Topical WIN 55 212-2 Confers Long-Term Intraocular Pressure–Independent Neuroprotection in the DBA/2J Mouse Model of Glaucoma

IF 4.6 Q1 OPHTHALMOLOGY

Ophthalmology science Pub Date : 2025-08-19 DOI:10.1016/j.xops.2025.100918

Gabriele Gallo Afflitto MD , Tsung-Han Chou PhD , Mascha Louisa Korsch PhD , Francesco Aiello MD, PhD , Annagrazia Adornetto PhD , Rossella Russo PhD , Giacinto Bagetta MD , Carlo Nucci MD, PhD , Vittorio Porciatti DSc

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Abstract

Objective

To evaluate the neuroprotective efficacy of topical WIN 55 212-2 (WIN) in the DBA/2J mouse model of chronic glaucoma.

Design

Preclinical controlled study.

Subjects

Ninety 6-month-old DBA/2J mice (180 eyes) grouped in Untreated (UN), WIN 1%, or WIN 1% + rimonabant (RIM).

Methods

In vivo recordings were performed at 6 (T6), 8 (T8), and 10 (T10) months. Two broken-stick generalized estimating equation models were fitted: model 1 treated Intraocular Pressure (IOP) as a covariate; model 2 treated IOP as an explicit predictor. Retinal lysates underwent Western blotting (LC3-II, p62, Parkin, Optineurin, RNA-binding protein with multiple splicing (RBPMS), α-spectrin breakdown products [SBDPs]), and untargeted proteomics for exploratory mechanistic granularity assessment.

Main Outcome Measures

Pattern electroretinogram (PERG), IOP, flash ERG (FERG), and photopic negative response amplitudes as well as ganglion cell complex (GCC) thickness.

Results

At T8, IOP was 11.5 ± 1.4 mmHg in WIN compared to 22.0 ± 2.0 mmHg in UN and 21.7 ± 2.3 mmHg in RIM (P < 0.001). Similar results were observed at T10 (WIN: 19.4 ± 3.0 mmHg; UN: 23.4 ± 2.0 mmHg; RIM: 22.7 ± 3.2 mmHg) (P < 0.001). Statistically significant differences in PERG amplitude were observed among groups at both T8 and T10. In model 1, WIN-treated eyes demonstrated a 6.1 μV higher PERG amplitude at T10 (P < 0.001). Model 2 showed no significant WIN×IOP×T10 interaction (P = 0.757), suggesting that WIN-mediated protection is largely independent of IOP. Time-dependent decline in FERG was similar among groups. Photopic negative response amplitudes and GCC thickness decreased in all groups, with significant intergroup differences favoring WIN at both T8 and T10 (both P < 0.001). Molecularly, WIN reduced LC3-II by 35% at T10 without p62 accumulation, doubled Parkin and lowered Optineurin at T8, and markedly blunted RBPMS downregulation and SBDP accumulation (SBDP-120 kDa: –65% at T10). Proteomics identified 15 downregulated proteins in WIN retinas associated with relief of lysosomal antiprotease and Ca²⁺-stress pathways and enhanced autophagy–mitophagy flux.

Conclusions

Topical WIN 1% transiently lowers IOP, preserves retinal ganglion cell function, and attenuates structural and molecular degeneration in a seemingly cannabinoid receptor 1–dependent and IOP-independent fashion, thus representing a promising neuroprotective strategy for glaucoma.

Financial Disclosure(s)

Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

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外用WIN 55 212-2对DBA/2J型青光眼小鼠模型具有长期眼压不依赖性神经保护作用

目的评价外用WIN 55 212-2 （WIN）对DBA/2J型慢性青光眼小鼠的神经保护作用。设计临床前对照研究。实验对象96只6月龄DBA/2J小鼠（180只眼），分为未治疗组（UN）、WIN 1%组和WIN 1%组+利莫那班组（RIM）。方法分别于6个月（T6）、8个月（T8）和10个月（T10）进行体内记录。拟合两个断棒广义估计方程模型：模型1以眼内压（IOP）作为协变量；模型2将IOP作为显式预测因子。视网膜裂解物采用Western blotting （LC3-II, p62, Parkin, optinurin， rna结合蛋白与多重剪接（RBPMS）， α-谱蛋白分解产物[sbdp]）和非靶向蛋白质组学进行探索性机制粒度评估。主要观察指标：视网膜电图（PERG）、眼内压（IOP）、闪速ERG （FERG）、光负反应幅度以及神经节细胞复合体（GCC）厚度。结果T8时，WIN组IOP为11.5±1.4 mmHg， UN组为22.0±2.0 mmHg， RIM组为21.7±2.3 mmHg （P < 0.001）。T10组也有类似的结果（WIN: 19.4±3.0 mmHg; UN: 23.4±2.0 mmHg; RIM: 22.7±3.2 mmHg）（P < 0.001）。T8、T10组间PERG幅度差异均有统计学意义。在模型1中，win处理的眼睛在T10时的PERG幅度高6.1 μV （P < 0.001）。模型2没有显示出显著的WIN×IOP×T10交互作用（P = 0.757），表明win介导的保护在很大程度上独立于IOP。各组间FERG随时间的下降相似。所有组的光负反应幅度和GCC厚度均下降，在T8和T10时，组间差异均有利于WIN （P < 0.001）。从分子上看，WIN在T10时使LC3-II降低35%，无p62积累，在T8时使Parkin加倍并降低optinurin，并显著减弱RBPMS下调和SBDP积累（SBDP-120 kDa: T10时-65%）。蛋白质组学鉴定了WIN视网膜中15个下调蛋白，这些蛋白与溶酶体抗蛋白酶和Ca2+应激途径的缓解以及自噬-有丝自噬通量的增强有关。结论临床上1%的WIN可短暂降低IOP，保持视网膜神经节细胞功能，并以一种看似依赖于大麻素受体1和眼压独立的方式减弱结构和分子变性，因此是一种很有前景的青光眼神经保护策略。财务披露专有或商业披露可在本文末尾的脚注和披露中找到。

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