

**Table 1** sums characteristics of endovascular devices according to their IFU, including diameter measurements recommendations.

### **4. The path to EVAR access sheaths: iliacs**

Traditionally, the vascular access for EVAR performance is warranted by the direct puncture of the femoral artery under open surgery dissection [44]. Alternatively, the access can be obtained by an ultra-sound guided puncture of the common femoral artery in a non-diseased arterial segment (percutaneous technique) [45].

However, the pathway to EVAR cannot always present the favorable properties for sheath progression. Unfavorable sizes of the device diameters, marked tortuosity, or calcifications can prevent sheath progression augmenting the procedure's complexity. These alterations, when not identified in the pre-operatory period, can lead to a significant rise in morbidity and mortality [46]. Iliac rupture is directly related to lethality and the necessity of immediate correction, which can jeopardize the intervention's final results. In the EUROSTAR registry, the most common cause of primary conversion is access failure, the main body graft sheath progression being the most responsible (the main body graft) [47].

 Thus, the EVAR graft choice shall take into count the delivery component size, especially the main body graft, compared to the size of the vessel access (iliacs) that should be compatible. Considering that infrarenal grafts available in the market have delivery sheaths up to 24 F (while thoracic grafts are up to 28-F) [48], the lesser iliac diameters acceptable are between 7mm and 8mm. In addition not to display pronounced tortuosity (over 90 degrees), iliacs and femorals should not have calcifications that may lead to plaque dislodgement, vessel-wall lacerations, or arterial embolizations.

Although less frequent in daily practice, strategic alternatives are proposed for these morphologic adverse presentations.

Conduits are designated for small or calcified arteries (femorals/external iliacs) and the necessity of larger delivery sheaths (from 22F). Through an extraperitoneal exposure a 10mm dacron graft is sewed terminal laterally to the common iliac (or bottom of the aorta, in cases of extreme iliac calcification) and externalized by counter opening in the groin, serving as a conduit for the device progression [49].

External iliac artery straightening: In very tortuous arteries, extraperitoneal dissection and manual rectification by traction of the iliac artery can favor sheath introduction and progression. By the end of the procedure, the redundant segment can be resected and the artery reconstructed [50].

Brachial artery catheterization: It consists of percutaneous access of the left brachial artery and a 035″ wire passage (extra stiff of 300cm or a 450cm of a hydrophilic) with exteriorization through femoral access. The wire is then caudally pulled at the femoral site rectifying the tortuosity [51].

Endoconduits: The use of an diameter oversized covered stent, followed by vessel angioplasty, promoting an iliac "controlled rupture," in a way to allow passage of the delivery system [50, 52].

Hydrophilic dilators (Coons, Cook Medical) are available up to 22F. They can be introduced by femoral access and gently progressed over a stiff guide wire under radioscopy. One can estimate the delivery sheath behavior in an adverse iliac anatomy without necessarily contaminating the endograft [53]. They also can, in exception, be used for careful and gradual dilation of limiting-size iliacs.

#### **5. Essential tools for planning and sizing with OsiriX/Horos**

**Table 2** sums the most important Region of Interest (ROI) tools for EVAR measuring and image manipulation that are most commonly used in OsiriX/Horos.

Some particularities of aneurysm measurements must be noticed: it is known that when twodimensional (2D) images are used to evaluate 3D structures, like necks, it induces observers' measurements' variations [15]. To diminish the divergence, authors recommend that diameters should be estimated under a 3D reconstruction of the centerline lumen (CLL) [15, 54]. Some software can perform the centerline reconstruction automatically (like the Aquarius iNtuition and OsiriX, when the specific *EVAR plug-in/sovamed.com*or unofficial plug-ins like *CMIV CTA Tools* are installed) but intrinsic errors of self-regulating algorithms may occur. Because an automatically constructed centerline always follows the middle of contrast line, it won't observe the aortic axis in a saccular aneurysm, for example, deviating its route. By doing so, the transversal images perpendicular to the CLL may sometimes not represent the actual size of the vessel.-

This is why concepts of central lumen flow (CLF) are used: It tends to considerate the preliminary location of the wire paths and endografts, along with aortic migration (**Figure 2**). For this

#### Planning and Sizing with OsiriX/Horos 69 http://dx.doi.org/10.5772/intechopen.78018


**Table 2.** Main basic tools of image manipulation for EVAR planning in OsiriX/Horos.

**Figure 2.** Central lumen line (CLL) construction (left) and Central lumen flow (CLF, right).-

**Figure 3.** Orthogonal exposure of the aorta perpendicular to its axis, when sagittal and coronal planes are corrected.-

reason, it seemsjustifiable that diameter measurements should instead be obtainable from orthogonal projections (CTA under 3D MPR reconstruction) (**Figure 3**) [6].

