Etanercept Prevents Glaucomatous Neurodegeneration with vehicle or Etanercept; {{P,0.01 comparing OHT cases treated with vehicle vs. Etanercept. Data are expressed as the mean 6 SEM (n = 6 for each group). Original magnification, 68 (A–C); Scale bar, 100 mm. OHT, ocular hypertension; Etan., Etanercept 1.0 mg/kg. doi:10.1371/journal.pone.0040065.g003 Neurofilament Expression Decreases with OHT To examine changes in proteins associated with the axonal cytoskeleton, we performed western blot analysis and immuno- histochemical staining to detect the light- and medium chains of neurofilaments (NF-L, NF-M, respectively). In the retina, both NF-L and NF-M antibodies stained axon fascicles around the ganglion cell layer (Fig. 7A). Protein analyses show that expression of both NF-L and NF-M decreased 14 days after induction of OHT, whereas Etanercept maintained the expression of both proteins close to normal levels (Fig. 7B–E). Etanercept Prevents Retinal Ganglion Cell Loss with OHT We next investigated whether elevation of IOP leads to the hallmark feature of glaucoma, a loss of RGCs, in the current model [26–28]. In the sham-operated control group, the number of RGCs visualized in retinal whole-mounts (Fig. 8A) that were co- 2 labeled with both TUJ1 and NeuN was 2004.4640.4 per mm , whereas the number in OHT retinas treated only with saline was 38% lower (Fig. 8B, Mann-Whitney U test, P,0.001). Etanercept at either 0.3 or 1.0 mg/kg prevented this decrease and maintained the number of RGC at near-normal levels (Fig. 8B, Mann- Whitney U test, P,0.001 for either dosage compared to saline- treated OHT controls). Discussion The current study confirms and extends previous findings linking retinal ganglion cell (RGC) loss in glaucoma to TNF-a. Our results demonstrate this link using a different method to induce ocular hypertension (OHT) and a different species than our earlier study, while for the first time identifying microglia at the optic nerve head (ONH) as the source of TNF-a. In addition, we show that Etanercept (EnbrelH), a widely used TNF-a antagonist, attenuates inflammation and RGC loss. In the present study, we elevated intraocular pressure (IOP) by episcleral vein cauterization (EVC), which led to axon degeneration in the optic nerve and eventual loss of RGCs. Elevated IOP caused levels of TNF-a to increase within 3 days, and by 7 days, levels were 17-fold higher than normal and remained elevated for at least 4 weeks. However, compared to proinflammatory eye disease such as experimental uveitis induced by lipopolysaccharide [29], the level of TNF-a is much lower in our model. Nevertheless, along with the increases of TNF-a in our studies, we observed a massive expansion of the macrophage/microglial population around the ONH after 5–7 days. This change is consistent with our previous proposal that the cytotoxic effect of OHT-induced TNF-a might be mediated by CD11b+ microglia in a mouse model of chronic OHT [20]. However, the previous study left several major questions unanswered. One limitation of that study was the use of laser-induced angle closure to elevate OHT, which could have caused an inflammatory response and an increase in proin- flammatory cytokines, including TNF-a, as a result of 100 confluent laser spots on the corneal limbus. By using three-point episcleral vein cauterization, we minimized the inflammatory effect associated with the model itself. Secondly, in the previous study we investigated the role of TNF-a using genetically altered mouse strains and immunodepletion. The present study brings us closer to clinical translation by showing that Etanercept, an agent that is already in wide clinical use for Figure 4. Confocal images of immunofluorescent staining for Iba-1 (green), TNF-a (red), and GFAP (cyan). Merged image demonstrates the colocalization of TNF-a and Iba-1 (C, F, I). Square insets show enlargements of selected cells (C’, F’, and I’). Original magnification: 610 (A–H), 663 (A9–H9); Scale bars: 100 mm at lower magnification, 25 mm at higher magnification. doi:10.1371/journal.pone.0040065.g004 PLoS ONE | www.plosone.org 5 July 2012 | Volume 7 | Issue 7 | e40065