Figure 3

Müller cell expression of growth factors and neuroprotective function following microglial co-culture. (A) Semi-quantitative RT-PCR comparing mRNA levels of growth factors in Müller cells following microglial co-culture. Expression of growth factors GDNF and LIF were significantly elevated in Müller cells co-cultured with activated microglia. Expression levels of NGF, bFGF, and BDNF were similar between the three groups, while CNTF levels were slightly reduced. Representative gel images for genes whose expression were significantly changed following co-culture are shown (right). (B) ELISA quantification of protein levels of GDNF and LIF in the conditioned media of Müller cells following microglial co-culture. Relatively elevated levels of these growth factors were found in conditioned media from Müller cells co-cultured with activated microglia. (C, D) Neuroprotective function of Müller cells following microglial co-culture were evaluated by assessing the ability of conditioned media from co-cultured Müller cells to rescue photoreceptor cells (661W cells) from H₂0₂-induced oxidative cell death. Following Müller cell-microglia co-culture, microglia-containing cell inserts were removed, fresh medium was added to the resulting Müller cell cultures and left to condition for 24 hours. These conditioned media were added to 661W cells in the presence of 0.1 M H₂0₂. 661W cells exposed to 0.1 M H₂0₂ in regular unconditioned media served as controls. Cell viability of H₂0₂-exposed 661W cells were evaluated with a tetrazolium-based cell counting Kit-8 assay (in C) and a differential cell-staining (Live/Dead) assay (in D). Müller cell-media from all co-culture conditions exerted significant neuroprotective effect relative to unconditioned medium (marked by * over individual bars) but that from Müller cells co-cultured with activated microglia exerted a greater neuroprotective effect relative to Müller cells cultured without microglia. (E) Neuroprotective effect of exogenous GDNF and LIF on photoreceptor cells undergoing oxidative stress. GDNF and LIF (100 pg/ml and 500 pg/ml) were added to 661W cells in the presence of 0.1 M H₂0₂. 661W cells exposed to 0.1 M H₂0₂ in regular unconditioned media served as a control. Additions of GDNF and LIF were able to significantly increase 661W cell survival as evaluated with a cell counting Kit-8 assay. No significant dose-dependent effect was observed in the range of concentrations used. (* indicates p < 0.05 for comparisons, one-way ANOVA with Tukey-Kramer multiple comparison test, n = 6-8 replicates from two independent experiments).