• nigrostriatal pathway
• mechanical stimuli
• cell therapy
• Parkinson
• organotypic

Nerve regeneration has always aroused much interest in the scientific community, both in terms of understanding the mechanisms behind this phenomenon and for possible application in the medical field. It has long been known that axonal growth can be stimulated and guided by chemical factors. More recent is the discovery that mechanical stimuli can also play a role in these processes. In particular, a growth model was proposed in 2004, called the 'stretch-growth model', according to which elongation can be induced by mechanical stimuli, whether these originate from the growth cone or from external stimuli such as body growth or exogenous mechanical forces. To study this process, the research group where I did my thesis internship developed a protocol involving the internalisation of magnetic nanoparticles (MNPs) by neurons and the application of an external magnetic field for the generation of very small forces, comparable to endogenous ones. This technology has made possible to show how the application of forces of the order of pico-Newtons, prolonged over time, is capable of stimulating axonal growth, both in morphological terms, with an increase in neurite length, and in functional terms, revealing accelerated cell maturation. These results are also very promising with a view to a possible stimulation of regeneration; however, validation on less complex models is necessary before an in vivo application. For this reason, this thesis project focused on the possibility of promoting the phenomenon of axonal growth by applying very small forces on microinjected cells within an ex vivo model of organotypic brain co-culture. Indeed, organotypic cultures preserve the architecture of a tissue and maintain many of its interactions. Recently, it has been seen that this is true not only within a single section, but also between sections of different portions through their co-culture. During my thesis residency I developed an organotypic co-culture protocol to reconstruct the nigrostriatal pathway, formed by dopaminergic neurons that project from the substantia nigra into the dorsal striatum. The culture was obtained by sectioning mouse encephalons, selecting sections comprising the dorsal striatum and the substantia nigra and placing them a gap apart. This co-culture provided the substrate for the microinjection of cells. Each co-culture was subjected to three microinjection spots in the area corresponding to the substantia nigra with the cell lines SH-SY5Y and PC12, which can be differentiated by retinoic acid and NGF, respectively, to assume a neuronal phenotype, and also the neuroepithelial cell line, which can be differentiated into mature neurons by BDNF. The co-culture protocol developed proved to ensure good tissue survival and to allow the connection between dorsal striatum and substantia nigra thanks to the migration of neurites from one area to the other. With regard to microinjection, the conditions set allowed good viability to be maintained, both of the tissue and the injected cells. Finally, the collected results laid the basis for the execution of some pilot experiments for the application of mechanical forces on the injected cells within the co-culture. Observing a more rapid and directional axonal growth of injected cells within a tissue section would bring us closer to a possible practical application. In particular, with regard to the treatment of Parkinson's disease, which is characterised by the degeneration of dopaminergic neurons constituting the nigrostriatal pathway, the possibility of a combinatorial approach between the implantation of healthy cells, now a state-of-the-art therapy, and the mechanical stimulation of their growth by means of minimally invasive techniques would open up very promising scenarios.