作者感谢阿尔康为这些研究提供财政支持。

1. 人类角膜上皮细胞培养
<ol><li>生长不朽的HCEC19在胶原蛋白涂层75厘米2烧瓶与20毫升杜尔贝科的改良鹰介质/营养混合物F-12（DMEM/F12）含有10%胎儿牛血清（FBS）和1%青霉素/链霉素在37°C与5%CO2。 注:不需要摇晃。</li>
	<li>每 2 −3 天更换一次媒体。</li>
</ol>2. 准备细胞进行测试
<ol><li>细胞几乎汇流后去除培养介质。</li>
	<li>在每个 75 厘米2 烧瓶中加入 4−6 mL 的细胞分离溶液。在37°C孵化细胞，直到细胞分离（约20−30分钟），定期在显微镜下检查。</li>
	<li>在每个 75厘米 2 烧瓶中加入 2−6 毫升的 DMEM/F12，其中包含 10% FBS。</li>
	<li>将烧瓶中的内容转移到 50 mL 离心机管中。</li>
	<li>以 450 −500 x g 的速度将细胞离心5分钟。将超自然物移出，并在预热介质中重新加热细胞。</li>
	<li>使用血细胞计数数细胞。计算包含共 1 x 105 个单元格的介质体积。</li>
	<li>在 48 口井胶原蛋白-1 涂层培养板的每口井中加入 1 x 10 5 个细胞。在井中添加足够的介质，使井中的最终媒体量为 0.5 mL 文化 DMEM/F12，FBS 为 10%。 注:人工撕裂配方可能导致细胞毒性，可以通过HCEC代谢活性的减少来测量。数据的一致性取决于在播种时向每口油井添加相同数量的 HCEC。建议48个井板的文化浓度为1×105。由于哺乳动物细胞在悬念后可以定居在离心管中，因此建议在用细胞播种板时经常通过搅拌来补充细胞，使细胞悬浮液的HCEC均匀分布。</li>
	<li>在 37 °C 和 5% CO2 中孵化细胞 24 小时。</li>
</ol>3. 无干燥协议
<ol><li>控制程序<ol>
<li>24 h 孵化后，将培养介质从盘子中删除。</li>
		<li>立即用 150 μL 的测试配方和介质控制解决方案进行治疗。</li>
		<li>在 37 °C 和 5% CO2 中孵化细胞 30 分钟。</li>
		<li>从细胞中取出测试解决方案。</li>
		<li>添加 0.5 mL 的 10% 代谢染料溶液，以测试代谢活性。在37°C和5%CO2下再孵化细胞4小时。</li>
		<li>4 h 潜伏期后，从每口井中取出 100 μL 的代谢染料溶液，并将每口 100 μL 放入 96 井板的井中。</li>
		<li>使用荧光板读卡器（材料表）测量每口油井的荧光度。将激发波长设置为 540 nm，将发射波长设置为 590 nm。</li>
	</ol>
</li>
	<li>无干燥协议（恢复程序）<ol>
<li>重复步骤 3.1.1−3.1.4。</li>
		<li>向每口井添加 0.5 毫升 DMEM/F12 介质。</li>
		<li>在 37 °C 和 5% CO2中孵化一组 18 h 的培养物。</li>
		<li>移除介质并添加 0.5 mL 的 10% 代谢染料溶液。重复步骤 3.1.6 和 3.1.7。</li>
	</ol>
</li>
</ol>4. 干燥协议
<ol><li>控制程序<ol>
<li>24 h 孵化后（步骤 2.8），从板中删除培养介质。</li>
		<li>立即用 150 μL 的测试配方和介质控制解决方案进行治疗。</li>
		<li>在 37 °C 和 5% CO2 中孵化细胞 30 分钟。</li>
		<li>从细胞中取出测试解决方案。</li>
		<li>将细胞放置在37°C和45%RH室中5分钟，以干燥细胞。</li>
		<li>添加 0.5 mL 的 10% 代谢染料溶液，以测试代谢活性。在37°C和5%CO2下再孵化细胞4小时。</li>
		<li>4 h 潜伏期后，从每口井中取出 100 μL 的代谢染料溶液，并将每口 100 μL 放入 96 井板的井中。</li>
		<li>使用荧光板读卡器测量每口油井的荧光度。将激发波长设置为 540 nm，将发射波长设置为 590 nm。</li>
	</ol>
</li>
	<li>恢复程序<ol>
<li>重复步骤 4.1.1-4.1.5。</li>
		<li>向每口井添加 0.5 毫升 DMEM/F12 介质。</li>
		<li>在 37 °C 和 5% CO2中孵化一组 18 h 的培养物。</li>
		<li>移除介质并添加 0.5 mL 的 10% 代谢染料溶液。重复步骤 4.1.7 和 4.1.8。</li>
	</ol>
</li>
</ol>5. 数据分析
<ol><li>计算媒体控制的平均荧光。使用以下公式计算样本的可行性百分比: 样品的存活率 = 测试样品荧光/介质控制荧光 x 100 注:媒体控制平均值为 100% 可行性。代谢染料本身有少量的荧光与它相关。代谢染料试剂的荧光可以在计算测试样品的活性之前从控制和测试样品荧光中减去。</li>
	<li>执行常态测试和对控制和测试样本的等值方差的测试。</li>
	<li>如果正常性测试通过，且方差相等，则执行单向 ANOVA。要确定特定组之间的显著差异，在临时测试后进行对比（即 Tukey 或 Dunnett 的多重比较测试）。</li>
	<li>如果正常性测试通过，方差不同，则执行韦尔奇的 ANOVA。要识别特定组之间的显著差异，在临时测试后进行对比（即 Dunnett 或 Games-Howell 多次比较测试）。</li>
	<li>如果正常性测试失败，请使用非参数测试（即克鲁斯卡尔-瓦利斯测试）。要识别特定组之间的显著差异，在特设测试后进行对比（即 Dunn 的测试）。</li>
</ol>

