Caratterizzazione funzionale del sistema linfatico intracraniale sfruttando la visualizzazione in vivo degli zebrafish.

Research is becoming increasingly dependent on the examination of biological processes in living cells and organism, since imaging approaches provide relevant insight in basic science, physiology, diseases, immune responses, and therapies. However, while imaging within a certain degree of accuracy through soft superficial tissue is well established, the brain still poses significant challenges, especially in mammals, due to its inaccessibility. The zebrafish model is emerging as a possible good alternative, as the fine tuning of existing microscopy techniques is helping us achieve cell level resolution in the brain without harming the specimen. In this thesis I illustrate recent reports in which zebrafish models were used to demonstrate its applicability in upscaled long term in vivo neuroscience studies. The first report managed to confirm the presence of an intracranial lymphatic network separate from the blood vascular one, and its involvement in the drainage of interstitial fluid from the brain. By using high resolution optical imaging, the authors visualised fluorescently tagged lymphatic cell lines in the zebrafish cranium, and demonstrated their sensitivity to the vascular endothelial growth factor VEGF-C. Finally, the development of this lymphatic network was documented along with its role in immune cell trafficking. In the second report, xenografts in Zebrafish were used to assess the in vivo response towards an array of drug treatments. The publication was aimed at monitoring glioblastoma response to drugs: fishes were engrafted with GFP-tagged patient-derived glioblastoma cell cultures. Researchers managed to monitor the xenografts with automated artificial intelligence analysis, and the results showed that tumour initiation correlated strongly with matched xenografts in mice. Additionally, the in vivo evaluation of the drug marizomib, a proteasome inhibitor currently under clinical trials, showed an effect on fish survival corresponding to previous in vitro and in vivo tests. Mouse xenografts, despite being the gold-standard, are costly, time-consuming, ethically not well accepted and do not enable observation of the growth of the tumour in living tissue. On the contrary, zebrafish models overcome this issue and additionally prove themselves to be a scalable alternative applicable to patient-specific drug evaluation. The third report addresses the problematics of in vivo imaging of adult animals. While larval stage zebrafish can be mounted in low melting agarose without major physiological changes, the risk of gill obstruction makes it prohibitive for juvenile and adult specimen. This limits the research applications since the adaptive immune system and intracranial lymphatics only fully develop past the larval stage. Researchers devised a support for adult specimen intubation under inverted confocal microscope, which allowed for high resolution imaging sessions of up to 20h, followed by total recovery. The zebrafish model will become advantageous for studies aimed at observing and understanding the involvement of cranial lymphatics in fluid homeostasis, cellular waste management, progress of degenerative diseases such as Alzheimer’s, inflammatory processes, and antitumoral response.