Stimulation of the P2X₇ receptor kills rat retinal ganglion cells in vivo

Abstract

The P2X₇ receptor is associated with the death of many cell types, and growing evidence supports its presence on neurons. Activation of the P2X₇ receptor on isolated retinal ganglion cells increases intracellular calcium levels and can kill the cells. Within the intact eye, however, glia and other cell types surrounding the ganglion cells may provide protection and attenuate the effects of receptor stimulation. This investigation thus asks whether stimulation of the P2X₇ receptor can actually kill retinal ganglion cells in vivo. Drugs were injected intravitreally into the superior/nasal region of Long Evans rats. Cell survival was determined by counting the number of remaining ganglion cells labeled with aminostilbamidine. The P2X₇ receptor agonist BzATP reduced ganglion cell survival as compared to eyes injected with saline solution. Ganglion cell death was inhibited by co-injection of the P2X₇ antagonists Brilliant Blue G and MRS 2540. The loss of ganglion cells following activation of the P2X₇ receptor was also prevented by the adenosine A₃ adenosine receptor agonist MRS 3558. In conclusion, stimulation of the P2X₇ receptor can kill retinal ganglion cells in vivo. The neuroprotective effects of A₃ receptor activation identified in isolated ganglion cells are also apparent in vivo. This implies that the balance between extracellular ATP and its protective metabolite adenosine can influence ganglion cell survival in the living eye.

Introduction

The purines ATP and adenosine act as transmitters throughout the nervous system, with ATP stimulating metabotropic P2Y and ionotropic P2X receptors, while adenosine acts at metabotropic A₁, A_2A, A_2B or A₃ receptors (Ribeiro et al., 2002, Abbracchio et al., 2009). The dephosphorylation of ATP into adenosine by extracellular enzymes can consequently influence signaling (Zimmermann, 2000, Zuo et al., 2008), and retinal ganglion cells highlight a particularly extreme example of these interactions. Previous work has demonstrated that stimulation of the P2X₇ receptor killed isolated retinal ganglion cells in vitro (Zhang et al., 2005) while stimulation of the A₃ adenosine receptor enhanced their survival (Zhang et al., 2006b). In these isolated ganglion cells, the lethal action of P2X₇ receptor stimulation corresponded with elevations in cytoplasmic Ca²⁺, while A₃ receptor activation reduced the Ca²⁺ response.

While these in vitro experiments clearly demonstrated the lethal potential of P2X₇ receptors and the contrasting neuroprotective potential of adenosine A₃ receptors on isolated ganglion cells, it is not currently known whether these findings can be reproduced in an intact animal. The mechanical and chemical dissociation of neuronal cells leaves them considerably more fragile than they are in their native environment. Even the removal of the inner limiting membrane may decrease ganglion cell survival (Halfter et al., 2005). Moreover, retinal ganglion cell somata lie close to Müller and astrocytic glial cells in addition to other cells that can release protective compounds (Newman, 2003, Bringmann et al., 2009). It is thus possible that ganglion cells may be more resistant to stress in vivo than in vitro.

This study asked whether stimulation of the P2X₇ receptor kills retinal ganglion cells in vivo as it does in vitro. In addition, the ability of the A₃ adenosine receptor to prevent ganglion cell death in vivo was examined. We demonstrate that in vitro findings are relevant to the intact retina, and that the balance of extracellular purines can alter ganglion cell health in vivo.