Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany.
Department of Ophthalmology, University Hospital, Ludwig-Maximilians-University Munich, Germany.
Mol Vis. 2020 Mar 5;26:135-149. eCollection 2020.
To analyze whether activation of endogenous wingless (Wnt)/β-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage.
Transgenic mice with a tamoxifen-dependent β-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal β-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed.
Following NMDA injection of wild-type mice, a statistically significant increase in retinal β-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific β-catenin deficiency. Furthermore, in mice with a β-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a β-catenin deficiency in Müller cells.
Endogenous Wnt/β-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.
分析 Müller 细胞中内源性 Wnt/β-连环蛋白信号的激活是否参与兴奋性损伤后视网膜神经节细胞 (RGC) 的保护。
通过玻璃体腔注射 N-甲基-D-天冬氨酸 (NMDA) 诱导一只眼的 RGC 兴奋性损伤，同时对另一只眼仅给予 PBS，建立 Müller 细胞依赖于他莫昔芬的β-连环蛋白缺陷的转基因小鼠模型，以研究其对 RGC 的影响。通过视神经横切片中 RGC 轴突总数的计数和子午线切片中视网膜各层厚度的测量来量化视网膜损伤。然后，通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记 (TUNEL) 测定法来识别两种基因型视网膜中的凋亡细胞。通过 Western blot 分析评估视网膜β-连环蛋白的水平，通过实时 RT-PCR 定量神经保护因子的视网膜表达。
与 PBS 注射对照组相比，野生型小鼠 NMDA 注射后视网膜β-连环蛋白蛋白水平明显升高，而 Müller 细胞特异性β-连环蛋白缺陷小鼠的这一效应被阻断。此外，在 Müller 细胞中β-连环蛋白缺陷的小鼠中，与 NMDA 处理对照组相比，NMDA 注射导致 RGC 轴突数量明显减少，以及 RGC 层中 TUNEL 阳性细胞显著增加。此外，在对照小鼠的视网膜中，检测到 NMDA 介导的白血病抑制因子 (Lif) mRNA 的统计学显著诱导，而在 Müller 细胞中β-连环蛋白缺陷的小鼠中，这一效应明显降低。
Müller 细胞中的内源性 Wnt/β-连环蛋白信号通过诱导神经保护因子（如 Lif）来保护 RGC 免受兴奋性损伤，这种效应很可能是通过这种机制介导的。
