AN APPROACH FOR VISUALIZATION OF THE INTERACTION BETWEEN COLLAGEN AND ELASTIN IN LOADED HUMAN AORTIC TISSUES A. Pukaluk 1 , H. Wolinski 2,3 , C. Viertler 4 , P. Regitnig 4 , G.A. Holzapfel 1,5 , G. Sommer 1 1 Institute of Biomechanics, Graz University of Technology, Austria 2 Institute of Molecular Biosciences, University of Graz, Austria 3 Field of Excellence BioHealth – University of Graz, Austria 4 Institute of Pathology, Medical University of Graz, Austria 5 Department of Structural Engineering, NTNU, Norway pukaluk@tugraz.at Abstract. Knowledge of the interaction between the constituents of loaded aortic tissues is crucial to ex- pand our understanding of load-bearing mechanisms in the aorta. We have therefore developed a proce- dure that enables simultaneous multi-photon micros- copy imaging of collagen and elastin in human aortic tissue during the biaxial extension test. The micros- copy images obtained were verified with the results of the histological staining. The mechanical response was also compared with findings from previously performed biaxial extension tests. The proposed pipeline has shown successful and has great poten- tial for structural analysis of human aortic tissue. Keywords: Human aorta, collagen, elastin, biaxial extension test, multi-photon microscopy Introduction The healthy aortic wall consists of three layers, namely the intima, media and adventitia [1]. Each of the layers is characterized by its own structure and function. From a mechanical point of view, the main role is played by the media responsible for the aortic response to loading and the adventitia, which pre- vents the aorta from overstretching and possible rupture. Both media and adventitia owe their passive mechanical properties mainly to two proteins, namely collagen and elastin. Although the arrangement of these proteins in the aortic layers in the unloaded state has already been described [1,2], little is known about the changes caused by the load. Therefore, this study proposes a method for the efficient visuali- zation of collagen and elastin in loaded aortic tissue. Methods The developed procedure was applied to one medial and one adventitial specimen from a non-atheroscle- rotic and non-aneurysmatic human abdominal aorta (52 yrs old, female). The aorta was received within 24 h of death and frozen at -20°C. Sample preparation. The aortic tube was thawed at 4°C prior to preparation for testing and imaging. Dur- ing preparation, all steps were carried out at room temperature and the samples were kept moist with phosphate buffered saline (PBS) at pH 7.4. Loose connective tissue was removed, and the intact aortic tube was cut open in the longitudinal direction. The intimal and adventitial layers were then carefully dis- sected from the media [3], and square samples measuring 20×20 mm were cut in order to obtain medial and adventitial patches. In addition, adjacent rectangular patches of dimensions of about 4×10 mm were cut for histological examinations. Particular care was taken to ensure that the edges of squares and rectangles match the anatomical longitudinal and circumferential directions of the aorta. The mean thickness of each sample was measured optically [3]. Each square sample was then pierced by four sets of hooks connected by sutures. A set of five hooks was used on each side [4]. Histology. Aortic specimens were embedded in paraffin and cut at 4 μm with the microtome Microm HM 335 (Microm, Walldorf/Baden, Germany). Next, the sections were stained with Picrosirius Red (PSR) to highlight fibrillar collagen and Elastica van Gieson (EvG) to highlight elastin fibers [5] to verify multi- photon microscopy images. Multi-photon microscopy. The imaging took place at the IMB-Graz Optical Imaging Resource with a tunable picosecond laser (picoEmerald; APE, Berlin, Germany), which was integrated into a Leica SP5 confocal microscope (Leica Microsystems, Mann- heim, Germany). The laser was tuned to 880 nm to induce both the second harmonic generation (SHG) signal from collagen and the two-photon excited (TPE) autofluorescence signal from elastin. A two- channel, non-descanned detector (NDD) in epi-mode was used to detect SHG and TPE signals simultane- ously (SP 680 nm barrier filter, i.e., excitation light filter; BP 460/50 nm for SHG signal; BP 525/50 nm for TPE signal; beamsplitter RSP 495 for two-channel separation of SHG and TPE signals). Z-stacks were acquired with the HCX IRAPO L 25× NA 0.95 water immersion objective with a large working distance of 1.5 mm for imaging the deep tissue and a sampling interval of 0.6×0.6×5.0 μm. As a compromise between image quality and acquisi- tion time, four-fold line averaging was used to reduce image noise. A coverglass and water as the immer- sion medium could not be used with samples mount- ed on the biaxial test device, since the coverglass Proc. Annual Meeting of the Austrian Society for Biomedical Engineering 2021 DOI: 10.3217/978-3-85125-826-4-06 CC BY Published by Verlag der TU Graz Graz University of Technology