Neuroprotective Effect of PACAP Against NMDA-Induced Retinal Damage in the Mouse

Abstract

Retinal excitotoxicity is one of the major causes of retinal ganglion cell (RGC) death in glaucoma. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic peptide with potent neuroprotective activity; however, whether it exerts such an effect in the retina and the mechanism by which RGCs are protected is still not well understood. In this study, we examined the effect of exogenous and endogenous PACAP on RGC death induced by N-methyl-D: -aspartate acid (NMDA). The vitreous body of anesthetized adult male mice (C57/BL6J) was injected with NMDA (40 nmol in a 2 μL saline solution). The number of RGCs decreased from days 1 to 7 after NMDA injection, and the number of dUTP end-labeling (TUNEL)-positive cells, an indicator of cell death, peaked at day 3. However, when PACAP38 (10(-8), 10(-10), 10(-12), 10(-14), or 10(-16)M) was co-administered with NMDA, the 10(-10)M dose resulted in significantly increased RGC survival at day 7, and a decrease in the number of TUNEL-positive RGCs at day 3. We next investigated the neuroprotective effect of endogenous PACAP using PACAP heterozygote(+/-) mice. Under normal circumstances, there was no significant difference in the number of RGCs in the PACAP(+/-) mice compared with their wild-type counterparts. However, the number of RGCs significantly decreased in the PACAP(+/-) mice 7 days after NMDA injection, relative to their wild-type counterparts. The number of TUNEL-positive RGCs peaked at day 1 in the PACAP(+/-) mice. These effects in the PACAP(+/-) mice were reversed by intravitreous injection of 10(-10)M PACAP38. This suggests that exogenous PACAP is able to counteract NMDA-induced toxicity, and that endogenous PACAP exerts a neuroprotective effect in the retina.

Protective Effects of PACAP in the Retina

“…The authors found, in accordance with our results, that under normal circumstances, there was no difference in the number of retinal ganglion cells in heterozygous PACAP defi cient mice and wild-type counterparts [ 107 ]. However, when these mice were subjected to NMDA-induced toxicity, PACAP heterozygous mice reacted with increased loss of ganglion cells [ 107 ]. The number of apoptotic cells, as measured by TUNEL staining, peaked on day 1 in heterozygous mice, while the peak was on day 3 in wild types, suggesting that apoptotic cell death began earlier in mice defi cient in PACAP.…”

“…The higher vulnerability of PACAP defi cient mice against lesions was confi rmed by another study by Endo et al [ 107 ]. The authors found, in accordance with our results, that under normal circumstances, there was no difference in the number of retinal ganglion cells in heterozygous PACAP defi cient mice and wild-type counterparts [ 107 ].…”

“…These effects were reversed by simultaneous injection of PACAP in heterozygous mice. These results confi rm that endogenous PACAP is protective in the retina and prove that even partial loss of PACAP-driven neuroprotection decreases resistance against harmful effects [ 107 ]. Our recent, preliminary results also confi rm that endogenous PACAP is protective in lipopolysaccharide (LPS)-induced retinal infl ammation, as shown by the increased infl ammatory status of PACAP KO.…”

Pleiotropic and retinoprotective functions of PACAP

“…We found that the neuroprotective effect of PACAP in response to N-methyl-D-aspartate (NMDA) occurred with a single peak at a nanomolar concentration, leading us to speculate that the NMDA-induced neuronal injury was mediated by a simple mechanism, whereby Ca 2? flowed directly into the cells via NMDA receptor activity (Endo et al 2011). Based on electroretinographic measurements, PACAP has been shown to improve the functional outcome in the inner retinal layers and photoreceptors following excitotoxicity (Varga et al 2011).…”

The Neuropeptide Systems and their Potential Role in the Treatment of Mammalian Retinal Ischemia: A Developing Story

“…Moreover, recent observations in an ex vivo mouse model of retinal ischemia by adding 10 mM sodium azide to the culture medium show that 1 µM PACAP reduces cell death, glutamate release and oxidative stress (D. Cervia and G. Casini, unpublished data). Since retinal neuronal damage in ischemic conditions is likely to depend, at least in part, by excessive glutamate release, it is interesting to note that PACAP protects against NMDA induced retinal damage [59]. In general, PACAP acts by activating antiapoptotic and inhibiting proapoptotic signaling pathways in the retina [56].…”