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M., Tong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relevance+of+Lipid-Based+Products+in+the+Management+of+Dry+Eye+Disease.">Relevance of Lipid-Based Products in the Management of Dry Eye Disease.</a> Journal of Ocular Pharmacology and Therapeutics. 33 (9), 647-661 (2017).</li><li>Lievens, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+an+enhanced+viscosity+artificial+tear+for+moderate+to+severe+dry+eye+disease:+A+multicenter,+double-masked,+randomized+30-day+study.">Evaluation of an enhanced viscosity artificial tear for moderate to severe dry eye disease: A multicenter, double-masked, randomized 30-day study.</a> Contact Lens &amp; Anterior Eye. 42 (4), 443-449 (2019).</li></ol>

三位作者（雷卡·兰加拉詹、霍华德·凯特尔森、拉克什曼·苏巴拉曼）是阿尔康的员工。阿尔康是该项目的唯一资金来源和赞助者。在过去的3年里，Lyndon W. Jones获得了以下公司的研究支持或讲座荣誉:阿尔康、阿勒甘、康塔马克、库珀维森、GL Chemtec、Inflamax研究、J&J视觉、梅尼康、自然之路、诺华、PS治疗、桑滕、夏尔、视觉玻璃和视觉。林登·琼斯也是阿尔康、库珀维森、J&J视觉、诺华和奥普特克的顾问和/或顾问委员会成员。以下作者名单无可披露:理查德·多、大卫·麦肯纳、阿德琳·苏科、达里尔·恩斯通、贾亚·丹塔姆。

