Neuropeptide Y induces neuroprotective effects and neurite outgrowth in rat retinal cell cultures

Abstract

Purpose : Neuropeptide Y (NPY) has been shown to exert neuroprotective effects in the brain and lately also in retinal tissue. We further explored the neuroprotective effects of NPY by studying actions of NPY on glutamate-induced toxicity in rat retinal cell cultures. In addition, we also for the first time examined effects of NPY on neurite outgrowth in retinal cells, a neuroplastic effect that might contribute to recovery of retinal cells after injury.

Methods : Rat retinal cell cultures were prepared from newborn Wistar rats (P3-P5). For neuroprotection cultures were cultivated for one week (n=11). Cell death was induced by addition of glutamate (300 μM) for 24h added 1h after pretreatment with NPY. To evaluate the specific NPY receptors involved in neuroprotection, cultures were pretreated with Y1, Y2, or Y5 receptor antagonists (n=5). Cell death was evaluated by a propidium iodide (PI) assay and calculated as a ratio of PI positive cells to total cell numbers. For neurite outgrowth, cultures were grown for 24h with NPY, immunostained with anti-GAP43 neuron-specific antibody and evaluated using specially developed software (n=4). Data were analyzed with GraphPad Prism software using repeated measures one-way ANOVA with Bonferroni-adjusted post-hoc tests.

Results : NPY induced neuroprotective effects in a dose-dependent manner, reaching significant levels at 0.3 and 1 μM NPY with maximal protection found with 1 μM (0.3 μM: p<0.05; 1 μM: p<0.01). This neuroprotective effect was inhibited by addition of selective NPY receptor antagonists and completely abolished by addition of all three antagonists (NPY vs NPY+Y1-Y2-Y5-antagonists: p<0.05). Addition of NPY antagonists suggested that the neuroprotective effect was mainly mediated via Y2 and Y5 receptors. [R1] Moreover, NPY induced neurite outgrowth at concentrations of 1 μM and 10 μM NPY with most pronounced effects seen at 10 μM (1 μM: p<0.05; 10 μM: p<0.05).

Conclusions : Consistent with previous work, we found that NPY exerts neuroprotective effects in rat retinal cell cultures predominantly mediated via Y2 and Y5 receptors. NPY was also for the first time found to stimulate neurite outgrowth in retinal cells. These neuroprotective and neuroplastic effects of NPY indicate that NPY could play a role as a neurotrophic and neuroprotective agent in future treatment of retinal diseases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.