# Heterogeneity of Ex Vivo and In Vivo Properties along the Length of the Abdominal Aortic Aneurysm
## 4 Discussion
A particularly marked effect of position along the aortic length, in contrast, was consistently observed in several ex vivo and in vivo properties, with the central regions corresponding to the aneurysm dilatation (particularly position 3) being strikingly distinct from the adjacent patches (i.e., showing significant difference with respect to the other identified regions). The aneurysm area was characterized by disturbed hemodynamics likely to drive the pathological remodeling that results in a changed mechanical response as observed in the heterogeneity of collagen-related behavior (HTM, Tzo, and TZe) and inflammatory markers content along the length of the artery. First, the consistency observed between ex vivo and in vivo properties clearly suggests that in vivo biomarkers can eventually improve aneurysm assessment and outcome prediction, with regional heterogeneity providing a direction for future work on non-invasive risk assessment. Second, while the central regions of the aorta corresponded to the position of the aneurysm sac and, therefore, included the location of maximum diameter, the present research clearly points out the shortcomings of the use of maximum diameter. The aortic size alone cannot fully characterize the regional variability and heterogeneity of the AAA tissue, especially when considering that rupture has been reported to often occur away from the location of maximum diameter [4; 7]. This study presents limitations that need to be addressed. The sample size was limited, in terms of both patients and AAA tissue specimens; present results would benefit from analyses performed on a larger cohort that can better represent the variability among different patients and aortic anatomies. A larger study cohort would possibly allow for the investigation of difference in female versus male AAA patients, as well as age-matched analysis, given the effect of sex and aging on the arterial structure and function. Similarly, access to healthy aortic tissue and imaging would provide a means for valuable considerations. There are shortcomings in the colorimetric analysis performed on histologies as only three colors on a pentachrome stain were analyzed: darker pixels may be recognized as black, ultimately affecting the elastin content results. Moreover, due to the scarcity of specimens containing measurable collagen in the media, along with the faint nature of the Musto/Movat staining for collagen, analysis for this constituent was not feasible. Further studies should be conducted to include Picrosirus Red staining to allow for collagen visualization, and Total Collagen Assay and ELISA to allow for quantitative assessment of collagen in the tissue. Additionally, the use of multiphoton microscopy would prove useful to investigate collagen fiber morphology (i.e., fiber thickness, direction, ondulation) as it relates to the mechanical properties of the tissue. While the Musto/Movat staining provided an insight into the elastin content of the specimens, it did not allow for any inference on the state of the elastin fibers, thus additional analysis would prove useful to access information on elastin fragmentation. Similarly, a look into extracellular matrix breakdown and matrix metalloproteinase would provide information on the proteolytic process associated with the degradation of the aortic media. The IHC analysis had limitations due to the presence of background staining that can lead to a misinterpreted and overestimated cell count. In this regard, manual cell counting can be prone to errors and may be insufficient as a stand-alone metric for inflammation as cytokines, signaling interactions and molecular mechanisms are also involved in the complex inflammatory process. The assumption of rigid aortic wall for CFD simulations also presented limitations.