2.10 RNA preparation and RT-PCR
Total RNA of brain tissues was isolated by using Trizol (Takara, Kusatsu, Japan) and then reverse-transcribed to cDNA using PrimeScriptTM RT Master Mix Kit (Takara). RT-PCR was conducted with SYBR Green master mixes (Takara) on Roche LightCycler480 System to measure mRNA levels of Trpm4 , interleukin (IL)-1β , tumor necrosisfactor-α (TNF-α) , inducible nitricoxide synthase (iNOS) , transforming growth factor-β (TGF-β) , Arg1 , IL-10 and glyceraldehyde phosphate dehydrogenase (Gapdh) . Relative changes of mRNA expressions were normalized to Gapdh [19].