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Endoplasmic reticulum stress is involved in retinal injury induced by repeated transient spikes of intraocular pressure

内质网应激参与反复瞬时高眼压诱导的视网膜损伤

Abstract

Clinically, a large proportion of glaucoma patients undergo repeated intraocular pressure (IOP) spike (Spike IOP) attacks during their sleep, which may facilitate retinopathy. In this study, we established a mouse model of repeated transient Spike IOP to investigate the direct damage to the retina following Spike IOP attacks, and elucidated the underlying molecular mechanism. We analyzed the changes in the number of retinal ganglion cells (RGCs) via immunofluorescence. Thereafter, we detected retinal cell apoptosis via terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) staining, and performed RNA sequencing (RNA-seq) to reveal the underlying molecular mechanism. Finally, we validated the expression of key molecules in the endoplasmic reticulum (ER) stress pathway using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis. Results revealed a time-dependent RGC loss in Spike IOP, evidenced by a reduction in the number of Brn3a-positive RGCs in experimental eyes following a 7-d continuous treatment with Spike IOP. In addition, TUNEL staining indicated that apoptosis of retinal cells started in the outer nuclear layer (ONL), and then spread to the ganglion cell layer (GCL) with time. RNA-seq analysis revealed that ER stress might be involved in Spike IOP-induced retinal injury. This result was corroborated by western blot, which revealed upregulation of ER stress-related proteins including binding immunoglobulin protein/glucose-regulated protein 78 (BiP/GRP78), phosphorylated inositol-requiring enzyme 1 (p-IRE1), unspliced X-box-binding protein 1 (XBP1-u), spliced X-box-binding protein 1 (XBP1-s), phosphorylated c-Jun N-terminal kinase (p-JNK), C/EBP-homologous protein (CHOP), and B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax). These findings indicate that repeated IOP transients are detrimental to the retina, while ER stress plays an important role in retinal cell apoptosis in this situation. Notably, repeated Spike IOP among glaucoma patients is a crucial factor for progressive retinopathy.

概要

目的

研究和探讨反复瞬时高眼压对视网膜的损伤作用及其潜在的分子机制.

创新点

模拟临床上青光眼患者夜间难以检测到的眼压峰值波动对视网膜的影响, 首次证明反复的瞬时高眼压对视网膜具有直接损伤效应, 其损伤机制与内质网应激通路激活有关.

方法

通过眼前房生理盐水灌注, 建立小鼠反复瞬时高眼压模型(50 mmHg, 1 min× 7次), 连续灌注处理1、 3和7天后, 采用视网膜铺片(Whole-mount retina)评估视网膜神经节细胞(RGC)损伤情况; 用TUNEL法检测视网膜全层细胞凋亡; 用转录组测序(RNA-seq)筛选参与视网膜损伤的分子通路; 用实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白免疫印迹(Western blot)进一步检测内质网应激通路相关分子的表达.

结论

反复瞬时高眼压可以损伤视网膜全层, 并呈时间依赖性的由外核层细胞(ONL)死亡进展至视网膜神经节细胞层(GCL)死亡. 内质网应激相关信号通路中肌醇酶1(IRE1)信号通路激活参与了视网膜的损伤过程.

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Acknowledgments

This work was supported by the Guangzhou Science and Technology Plan Project (Nos. 201803040020, 201903010065, and 202102021099), the Guangdong Natural Science Foundation (No. 2020A151501168), and the Research Funds of the State Key Laboratory of Ophthalmology (No. PT1001022), China.

Author information

Affiliations

Authors

Contributions

Xue YANG performed this study, collected the data, and drafted the manuscript. Zhenni ZHAO, Jiamin ZHANG, and Xiaoqian SU assisted in carrying out the research. Xiaowei YU and Yuqing HE assisted in analyzing the data. Nannan SUN and Zhigang FAN conceived the study and proofread the manuscript. All authors have read and approved the final manuscript and, therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.

Corresponding authors

Correspondence to Nannan Sun or Zhigang Fan.

Ethics declarations

Xue YANG, Xiaowei YU, Zhenni ZHAO, Yuqing HE, Jiamin ZHANG, Xiaoqian SU, Nannan SUN, and Zhigang FAN declare that they have no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed.

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Yang, X., Yu, X., Zhao, Z. et al. Endoplasmic reticulum stress is involved in retinal injury induced by repeated transient spikes of intraocular pressure. J. Zhejiang Univ. Sci. B 22, 746–756 (2021). https://doi.org/10.1631/jzus.B2100053

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Key words

  • Endoplasmic reticulum (ER) stress
  • Intraocular pressure spike (Spike IOP)
  • Retinal injury
  • Neuron apoptosis
  • Glaucoma

关键词

  • 内质网应激
  • 瞬时眼压峰值(Spike IOP)
  • 视网膜损伤
  • 视网膜神经细胞凋亡
  • 青光眼