Polyamines Exacerbate Excitotoxic Death of Adult Retinal Ganglion Cells in vivo

Abstract

Purpose: : The polyamines, putrescine, spermidine and spermine, are present in most eukaryotic cells and are involved in multiple biological processes including cell proliferation and survival. Polyamines are natural co–activators of N–methyl–D–aspartate (NMDA) receptors. Here, we tested if polyamines potentiate NMDA–induced retinal ganglion cell (RGC) death.

Methods: : The cellular source of polyamine production in the adult retina was investigated by immunocytochemistry and western blot analysis of Arginase I, the rate limiting enzyme for polyamine biosynthesis. For analysis of neuronal survival, RGCs were backlabeled with FluoroGold one week prior to intravitreal injection of: i) NMDA (5 µl: 5 mM) alone, or ii) NMDA in combination with putrescine (10 mM) or spermine (0.1 mM). To block polyamine action, we used the polyamine synthesis blocker di–fluoro–methyl–ornithine (DFMO, 10 mM, 100 mM). The density of surviving RGCs was quantified in 12 standard areas in flat–mounted retinas at 3 hrs, 6 hrs, 12 hrs, 24 hrs, 3 days or 7 days after intraocular injections.

Results: : Robust Arginase I immunostaining was found primarily in adult RGCs in the intact retina. Following NMDA injection, Arginase I immunostaining dramatically increased in Müller glial cells. Analysis of neuronal survival demonstrated that co–administration of putrescine or spermine with NMDA potentiated excitotoxic RGC death. For example, combination of putrescine or spermine with NMDA reduced the number of surviving RGCs by 29% and 20%, respectively, compared to eyes treated with NMDA alone. In contrast, administration of putrescine or spermine only did not cause RGC death, indicating that polyamines are not toxic by themselves. Importantly, blockade of polyamine synthesis with DFMO markedly reduced NMDA–induced RGC death at 3 days after injection: 900 RGCs/mm² survived in the presence of NMDA and DFMO, in contrast to 500 RGCs/mm² in control retinas treated with NMDA and saline.

Conclusions: : We demonstrate, for the first time, that polyamines exacerbate NMDA–induced RGC death in vivo. The finding that polyamine synthesis is strongly upregulated in Müller cells following excitotoxic damage suggests that these cells may play an important role in RGC death. Our study also shows that DFMO, a blocker of polyamine synthesis, can be used to protect RGCs from excitotoxic death.

Keywords: ganglion cells • excitatory neurotransmitters • neuroprotection