Interazione tra le vie di segnalazione Neuregulin1 e c-Jun in topi KO JNK1 (c-Jun N terminal chinasi 1) a seguito di lesioni dei nervi periferici e ottimizzazione della tecnica iDISCO per la chiarificazione dei tessuti nervosi

Peripheral nerve damage resulting from trauma are potentially devastating injuries, which compromise the patient’s life quality. Despite the intrinsic capacity of nerves to regenerate and much surgical advancement, most of peripheral nerve injuries demonstrate incomplete recovery with poor functional outcome. Thus, a better understanding of the different cellular interactions and the molecular pathways regulated in peripheral nerves following an injury is essential for developing new strategies to improve nerve regeneration. For this aim, this study is focused into two correlated aspects. First, the role of molecular pathways involved in nerve regeneration, to further elucidate their mechanism of action. NRG1/ErbB signaling plays an important role in Schwann cell dedifferentiation and peripheral nerve regeneration. In addition, c-Jun signaling is another pathway that is known to have an important role in Schwann cell reprogramming; one of the most important upstream regulators of c-Jun are c-Jun N terminal kinases (JNKs). In our study we were interested in analyzing the possible interaction and regulation between these two pathways; therefore, we used a JNK1 KO mice model to detect if the absence of JNK1 has any possible effects on the NRG/ErbB system and interpret how this would affect nerve repair. Indeed, as we expected, there is an interaction between the two pathways; here we report that the lacking of c-Jun phosphorylation upregulates the expression of soluble NRG1. We also found a higher activation of Erk, a downstream molecular pathway known to be activated after NRG1 signaling. These data suggest that c-Jun phosphorylation might somehow regulate NRG1 expression, but further studies are needed to elucidate the interplay between c-Jun and NRG1. In the second part of this work, we were interested in optimizing a tissue clearing technique that could be helpful in analyzing regenerating nerves. The current gold standard technique for nerve morphological analysis is confocal microscopy. However, this technique needs sectioning of the nerve to obtain thin slices, which is laborious and time consuming; moreover, to create a reconstruction of the whole tissue, the analysis of several slices is needed. In consequence, our second aim was focused on applying a tissue clearing technique (iDISCO), and further optimizing it for nerve tissue study, which will allow a rapid and whole analysis of the tested nerve samples by the use of light sheet fluorescence microscopy. Here we set up iDISCO protocol to morphologically study the process of peripheral nerve regeneration at different time points following injury and repair (7, 14, 21 and 28 days after injury), focusing our attention on migrating Schwann cells, growing axons and endothelium. Our results showed that iDISCO is an optimal approach to obtain 3D reconstruction of a whole regenerated nerve, and seems a promising technique to elucidate cellular interactions occurring during nerve repair; however, to achieve this, iDISCO and light sheet microscopy should be further developed to increase the imaging resolution and facilitate the data analysis.