Neuropeptide Y protects ganglion cells against excitotoxicity and modulates microglia activation in the rat retina

Abstract

Purpose: Neuropeptide Y (NPY) exerts neuroprotective effects in CNS. Evidence also shows that NPY is a regulator of inflammation. Microglia activation has been associated with several retinal degenerative diseases, including glaucoma, characterized by apoptosis of retinal ganglion cells (RGCs). Thus, neuroprotective and anti-inflammatory strategies aiming to rescue RGCs have been investigated. We evaluated the potential protective effect of NPY and NPY receptor agonists against RGC death induced by excitotoxicity and assessed if the manipulation of NPY Y1 receptor can prevent retinal microglia activation.

Methods: The protective effect of NPY was assessed in cultured retinal explants using propidium iodide (PI) and TUNEL assays. PI+ cells were counted in the RGC layer at DIV2 (before NMDA incubation) and DIV4. TUNEL+ cells were counted at DIV4. The iNOS-ir was quantified in microglial cells in retinal explants. The levels of IL-1β and TNF-α were quantified by ELISA in culture medium of retinal explants.

Results: Exposure to NMDA for 48 h increased the PI+ cells ratio (DIV4/DIV2). Pre-treatment with 1 μM NPY decreased PI+ cells ratio and the co-exposure with 1 µM Y1 or Y5 receptor antagonists (BIBP 3226 and L-152,804, respectively) abolished the protective effect of NPY. Pre-treatment with 1 µM Y1 ([Leu31, Pro34]-NPY, LP-NPY) and Y5 receptor agonists ([Gly1,…,Aib32]-NPY) also inhibited the toxic effect of NMDA. Similarly, NMDA increased the number of TUNEL+ cells in RGC layer, which was inhibited by NPY. Co-incubation with Y1 or Y5 receptor antagonists prevented the protective effect of NPY. Retinal microglial cells express NPY and NPY Y1, Y2 and Y5 receptors. Exposure to LPS for 24h increased iNOS-ir in microglial cells in retinal explants. Pre-treatment with LP-NPY inhibited the increase in iNOS-ir. Treatment with BIBP 3226 abolished the effect of LP-NPY. LPS also increased the concentration of IL-1β and TNF-α in culture medium. Pre-treatment with LP-NPY inhibited the increase in IL-1β and TNF-α, and BIBP 3226 prevented the effect of LP-NPY.

Conclusions: NPY exerted a neuroprotective effect against RGC death triggered by NMDA, involving Y1 and Y5 receptors. NPY Y1 receptor activation induced an anti-inflammatory effect in retinal microglial cells. Thus, NPY system can be targeted as a strategy to delay the progression of retinal degenerative diseases with a neuroinflammatory component.

Keywords: 695 retinal degenerations: cell biology • 531 ganglion cells • 699 retinal glia