Annika Kiel (1), Laureen P Helweg (1), Angela Kralemann-Köhler (1), Jan Schulte am Esch (1,2)

(1) General and Visceral Surgery – Liver and Tumour Biology, Medical School OWL, Bielefeld University

(2) Protestant Hospital of Bethel Foundation v. Bodelschwinghsche Stiftungen Bethel

The liver is the metabolic center of the human body and an integral part of our vascular system. An abundant cell type within the liver are the liver sinusoidal endothelial cells (LSECs), which are interspersed with small transcellular pores (fenestrations) located within sieve plates. These fenestrations allow the passage of biomolecules and drugs for later processing in the hepatocytes and therefore provide insights about the functionality of LSECs. However, due to the size of the fenestrations with an average diameter of 100 nm, these can only be visualized using super-resolution techniques such as 3D fluorescent structured illumination microscopy (SIM). Especially live cell imaging offers the possibility to capture the dynamics of fenestrations during treatments. Nonetheless, the phototoxicity of the light leads to the closing of the fenestrations, making live imaging of LSECs challenging. The formation of reactive oxygen species (ROS) due to the irradiation could be one reason for this phototoxic effect, as ROS leads to intracellular stress followed by the closing of the fenestrations. In order to counteract ROS, murine LSECs were treated with the antioxidant N-acetylcysteine (NAC) and the oxygen depletion enzyme OxyraseTM. Given the role of fenestrations in physiology and homeostasis, we aim to gain a better understanding of the regulation of these structures, which will help to develop therapeutic approaches in the context of liver injury. Images of murine LSECs using live SIM depict a phototoxicity-mediated morphologic change of LSECs due to multiple exposures. Therefore, they were treated with the antioxidant N-acetylcysteine (NAC) and the oxygen depletion enzyme OxyraseTM during life SIM. While low concentrations did not negatively affect cell viability, the effect was significantly lower with 2 mg/mL NAC and 2% Oxyrase. A positive effect of the NAC concentrations 0.75 and 1 mg/mL was also observed in live cell imaging, where multiple exposures over 45 min caused only slight morphological changes. At 2 mg/mL, the cells showed the most distinct morphological changes, such as deformation of the cell structure. Conclusively, the application of ‘oxygen scavengers’ has a positive effect on the phototoxic effect without affecting the morphology of the cells. This offers the possibility to observe ‘live’ cell dynamics under treatment, i.e. with regard to polypharmacy studies.

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