Biaxial loading of arterial tissues with 3D in situ observations of adventitia fibrous microstructure: A method coupling multi-photon confocal microscopy and bulge inflation test

Disorders in the wall microstructure underlie all forms of vascular disease, such as the aortic aneurysm, the rupture of which is necessarily triggered at the microscopic level. In this context, we developed an original experimental approach, coupling a bulge inflation test to multiphoton confocal microscopy, for visualizing the 3D micro-structure of porcine, human non-aneurysmal and aneurysmal aortic adventitial collagen under increasing pressurization. The experiment complexity on such tissues led to deeply address the acquisition major hurdles. The important innovative features of the methodology are presented, especially regarding region-of-interest tracking, definition of a stabilization period prior to imaging and correction of z-motion, z being the objective's axis. Such corrections ensured consistent 3D qualitative and quantitative analyses without z-motion. Qualitative analyses of the stable 3D images showed dense undulated collagen fiber bundles in the unloaded state which tended to progressive straightening and separation into a network of thinner bundles at high pressures. Quantitative analyses were made using a combination of weighted 2D structure tensors and fitting of 4 independent Gaussian functions to measure parameters related to straightening and orientation of the fibers. They denoted 3 principal fibers directions, approximately 45 degrees, 135 degrees and 90 degrees with respect to the circumferential axis in the circumferential-axial plane without any evident reorientation of the fibers under pressurization. Results also showed that fibers at zero-pressure state were straighter and less dispersed in orientation for human samples - especially aneurysms - than for pigs. Progressive straightening and decrease in dispersion were quantified during the inflation. These findings provide further insight into the micro -architectural changes within the arterial wall.