该协议旨在确定人工撕裂溶液对细胞干燥的保护作用。最初，细胞暴露在干眼药水中，以涂覆细胞。接下来，细胞干燥，代谢活动被监测，以评估配方是否可以减轻干燥应激的有害影响。这些步骤对于了解干眼配方对角膜上皮细胞的整体生存能力的总体影响至关重要。
在从培养井中取出大部分干眼配方后，与细胞接触的剩余分子的化学成分将影响细胞干燥。有些产品含有微乳油，最大限度地减少细胞蒸发20。一项在冲洗后评估摩擦分析的研究表明，一些干眼配方的摩擦力较低，这可能是干眼配方21增强居住时间的指标。此测定的局限性之一是，虽然与其他眼药水配方相比，可以评估干燥保护，但由于评估的干燥时间短，无法确定此保护的持续时间。在体外测定中，较长的干燥时间可能需要使用比细胞单层具有整体水分含量的多层培养物。
如果温度和湿度值发生变化，可能需要调整细胞上配方的特定孵化时间和干燥时间的持续时间。选择温度，RH和气流模拟环境压力条件，可以促进干眼可能很困难，因为季节性和局部环境条件是相当可变的21。现代人类研究的环境室可以改变温度在5°C和35°C之间，RH在5%到95%22之间。使用蒸发计和紧密安装的护目镜，确定在 RH 40−45% 角膜蒸发率为 0.037 μL/cm2/min，在 RH 为 20−25%， 蒸发率为 0.065 μL/cm2/min23。如果体外模型在极低的RH条件下（如飞机15、沙漠或干燥的室内环境16）中观察配方的保护作用，可能需要缩短孵化时间，以适应细胞的增强蒸发率。在人类泪液中，存在各种来自肌瘤和其他腺体的脂质，保护眼泪免受蒸发。这些脂质有不同的熔点24。温度变化可能会影响泪液的流动性，因此在环境温度下进行的测试的蒸发率可能与在 37 °C 下进行的测试显著不同。 由于眼表面的平均温度范围为 32.9 至 36 °C25，建议在此温度范围附近进行测试。
除了本协议中使用的代谢染料（阿拉马蓝）外，还有其他化学试剂可用于评估产品配方对细胞代谢活动的影响。四环盐（MTT、XTT、MTS、WST）是可使用的替代化学品。四环盐的区别在于MTT是一个带正电荷的分子，所以它可以进入活细胞的细胞质26。MTT 在减少 NAD（P）H27后变得不溶。在阅读读盘器上的吸收性之前，必须进行溶解。其他四环盐的负电荷为28。它们必须被活细胞外的次生分子减少，因为它们不能进入细胞，不能直接与细胞内分子29相互作用。对于 XTT、MTS 和 WST-1 检测，添加电子耦合剂 1-甲氧苯丙胺甲硫酸酯 （PMS） 可提高检测30的性能。与使用四环盐的代谢检测相比，阿拉马蓝不需要额外的溶解剂或电子耦合剂。此外，与XTT、MTS或WST-1不同，阿拉马蓝穿透细胞膜，可直接通过细胞酶27、29减少。将阿拉马蓝与四色盐直接比较表明，阿拉马蓝对评估包括HCEC3、27、31、32在内的各种细胞系的代谢活动更为敏感。
其他生化终点也可以考虑通过干燥来评估人类角膜上皮细胞的保护。荧光染料可用于检测细胞膜渗透性和细胞酯酶活性1。此外，通过在细胞表面染色凋亡标记或测量细胞酶活性1，33来检测凋亡。其他检测已经确定眼科产品对HCEC紧密路口34的影响。这些检测可以利用本文中描述的干燥程序纳入未来的评估。
眼睛干涸的原因有很多。干眼可由眼泪分泌减少、眼部表面炎症增加或眼部脱水增加引起。对于蒸发性干眼使用干眼滴，防止其细胞在干燥条件下干燥的个人，可以减轻干眼的症状。一些干眼配方中的问题之一是存在可能损害眼部表面的防腐剂。伤害细胞对干眼病人没有帮助，因为它会导致更多的细胞死亡，因为受伤的细胞更容易干燥应激35。
Garrigue等人最近发表的一篇评论文章对目前 基于脂质的产品如何作用于缓解蒸发性干眼的知识进行了评估。在脂质不足的眼泪中加入脂质可以防止蒸发，从而保护细胞免受干燥。此外，腐殖质是吸引和保留水在眼表37的分子。溶液中的甘油#1和溶液中的丙二醇#3都是腐殖质。溶液#3显示了HCEC更大的保护作用，这可能是由于配方成分的脂质和腐殖质特性。
此协议旨在评估接触干眼配方和干燥应激后HCEC的代谢活性。通过使用本文中描述的方法，可以为蒸发性干眼患者开发新的非刺激性和保护性眼药水。

