PCR products yielded fragments smaller than 150-bp length. Real-time PCR (ABI prism 7700 detection system, PE

Applied Biosystems, Foster City, CA) was performed using Brilliant III Ultra-Fast SYBR® Green qPCR master mix (Agilent Technologies, Santa Clara, CA, USA). We previously fixed the optimal concentration of the cDNA to be used as template for each gene analysis to obtain reliable CT (threshold cycle) values for quantification. Four samples were used per condition and each sample was run in triplicate. The thermal cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C Inhibitors,research,lifescience,medical for 15 sec, 60°C for 1 min. CT values were obtained and analyzed with the ABI prism 7700 SDS Software. Fold change in gene expression was estimated using the CT comparative method (2−ΔΔCT) Inhibitors,research,lifescience,medical normalizing to Gapdh CT values and relative to the average of control samples. Melting curves confirmed amplification of solely one PCR product for all qPCRs. Statistical analysis Data are expressed as the mean ± SEM. Comparisons between

groups of mice of different ages were made Inhibitors,research,lifescience,medical by one-way analysis of variance (ANOVA) with post hoc Dunnett’s multiple comparison test for IHC analysis using Lumacaftor nmr GraphPad Prism 5.01 software. For qPCR analysis, it was used a nonparametric Mann–Whitney test. Statistical significance was set at P < 0.05. The number of analyzed MNs and number of animals are indicated in the results section, as well in the figure legends. Results ChAT immunoreactivity Inhibitors,research,lifescience,medical In the WT mice at all ages analyzed, normal ChAT expression was located in the perikaryon, nucleus and processes as well as in presynaptic terminals apposed onto MNs at the ventral horn of the spinal cord. We also observed ChAT within cholinergic interneurons placed Inhibitors,research,lifescience,medical around the central canal (lamina X) and extended to the lateral

edge in the gray matter. When analyzing its temporal expression, we observed a transient reduction in CHAT immunoreactivity within the soma of MNs in transgenic mice carrying Edoxaban the mutation G93A in SOD1 gene (SOD1G93A) compared with the WT littermates (Fig. 1). We analyzed separately lumbar and thoracic segments as this mouse model is known to present a progressive caudal to rostral degeneration of the MNs (Gurney et al. 1994). We found that ChAT immunoreactivity was significantly decreased in ventral MNs at 1 month of age but close to normal at 2 and 3 months (Fig. 1A–C). This reduction was observed in practically all MNs located either lateral or medially in the ventral horn at different thoracic (decrease of 80 ± 2% at 1 month, n = 13–31) and lumbar (69 ± 3%, n = 13–64) levels.