La formation de synapses par les neurones périphériques sur des surfaces synthétiques

Background: Implantable intraneural electrodes as a neurotechnological interface with the peripheral nervous system are a promising solution to overcome the limitations caused by injury or disease. Their long-term viability could be enhanced by coating electrodes with synthetic substances which are well tolerated by the host. Synaptogenesis onto artificial material can be seen as an indirect marker of biocompatibility as it demonstrates the recognition of the said substance as a viable post-synaptic target. Our objective is to demonstrate the synaptogenic properties of poly-D-lysine (PDL) and an experimental hyperbranched polyglycerol (dendrimer, DND) with human peripheral neurons in vitro and rat neurons in vivo. Ultimately, we aim to develop coatings that promote synaptogenesis onto a synthetic object embedded in a peripheral nerve with the objective of increasing its longevity.   Methods: Uncoated (control) and coated (poly-D-lysine or dendrimer) microspheres were used. In vitro, motor neurons derived from human induced pluripotent stem cells (iPSCs) were grown with microspheres for 1, 3, 5, 7 or 9 days. In vivo, a nerve crush injury was introduced to adult rat sciatic nerve to induce a regenerative state before injecting microspheres proximally, distally and at the crush site, which were left for 2, 7 and 14 days. Cells and nerves were immunostained for synaptophysin and VAchT (presynaptic markers). All experiments were visualized using confocal microscopy and images were analyzed for staining intensity surrounding the microspheres using Fiji. Live cell imaging of human motor neurons with FM-143 was performed to verify the presence of exocytosis at contact sites with the microspheres.  Results: In vitro, our results show significantly more staining for synaptophysin (p<0.0001) and VAchT (p<0.0001) in both experimental conditions when compared to the control for all five time points. Preliminary results of live cell imaging show a significantly more important exocytosis of FM-143 labelled vesicles in DND-coated microspheres than PDL-coated ones (p=0.006) at 3 days of incubation. In vivo, the presence of synaptophysin is significantly more important at 14 days surrounding PDL-coated microspheres at the crush site and distal to it when compared with controls. VAchT staining is also significantly superior at 14 days at the crush site. Other time points and locations tested do not show significant difference when comparing coated microspheres with respect to its control.  Conclusions: Our study shows for the first time that poly-D-lysine and dendrimer are synaptogenic substances for human motor neurons in vitro and rat peripheral neurons in vivo. These results support the future use of these substances in improving the longevity of implantable electrodes for various clinical uses in neurosciences, such as for nerve stimulation or recording in chronic pathologies.

Keyphrases: synaptogenèse, poly-D-lysine, polyglycérol hyperbranché, VAchT, synaptogenesis, microsphere, hyperbranched polyglycerol, synaptophysin, iPSC, motor neurons, sciatic nerve, peripheral nervous system