Efecto de la melatonina sobre la apoptosis y activación de la microglia en modelos experimentales de la infección por el virus de encefalitis equina venezolana

Abstract

The Venezuelan equine encephalitis virus (VEE) is an infection of economical and clinical relevance in northern South America. It affects, with high morbidity, the central nervous system (CNS) in humans and equines. It has been demonstrated that melatonin (MLT) has a protective effect in viral infections and its role has been evaluated in vivo and in vitro. The interest in MLT has increased due to its influence on apoptosis and microglia activation as an important factor in viral pathogenesis. This study evaluated the effect of MLT on apoptosis and microglia activation in experimental models of VEE virus infection. In vivo assays were performed in albino NMRI mice that were treated subcutaneously with 500 g of MLT /Kg body weight. Mice were inoculated intra-peritoneally with the Guajira strain of the virus, following three schemes of treatment with MLT: Preventive and Precocious: MLT was administered daily 3 days before and 10 days after viral inoculation. Precocious Treatment: Mice were infected and immediately treated with MLT. Late Treatment: Mice were infected with the virus and treated with MLT 24 hours after the infection. The animals were sacrificed in different periods post infection, five for each experimental group for each assay. Whole blood samples were extracted from the internal angle of the eyes to obtain serum to determine IgM anti VEE antibodies. Then the brain was extracted, previous intracardiac perfusion to determine microglia activation by indirect immunohistochemistry using monoclonal antibody anti mouse CD-200. Apoptosis was determined by TUNEL. For the in vitro assays, murine neuroblastoma (Na2) cells infected with VEE virus in concentration of 1x10-6 UFP/ml for 2, 4 y 6 hours at 37°C and treated with different concentrations of MLT (0,1mM; 0,5mM and 1,0mM) were used. In the present study it was demonstrated that preventive treatment with MLT, had protective effect in mice infected with VEE virus, when compared to mice exposed to previous and late treatment with MLT. This was evident on the sixth and seventh day after intraperitoneal injection, when the mortality rate was reduced to 25% and 45% in contrast to the 75% and 100% of mortality observed in the previous and late treatment, respectively. MLT had an important anti apoptotic effect at day 5 post infection reducing de number of brain apoptotic cells in mice infected by EEV virus. This effect was time dependent. Similarly, the anti apoptotic effect of MLT was demonstrated in neurobastoma cell cultures at 4 and 6 hours of viral infection. The effect of MLT on microglia activation in viral infection was also observed. Anti-CD200 antibodies positive brain cells were detected in different days post infection. MLT decreased the microglial activation in the brain of infected mice. In conclusion, MLT has experimental therapeutic effect on VEE virus infection, but further clinical approaches has to be done to determine its clinical beneficial effects in humans.