需要多剂量眼科解决方案中的许多不同成分来保持稳定性、pH、渗透性和有效性。然而，一些化学物质，如防腐剂或表面活性剂在眼溶液中可能会造成损害角膜上皮1，2。可使用人类角膜上皮细胞 （HCEC） 进行细胞生存能力测试，以评估这些成分在眼科配方中的潜在损害。体外检测，是特别有用的评估细胞损伤之一是阿拉马蓝检测3。在本测定中，代谢染料（即阿拉马蓝）含有雷萨祖林，作为细胞生存能力指标。在细胞呼吸过程中，通过接受电子4，雷萨祖林被简化为再苏鲁芬。再糖精的减少导致从非荧光分子转变为荧光分子，可以使用光谱荧光计5检测。
此调查使用两种不同的角膜上皮细胞治疗方法，以确定干眼产品在体外模型中的表现。在第一次评估中，细胞用这些产品治疗30分钟。治疗后，细胞在接触干眼产品后和恢复时间间隔后立即评估代谢活性。此评估确定这些产品在湿度室干燥前对细胞的影响。这些溶液中的溶液防腐剂、表面活性剂或缓冲剂可能对使用代谢染料检测到的细胞产生一些细胞毒性作用。
第二个评估确定暴露在干眼产物和干燥后细胞的代谢活性。在这项评估中，如果产品处理为细胞提供防干燥损伤的保护，细胞的代谢活性将得到维持。如果干眼产品可以涂上脂质的细胞，或者产品可能吸收周围增加水分的液体，则可能会对干燥的有害影响进行细胞保护。
该协议旨在模拟细胞环境压力的严重状况。其他研究也使用各种模型来制造这种外部压力。一些研究已经干燥了层流罩6，7，8的细胞，而其他研究已经使用了室温和各种相对湿度（RH）值9，10，11。模拟干眼状况的方法是增加孵化溶液12，13的渗透性。另一种模拟不良环境条件的体外方法是向细胞介质中加入细胞因子，以模拟干眼患者的泪液。虽然这些测试是有趣的模型，可以评估化学物质如何减少渗透应激或刺激免疫系统，但这些模型并没有直接显示化学配方如何保护细胞免受干燥压力。
干燥模型使用温度/湿度室 37 °C 和 45% RH。它模拟可能导致眼部表面蒸发的环境条件。也可以使用这种方法模拟其他极端环境条件，例如15 号机舱或16 号冬季室内环境中的低湿度。
细胞培养建模用于模拟干燥应激对人体角膜的影响。为了检测细胞应力，进行细胞生存能力测试，以确定对细胞健康的影响。可以在细胞上进行几种不同的生理测试，以确定它们是否受到压力。本测试过程中进行的分析使用市售的代谢染料（材料表）。它比许多其他细胞应力测试有优势，因为它是无毒的，可溶于生长介质，可以评估细胞没有细胞固定18。在此过程中，每次治疗后立即对细胞进行代谢活性测试，第二组细胞被放回生长介质，并在 18 小时后恢复后进行评估。恢复期后立即进行测试的原因是确定对代谢活动和导致延迟效应的细胞损伤的直接影响。此外，它提供了一个机会来评估这些配方对短期与长期损害的影响，以及配方从最初的侮辱中恢复过来/不恢复的能力。
本调查用于比较各种含脂干眼产品对HCEC代谢活动的影响，无论有没有干燥。所测试的干眼产品的成分在 材料表中描述。溶液中的成分#1是碳化甲基纤维素钠（0.5%）、甘油（1%）和多糖酸盐80（0.5%），溶液中#2它们是轻矿物油（1.0%）和矿物油（4.5%），并在溶液中#3脂质是矿物油和丙二醇是脱硫。这些脂质是配方成分，有望为HCEC提供防干燥保护。

