Caracterización bioquímica del nervio óptico en el ratón que sobreexpresa el gen p53. Análisis de estrés oxidativo / Biochemical characterization of the optic nerve in mice overexpressing the P53 gen. Oxidative stress assays

Objetivos: El gen supresor tumoral p53 detiene la proliferación y la muerte celular por apoptosis subsecuente a la acción de diversos factores, entre ellos el estrés oxidativo. La proteína p53 es fundamentalmente un regulador de la expresión génica. Utilizando ratones genéticamente manipulados para presentar una copia extra del gen p53 (transgénicos super p53) frente a ratones controles, hemos investigado el estado oxidativo y antioxidante en los nervios ópticos, en relación a p53. Método: Se han utilizado ratones de la cepa C57BL/6 de 12 meses de edad en dos grupos: 1) grupo super p53 (Sp53) y 2) grupo de controles wild-type (GC). Los ratones fueron anestesiados en atmósfera de éter, extrayendo los globos oculares y nervios ópticos que se lavaron en PBS, manteniendo las muestras en nitrógeno líquido y en congelador de –85ºC hasta su procesamiento. Se homogeneizaron 3-4 nervios ópticos por cada eppendorf, clasificando por grupos y determinando mediante métodos enzimático-colorimétricos la actividad traperoxidativa y actividad antioxidante total y la concentración de oxido nítrico. Resultados: Existe aumento significativo en la formación de radicales libres via peroxidación lipídica (p<0,001), de la actividad antioxidante (p<0,001) y síntesis de óxido nítrico (p<0,05) en los nervios ópticos de los ratones transgénicos super p53, frente a los ratones controles. Conclusiones: La presencia de una copia extra del gen p53 está ligada a modificaciones de la actividad redox en el nervio óptico del ratón, sugiriendo que p53 otorga una mayor resistencia a la agresión oxidativa. Valoramos la utilización de este modelo de ratón transgénico en procesos neurodegenerativos relacionados con el estrés oxidativo y la inducción de la apoptosis, como la neuropatía óptica glaucomatosa o la degeneración macular asociada a la edad

Purpose: The tumour inhibitor p53 gene has the ability of triggering proliferation arrest and cellular death by apoptosis subsequent to several factors, among them oxidative stress. The p53 protein is a major regulator of gene expression. Using genetically manipulated mice carrying an extra copy of gene p53 (transgenic mice super p53) versus control mice, we have investigated the generation of reactive oxygen species and antioxidant activity in the optic nerve of mice in relation to p53 availability. Methods: We studied two groups of 12-month-old mice of the strain C57BL/6: 1) super p53 group (Sp53) and 2) wild-type control group (CG). Mice were anesthetized in ether atmosphere and the eyeball and retrobulbar optic nerves were excised, washed, soaked in PBS, and stored in liquid nitrogen at –85ºC until processing. Three-four optic nerves from the same group were placed in an eppendorf tube, homogenized and enzymatic-colorimetric methods used to determine oxidative and antioxidant activities and the nitric oxide synthesis. Results: A significant increase in free radical formation (via lipid peroxidation; p<0.001), antioxidant activity (p<0.001) and nitric oxide synthesis (p<0.001) was found in the optic nerves from transgenic super p53 mice compared to respective controls. Conclusion: The presence of an extra copy of the p53 gene correlated with redox status in the mouse optic nerve. This transgenic mouse could be useful as an experimental model to study cell resistance to neurodegenerative processes in relation to oxidative stress and to apoptosis induction, such as glaucomatous optic neuropathy or age-related macular degeneration

Strain-dependent effects of morphine injected into the periaqueductal gray area of mice

Microinjection of morphine (0.31,1.25 and 5.0 *g) into the periaqueductal gray area (PAG) of C57BL/6 (C57) and DBA/2 (DBA) mice increased the pain threshold in the tail-flick test. The highest dose also caused a behavioral reaction in both strains characterized by periods of immobility laternating with explosive motor behavior. In the DBA strain, the analgesic effect was demonstrated with all doses of morphine,with in the C57 strain only the highest dose induced analgesia. DBA mice presented a decrease in activity with the lowest dose of morphine, whereas in the C57 strain, this effect was obtained only with the highest dose of morphine. These data corroborate at the PAG level the results of other studies which have shown that central and peripheral injections of morphine procedure analgesia and alter motor activity in C57 and DBA strains. They also confirm that these two strains of mice present genotype-dependent differences in sensitivity to opioids as determined after injections of morphine into the PAG