microglia in the glaucomatous retina [52], and that the membrane-bound form of FasL acts as a key effector of RGC death downstream from TNF- a elevation [53]. Another finding in our study was the early dramatic decrease in retinal levels of NF-L and NF-M 14 days after OHT induction (Fig. 7), which preceded the loss of RCGs at day 28 (Fig. 8). These findings are consistent with earlier studies showing that axonal dysfunction precedes neuronal loss [54]. The effect of TNF-a inhibition in maintaining near-normal levels of neurofilament and RGCs in rats with OHT suggests that Etanercept may protect axons and secondarily RGC somata even in the presence of persistent ocular hypertension. In conclusion, our results suggest that elevation of the pro- inflammatory cytokine TNF-a and ONH inflammation play an important role in linking elevated intraocular pressure to the loss of optic nerve axons and RGCs that are the hallmark features of glaucoma (Fig. 9). Lowering of IOP is currently the most effective clinical approach to reducing the progression of glaucomatous visual loss [55,56]. However, some patients continue to lose vision despite lowering of IOP, suggesting the need for adjunct treatments. The present results, supported by considerable evidence from other studies, suggest that Etanercept or other TNF- a antagonists or suppressors of inflammation should be considered as an adjunct therapy in the treatment of glaucoma. Materials and Methods Animals All animal experiments followed the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Animal Care Committee of The Massachusetts Eye and Ear Infirmary. Male Brown Norway rats (8–10 weeks) were purchased from Charles River Laboratories (Wilmington, ME). A total of 140 rats between 200 and 300 g weight were used. Animals were housed under a 12-h light/dark cycle with free access to water and food. For general anesthesia, a mixture of ketamine (100 mg/kg; Phoenix Scientific, St. Joseph, MO) and xylazine (10 mg/kg; Phoenix Scientific) was adminis- tered intramuscularly. Etanercept Prevents Glaucomatous Neurodegeneration Experimental Ocular Hypertension Chronic ocular hypertension (OHT) was induced in the right eye (total n = 140) by episcleral vein cauterization (EVC) according to published procedures with minor modifications [57,58]. Briefly, after animals were deeply anesthetized, an incision was made through the conjunctiva and Tenon’s capsule on the limbal periphery. The episcleral veins in the right eye were identified by their location in relation to the extraocular muscles; three of them (two dorsal veins and one temporal ventral vein) were cauterized with a hand-held ophthalmic cautery (Electric Eye Cautery, Rumex International Co., St. Petersburg, FL) under a surgical microscope. The left eye received sham surgery (conjunctival incisions without cauterization). Successful OHT was defined as intraocular pressure (IOP) elevation greater than 30% of baseline IOP and no cauterization-associated complications after EVC. Measurement of Intraocular Pressure IOP was measured in both eyes with the TonoLab tonometer TV02 (Helsinki, Finland) with rats under topical anesthesia with 0.5% proparacaine hydrochloride eye drops. IOP was measured before EVC, immediately after cauterization, and then on days 1, 7, 14, 21, and 28. The TonoLab acquires six valid rebounds of the device from the eye and takes the mean of the middle four readings to make one summary measurement. The instrument indicates the reliability of the reading by the position of a bar next to the reported value. A single set of readings was collected with the best reproducibility indicator (no bar) at each time point during the IOP measurements. Rats were excluded from further analysis if the peak IOP was ,6 mm Hg above baseline or if the treated eye developed corneal ulceration, hyphema, or other ocular pathology. In total, 13 rats were excluded based on these criteria. Treatment with a Soluble TNF-a Receptor Etanercept, a soluble 75 kDa TNF-a receptor/Fc fusion protein (EnbrelH, Amgen, Thousand Oaks, CA, USA), was reconstituted with sterile water according to the manufacturer’s instructions. Rats were separated into three groups. Starting one day after induction of chronic glaucoma, two groups (n = 10 each) were given subcutaneous injection of Etanercept at either 0.3 mg/kg or Figure 9. Schematic illustration of retinal ganglion cell (RGC) death following elevation of intraocular pressure (IOP). A, Ramified quiescent microglia in eyes with normal IOP. B, Elevated IOP leads to increased numbers of activated microglia with amoeboid morphology around the optic nerve head. These microglia appear to secrete TNF-a leading to RGC death. Other molecules, including FasL on microglia, nitric oxide (NO), and reactive oxygen species (ROS) may also play a role in RGC death. doi:10.1371/journal.pone.0040065.g009 PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e40065