<table><tbody><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Vented Flask</td><td>Corning</td><td>354485</td><td>This is the BioCoat brand collagen coated</td></tr><tr><td>96 well plate</td><td>costar</td><td>3370</td><td></td></tr><tr><td>alamarBlue</td><td>Fisher Scientific</td><td>dal 1025</td><td></td></tr><tr><td>Corning 48 Well plates</td><td>Corning</td><td>354505</td><td>This is the BioCoat brand collagen coated</td></tr><tr><td>Cytation 5</td><td>BioTek</td><td>CYT5MPV</td><td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td></tr><tr><td>DMEM/F12 with L-glutamine and 15 mM HEPES</td><td>Gibco</td><td>11039-021</td><td>There is No Phenol Red in this media</td></tr><tr><td>Fetal Bovine Serum</td><td>Hycone</td><td>SH30396.03</td><td></td></tr><tr><td>Human Corneal Epithelial Cells</td><td>University of Ottawa</td><td>N/A</td><td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169&amp;ndash;72.</td></tr><tr><td>Humidity/Temperature Chamber</td><td>Associated Environmental Systems</td><td>LH-1.5</td><td>Economy Line Humidity Chamber</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>100X concentration so add 1ml to each 99ml of media</td></tr><tr><td>Refresh Optive Advanced (solution #1)</td><td>Allergan, Inc</td><td></td><td>Carboxymethylcellulose Sodium (0.5%),Glycerin (1%),polysorbate 80 (0.5%),-Lubricants- Inactive ingredients-boric acid; carbomer copolymer type A; castor oil; erythritol; levocarnitine; purified water; Purite (stablized oxychloro complex)and may contain sodium hydroxide to adjust pH.</td></tr><tr><td>Soothe XP (solution #2)</td><td>Bausch &amp;amp; Lomb</td><td></td><td>Light mineral oil (1.0%), Mineral Oil (4.5%)-emolients-Inactive ingredients-boric acid, edetate disodium, octoxynol-40, polyquaternium-1 (preservative), polysorbate 80, purified water, sodium borate decahydrate. May contain hydrochloric acid and/or sodium hydroxide (to adjust pH).</td></tr><tr><td>Systane Complete (solution #3)</td><td>Alcon Inc.</td><td></td><td>Propylene Glycol 0.6% Lubricant Inactive ingredients- boric acid, dimyristoyl phosphatidylglycerol, edatate disodium, hydroxypropyl guar, mineral oil, polyoxl 40 stearate, POLYQUAD&amp;reg; (polyquaternium-1) 0.001% preservative, sorbitan tristearate, sorbitol and purified water. May contain hydrochloric acid and/or sodium hydroxide to adjust pH.</td></tr><tr><td>TrypLE Express (cell disassociation solution)</td><td>Fisher Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/12605036&quot; target=&quot;_blank&quot;&gt;12605036&lt;/a&gt;</td><td></td></tr></tbody></table>

effect of artificial tear formulations on the metabolic activity of human corneal epithelial cells after exposure to desiccation

开发人工含脂催泪配方，通过恢复缺损的泪脂层来减少撕裂蒸发。防止细胞干燥的人工撕裂配方将产生眼部表面保护和细胞代谢活性的维持。在脱水期间，细胞经历代谢活动丧失的过程，随后细胞死亡。这项工作描述了一种评估人工撕裂制剂功效的方法。代谢染料（即阿拉马蓝）从低荧光分子雷萨祖林变为可行细胞中的荧光分子再糖素。人工撕裂制剂的生物性能被测量为配方维持细胞生存能力的能力，并且 （b） 提供细胞防干燥。生长介质和盐水用于细胞生存能力/干燥测试的控制。细胞用测试溶液孵育30分钟，然后干燥0或5分钟，在37°C和45%的相对湿度。细胞代谢活动后，初始接触和细胞干燥后，然后确定。结果表明，眼药水配方对细胞代谢活动和干燥保护的比较作用。这种方法可用于测试干眼配方，旨在用蒸发性干眼治疗个体。

一项比较三种干眼配方的研究结果见 图1。这个数字表明，这三种产品、解决方案#1、解决方案#2和解决方案 #3（材料表）对HCEC的可行性的影响存在差异。溶液#1和溶液#2对干燥前HCEC的代谢活性有显著影响。这意味着这些产品中有配方组件可以破坏 HCEC 的代谢活动。暴露在溶液中的细胞恢复18小时后细胞代谢活动进一步下降，#1表明HCEC最初受伤，18小时后损伤没有修复。相比之下，解决方案#3对HCEC的代谢活动只有轻微的影响。
图2 显示了这些含脂干眼配方对细胞保护的影响。在18小时恢复间隔内，HCEC代谢活动接近0%，溶液#1和溶液#2不能保护细胞免受干燥应激。然而，解决方案#3提供了一些防干燥压力的保护。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60812/60812fig1.jpg"> 图1:干眼脂增强产品对细胞生存能力的影响。 使用含脂干眼产品治疗后，由HCEC代谢活动测量的相对生存能力。* 与解决方案#3相比，统计学意义（p < 0.05），Δ = 相对于适当恢复期的媒体控制的统计意义（p < 0.05）。条的高度表示平均值，错误栏为 SD。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60812/60812fig1large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60812/60812fig2.jpg"> 图2:干眼脂增强产品对干燥保护的影响。 含脂干眼配方对细胞保护的影响。溶液#1和溶液#2不能保护细胞免受干燥压力的影响，而且随着时间的推移，它们不会从干燥效果中恢复过来。相比之下，解决方案#3提供了一些防干燥压力的保护。*=p< 0.05相对于解决方案#3，Δ=p<0.05相对于未经处理（控制）介质为适当的恢复时间。条的高度表示平均值，错误栏为 SD。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/60812/60812fig2large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation at JoVE.com

