Figure 4.

hμG-derived TNF-α and IL-1β influence neuritogenesis in and function of developing hRGCs

Schematic representation of the hμG-hRGCs co-culture to examine the effect of the former on hRGC generation, neurite growth, and function (A). Immunocytochemical analysis revealed no difference in the number of tdT⁺ and PAX6⁺ hRGCs, generated in three different culture conditions (control, hμG CM, and hμG CM with anti-TNF-α and anti-IL-1β antibodies) demonstrating that proinflammatory cytokines secreted by hμG have no effects on hRGC generation (B–E). Analysis of SMI32⁺ TAU1⁺ axons of hRGCs revealed a significant increase in the complexities and length (F–J) of the axons in the presence of hμG CM versus controls and their abrogation in antibody-neutralized hμG CM. Analysis of MEA recording at day 7 of hRGCs revealed a significant increase in the number of active electrodes (K) and mean firing rate (L); when cells are exposed to hμG CM versus controls, the effects significantly abrogated in antibody-neutralized hμG CM (K, L). Raster plots of representative hRGC recordings demonstrated the effects of the three different culture conditions on the spontaneous activities (M–O). Representative extracellular spike waveforms revealed the facilitatory effects of hμG CM on the neural function versus control, which is abrogated in antibody-neutralized hμG CM (M–O). Western blot analysis carried out on the cell lysates of hRGCs showed hμG CM influences the sodium channel (SCN2A) expression (P). SCN2A levels increased significantly in the presence of hμG CM versus controls, which is abrogated in antibody-neutralized hμG CM (Q). Experiments were carried out in triplicates per group from 3 to 4 independent biological replicates. Values expressed as mean ± SEM. Scale bar: 50 μm. MEA, microelectrode array.