TNF-a, whether the observed RGC loss was due to the particular method of OHT induction that was used, whether the findings would generalize to other species, and whether RGC loss could be attenuated using clinically available treatments. Etanercept (EnbrelH) is a decoy receptor consisting of the ligand-binding domain of the TNF type II receptor and the Fc component of human immunoglobulin G1. Etanercept competitively inhibits the binding of free TNF-a and TNF-b to cell surface receptors, and is used clinically for rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, and psoriatic arthritis [21,22]. In rats with endotoxin-induced uveitis, subcutaneous injection of Etanercept reduced the level of TNF-a and decreased intraocular inflammation [23]. The aims in the present study were to examine the expression of TNF-a in a rat model of chronic OHT, determine the cellular localization of TNF-a, and evaluate whether Etanercept would decrease TNF-a levels and reduce optic nerve degeneration and RGC loss. Results Systemic Treatment with Etanercept does not Affect Intraocular Pressure We induced OHT in the right eyes of rats (n = 40) by cauterizing the episcleral vein, leaving the left eye as a control. Whereas the average IOP in the control eye was 14.460.3 mm Hg, IOP rose to 47.6612.7 mm Hg immediately after cauterization and remained elevated for the duration of the study in 80% (n = 32) of the eyes at 4 weeks after EVC; 12.5% (n = 5) fell into phthisis and 7.5% (n = 3) did not meet the criteria for successful OHT induction. In the OHT groups, Etanercept (0.3 or 1.0 mg/kg) or saline was injected intraperitoneally three times a week from the time of initial IOP elevation until the time of euthanasia. Over the next 4 weeks, IOP remained stable at approximately 2.2-fold above baseline in the group treated with 0.3 mg/kg Etanercept, 1.9 -fold in the group treated with 1.0 mg/ kg Etanercept, and 2-fold in the sham-operated control group; these values did not differ from one another (Kruskal-Wallis test, all P.0.05 at each time point: Fig. 1). Thus intraperitoneal injection of Etanercept had no effect on IOP. Detection of Etanercept in the Eye Etanercept is a recombinant chimeric protein generated by fusing the ligand-binding portion of human TNF- a receptor TNFR-2 and the Fc portion of human IgG1. To investigate whether intraperitoneally injected Etanercept reaches the retina, we immunoprecipitated retinal lysates treated with Etanercept or normal saline with an antibody against human IgG1. The immunoprecipitate from animals treated with Etanercept con- tained one strong band at the appropriate size that was absent in immunoprecipitates from mice receiving normal saline (Fig. S1). TNF-a Levels Increase with OHT Previous studies have reported elevated levels of TNF-a in the retina and ONH of glaucoma patients [11,12,17,24]. We investigated whether this elevation could be detected in the present rat model by measuring TNF-a levels by ELISA in retinal lysates. The ELISA results are normalized by the level of protein per retina. ELISA showed that the level of TNF-a increased 3.9- fold three days after OHT induction relative to sham-operated control rats and continued to rise. Seven days after OHT induction, TNF-a levels were 1.060.5 pg/mg, approximately 17 times higher than in sham-operated control rats. Levels declined thereafter but remained elevated for at least 4 weeks. Treatment with 0.3 mg/kg of Etanercept reduced the level of TNF-a 7 days Etanercept Prevents Glaucomatous Neurodegeneration Figure 1. Etanercept does not affect intraocular pressure (IOP) after episcleral vein cauterization (EVC). EVC increases IOP over a 4-week period, and Etanercept does not block this increase at either 0.3 or 1.0 mg/kg (n = 10 rats for each time point). doi:10.1371/journal.pone.0040065.g001 after treatment initiation (Mann-Whitney U test, P = 0.001), and the higher dose (1.0 mg/kg) led to an even stronger reduction (Mann-Whitney U test, P,0.001: Fig. 2A). Western blot analysis confirmed the elevation of TNF-a 14 days after the induction of OHT and the reduction in TNF-a after Etanercept treatment (0.3 mg/kg, Fig. 2B). Cellular Source of TNF-a To identify the source of TNF-a, we carried out immunostain- ing on retinal whole-mounts. Immunostaining with the Iba-1 antibody revealed differences in the appearance of microglia in the ONH area 5 days after OHT induction. Microglial activation is characterized by morphological changes such as swelling of the cell body, thickening of the proximal processes, and a reduction of distal ramifications [25]. In control eyes with sham surgery, most Iba-1-positive cells were stage 1 resting microglia, with very few stage 3 and 4 cells around the ONH (28.362.6 cells/mm2: Fig. 3A). With OHT, we observed increased numbers of stage 3 and 4 microglia with round-shaped cell bodies and thick, stout processes or no processes at all around the ONH (167.8610.3 2 cells/mm , Mann-Whitney U test, P,0.001: Fig. 3B). Etanercept treatment resulted in a 53% decrease in the number of infiltrative stage 3 and 4 microglia around the ONH compared to untreated 2 OHT rats (7869 cells/mm , Mann-Whitney U test, P,0.001: Fig. 3C, D). We also carried out confocal microscopy on tissue double- labeled with antibodies against microglia (Iba-1) and astrocytes (GFAP). In sham-operated (control) retinal whole-mounts, we observed resting microglia with ramified processes distributed in a PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e40065