Purpose

Dry eye disease (DED) is characterized by loss of tear film stability that becomes self-sustaining in a vicious cycle of pathophysiological events. Currently, desiccation stress (DS) is the dominant procedure for inducing DED in mice, however its’ effect on limbal epithelial stem cells (LESCs) has been overlooked. This study aimed to establish a DS model via the use of a novel hardware to investigate the impact on the ocular surface including LESCs.

Methods

A mouse transporter unit was customized to generate a dehumidified environment. C57BL/6J mice were exposed to mild DS and injected with scopolamine hydrobromide (SH) or remained untreated (UT) under standard vivarium conditions for 10 consecutive days (n = 28/group). Clinical assessments included phenol red tear-thread test, fluorescein staining and optical coherence tomography assessments. Histopathological and immunofluorescence was used to evaluate tissue architecture, goblet cell (GC) status, lacrimal gland (LG) inflammation and epithelial phenotype on the ocular surface. Whole flat-mounted corneas were immunostained for keratin-14 (K14), then imaged by confocal microscopy and analyzed computationally to investigate the effect of DS on LESCs.

Results

Custom modifications made to the animal transporter unit resulted in dehumidified cage relative humidity (RH) of 43.5 ± 4.79% compared to the vivarium 53.9 ± 1.8% (p = 0.0243). Under these conditions, aqueous tear production in mice was suppressed whilst corneal permeability and corneal irregularity significantly increased. H&E staining indicated stressed corneal basal epithelial cells and increased desquamation. DS-exposed mice had reduced GC density (41.0 ± 5.10 GC/mm vs 46.9 ± 3.88 GC/mm, p = 0.0482) and LGs from these mice exhibited elevated CD4⁺ cell infiltration compared to controls. DS elicited K14⁺ epithelial cell displacement, as indicated by increased fluorescence signal at a distance of 50–100 μm radially inwards from the limbus [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p = 0.0317].

Conclusions

Application of mild DS using customized hardware and SH injections generated features of DED in mice. Following DS, ocular surface epithelial cell health decreased and LESCs appeared stressed. This suggested that potential downstream effects of DS on corneal homeostasis are present, a phenomenon that is currently under-investigated. The method used to induce DED in this study enables the development of a chronic model which more closely resembles disease seen in the clinic.

中文翻译：

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使用定制工程干燥系统诱发干眼病：对眼表的影响，包括角蛋白 14 阳性角膜缘上皮干细胞

目的

干眼病 (DED) 的特征是泪膜稳定性丧失，在病理生理事件的恶性循环中变得自我维持。目前，干燥应激 (DS) 是诱导小鼠 DED 的主要方法，但其对角膜缘上皮干细胞 (LESC) 的影响已被忽视。本研究旨在通过使用新型硬件建立 DS 模型，以研究对包括 LESC 在内的眼表的影响。

方法

鼠标转运单元被定制以产生除湿环境。C57BL/6J 小鼠暴露于轻度 DS 并注射氢溴酸东莨菪碱 (SH) 或在标准动物饲养条件下连续 10 天保持未治疗 (UT)（n = 28 / 组）。临床评估包括酚红泪线测试、荧光素染色和光学相干断层扫描评估。组织病理学和免疫荧光用于评估组织结构、杯状细胞 (GC) 状态、泪腺 (LG) 炎症和眼表上皮表型。对整个平面角膜进行角蛋白 14 (K14) 免疫染色，然后通过共聚焦显微镜成像并进行计算分析以研究 DS 对 LESC 的影响。

结果

对动物转运装置进行的自定义修改导致除湿笼相对湿度 (RH) 为 43.5 ± 4.79%，而动物饲养箱为 53.9 ± 1.8% ( p  = 0.0243)。在这些条件下，小鼠的水性泪液产生受到抑制，而角膜渗透性和角膜不规则性显着增加。H&E 染色表明角膜基底上皮细胞受压和脱屑增加。DS 暴露小鼠的 GC 密度降低（41.0 ± 5.10 GC/mm 与 46.9 ± 3.88 GC/mm，p  = 0.0482），与对照相比，这些小鼠的 LG 表现出升高的 CD4 ⁺细胞浸润。DS引出K14 ⁺上皮细胞位移，如从边缘径向向内 50-100 μm 距离处荧光信号增加所表明的 [0.63 ± 0.053% (DS) vs 0.54 ± 0.060% (UT), p  = 0.0317]。

结论

使用定制硬件和 SH 注射应用轻度 DS 会在小鼠中产生 DED 的特征。DS 后，眼表上皮细胞健康下降，LESC 出现压力。这表明 DS 对角膜稳态的潜在下游影响是存在的，这一现象目前正在研究中。本研究中用于诱导 DED 的方法能够开发一种更类似于临床中所见疾病的慢性模型。