Extracellular Hsp70 promotes RGC death by mediating production of cytotoxic levels of Tnf by glial cells. A) Recombinant Hsp70 contributes to RGC death in vitro. RGC/glia co-cultures were treated 24 hours with 5 and 20 μg/ml of recombinant Hsp70, and RGC death was detected by using the Apoptosis Assay Kit #2; (Invitrogen, United States). B) Inhibition of PKC activity in RGC/glia co-cultures with PMB helps to protect RGCs from neurotoxicity mediated by Hsp70. RGC/glia co-cultures were treated 24 hours with 20 μg/ml of Hsp70 in the presence of 10 μg/ml of PMB or PBS (control) and then RGC death was detected. C) Representative confocal images of beta III Tubulin-labeled RGCs in the GCLs (green) of flat-mounted retinas of eyes intravitreally injected with Hsp70 (1 μg per injection) and control eyes (vehicle treated; VT) were obtained seven days after treatment. D) The percent of RGC death in control and Hsp70-treated eyes of WT and Tlr4KO animals (**P < 0.01, n = 6). E) Glial cells treated with Hsp70 produce sufficient amounts of Tnf to initiate neuronal death. F) Treatment with Hsp70 increased RGC death in the RGC/glia co-cultures, while treatment with Hsp70 in the presence of Tnf inhibitor (XPro1595) resulted in RGC protection. Hsp70, heat shock protein 70; PMB, polymyxin B, PKC, protein kinase C; RGC, retinal ganglion cells.