Mice were killed at specific time points by CO2 asphyxiation. Whole brains were dissected in block from the skull, and spinal cords were harvested by extrusion. Using low-power binocular magnification, tissues were dissected on a chilled surface. Brain tissue was separated into cortex, cerebellum, and brainstem. Dissected tissue was then quick frozen in liquid nitrogen and stored at −80°C until use. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Valencia, California, USA) according to the manufacturer's instructions, its purity and concentration were determined spectrophotometrically as described previously for brain and spinal cord samples [26]. cDNA was synthesized from total RNA using random hexamer priming and a First Strand cDNA Synthesis Kit (Invitrogen, Carlsbad, California, USA). Briefly, 1 μg of total RNA was mixed with 4 μl of 10x reverse transcription buffer, 8 μl of 25 mmol/l MgCl2, 4 μl 0.1mol/l dithiothreitol, 1 μl RNasin, 2 μl SSII (50 u/μl), 5 μl hexomers and RNase-free water to 40 μl. Incubation was then carried out at 42°C for 60 min followed by heat inactivation at 70°C. Finally, 1 μl of
