Desenvolvimento de um modelo experimental in vitro para o estudo da excitotoxicidade glutamatérgica e do estresse oxidativo na neuromielite óptica

Abstract

Neuromyelitis optical spectrum disorders (NMOSD) is an inflammatory/autoimmune disease, characterized by the production of autoantibodies (IgG) against the aquaporin-4 water channel (AQP4), a protein highly expressed in the astrocyte cell membrane. The AQP4-IgG binding induces the opening of the blood-brain barrier, causing astrocytopathy, tissue destruction and demyelination in structures such as the optic nerves, spinal cord, brainstem and cortex. It is known that astrocytes are cells that play important roles in the CNS, such as regulation of cerebral homeostasis and glutamatergic synapses. Therefore, any astrocytic damage can compromise the cell function and interfere in biological processes, such as glutamate uptake. Regulation of extracellular glutamate levels is performed by glutamate transporters present in astrocytes. When these cells are damaged, extracellular glutamate levels increase, and the neurotransmitter becomes toxic to nerve cells, conferring excitotoxicity and generating oxidative stress. For this reason, it is believed in addition to antigen-antibody interaction, excitotoxicity and oxidative stress are factors that may be related to the physiopathology of NMOSD. The objective of this work was to develop in vitro experimental model of NMOSD, using astrocytes from 36 adult rats, to investigate the influence of glutamatergic excitotoxicity and oxidative stress on NMOSD. For this, astrocytes were exposed to serum AQP4+ and purified IgG from patients with NMOSD. Characterization of the cellular phenotype was evaluated by the immunocytochemistry technique, and the cytotoxic effects of the exposure of serum AQP4+ and purified IgG in the cell culture were evaluated by the cell viability assay (MTT). The ability of cells to capture glutamate and the relation of oxidative stress were also evaluated through exposure tests with L-[2,3-3H] glutamate and DCFHDA. The data obtained showed that autoantibodies against AQP4 are cytotoxic to astrocytes mainly for astrocytes exposed to serum AQP4+ and purified IgG in high concentrations, impairing glutamatergic transport and inducing the production of intracellular oxidants. In this work, we conclude that autoantibodies are deleterious to astrocytes and capable of inducing cytotoxicity. In addition, impair in the astrocytic glutamate uptake and generate oxidative stress. Therefore, it can be inferred that these factors may 8 be associated with the development of CNS lesions observed in patients with NMOSD.