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

本协议的目的是评估不同的人工撕裂配方是否能使用体外模型保护人类角膜上皮细胞免受干燥。在接触人工撕裂配方和干燥后，对人体角膜上皮细胞进行代谢活性评估。

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

effect-artificial-tear-formulations-on-metabolic-activity-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

3.8K 次观看.  Alcon Vision, LLC. 本协议的目的是评估不同的人工撕裂配方是否能使用体外模型保护人类角膜上皮细胞免受干燥。在接触人工撕裂配方和干燥后，对人体角膜上皮细胞进行代谢活性评估。

视频: Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

诱导眼表炎症和受累组织收集

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

科研

免疫与感染

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

医学

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

在兔子中使用完全的白眼切除术建立严重干眼模型

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮机械和化学损伤的离体和体内动物模型

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

生物学

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

<meta charset="utf8"></head><body>用于毒性检测和伤口愈合的离体猪角膜培养系统

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 &#176;C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

<meta charset="utf8"></head><body>作者聚焦：探索糖尿病患者的角膜先天免疫和伤口愈合延迟

Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.

使用器官培养的猪角膜评估治疗和化学毒性以及上皮伤口愈合

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular ...

Nasolacrimal Lavage as a Treatment for Ocular Surface Toxic Soup Syndrome

Ocular surface toxic soup syndrome (OSTSS) is characterized by inadequate tear drainage through the nasolacrimal duct system, leading to the accumulation of inflammatory mediators in the tear film. This condition can result in toxic keratoconjunctivitis, with symptoms such as conjunctival hyperemia, itchiness, discomfort, and epiphora. Dilation and irrigation are common diagnostic procedures in both optometry and ophthalmology, used to assess nasolacrimal duct obstruction in cases of epiphora. This technique involves the injection of saline into the nasolacrimal duct system through the puncta, followed by the evaluation of reflux, which indicates obstruction. Although intended to be diagnostic, many patients report significant improvements in epiphora and ocular comfort after the procedure. In this paper, we present a case study in which a patient achieved complete resolution of epiphora and itchiness following therapeutic nasolacrimal lavage performed every 2 months. Subjective improvements in symptoms in 3 additional patients are also reported. We propose nasolacrimal lavage not only as a diagnostic tool but also as an effective therapeutic intervention for managing OSTSS.

OSTSS leads to pooled inflammatory mediators in the tear film, causing symptoms like epiphora and discomfort. Here, we present a case where therapeutic nasolacrimal lavage every 2 months resolved epiphora and itchiness, suggesting its efficacy as a therapeutic intervention for OSTSS. Additionally, we report subjective improvements in symptoms in 3 additional patients.

Ocular surface toxic soup syndrome (OSTSS) is characterized by inadequate tear drainage through the nasolacrimal duct system, leading to the accumulation ...

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

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诱导眼表炎症和受累组织收集

由康卡纳林注射到乳原区引起的水性干眼病的兔子模型:药物功效研究的应用

在兔子中使用完全的白眼切除术建立严重干眼模型

角膜上皮机械和化学损伤的离体和体内动物模型

确定紫外线辐射和化学物质对原代和永生化的人角膜上皮细胞的毒性

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

使用器官培养的猪角膜评估治疗和化学毒性以及上皮伤口愈合

使用器官培养的猪角膜评估治疗和化学毒性以及上皮伤口愈合

使用器官培养的猪角膜评估治疗和化学毒性以及上皮伤口愈合

Nasolacrimal Lavage as a Treatment for Ocular Surface Toxic Soup Syndrome