New tools for the evaluation of toxic ocular surface changes in the rat
New tools for the evaluation of toxic ocular surface changes in the rat
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DOI：
10.1167/iovs.06-0728
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发表时间：
2007-12-01
影响因子：
4.4
通讯作者：
Baudouin, Christophe
中科院分区：
文献类型：
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作者：
Pauly, Aude;Brignole-Baudouin, Francoise;Baudouin, Christophe
目的。为了评估非侵入性联合技术在毒理学研究中观察眼表变化的有效性，方法将0.01%、0.1%、0.25%和0.5%的苯扎氯铵(BAC)分别滴入大鼠角膜上11天。从第0天到第52天对眼进行大体评估。用裂隙灯检查角膜，进行荧光素试验，并在体内使用共聚焦显微镜计算角膜厚度，计算角膜上皮和内皮缺损的评分，并对角膜基质炎症和新生血管进行定量。分别取结膜印迹和刷状细胞学标本进行MUC-5AC抗体标记和流式细胞仪亚G1峰分析。结果肉眼和裂隙灯检查仅在0.25%和0.5%BAC组有眼刺激迹象，但体内共聚焦显微镜显示0.01%和0.1%BAC组角膜上皮存在缺陷，亚G1峰分析显示所有浓度的BAC组在D8和D11均有细胞凋亡增加。0.25%和0.5%的BAC可导致角膜厚度增加、杯状细胞丢失、可逆性角膜炎症和持续性新生血管形成。结论：结膜刷的亚G1峰分析，结合体内角膜共聚焦显微镜和结膜印迹的免疫标记，构成了一种非侵入性、可靠和敏感的工具，除了标准的临床评估外，还可以在广泛的浓度范围内(包括最低浓度)评估有毒药物引起的大鼠眼表损伤。这项研究与旨在减少动物使用和完善动物毒理学模型的国际战略是一致的。
PURPOSE. To assess the usefulness of noninvasive combined technologies used to observe ocular surface changes in toxicology studies.METHODS. Benzalkonium chloride (BAC) at 0.01%, 0.1%, 0.25%, and 0.5% was applied to rat corneas for 11 days. The eye was evaluated macroscopically from day (D)0 to D52. The cornea was examined with the slit lamp, a fluorescein test was performed, and a confocal microscope was used in vivo to calculate corneal thickness, score corneal epithelial and endothelial defects, and quantify corneal stromal inflammation and neovascularization. Conjunctival impression and brush cytology specimens were taken for labeling with MUC-5AC antibodies and sub-G1 peak analysis by flow cytometry, respectively. Histologic analyses were performed on D11.RESULTS. Although macroscopic and slit lamp examinations revealed signs of ocular irritation in the 0.25% and 0.5% BAC-treated eyes only, in vivo confocal microscopy revealed epithelial defects in the 0.01% and 0.1% BAC-treated corneas, and sub-G1 peak analyses showed increased apoptosis for all the BAC concentrations on D8 and D11. BAC at 0.25% and 0.5% induced increased corneal thickness, loss of goblet cells, reversible corneal inflammation, and persistent neovascularization.CONCLUSIONS. Sub-G1 peak analysis of conjunctival brushings, in conjunction with in vivo confocal microscopy of the cornea and immunolabeling of conjunctival imprints, constitutes a noninvasive, reliable, and sensitive tool to evaluate toxic drug-induced ocular surface damage in rats, in addition to standard clinical assessments and at a wide range of concentrations, including the lowest ones. This study is consistent with the international strategy aimed at reducing the use of animals and refining animal toxicologic models.