Intensification of motor regeneration with HNK-1 mimetic and ursolic acid peptide after avulsion and reimplantation of spinal ventral roots in C57BL/6J mice

Injuries to the spinal ventral roots lead to significant degeneration of motoneurons, preventing significant functional recovery. In such cases, HNK-1 and ursolic acid (UA) are neuroprotective molecules a that stimulate neurite outgrowth both in the central and peripheral nervous systems. Thus, the present study used the association between the mimetic peptide of HNK1 (pm-HNK-1) and UA with the reimplantation of avulsed nerve roots, to optimize neuronal plasticity, synaptic preservation, and motor functionality after the injury. The CNS/PNS repair was performed using a fibrin sealant (FS), taking into account previous studies in our laboratory. For this, 71 female mice of the isogenic surgical strain C57BL/6J were subjected to the avulsion of motor roots (L4-L6) followed by reimplantation, with the application of FS. The animals were divided into four experimental groups: Avulsion = avulsion with FS, Reimplantation = Avulsion + reimplantation + FS, pm-HNK-1 = Avulsion + reimplantation with FS + pm-HNK-1 and AU = Avulsion + reimplantation with FS + UA. The experimental groups were evaluated at 2 and 12 weeks after surgery. Functional assessment was performed weekly using the Walking Track Test (Catwalk platform, Noldus). Neuronal survival was performed by histochemistry (Toluidine-blue staining and retrograde tracing using Fluoro-Ruby). Reactive gliosis and synaptic covering evaluation were performed by immunofluorescence using anti-Iba1 (microglia and macrophages marker), anti-GFAP (astrocytic marker), anti-GAD65 (GABAergic synapses marker), and anti-VGLUT1 (glutamatergic synapses marker). Ultrastructural analysis of the synaptic terminals opposed to the alpha motoneurons was performed using transmission electron microscopy. Motoneuron survival results show 60% of motoneuron survival in pm-HNK-1 group, 52% in the UA group, and 39% in the Avulsion group. Such effect is also observed in the total number of motoneurons counted after retrograde tracing, which showed 27% in the pm-HNK-1 group, 2.49% in the Reimplant, and 5.2% in the Avulsion, which suggests a neuroprotective and regenerative effect of the pm- HNK-1. Regarding the microglial reactivity, the UA, pm-HNK-1, and Reimplantation groups had lower glial reactivity when compared to the Avulsion group. The ultrastructural analysis of synaptic preservation demonstrated a significant improvement in the pm-HNK-1 and the Reimplant groups, compared to the Avulsion counterpart. However, the functional analysis showed that the pm-HNK-1 group did not outperform the UA group, which showed better results (AU)