一种新型脂多糖诱导的斑马鱼葡萄膜炎模型。

A novel uveitis model induced by lipopolysaccharide in zebrafish.

机构信息

Henan International Joint Research Laboratory for Ocular Immunology and Retinal Injury Repair, Henan Province Eye Hospital, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.

The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology and Chongqing Eye Institute, Chongqing Branch (Municipality Division) of National Clinical Research Center for Ocular Diseases, Chongqing, China.

出版信息

Front Immunol. 2022 Dec 1;13:1042849. doi: 10.3389/fimmu.2022.1042849. eCollection 2022.

DOI:10.3389/fimmu.2022.1042849

PMID:36532084

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9751191/

Abstract

OBJECTIVE

Endotoxin-induced uveitis (EIU) is an important tool for human uveitis study. This study was designed to develop a novel EIU model in zebrafish.

METHODS

An EIU model in zebrafish was induced by intravitreal lipopolysaccharide (LPS) injection and was assessed dynamically. Optical coherence tomography (OCT) was used to assess infiltrating cells in the vitreous body. The histological changes wereevaluated using HE staining and immune cells were measured by immunofluorescence. The retinal RNA Sequencing (RNA-Seq) was used to explore the transcriptional changes during inflammation. RNA-Seq data were analyzed using time-course sequencing data analysis (TCseq), ClueGO plugin in Cytoscape, and Gene Set Enrichment Analysis (GSEA) software. Flow cytometry and retinal flat mounts were used to dynamically quantify the immune cells.

RESULTS

EIU was successfully induced in zebrafish following intravitreal LPS injection. Inflammation appeared at 4 hours post injection (hpi), reached its peak at 24 hpi, and then resolved at 72 hpi. Immunofluorescence confirmed that massive influx ofneutrophils into the iris and vitreous body, and activation of microglia as evidenced by ameboid-shaped appearance in the retina. Retinal RNA-seq during the EIU course identified four gene clusters with distinct expression characteristics related to Toll-likereceptor signaling pathway, cytokine-cytokine receptor interaction, NOD-like receptor signaling pathway, and extracellular matrix (ECM)-receptor interaction, respectively. Prednisone immersion inhibited the inflammatory response of EIU in zebrafish, whichwas confirmed by decreased neutrophils detected in flow cytometry and retinal flat mounts.

CONCLUSIONS

We developed a novel EIU model in zebrafish, which may be particularly useful for gene-editing and high-throughput screening of new drugs for the prevention and treatment of uveitis.

摘要

目的

内毒素诱导的葡萄膜炎（EIU）是研究人类葡萄膜炎的重要工具。本研究旨在建立一种新型斑马鱼 EIU 模型。

方法

通过玻璃体内注射脂多糖（LPS）诱导斑马鱼 EIU 模型，并进行动态评估。光学相干断层扫描（OCT）用于评估玻璃体浸润细胞。采用 HE 染色评估组织学变化，免疫荧光法检测免疫细胞。采用视网膜 RNA 测序（RNA-Seq）探讨炎症过程中的转录变化。使用时间序列测序数据分析（TCseq）、Cytoscape 中的 ClueGO 插件和基因集富集分析（GSEA）软件分析 RNA-Seq 数据。采用流式细胞术和视网膜铺片动态定量免疫细胞。

结果

玻璃体内 LPS 注射后成功诱导斑马鱼 EIU。炎症在注射后 4 小时（hpi）出现，24 hpi 达到高峰，72 hpi 缓解。免疫荧光证实大量中性粒细胞涌入虹膜和玻璃体，并激活小胶质细胞，表现为视网膜中阿米巴样外观。EIU 过程中的视网膜 RNA-seq 鉴定出四个具有不同表达特征的基因簇，分别与 Toll 样受体信号通路、细胞因子-细胞因子受体相互作用、NOD 样受体信号通路和细胞外基质（ECM）-受体相互作用有关。泼尼松浸浴抑制了斑马鱼 EIU 的炎症反应，这通过流式细胞术和视网膜铺片中检测到的中性粒细胞减少得到证实。