Abstract

Cerebral aneurysm rupture causes acute and often fatal brain injuries. At cerebral arterial bifurcations, the vasculature is highly responsive to specific impinging blood flow parameters. Previous in vivo studies have demonstrated a relationship between hemodynamic parameters at bifurcations and aneurysm development (Meng et al., 2006) Though in vivo experiments can test general hypotheses and yield invaluable results, obtaining definitive information is difficult due to the myriad of complex biological pathways that exist in animal models. In vitro experiments can isolate and test specific variables however, their utility is limited due to their two-dimensional nature and over simplification of biological systems. We have developed an ex vivo system in which explanted arteries can be subjected to precisely controlled hemodynamic variables. In this system, tissue can be sustained in an in vivo-like environment with specifically defined variables and the ability to obtain biological and corresponding hemodynamic data at any time interval. Using 3D angiography and computational fluid dynamics (CFD) we can correlate the bifurcation hemodynamics with the morphological and biological mechanistic changes. The aim of this study is to establish a model system to study the association of specific hemodynamic variables and biochemical pathways involved in the development of vascular disease.

Method

System setup

· During surgery, attach ovine mesenteric arteries to cylindrical piping to create fully developed flow. · Insert vessels into ex vivo flow loop (Inoguchia et al., 2007) · Immerse vessels in flow media and circulate media within loop. Media mimics blood nutrient content and viscosity · Flow rate and pressure are modified to simulate normal physiological and diseased conditions · Vessels exposed to specified hemodynamics for 1 to 6 days

Fig. 1: Schematic of Ex Vivo Flow System

Hemodynamic and Histological Analysis · Vessel is imaged with rotational angiography to obtain 3D orientation · Tissue is fixed in situ to maintain geometry and processed for histological and quantitative analysis · Computational fluid dynamics (CFD) is used to create 3D analysis of vessel hemodynamics · 2D hemodynamic flow-field extracted from 3D analysis and mapped to corresponding histology Fig. 2: 2D hemodynamic flow field indicating velocity vectors            Histology (In vivo) Fig. 3: Artery bifurcation (In Vivo Control, 40x and 200x) Stains from left to right   A)   Haematoxylin & Eosin (H&E): The distribution of cell nuclei is evenly spread throughout the intimal and medial layers. Nuclei are displayed in blue. B)  Trichrome: The medial layer thickness is constant on the main and side branches. Collagen is displayed in blue-green and cellularity in magenta. C)  Van Gieson's (VG): Elastin is displayed in black. The internal elastic lamina dividing the intimal and medial layers is continuous and uninterrupted.     Histology (Ex vivo, Normal Flow) Fig. 4: Artery bifurcation (Ex Vivo, Normal Flow, 40x and 200x)   The geometric difference between the vessels in Figures 2 and 3 is a natural variation that may occur among specimens. In vivo natural bifurcations under normal flow strongly resemble ex vivo vessels under normal flow. There is little to no effect on vessel morphology from excising the in vivo bifurcation and placing it into the ex vivo flow system.     Histology (Ex vivo, High Flow) Fig. 5: Artery bifurcation (Ex Vivo, High Flow, 40x and 200x)   A)   There is significantly reduced cell density (indicated by the absence of cell nuclei in the medial layer on the side branch). B)  The medial layer (in magenta) shows major deterioration and loss. There is also significant medial layer thickening on the main branch. C) The arrow indicates significant intimal thickening on the side branch.

Hemodynamic Mapping

Fig. 6: Progression from explanted vessel to hemodynamic and histological mapping

A)    Explanted mesenteric vessel placed into ex vivo system B)    Reconstructed geometry obtained from rotational angiography C)    3D analysis of hemodynamic wall shear stress (WSS) on vessel walls D)    2D slice of mesenteric vessel stained with trichrome (magnified 25x) E)    2D hemodynamic flow field indicating velocity vectors F)    Hemodynamic velocity flow field mapped with histology section Data for specific correlation of hemodynamics to morphological changes is not yet available.

Conclusions

Using this system, we demonstrated the ability to maintain normal physiological morphology in an ex vivo flow system. Similar to previous studies, under increased flow we observed vascular remodeling. (Lehoux et al. 2002) Through the use of CFD, we obtained 2D and 3D representations of vessel hemodynamics and mapped them to corresponding histology.

In the future, we plan on conducting studies to correlate specific hemodynamic parameters and with histological changes. As a part of the study, we will examine mechanistic pathways involved and their role in vascular remodeling.

References

1.  Meng H, Swartz, DD, Wang, Z, Hoi, Y, Kolega, J, Metaxa, EM, Szymanski, MP, Yamamoto, J, Sauvageau, E, Levy, EI. A model system for mapping vascular responses to complex hemodynamics at arterial bifurcations in vivo. Neurosurgery. 2006; 59:1094-1101.

2.   Inoguchia H., Tanakac T., Maeharab Y., Matsuda T., Biomaterials, 2007. 28: p. 486 - 495

3.  Lehoux, S., Tronc, F., Tedgui, A.,  Biorheology, 2002. 39(3-4): p. 319 - 324

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