However, it is difficult to recognize the correspondent vessel segment assessed in MPR on a CLF-reconstructed image. This causes the length measurements not so precise if related directly to the different levels of aortic dimensions in an orthogonal view. For that reason, it is proposed that the orthogonal projections on MPR should be marked with the *3D Point* while determining aortic's widths. By doing so, the exact corresponding aortic segment is mark represented posteriorly in a curved-MPR centerline reformatting, promoting precise longitudinal dimensions (achievable by moving the rulers between two previously marked 3D points).

### **6. Planning EVAR: Neck total length exposure—The renal artery ostial projection technique**

The intraoperative assessment for the stent-graft deployment is usually guided by aortic neck's angiography, which provides a 2D view prone to the parallax effect (an artifact caused by overlaying structures of different levels in a single image). Therefore, the proximal neck of AAA and/or too angulated iliac arteries may hinder accurate visualization of the ostium of the renal artery. A suboptimal positioning of the X-ray equipment for image capture can cause an overlapping of branches along with neck tortuosity, restraining the correct judgment and use of the entire neck's length for graft fixation and proximal sealing.-

Thus, the finest way to prevent this artifact is by determining preoperatively the optimal intraoperative disposition of the fluoroscopy unit, with a perfectly perpendicular view to the origin of the LRA [22]. The technique described here is intended to promote the LRA visualization, exposed orthogonally to its emergence and perpendicularly to the aortic axis of aneurysm's neck [55]. It offers an alternative to the Broeders and Blankensteijn technique [56], to the foreknowledge of the C-arm optimal positioning.

Using concepts of geometric correction and through the manipulation of DICOM images in OsiriX/Horos software, it is possible to trace the same angle of renal artery's ostial exposure in intraoperative 2D imaging (contrast angiography). The LRA ostial perpendicular view is obtained by 3D-MPR CTA reconstruction and the manipulation of sagittal and coronal layers in order to obtain the true axial image of the aorta. This exposure practically corrects any rotational effects of the aortic neck caused by tortuosity of AAA,with a near-perfect circumferential slice of the aorta.

The frame, that displays a slice 90 degree to the aortic axis, is then marked with the *3D point-* tool that allows a permanent voxel signal to the CTA volume. Three *points*are settled in an equilateral circumscribed triangle shape array [57] (one point in the anterior wall and two in the posterior), of which the anterior point is oriented by the tangent line of the LRA ostium. The *points-*marked voxels are then reproduced under a 3D-by-volume rendering, preserving their spatial properties [58]. As in spatial geometry, three points are always coplanar; and if a rotation of the 3D by volume promotes the *points*alignment along a single axis (and equidistant), an orthogonal exposure of aneurysm's neck related to the LRA is achieved (**Figure 4**) [55]. The angles that are necessary to reproduce the same ostial LRA exposure intraoperatively are automatically provided by the software (right corner of the 3D-by-volume rendering image). When these angles are recreated during radioscopic contrast angiography the images are alike (**Figure 5**). The deployment of endografts that has proximal markers (at least three) under this optimal angulation demonstrates them visible in a straight-line formation, just as when these markers are used to the C-arm gantry-anglefine-tuning [15]. Therefore, this technique allows the software to simulate these proximal marks (with the advantage of also exposing the renal artery ostia free of parallax).

**Figure 4.** Above: tangent targeted from the projection of the LRA and intraluminal positioning for beginning 3D *point-* marking. Below: construction of the equilateral circumscribed triangle and geometric representation of the points triangular array (for the exemplified case).-

**Figure 5.** Alignment of the 3D *points* in 3D-by-volume rendering. The automatic angulation is provided automatically by the software (green arrow, right corner). When reproduced in the radioscopic device, the foreseen image and the intraoperative angiography are the same.

