3 Minute 3Rs September 2019

The September episode of 3 Minute 3rs, brought to you by the NC3Rs (www.nc3rs.org.uk), the North American 3Rs Collaborative (www.na3rsc.org), and Lab Animal (www.nature.com/laban)Papers behind the pod:1. http://jpet.aspetjournals.org/content/371/1/15.long2. https://www.sciencedirect.com/science/article/pii/S001216061830006X3. https://www.nature.com/articles/s41467-019-11259-w[NC3Rs] Accurate measurements of drug and metabolite concentrations in the blood are vital to estimate exposure to the target in humans and animals during drug development. Zebrafish larvae are increasingly used for pharmacological research, but measurements of blood drug concentrations in these small animals have been technically challenging.A team at Leiden University have developed a method for nanoscale blood sampling from the posterior cardinal vein of zebrafish larvae at five days post fertilisation. A median volume of 1.12 nL of blood can be collected from each embryo and samples pooled to form a single replicate, which can be analysed by Liquid chromatography–mass spectrometry. While drug and metabolites could be successfully measured in the pooled samples, the authors suggest improvements to the sensitivity of the technique, which could reduce the number of embryos needed for each replicate. In addition, using microfluidic embryo handling techniques, blood sampling could be further automated and yield improved while reducing the amount of drug required. Further development of this microsampling technique has the potential to increase the use of the zebrafish embryo model to define drug pharmacokinetic properties. [NA3RsC] Josephine Morris and her colleagues at Bristol University used transgenic lines of zebrafish to study mechanisms of collagen formation and repair. This work was published in the journal Developmental Biology, Vol 441. They crossed transgenic zebrafish lines which integrated green fluorescent protein, expressed in the epidermis and mCherry collagen which is specifically expressed in the basal epidermis which allowed them to understand the dynamic nature of collagen 1 fibril deposition. The authors used Transmission Electron microscopy to demonstrate the intricate pattern integration of the fluorescent proteins in embryonic development. In other studies, a wound was created with a 30 g needle on the flank of 4 day old larvae and collagen repair was documented pictorially. By using the GFP collagen and mCherry collagen lines together, they were able to exploit the unique live imaging in larval fish to probe the process of collagen deposition and wound remodeling specifically in the basal epidermis and deeper layers. It is hoped that these transgenic lines will enable live imaging of collagen deposition and remodeling in various other organs and diseases.[LA] And finally, say hello to LipoGlo, a new reporter system for keeping track of lipoproteins in vivo. These proteins ferry fats throughout the body – you may be familiar with HDLs and LDLs, the latter of which can contribute to cardiovascular disease in people. A particular particle, Apolipoprotein B-containing lipoprotein, is particularly problematic and while it has been studied in mammals, such animals don’t lend themselves to the large numbers needed for high-throughput drug discovery work. Enter the larval zebrafish. In order to visualize APoB-lipoproteins across the whole translucent organism, the research team took advantage of a glowing enzyme called NanoLuc, which they attached to APoB in the larvae. This bioluminescent reporter is highly sensitive and quite bright, allowing the team to follow the distribution and concentrations of the lipoprotein as it traversed the vascular system of the tiny little zebrafish. Additional details about the new tool can be found in the journal Nature Communications.

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3 Minute 3Rs September 2019

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