CNC - Center for Neuroscience and Cell Biology, University of Coimbra; Largo Marquês de Pombal, Coimbra, Portugal.
Cell Death Dis. 2013 May 16;4(5):e636. doi: 10.1038/cddis.2013.160.
It has been claimed that glutamate excitotoxicity might have a role in the pathogenesis of several retinal degenerative diseases, including glaucoma and diabetic retinopathy. Neuropeptide Y (NPY) has neuroprotective properties against excitotoxicity in the hippocampus, through the activation of Y1, Y2 and/or Y5 receptors. The principal objective of this study is to investigate the potential protective role of NPY against glutamate-induced toxicity in rat retinal cells (in vitro and in an animal model), unraveling the NPY receptors and intracellular mechanisms involved. Rat retinal neural cell cultures were prepared from newborn Wistar rats (P3-P5) and exposed to glutamate (500 μM) for 24 h. Necrotic cell death was evaluated by propidium iodide (PI) assay and apoptotic cell death using TUNEL and caspase-3 assays. The cell types present in culture were identified by immunocytochemistry. The involvement of NPY receptors was assessed using selective agonists and antagonists. Pre-treatment of cells with NPY (100 nM) inhibited both necrotic cell death (PI-positive cells) and apoptotic cell death (TUNEL-positive cells and caspase 3-positive cells) triggered by glutamate, with the neurons being the cells most strongly affected. The activation of NPY Y2, Y4 and Y5 receptors inhibited necrotic cell death, while apoptotic cell death was only prevented by the activation of NPY Y5 receptor. Moreover, NPY neuroprotective effect was mediated by the activation of PKA and p38K. In the animal model, NPY (2.35 nmol) was intravitreally injected 2 h before glutamate (500 nmol) injection into the vitreous. The protective role of NPY was assessed 24 h after glutamate (or saline) injection by TUNEL assay and Brn3a (marker of ganglion cells) immunohistochemistry. NPY inhibited the increase in the number of TUNEL-positive cells and the decrease in the number of Brn3a-positive cells induced by glutamate. In conclusion, NPY and NPY receptors can be considered potential targets to treat retinal degenerative diseases, such as glaucoma and diabetic retinopathy.
有人声称，谷氨酸兴奋性毒性可能在几种视网膜退行性疾病的发病机制中起作用，包括青光眼和糖尿病性视网膜病变。神经肽 Y（NPY）通过激活 Y1、Y2 和/或 Y5 受体，对海马中的兴奋性毒性具有神经保护作用。本研究的主要目的是研究 NPY 对大鼠视网膜细胞（体外和动物模型）中谷氨酸诱导的毒性的潜在保护作用，阐明所涉及的 NPY 受体和细胞内机制。从小鼠（P3-P5）中制备视网膜神经细胞培养物，并将其暴露于谷氨酸（500μM）24 小时。通过碘化丙啶（PI）测定法评估坏死性细胞死亡，通过 TUNEL 和 caspase-3 测定法评估凋亡性细胞死亡。通过免疫细胞化学鉴定培养物中存在的细胞类型。使用选择性激动剂和拮抗剂评估 NPY 受体的参与。用 NPY（100nM）预处理细胞可抑制谷氨酸触发的坏死性细胞死亡（PI 阳性细胞）和凋亡性细胞死亡（TUNEL 阳性细胞和 caspase-3 阳性细胞），神经元是受影响最严重的细胞。NPY Y2、Y4 和 Y5 受体的激活抑制了坏死性细胞死亡，而仅激活 NPY Y5 受体可预防凋亡性细胞死亡。此外，NPY 的神经保护作用是通过激活 PKA 和 p38K 介导的。在动物模型中，在向玻璃体中注射谷氨酸（500nmol）前 2 小时，向玻璃体中注射 NPY（2.35nmol）。通过 TUNEL 测定法和 Brn3a（神经节细胞标志物）免疫组织化学评估 NPY 抑制谷氨酸（或生理盐水）注射后 24 小时的保护作用。NPY 抑制了 TUNEL 阳性细胞数量的增加和 Brn3a 阳性细胞数量的减少。总之，NPY 和 NPY 受体可被视为治疗青光眼和糖尿病性视网膜病变等视网膜退行性疾病的潜在靶点。
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