BACKGROUND Dry eye disease (DED) is a multifactorial disease associated with ocular surface inflammation, pain, and nerve abnormalities. We studied the peripheral and central neuroinflammatory responses that occur during persistent DED using molecular, cellular, behavioral, and electrophysiological approaches. METHODS A mouse model of DED was obtained by unilateral excision of the extraorbital lachrymal gland (ELG) and Harderian gland (HG) of adult female C57BL/6 mice. In vivo tests were conducted at 7, 14, and 21 days (d) after surgery. Tear production was measured by a phenol red test and corneal alterations and inflammation were assessed by fluorescein staining and in vivo confocal microscopy. Corneal nerve morphology was evaluated by nerve staining. Mechanical corneal sensitivity was monitored using von Frey filaments. Multi-unit extracellular recording of ciliary nerve fiber activity was used to monitor spontaneous corneal nerve activity. RT-qPCR and immunostaining were used to determine RNA and protein levels at d21. RESULTS We observed a marked reduction of tear production and the development of corneal inflammation at d7, d14, and d21 post-surgery in DED animals. Chronic DE induced a reduction of intraepithelial corneal nerve terminals. Behavioral and electrophysiological studies showed that the DED animals developed time-dependent mechanical corneal hypersensitivity accompanied by increased spontaneous ciliary nerve fiber electrical activity. Consistent with these findings, DED mice exhibited central presynaptic plasticity, demonstrated by a higher Piccolo immunoreactivity in the ipsilateral trigeminal brainstem sensory complex (TBSC). At d21 post-surgery, mRNA levels of pro-inflammatory (IL-6 and IL-1β), astrocyte (GFAP), and oxidative (iNOS2 and NOX4) markers increased significantly in the ipsilateral trigeminal ganglion (TG). This correlated with an increase in Iba1, GFAP, and ATF3 immunostaining in the ipsilateral TG of DED animals. Furthermore, pro-inflammatory cytokines (IL-6, TNFα, IL-1β, and CCL2), iNOS2, neuronal (ATF3 and FOS), and microglial (CD68 and Itgam) markers were also upregulated in the TBSC of DED animals at d21, along with increased immunoreactivity against GFAP and Iba1. CONCLUSIONS Overall, these data highlight peripheral sensitization and neuroinflammatory responses that participate in the development and maintenance of dry eye-related pain. This model may be useful to identify new analgesic molecules to alleviate ocular pain.

中文翻译：

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慢性干眼在小鼠三叉神经干中诱发角膜超敏反应，神经炎症反应和突触可塑性。

背景技术干眼病（DED）是与眼表炎症，疼痛和神经异常有关的多因素疾病。我们使用分子，细胞，行为和电生理学方法研究了持续性DED期间发生的周围和中枢神经炎症反应。方法通过单侧切除成年雌性C57BL / 6小鼠的眶外泪腺（ELG）和哈德氏腺（HG），获得DED小鼠模型。手术后第7、14和21天（d）进行体内测试。通过酚红试验测量泪液产生，通过荧光素染色和体内共聚焦显微镜评估角膜改变和炎症。通过神经染色评估角膜神经形态。机械角膜敏感性使用冯·弗雷丝监测。睫状神经纤维活性的多单元细胞外记录用于监测自发性角膜神经活性。RT-qPCR和免疫染色用于确定d21时的RNA和蛋白质水平。结果我们观察到DED动物手术后d7，d14和d21时泪液产生和角膜发炎的明显减少。慢性DE引起上皮内角膜神经末梢的减少。行为和电生理研究表明，DED动物出现了时间依赖性的机械性角膜超敏反应，并伴有自发性睫状神经纤维电活动的增加。与这些发现一致，DED小鼠表现出中央突触前可塑性，这在同侧三叉神经干脑干感觉复合物（TBSC）中具有较高的短笛免疫反应性得到证实。在d21手术后，同侧三叉神经节（TG）中促炎性（IL-6和IL-1β），星形胶质细胞（GFAP）和氧化性标志物（iNOS2和NOX4）的mRNA水平显着增加。这与DED动物同侧TG中Iba1，GFAP和ATF3免疫染色的增加有关。此外，在第21天，DED动物的TBSC中的促炎细胞因子（IL-6，TNFα，IL-1β和CCL2），iNOS2，神经元（ATF3和FOS）和小胶质细胞（CD68和Itgam）标志物也被上调，以及针对GFAP和Iba1的增强的免疫反应性。结论总体而言，这些数据突出了外周敏感性和神经炎性反应，这些反应参与了干眼相关性疼痛的发生和维持。该模型对于识别减轻眼痛的新止痛剂分子可能有用。