Fig. 3

Microglial activation was predominantly neurotoxic. Microglial NO (a ***p < 0.001; n = 5 in duplicates) and ROS (b–b”, c *p < 0.05, NC vs. ALS; ^## p < 0.01, NALS vs. ALS; n = 5 in triplicates) levels were upregulated on exposure to ALS-CSF when compared to NC. iNOS mRNA (d ***p < 0.001; n = 5 in triplicates) and protein (e–e”, f **p < 0.01, NC vs. ALS; ^### p < 0.001, NALS vs. ALS; n = 5 in duplicates) expression was upregulated in a similar manner. However, arginase mRNA (g ***p < 0.001; n = 3 in triplicates) and protein (h–h”, i *p < 0.05; n = 5 in duplicates) expression was downregulated in the ALS group as compared to NC. A comparison between the iNOS and arginase protein expression clearly demonstrated a ratio skewed towards more pro-inflammatory microglial cells in the ALS group as compared to the controls (j **p < 0.01; n = 5 in duplicates). Compare the morphology of the NSC-34 cells grown in the conditioned media from the microglial cultures exposed to the control and ALS-CSF groups (ALS-MCM). Note the excessive vacuolation (k’ white arrowhead) and clumping (k” white arrow) in response to the ALS-MCM. MTT reduction assay clearly showed reduced cell viability in response to the ALS-MCM (l ***p < 0.001; n = 5 in triplicates). Statistical analysis was carried out using one-way ANOVA followed by Tukey’s post hoc test