Fig. 4.

Activated microglia secrete factors that trigger retinal gliosis. Conditioned medium from microglial cells incubated with BMP7 or vehicle for 24 h was added to the medium of the retinal astrocytes, directly or pretreated with LDN193189 (c, d). RNA was isolated from these cells 24 h posttreatment and analyzed via RT-qPCR for a panel of gliosis markers. Statistically significant increase in levels of Gfap, Gs, S100-β, Pcan, Egfr, and Tlr4 was observed in astrocytes incubated with conditioned medium added directly or pretreated with LDN193189 (c, d). Cells treated with DMSO (carrier for LDN193189) or LDN only (a) or cells pretreated with DMSO and conditioned medium from BMP7 or vehicle-treated microglia (b) were used as experimental controls. Data shown in graphs represent relative expression levels of RNA in retinal astrocyte cells treated with LDN193189 relative to DMSO (a) or with conditioned media from BMP7-treated microglial cells relative to retinal astrocyte cells treated with conditioned media from vehicle-treated microglia (b–d). Bars above a level of 1.0 (solid black line) represent an increase in mRNA levels while bars below the level of 1.0 represent a decrease in mRNA levels relative to the corresponding vehicle control. Statistical analysis was performed by unpaired Student’s t test. *p value <0.05. Abbreviations: Egfr epidermal growth factor receptor, Gfap glial fibrillary acidic protein, Pcan phosphacan, Tlr toll like receptor