Fig. 3

Astrocytes secrete increased levels of glutamate, ROS, and NO in response to ALS-CSF and impart neurotoxicity to NSC-34 cells. a–c Graphs representing the up-regulation in the glutamate synthesis and secretion by astrocytes. Glutamate secretion was up-regulated in the astroglial supernatants 48 h after the exposure to ALS-CSF as compared to NC (*p < 0.05 NC v/s ALS; n = 5 in duplicates; graph a). Also, note the up-regulated expression in the cellular glutamate (**p < 0.01 NC v/s ALS; n = 5 in duplicates; graph b). Taken together, the total increase in the astroglial glutamate levels is evident as plotted in graph c (**p < 0.01 NC v/s ALS; n = 5 in duplicates). Similarly, the secreted (**p < 0.01 NC v/s ALS 48 h, n = 5 in duplicates, graph d), cellular (**p < 0.01 NC v/s ALS, n = 5 in duplicates, graph e), and total expression levels (***p < 0.001 NC v/s ALS; n = 5 in duplicates; graph f) of NO showed up-regulation in response to a 48-h exposure to ALS-CSF as compared to NC. The confocal images g, h (CY3, red) represent the expression patterns of iNOS in the NC and ALS subsets, respectively. Note the enhanced expression of iNOS in ALS group, as determined by the MFI (***p < 0.001 NC v/s ALS; n = 5 in duplicates, graph k). Also note the increased mRNA expression of iNOS in response to the ALS-CSF as compared to the normal controls (**p < 0.01 NC v/s ALS; n = 3 in triplicates; graph l). Representative confocal images for the cellular levels of (DCFDA, green, i, j) as well as quantitative fluorometric analysis (graph m) denote an increased ROS expression in ALS subsets as compared to the NC (***p < 0.001 NC v/s ALS; n = 6 in triplicates). Analysis of significance was carried out using Student’s t test. Scale bars are indicated