The closer is this fluoroscopic incidence correction to the software's tomographic reproduction, the more careful is the LRA visualization and the better the exploitation of aortic's neck for anchorage and sealing—and the more accurate is the endograft deployment. By applying these concepts of spatial geometry in order to systematically achieve the best angle for LRA ostial exposure, it is possible to reduce variations between different CTA examinators during EVAR planning. When ensuring the reproducibility of the technique, errors of personal interpretation are reduced.

#### **7. Planning EVAR: The virtual fluoroscopy preset-**

In addition to precise measurement—such as diameters, lengths, and angles [7]—and the analysis of the characteristics of the aneurysm, it is possible to get a better use of information such as topographic positioning of visceral arteries and their respective references under a radioscopic view.

This technique grants the intraluminal placement prediction of angiographic catheters and radioscopic analysis during EVAR [58]. In a 3D-by-volume rendering, using the pre-defined- bone CT reconstruction and the pencil preset, one can adapt and modify the tomographic values of windowing, color lookup table (CLUT), and shading which in turn define brightness, contrast,- and color range of the image. At this new setting, the name of virtual fluoroscopy (VRF) can be- assigned and recreated in other OsiriX/Horos platforms, becoming a replicable format (**Figure 6**).

Then, markings of the renal arteries on axial projections are performed (with the *3D point-* command). Again, the *points-*marked voxels (now embodied as renal vessels) are reproduced when submitted under any CTA reconstruction, preserving their volumetric properties. Once the exam is subjected to a 3D-by-volume rendering using this VRF preset, the renal arteries (and other visceral branches of interest that could be previously *pointed*) may be assessed in relation to a simulated fluoroscopy image (**Figure 7**).

Then, additional annotations can be made regarding the renal arteries' position referring to the vertebral axis. Also, guided by these images, it is possible to minutely predict aneurysm


**Figure 6.** The Virtual Fluoroscopy Preset.-

**Figure 7.** The Virtual Fluoroscopy and its correlation to the intraoperative fluoroscopy.-

neck location (when under intraoperatory fluoroscopy acquisition), as well as estimate the ideal positioning of the diagnostic catheters for digital subtraction angiography (DSA) at the moment of stent-graft deployment. Vertebral osteo-degenerative alterations identified in VRF can easily be recognized intraoperatively, enhancing vessel navigation without the necessity of consecutive angiographies so as to identify visceral branches' position. In addition, an improved position of the C-arm unit so as to reduce the interference from the parallax effect is foreseen (**Figure 8**).

However, the ideal positioning of the X-ray equipment during the surgical procedure may be different than expected during the preoperative study. It is considered that the aneurysm neck- can possibly shorten or lengthen higher than expected in the intraoperatory, because of the influence of inserted extra-support guide wires or the endograft itself [59]. Even so, although aneurysm's neck angulation, can change the ostial position of renal arteries/visceral branches does not expressively shift [22, 60]. This does not compromise comparisons between images formed by the VRF preset from those radioscopically composed, but this difference can be significant- if fusion of pre procedural images are overlayed to real time fluoroscoy (VRF vs. fluoro) [60].

The 3D-by-volume rendering adjusts the voxel's attenuation coefficient at a scale of color and degree of opacity (transparency) along the axes. Thus, it preserves information of depth and shows better spatial distribution of structures along with an enhanced-by-light (shading) 3D effect [7]. The manipulation of these data (the dose distribution radiated to a surface) allows the visualization of the maximum intensity projection (MIP), which demonstrates the densest voxel (higher attenuation coefficient). They are displayed as opaque areas of high contrast (as bone surfaces) and as transparent values of low attenuation (soft tissues). Even if there are overlaid images of different depths in the same drawing (i.e., structures that when superimposed compete with the density of others of interest, like the aorta), this is a "desirable" effect when the aim is to simulate a gray-scale CTA image of a single bi-planar fluoroscopy.-

Carefully, by studying the CTA under VRF, one can reduce the number of intraoperative angiographies in an attempt to obtain the best angiographic capture that provides the location of renal arteries and aneurysm neck for graft deployment. The closer is this angiographic reproduction to virtual fluoroscopy, the more careful is the surgeon's inspection of the renal arteries' location and the better will be the use of aneurysm neck for fastening and sealing

**Figure 8.** The complete "beyond basics" study of CTA.-

endoprosthesis; being more accurate EVAR execution while the total volume of contrast used is smaller and reducing renal overload in vulnerable patients. Consequently, optimizing these surgical steps comes also with lesser radiation dose exposure.

