On the integrity of endothelial cells junctions at Mycobacterium marinum-induced granulomas in the zebrafish embryo model

Abstract

Important drug delivery discoveries have been made in the field of drug carrying NPs. The penetration and accumulation of nanoparticles (NPs) at the granulomatous area was shown in a zebrafish and mice study conducted by the laboratory of Prof. Gareth Griffiths. Understanding the endothelial cells (ECs) junctions could prove to be the key for improving drug delivery systems. The work described in this thesis investigates the ECs junctional integrity using zebrafish embryos. We compared the ECs junctional integrity in healthy -and Mycobacterium marinum (M.m) infected zebrafish embryos. We chose to study the adherens junctions (AJs) protein VE-cadherin, and the tight junctions (TJs) proteins Claudin5 and ZO1. In order to study the ECs junctions we developed a whole mount immunohistochemistry (IHC) protocol. Further, we quantified the ECs junctional labeling intensity. Our data suggests that there was only a big difference in the integrity of Claudin5, being higher in the healthy fish compared to the M.m infected fish. We tried to reconfirm the data by using another technique. At last we investigated the ECs junctional integrity in live zebrafish embryos. Our data here suggests that the endothelium in live zebrafish embryos endocytose the antibodies upon intravenous injection, and that the investigation of possible junctional labeling requires higher resolution level than the confocal microscope we used. Additionally, the developed IHC protocol proved to be reliable as an all-round protocol for antibodies, this allowed me, besides my masters thesis, to generate data for 3 different scientific publications from the host laboratory of my masters.