Figure 2

GCSF is neuroprotective in vitro. Primary spinal cord neuronal culture expressed >90% neurons, majority of which were interneurons, positive for NeuN (A) while a small fraction of cells were motoneurons, positive for SMI-32 (B). When spinal cord neurons were treated with GCSF for 24 h, the Akt phosphorylation (pAkt) was increased (C). β-actin is shown as a loading control and the data was normalized to β-actin control in quantification. GCSF increased pAkt as quantified from western blot data (C, p <0.05, n = 3). The molecular weight of pAkt and β-actin are 60 and 42 kDa, respectively. When spinal cord neurons were exposed to glutamate, GCSF protected the neurons from glutamate excitotoxicity (D, p <0.001, n = 18-20). GCSF did not have any effect of neuron survival in control conditions (D, n = 14). When spinal cord neuron culture was prepared from mutant SOD1 mice, we discovered that the general cell viability was slightly reduced in mutant SOD1 neurons (E, p <0.001, n = 16 embryos). GCSF increased the cell viability in mutant SOD1 neurons (E, p <0.05, n = 16 embryos). The scale bar is 50 μm.