Longitudinal MRI brain studies in live adult zebrafish

Zebrafish (Danio rerio) has been successfully used for decades in developmental studies and disease modelling. The remarkable uptake of zebrafish as a model system is partly due to its transparency during the early weeks of its development, allowing in vivo imaging of cellular and molecular processes. However, this key advantage wears off when tissues become opaque as the animal reaches juvenile and adult stages, rendering access to tissues for live imaging and longitudinal studies difficult. Here we provide a novel approach to image and assess tissue integrity of adult zebrafish using MRI on live zebrafish suitable for longitudinal studies. We built a 3D-printed life support chamber and designed a protocol-directed sedation regime to recover adult zebrafish after scanning in a 9.4 T MRI scanner. Our life support chamber is cheap and easy to create using 3D printing, allowing other groups to copy our template for quick setup. Additionally, we optimized the delivery of contrast agent to enhance brain signals in order to refine current delivery, usually delivered intravenously in rodents. We show here that immersion in gadolinium was a viable alternative to intraperitoneal injection to reduce T₁ relaxation times. This resulted in protocol refinement as per the 3Rs guidelines and improved image contrast in adult zebrafish disease models. In conclusion, we provide here a detailed methodology to allow longitudinal studies of brain tissue integrity of adult zebrafish, combining safe and efficient delivery of contrast agent and live MRI. This technique can be used to bridge the gap between in vivo studies and longitudinal brain analysis in adult zebrafish, and can be applied to the ever-growing number of adult zebrafish models of ageing and neurodegenerative diseases.