Fig. 1

Microglial cells are activated in response to ALS-CSFCSF: When compared to NC (c,d) and NALS (a’, b’), note the increased number of amoeboid microglia upon exposure to ALS-CSF (a’’, b’’). Representative phase contrast (c–c””) and confocal images immunolabeled for Iba-1 (d–d””) of the different morphologies of the microglial cells in cultures. c and d represent the ramified microglial cells, while c’–c”’ and d’–d”’ the intermediate stages between ramified and amoeboid morphology. c”” and d’” represent the fully activated amoeboid micro glial cells. The number of amoeboid microglial cells (g, ****p <0.0001 NC and NALS vs. ALS; n=6 in triplicates), the soma area (h, ****p <0.0001 NC vs. ALS; n=5 in duplicates) and Iba-1 expression (i, **p <0.01 NC and NALS vs. ALS; n=5 in duplicates) was significantly higher in the ALS group as compared to the NALS and control groups. The microglial cells exposed to ALS-CSF showed an increase in the no. of viable cells as seen by the MTT reduction assay, while no significant change was observed in the secreted LDH levels (j and k, respectively). Apart from the amoeboid morphology, many cells also showed multinucleation (e’, e’’ white arrow). Nuclear labelling was carried out with TO-PRO (Blue, e’ f’). The arrows represent the nuclei present in a single microglial cell. Also, cells with the disintegrating cell membrane (cytorrhexis) were fairly evident in random fields at 48h of ALS-CSF exposure (e-f’’ white arrowheads). Scale bars are indicated. MFI=mean fluorescence intensity