Etanercept Prevents Glaucomatous Neurodegeneration Figure 7. Etanercept prevents the loss of cytoskeletal proteins following OHT induction. A, Immunohistochemical staining for neurofilament light chain (NF-L) and middle chain (NF-M) proteins around the ganglion cell layer. Western blot analysis for NF-L (B) and NF-M (C) after OHT in rats treated with or without Etanercept (n = 4 in each lane). Differences in loading were normalized by the level of b-actin. Bar graphs indicating relative levels of NF-L (D) and NF-M (E) to b-actin by densitometry, reflecting the results from four independent experiments. Mann- Whitney U test, **P,0.01 comparing sham-operated control and vehicle-treated OHT; {{P,0.01 comparing OHT rats treated with vehicle vs. Etanercept. Original magnification : 620, Scale bar, 100 mm. OHT, Ocular hypertension; Etan., Etanercept; NF-L, neurofilament-L; NF-M, neurofilament- M. doi:10.1371/journal.pone.0040065.g007 Figure 8. Etanercept prevents the loss of RGCs following OHT induction. A, Flat-mounted retina double immuno-labeled with TUJ-1 and NeuN antibodies to visualize RGCs and their axons. OHT resulted in a loss of RGCs (b) that was prevented with Etanercept (c, d). B, Bar graph showing quantitation of RGC survival. Data are expressed as mean 6 SEM; n = 10 at each time point. Kruskal-Wallis test, **P,0.01 comparing three groups (A), Mann-Whitney U test, *P,0.05, **P,0.01 comparing sham-operated control and vehicle-treated OHT; {{P,0.01 comparing OHT treated with vehicle vs. Etanercept. NeuN (red), b-tubulin (green), Original magnification: 620; Scale bar, 50 mm. OHT, ocular hypertension; Etan., Etanercept. doi:10.1371/journal.pone.0040065.g008 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e40065