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. 2018 Mar 15;8:4641. doi: 10.1038/s41598-018-22848-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Tnf deficiency does not prevent RGC death after excitotoxic insult. To determine if TNF is required for NMDA induced RGC death, 2 μl PBS or 20 mM NMDA was injected into the vitreous of wildtype (Tnf^+/+) and Tnf deficient (Tnf^−/−) mice. The number of surviving RGCs (TUJ1 + cells) were counted 6 hours (hr) and 7 days (d) after intravitreal NMDA injection (n = 5 for 6 hr PBS Tnf^+/+, n = 5 for 6 hr NMDA Tnf^+/+, n = 5 for 6hr PBS Tnf^−/−, n = 5 for 6hr NMDA Tnf^−/−, n = 6 for 7d PBS Tnf^+/+, n = 7 for 7d NMDA Tnf^+/+, n = 5 for 7d PBS Tnf^−/−, n = 5 for 7d NMDA Tnf^−/−). (A) Representative images from flat mounted retinas stained with anti-TUJ1 show clear RGC loss in both wildtype and Tnf deficient mice after NMDA insult at both time points examined. (B) Quantification of TUJ1+ RGCs showed a significant loss of TUJ1 + cells in both wildtype and Tnf deficient mice at both 6 hours and 7 days after injury (*p < 0.001 for PBS to NMDA comparisons, P = 0.9694 comparing WT to Tnf^−/− NMDA). Note, there was no difference in RGC number between Tnf wildtype and deficient mice at either time (p > 0.9 for each time point). Scale bar: 50 μm.