Zebrafish (Danio rerio) have become an important model system to study a wide variety of disease processes. Ample molecular and genetic tools, alongside live imaging that is facilitated by the optical transparency of the larvae, have enabled significant insight into in vivo dynamics. However, Danio rerio does not maintain its transparency after larval stages; therefore, internal organ systems, later-onset diseases and the neuronal basis of complex behaviors, such as courtship, become largely inaccessible to imaging. To circumvent this issue, Danionella cerebrum, a close relative of D. rerio that is smaller and remains essentially transparent in adulthood, has been recently employed as a model for neuroscience and behavior studies. Importantly, although molecular genetic methods had been successfully used with D. cerebrum, longitudinal studies, including timelapse imaging of adults over extended periods of time, have not yet been described in D. cerebrum.

To this end, Pui-Yin Lam set out to develop reliable, long-term imaging methodology for adult D. cerebrum. Building from methods recently developed to enable long-term imaging of adult D. rerio, the author designed imaging chamber systems to house intubated D. cerebrum for imaging on either upright or inverted microscopes. With a particular interest in immune responses in the brain, the author generated new transgenic D. cerebrum lines to label macrophages and microglia [Tg(mpeg1:Dendra2)] and endothelial cells [Tg(kdrl:mCherry-CAAX)], using constructs originally generated for use in D. rerio. The author then went on to establish two experimental paradigms for brain injury in D. cerebrum: stab wound and laser-induced intracerebral hemorrhage to mimic this form of stroke. Macrophage and microglia behaviors were imaged under baseline conditions and after injury and, by using endothelial structures for orientation and registration, the same position in the brain was imaged repeatedly over the course of several days. In this way, immune cell response and resolution specifically around the injury site was visualized.

Together, this work demonstrates the feasibility of long-term, repeated imaging of adult D. cerebrum. With these new transgenic tools, D. cerebrum is poised to be a valuable model for brain injury and intracerebral hemorrhage. More broadly, the author has demonstrated the practicality of D. cerebrum for studying the immune response in living adult tissue during wound healing, repair and regeneration processes. Therefore, the tools developed here might impact research into many additional disease processes and organ systems beyond the brain.

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Longitudinal in vivo imaging of adult Danionella cerebrum using standard confocal microscopy
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