Neuroprotective and immunomodulatory effect of dimethyl fumarate (DMF) after avulsion and reimplantation of spinal motor roots associated with cell therapy

Spinal cord injury causes critical loss of motor and sensory function. Ventral root avulsion (VRA) is an experimental model in which there is the tearing of the ventral (motor) roots from the surface of the spinal cord. This rupture, at the interface between the peripheral (PNS) and the central nervous system (CNS), interrupts the contact between the motoneuron and the target muscle fibers. As a result, the majority of axotomized motoneurons degenerate between the second and third week after avulsion. This is worsened by enhanced glial reaction, triggering a chronic inflammatory state. However, some motoneurons present regenerative potential after lesion, serving as a regenerative model for CNS/PNS studies. Therefore, our goal was to test if the combination of therapies with neuroprotective and immunomodulatory properties could improve regenerative response. For that we used surgical repair with fibrin sealant (FS), a biological glue, which is citoprotective and allows for the restablishment of the contact between the neuron body and its target muscle fibers. We also used dimethyl fumarate (DMF), an FDA approved drug for psoriases and multiple sclerosis, which promotes cytotoxic protection, reestablishment of cellular homeostasis and also presents immunomodulatory effects. And for last, we used mesenchymal stem cells derived from adipose tissue (MSCs-AT) for cell therapy, since these cells provide several trophic factors and cytokines that can result in neuroprotection and immunomodulation. Thus, we had two experimental sets, (1) the combination between FS and DMF and (2) the combination between FS, MSCs-AT and DMF, being n = 5/group/technique. For both experimental settings, adult female Lewis rats were subjected to unilateral VRA of L4-L6 roots followed by reimplantation with FS, MSCs-AT transplantation and daily treatment with DMF (15 mg/Kg; gavage) for 4 weeks, being the survival time post-surgery 12 weeks. Treatments were evaluated by light and fluorescence microscopy for spinal cord and sciatic nerve, morphometry of sciatic nerve, transmission electron microscopy for neuronal coverage, RT-PCR for gene transcription and the Catwalk system for motor function recovery. Data are presented as mean ± SEM and were subjected to one- or two-way ANOVA, followed by Bonferroni’s post hoc test (p < 0.05). Our results indicate that the combination between FS and DMF was neuroprotective since it was able to preserve most of the axotomized motoneurons and also led to synapse preservation at the microenvironment and at neuronal membrane of the alfa motoneurons. Furthermore, we observed a significant decrease in astrogliosis and microglial reaction and nerve regeneration. Such parameters were combined with gait recovery, which was translated to up to 50% of motor functional recovery. As for the combination between FS, DMF and MSCs-AT we observed similar results, but glial reaction was further reduced and motor functional recovery was up to 70%. Altogether, our results indicate that both therapy combinations can enhance motoneuron survival through its neuroprotective and immunomodulatory effects, showing the potential of employing combined regenerative approaches following spinal cord root injury (AU)