Name: a chronic autoimmune dry eye rat model with increase in effector memory t cells in eyeball tissue
Uploaded: 2017-06-07T13:00:00.000+00:00
Duration: 9 min 42 s
Description: Watch this Scientific Journal Video about A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue at JoVE.com

作者感谢Tin Min Qi女士和Veluchamy Amutha Barathi博士及其小组协助处理这些动物。这项工作得到NHIC-I2D-1409007，SingHealth Foundation SHF / FG586P / 2014和NMRC / CSA / 045/2012的支持。

根据机构指导和ARVO"眼科和视力研究中使用动物声明"处理动物。研究方案由SingHealth机构动物保护和使用委员会批准。 泪液提取物的制备注意:腹腔注射氯胺酮（75mg / kg）和赛拉嗪（10mg / kg）麻醉大鼠。通过趾捏和尾was确认正确的麻醉。将眼用凝胶施用于大鼠眼睛，以防止每次手术后的干燥。将麻醉的大鼠置于远红外线下，使动物保持温暖，直至完全恢复。在手术和恢复期间，动物被研究人员密切监测。所有的材料和手术工具在使用前是无菌的。在实验结束时，通过腹膜内注射戊巴比妥（80mg / kg）将大鼠安乐死。完全的安乐死是通过缺乏心脏脉搏和触摸眼球无眨眼反射来证实的。大鼠在标准条件下饲养:室温，21-23℃;相对湿度30-70％;亮暗循环，交替12小时（上午7点至晚上7点）。 <ol><li> 将安乐死的女性Sprague-Dawley大鼠（年龄范围从8周到16周）平坦放置，一只耳朵靠在桌子上，另一只耳朵朝上。在一个弹簧剪刀下，在暴露的耳朵下面做一个10mm切口。通过将其从周围的结缔组织和排水管中分离出来去除泪腺。 <ol><li>将腺体存放在-80°C，直到需要。在冰上解冻腺体。用剪刀在冰上尽可能的微妙。每个泪腺加入150μL含有1x蛋白酶抑制剂的PBS。 </li></ol></li><li>超声波样品在冰上5分钟，20kHz，超声仪设定为10秒，10秒关闭，30％振幅。离心子冰醋酸样品在13,000 xg和4℃下20分钟。 </li><li>移取上清液并将其转移到新管中。将上清液等分至1.5 mL管，并将其储存在-80°C。根据制造商的说明书，用二喹啉酸测定法22测定蛋白质浓度。 </li></ol> 2.乳液和百日咳毒素的制备<ol><li> 乳胶 <ol><li>将40mg热杀死的结核分枝杆菌H37Ra加入到10mL含有1mg / mL 结核分枝杆菌H37Ra的完全弗氏佐剂中并充分混合。 注意:最终的完全弗氏佐剂含有5mg / mL 结核分枝杆菌H37Ra。 </li><li>称量卵清蛋白，溶解在PBS中，制备含有2mg / mL卵清蛋白和10mg / mL泪腺提取物的抗原混合物。针对每只动物注射体积为200μL，制备主混合物b根据动物数字。 </li><li>将不完全弗氏佐剂或完全弗氏佐剂转移到50mL管中，确保抗原混合物的佐剂体积为1:1比例。将抗原混合物逐滴加入佐剂，同时以不会导致溢出的最高速度涡旋。在所有抗原加入后继续涡旋5分钟。 </li><li>将乳液转移到5 mL注射器，并通过注射器连接器与另一个5-mL注射器连接。将乳液从一个注射器推到另一个注射器进行混合。 注意:如果在放置在水中时单个液滴保留为球体，则乳液已准备就绪。应使用新鲜制备的在4℃下储存过夜的乳液或乳液。 </li></ol></li><li> 百日咳毒素 <ol><li>在500μL水中重建50μg百日咳毒素，使终浓度达到100 ng /μL。 Votex容器30秒以确保t牛油完全溶解。 注意:不要过滤灭菌，否则会导致材料损失。不要冻结该溶液在4℃下保持活性至少6个月。 </li><li>使用PBS稀释100 ng /μL百日咳毒素至3 ng /μL。将100μL稀释的百日咳毒素转移到带有27 G针的1 mL Luer-lock注射器中。 </li></ol></li></ol>刘易斯大鼠的免疫<ol><li>在第0天，将乳液混合几次。将含有1毫克泪腺提取物和200克卵清蛋白的完全弗氏佐剂的200μL乳液分配到具有27G针的1mL鲁尔锁定注射器中。在没有麻醉的情况下，在大鼠尾巴的底部皮下注射乳剂。 注意:注射前不需要预热。每只大鼠用完全弗氏佐剂中的1mg泪腺提取物和200μg卵白蛋白免疫h500μg结核分枝杆菌H37Ra。 </li><li>在第14天，以与第0天相同的方式，在不完全弗氏佐剂中注入200μL含有1mg泪腺提取物和200μg卵清蛋白的乳液。腹膜内注射100μLPBS中的300ng百日咳毒素每只老鼠在同一天。 </li></ol> 4.将抗原混合物注射入穹隆结膜下和泪腺以招募抗原特异性免疫细胞并引起局部炎症<ol><li>基于实验中的大鼠数量计算卵清蛋白和泪腺提取物的量。称量所需量的卵清蛋白，并将其与步骤1中制备的解冻的泪腺提取物组合，制备含有1mg / mL卵清蛋白和1mg / mL泪腺提取物的抗原溶液。 </li><li>用氯胺酮（75mg / kg）和甲苯噻嗪（10mg / kg）通过腹膜内注射麻醉。捏捏到以确保已经实现适当的麻醉（ 即，在捏合后没有观察到反应性运动）。将5μL的抗原注射到眼睛的穹隆结膜下。将20μL抗原注入泪腺。 </li></ol> 5.干眼功能评估注意:对于步骤5.1-5.3，麻醉的大鼠应该用手套轻轻握在平坦表面上的直立位置以避免移动。 <ol><li> 用酚红线测量泪液体积。 <ol><li>使用一对镊子夹住线，另一镊子拉扯大鼠的下眼睑。将螺纹放在下穹窿的近端拐角处1分钟，然后取下螺纹。 注意:眼泪使线的润湿部分的颜色变红。 </li><li>用毫米标记拍摄一个尺子的湿润部分的长度的图像。测量湿润长度到第十个ofa毫米使用图像软件（ 例如， ImageJ）。 </li></ol></li><li> 测量角膜/撕裂光滑度。 <ol><li>将大鼠放在装有环形照明器和相机的立体显微镜下。向大鼠角膜涂抹5μL生理盐水。通过用戴手套的手指移动上下眼睑〜5次，以便传播盐水被动地眨眼。 </li><li>将环形照明器聚焦在角膜的中间，放大1.6倍。 10秒后拍摄照片。 注意:环形照明器在动物的角膜上投射两列圆点图像。无失真点的规则间距表示平滑的角膜/泪液层。 </li></ol></li><li> 用荧光素染色测量角膜损伤。 <ol><li>向大鼠角膜中加入0.2μL的0.2％荧光素。用戴手套的手指将大鼠眼睑动物打开并闭合3次，将荧光素染料铺展在眼睛表面。 </li><l1分钟后，用3 mL注射器（无任何针头附着）取1 mL生理盐水，将注射器置于角膜前方2-3 mm处，轻轻将盐水推入眼睛。 </li><li>在背景灯关闭的眼睛成像显微镜的钴蓝色滤光片（ 即 〜400 nm）下获取图像。 注意:随后由以前的出版物23修改的分级系统分析图像。然后通过主观分级绿点的数量，面积和强度从0到2进行分析，其中0表示不存在，1表示点状染色小于50个点，2表示多于50个点的点状染色。 </li></ol></li><li>通过腹膜内注射戊巴比妥（80 mg / kg）安乐死大鼠。用剪刀去除上下眼睑。通过用镊子推下眼睑组织来修复和脱垂眼球。通过切断t来释放地球他的眼外肌，视神经和穹窿结膜。 </li><li>用镊子固定大鼠眼球的位置，并沿赤道圆周切口打开。取出镜片和玻璃体。将解剖的眼球放入冰中的1.5 mL管中。 </li><li>收集泪腺，如步骤1.1所述。立即将解剖的眼球组织和泪腺转移到实验室以准备流式细胞术分析。 </li><li>用胶原酶和Dispase II消化法分离免疫细胞24,25 。继续使用流式细胞仪分析眼球组织中的T细胞亚群26 。 注意:抗体组为:抗CD45APC-Cyanine7（OX-1），抗CD3 BV421（1F4），抗CD4 PE-Cyanine7（OX-35），抗CD45RC Alexa647（OX-22），抗-CD62 PE（HRL1），抗CD44 FITC（OX-50）和活细胞染料7-AAD。在CD45 + CD3中<sup&gt; + 7AAD-群体，初始（CD3 + CD45RC + ），效应记忆（T EM， CD3 + CD45RC - CD44 + CD62L - ）和中央记忆（T CM， CD3 + CD45RC - CD44 + CD62L + ）T细胞。 </li></ol>

