Etanercept Prevents Glaucomatous Neurodegeneration Figure 2. Etanercept suppresses TNF-a elevation following IOP elevation. A, ELISA for TNF-a protein in the retina. Mann-Whitney U test, *P,0.05, **P,0.01 comparing sham-operated control and vehicle-treated OHT; {P,0.05, {{P,0.01 comparing OHT rats treated with vehicle vs. Etanercept (n = 6 for each group at each time point). B, Western blot analysis of TNF-a 14 days after OHT induction with and without Etanercept (n = 4 for each group). OHT, Ocular hypertension; Etan., Etanercept. doi:10.1371/journal.pone.0040065.g002 mosaic pattern around the ONH, with very few amoeboid-shaped cells and minimal GFAP expression (Fig. 4C, C9). Five days after OHT induction, increased numbers of microglia were found around the ONH along with increased expression of the astrocyte marker, GFAP (Fig. 4F). Iba-1-positive microglia were mostly amoeboid-shaped and were double-stained with antibodies to TNF-a (Fig. 4F9). Etanercept prevented the infiltration of amoeboid-shaped microglia and GFP expression. In addition, few Iba-1-positive microglia showed TNF-a staining in Etaner- cept-treated eyes (Fig. 4I9). Etanercept Prevents Pathologic Changes in the Optic Nerve We next compared axon morphology and density in cross- sections through the optic nerve. After 28 days of elevated IOP, the nerve showed marked axonal disorder compared to sham- operated controls, with a heterogeneous caliber of myelinated axons, increased vacuolization, and ballooning (Fig. 5B). Etanercept prevented this degeneration, resulting in an appear- ance similar to that of the control nerves (Fig. 5C). In terms of total axons, the axon density in sham-operated control nerves was 62,553612,966 axons/mm2, and this number decreased by 40.2% in OHT eyes (Mann-Whitney U test, P,0.001). Etanercept (0.3 mg/kg) maintained axon density at considerably higher levels than were seen in saline-treated controls (Mann- Whitney U test, P = 0.001: Fig. 4D) with only minor degener- ative changes. We next examined the morphology of axons by transmission electron microscopy (TEM). The normal sham- operated optic nerve shows a compact arrangement of myelinated axons (Fig. 6A, D). With OHT, the major change was the appearance of swollen axons (Fig. 6B, arrowhead) with occasional cytoskeletal disintegration (Fig. 6E, asterisk) and loosely organized myelin with multilayered, whorled masses (Fig. 6B, arrow). Axons in the optic nerves of Etanercept-treated OHT rats appeared nearly normal (Fig. 6C, F). PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e40065