Figure 6

Influence of microglia on Müller cell expression of adhesion molecules and adhesion properties. (A) Semi-quantitative RT-PCR comparing mRNA levels of adhesion molecules, VCAM-1 and ICAM-1, in Müller cells cultured alone (control, white bars), co-cultured with unactivated (gray bars) or activated (black bars) microglia. The mRNA levels of VCAM-1 were significantly elevated in Müller cells co-cultured with unactivated and activated microglia, compared with Müller cells cultured alone. ICAM-1 was significantly elevated only in Müller cells that were co-cultured with activated microglia. Representative gel images for genes whose expression were significantly changed following co-culture are shown (right). (B) Protein levels of adhesion molecules in Müller cell-conditioned media were measured with ELISA. Relatively elevated levels of VCAM-1 and ICAM-1 were found in conditioned media from Müller cells co-cultured with activated microglia. (C) The ability of Müller cells following co-culture to function as an adhesive substrate for microglia cells was assessed by a cell-adhesion assay. Unactivated microglia, labeled vitally with Calcein-AM (left panel, in green), were seeded onto Müller cells following 48-hr co-culture and allowed to adhere (right panel, Müller cells seen in bright-field). Non-adherent microglia were washed off and the remaining cells were counted and expressed as a fraction of total microglial cells added. Müller cells that had previously been co-cultured with activated microglia were found to induce the highest levels of adhesion, followed by Müller cells that had previously been co-cultured with unactivated microglia. (* indicates p < 0.05 for comparisons, one-way ANOVA with Tukey-Kramer multiple comparison test, n = 6-8 replicates from two independent experiments).