### **8. Routine for EVAR planning and sizing**

These steps are the same used at the Vascular Surgery Department (the University of Campinas, Brazil) and validated prospectively in 2015 [61].

	- the aortic width above the uppermost renal artery;-
	- diameters along aneurysm's neck, starting immediately after the LRA—from 3 to 5 measurements (for non-cylindrical necks). Segments where the difference between proximal and distal widths are above 15% for cylindrical and 20% for reverse-tapered shapes should not be considered as part of the necks;-
	- the largest AAA diameter;-
	- the distal aortic diameter (pre-bifurcation) and distal lumen aortic width;-
	- diameters along the right-common iliac artery, from 3 to 5 measurements (for noncylindrical iliacs);-
	- diameters along the left-common iliac artery, from 3 to 5 measurements (for non-cylindrical iliacs);-
	- diameters of external iliacs and common femorals (access vessels study);-
	- immediately after the LRA (aortic neck's first measurement);-
	- the lowest aortic neck's diameter;-
	- at the aortic bifurcation;-
	- at the emergence of hypogastric ostium bilaterally;-
	- Creation Mode: It is an outline draft disposure of aortic flow essential to the path oriented by the slightly inner curve in angulated aneurysms.
	- Editing Mode: It is the fine tuning of the centerline, between two previously constructed orientation marks.
	- Length measurements are obtained by positioning the vertical rulers along the centerline- (from A to B, from B to C, or from A to C). When the mouse cursor moves along the reformatted centerline image a highlighted correspondent dot moves along the CPR path in the- three planes (axial, sagittal, and coronal). The measuring bars A/B/C are positioned over- the previously 3D *pointed* (marked) references (previous step): *points* after the LRA and the- lowest aortic neck measurement/the aneurysm extension/the common iliac segments/the infrarenal distance to aortic bifurcation and to hypogastric ostium, bilaterally (**Figure 9**).

**Figure 9.** Summary of EVAR sizing.

• The positioning of renal arteries and aneurysm's neck itself is related to the vertebral axis, as long as one can anticipate the optimal positioning of angiographic catheters for image acquisition, by the VRF preset.

#### **9. Conclusions**

The use of OsiriX/Horus as a complementary tool allows doctors to assist in the preparation of surgeries (as endovascular) extending it beyond the field of diagnostic radiology. These tasks can be easily incorporated into the armamentarium of the surgeon to avoid pitfalls and unforeseen situations that are identified intraoperatively, increasing the operatory risk and often times leading to intervention failure.

This chapter presents simple techniques which are of great practical importance in planning interventional treatments. The ability to manipulate digital formats of medical images allows the recovery of a larger volume of data and grants that interventional procedures can be performed more efficiently, with less time for image projection adjustment, contrast injections, and exposure to ionizing radiation. As a result, one can obtain the impact in relation to the improvement of the surgical technique, translated into the less use of contrast, reduced surgical time, and intraoperative bleeding.

New ways to adapt this software have increased by expanding its use to new tasks. Our proposal is to create the familiarity of professionals and encourage demystified practice of this computer program, an essential tool in surgical planning, where more and more procedures are guided by images.

### **Acknowledgements**

We thank Dr Ana Terezinha Guillaumon, M.D., Ph.D., Chief of the Division of Vascular Surgery of the University of Campinas Surgery Department, because of whom the implantation of these protocols was possible and we had full unrestricted support.

#### **Conflict of interest-**

None.

#### **Author details**

Giovani José Dal Poggetto-Molinari-

Address all correspondence to: drgiovani.molinari@uol.com.br

University of Campinas, Campinas, Brazil-

### **References**

