Etanercept Prevents Glaucomatous Neurodegeneration Figure 6. Etanercept prevents axon degeneration after IOP elevation. A, D, Electron micrograph showing normal compact axoplasm and myelination in sham-treated control optic nerve. B, E, Axonal swelling with separation of myelin sheath (black arrowhead), degraded collection of altered tubulovesicular structures (E, asterisk), and a whorl-shaped mass (C, black arrow). C, F, Normal-appearing axons in OHT rat treated with Etanercept. Original magnification: 63400 (A–C), 65800 (D–F); Scale bar, 5 mm. OHT, Ocular hypertension; Etan., Etanercept. doi:10.1371/journal.pone.0040065.g006 gathering toward the ONH after OHT induction, in contrast to the mosaic distribution of ramified ‘‘resting’’ microglia seen in sham-operated control retinas (Figs. 3A, 4A). It is possible that elevated IOP with subsequent hypoxia may break down the blood- retina-barrier at the ONH [37–39] or cause local stress at the ONH [40], eliciting a strong inflammatory and glial response. Our colocalization studies indicate that Iba-1-positive microglia located around the ONH are the likely source of TNF-a seen after OHT (Fig. 4D–F). In Alzheimer’s disease (AD), microglia are activated at sites of neuronal degeneration [41]. Co-culture of amyloid b protein with activated microglia produces TNF-a [42] and chronic neuronal TNF-a expression in an AD mouse model, and results in neuronal cell death [43], suggesting that microglia are the source of TNF-a. However, the source of TNF-a in the eye has been controversial. Although it is widely acknowledged that increased IOP can trigger an inflammatory response involving the ONH area [11,20,44], various reports have suggested that glial cells [8,45], astrocytes [46] and M u¨ller cells [47] are the source of TNF- a. Here, OHT induction resulted in extensive microglia and astrocyte activation as visualized with Iba-1 and GFAP staining, respectively. However, TNF-a colocalized only with microglia, and not with either astrocytes or M u¨ller cells (data not shown) 5 days after EVC. Since TNF-a was seen to be elevated from day 3 onwards after OHT induction, it is possible that astrocytes and Mu¨ ller cells may express cytokines and chemokines at an earlier point that lead to microglial activation and further enhancement of the neural injury. Microglial activation and clustering at the ONH and central retina have been shown to be early events, rather than late consequences, of RGC degeneration in another animal model of glaucoma [48]. The morphology of Iba-1 positive microglia changes to an activated phenotype as early as 3 days after IOP elevation [49], and our results of elevated expression of TNF-a on or before day 3 (Fig. 2B) implicates microglia as the major source of TNF-a expression after OHT. This is also supported by studies by Kreutz et al. [50] who demonstrated that in response to optic nerve crush injury, macrophages are recruited to the optic nerve and their phagocytic activity is accompanied by demyelination of RGC axons, which was abolished with the blockade of TNF-a signaling. Another recent study [51] showed that soluble TNF- a production from microglia is required for the death of co-cultured dopaminergic neurons, and that Etanercept could attenuate cell death without affecting other possible effects of microglia. In our previous study, we showed that the effect of TNF-a on RGC death is indirect and is dependent upon TNFR2- mediated activation of microglia [20]. In addition, other studies have shown that FasL, a TNF family member, is upregulated in PLoS ONE | www.plosone.org 7 July 2012 | Volume 7 | Issue 7 | e40065