Decreased Nicotinamide Adenine Dinucleotide (NAD) Level and the Effect of Exogenous NAD on Microglia Activation N TNF--Induced Optic Nerve Degeneration

Abstract

Purpose: : To examine the level of NAD and the distribution of nicotinamide mononucleotide adenylyltransferase1 (Nmnat1) in TNF-α-induced optic nerve degeneration. To evaluate the effect of NAD on activation of microglia induced by TNF-α.

Methods: : Rats were euthanized at 1 day, or 1 and 2 weeks after PBS or TNF-α (10 ng) intravitreal injection. In some animals, exogenous NAD was injected intravitreally 24 hr before TNF-α injection. Nmnat1 protein in the optic nerve and retina was evaluated by Western blot analysis and immunohistochemistry. The level of NAD was determined by HPLC analysis. Microglia activation was evaluated by Iba1- and ED1-immunopositive cell counting.

Results: : Western blot analysis showed a significant decrease in Nmnat1 protein levels in the optic nerve at 1 and 2 weeks after TNF-α injection. Although Nmnat1 protein levels in the retina tended to be decreased after TNF-α injection compared to PBS injection, these changes were not statistically significant. Double labeling immunohistochemical studies showed substantial co-localization of Nmnat1 and neurofilament in the optic nerve in normal subject. Moreover, most Nmnat1 positive cells were colocalized with Thy-1 positive cells in the retina, but not with GFAP. HPLC analysis demonstrated a significant decrease in NAD level in the optic nerve 2 weeks after TNF-α injection. Morphometric analysis of Iba1- and ED1-immunopositive cell showed a significant increase in these positive cells by TNF-α and that exogenous NAD significantly suppressed this activation.

Conclusions: : Nmnat1 is found in both the retinal ganglion cell body and axon. Decreases in Nmnat1 and NAD in axons occur in association with TNF-α induced optic nerve axonal degeneration. The prevention of axonal degeneration with NAD may be with a consequence of its inhibitory effect on microglia activation.

Keywords: neuro-ophthalmology: optic nerve • neuroprotection • microglia