Figure 7. Retinal neuronal monocyte chemoattractant protein-1 (MCP-1) induced by advanced glycation end products (AGEs) stimulates tumor necrosis factor-α (TNF-α) expression in rat microglia via the p38, extracellular signal-regulated kinase (ERK), and nuclear factor-κB (NF-κB) pathways, but not the c-Jun N-terminal kinase (JNK) pathway. A: Real-time PCR was used to measure MCP-1 mRNA expression. B: Enzyme-linked immunosorbent assay (ELISA) was used to measure the soluble MCP-1 concentration. C, D, E: To exclude the effects of MCP-1 and/or TNF-α from the microglia exposed to AGEs in the Transwell apparatus, the primary cultured retinal neurons were pretreated with AGEs (750 μg/ml) in the culture medium for 24 h, then washed with PBS three times and removed AGEs, followed by coculture with the previously described isolated microglia in the Transwell apparatus for another 24 h. C: Real-time PCR was used to measure TNF-α mRNA expression. D: ELISA was used to measure the soluble TNF-α concentration (*p<0.05). E: The levels of phospho-p38, phospho-extracellular signal-regulated kinase (ERK), and the p50 and p65 subunits of NF-κB from the microglial cells increased. However, the levels of phospho-p38, phospho-ERK, and the p50 and p65 subunits of NF-κB from the microglial cells decreased accompanied by CC-chemokine receptor 2 (CCR2) knockdown to retinal microglia in the Transwell culture system with western blotting. Phospho-c-Jun N-terminal kinase (JNK) levels from the microglial cells remained unchanged over the entire experimental period in the retinal neuron–microglia Transwell culture system, and CCR2 knockdown did not lead to downregulation of the phospho-JNK levels.