Immunomodulation and neuroprotection via tumor necrosis factor (TNF-alpha) blockade after spinal cord ventral roots avulsion and reimplantation in mice.

Abstract

The Central Nervous System (CNS) is responsible for the animal integration into the environment, performing transmission of the stimuli between the supra segmental system and the periphery. In this tissue, lesions, usually due to car accidents or falls, are related to neuronal death, axon disruption, synapse loss, inflammation etc., which makes difficult the recuperation and the standardization of a treatment, so far non-existent. In this context, the inflammatory process and the tumor necrosis factor alpha (TNF-±) are directly related to the ability of regeneration after damage to the central (CNS) and peripheral (PNS) nervous systems. Etanercept (Enbrel) is a drug used in the treatment of autoimmune diseases, which acts by binding to TNF-±, blocking its activity and, consequently, decreasing inflammation, apoptosis and secondary functional loss. The use of Etanercept for regenerative proposes in the CNS after damage has been considered, since the exacerbated local inflammation, mediated by astrogliosis and microglial reaction, reduces the chance of recovery. Thus, we will evaluate the neuroprotection and immunomodulation potential of Etanercept after avulsion and reimplantation of spinal cord ventral roots, using a fibrin biopolymer, in C57BL/6J mice. The mice will be divided into 4 experimental groups: avulsion + root replantation; avulsion + root replantation + etanercept treatment 3mg/kg; avulsion + root replantation + etanercept treatment 6mg/kg; avulsion + root replantation + etanercept treatment 9mg/kg. Treatment will be carried out by intraperitoneal injections, applied on postoperative days 3, 6 and 9. The animals will be perfused 4 and 12 weeks after the injury, the spinal cords will be dissected out, cryopreserved and frozen for cryosections obtention. Neuronal survival (by Nissl staining), and reactive astrogliosis, microglial reaction and synaptic coverage (by immunohistochemistry for GFAP, Iba-1, synaptophysin VGLUT1 and GAD65) will be comparatively evaluated. The functional recovery will be monitored weekly until the 12th postoperative through the "walking track test" (CatWalk system). In the same mice, motor reinnervation will be evaluated by electroneuromyography every 15 days, until the 12th week. Complementarily, through the RT-qPCR technique, we will evaluate, one week post-lesion and repair, the expression levels of TNF-±, NF-º², IL1², IL-6 and IL-10 gene transcripts.