Resumen de 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

Milagros del Valle Montiel Aguilar

• español

  El virus de la encefalitis equina venezolana (EEV) afecta el SNC en humanos y équidos provocando las encefalitis equinas. La melatonina (MLT) posee efecto protector ante las infecciones virales, y su influencia sobre la apoptosis y activación de la microglía ha aumentado su interés como factor en la patogénesis viral. El presente estudio pretende evaluar el efecto de la MLT sobre la apoptosis y activación de la microglia en modelos experimentales de la infección por EEV. Los ensayos in vivo fueron realizados con ratones machos albinos tratados con 500 [my]g MLT/Kg de peso e infectado con 10 dl50 del virus de EEV, siguiendo tres esquemas de tratamiento con MLT: preventivo y precoz, precoz, y tardío. Los animales se sacrificaron (n=5) a diferentes días post infección (P.I.) por cada grupo experimental. Se recolectó sangre completa, para determinación de igm anti EEV sérico. Y se les extrajo el cerebro para determinar la activación de la microglia utilizando anticuerpo monoclonal anti cd-200 de ratón, y se detectó apoptosis por túnel. Para los ensayos in vitro, se utilizaron células de neuroblastoma múrido (na2) tratadas con MLT (0,1mm; 0,5mm y 1,0mm), las cuales se infectaron con EEV (1x10-6 ufp/ml) por 2, 4 y 6 h, y posterior determinación de apoptosis por túnel. Se demostró que el tratamiento preventivo y mantenido con MLT así como el tratamiento precoz, logran un efecto benéfico en los ratones infectados por EEV con significativa reducción de la mortalidad al 7mo día de seguimiento. Ambas modalidades de tratamiento con MLT consiguen mantener un porcentaje significativo de supervivencia del 25%. Se demostró el efecto anti-apoptótico de la MLT en la infección por EEV, al observar al 5to día P.I. en los cerebros de ratones infectados por el virus un aumento en el número de células apoptóticas; y, disminución cuando fueron tratados con 500 [my]g/Kg de MLT. La MLT disminuyó la activación de la microglía en cerebro de ratones infectados por EEV, evidenciada por la expresión de anticuerpos anti- cd200. Se concluye, que la MLT posee un efecto terapéutico sobre la infección por EEV, cuya importancia clínica deberá establecerse en futuros trabajos.

• English

  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.