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作者没有利益冲突。 LT收到了Alcon，Allergan，Santen，Bausch和Lomb，Eyelens和Eyedetec之前的资金和/或礼物。

该方案的关键步骤是确保乳液的均匀性。在精心制备的乳剂中，抗原完全涂覆油，确保注射的抗原的缓慢释放和持续的免疫刺激。该方案的另一个关键特征是使用Lewis大鼠。 Lewis大鼠比其他菌株更敏感于自身免疫性疾病27 。 该协议已经从以前发布的两种方案中进行了修改，该协议既可以使用泪腺提取物，也可以使用重组Klk1b22 20,21 。在目前的方案中，使用卵白蛋白加泪腺提取物作为抗原，抗原特异性免疫细胞被吸引到眼表和泪腺，诱发局部组织损伤。干眼发展缓慢，初次免疫后第48天达到〜85％。抗原性挑战眼睛和泪腺在第48天加剧干眼症，并确保其长达七十岁的长期性。 与Klk诱导的DED模型中的重组Klk1b22相比，当前模型中使用的泪腺提取物和卵清蛋白更便宜，更容易获得。泪腺提取物还含有除了Klk之外的其他可能诱导自身免疫的蛋白质，因此在诱导DED时，该提取物在理论上比Klk方法更有效。我们也尝试只用泪腺提取物免疫大鼠;尽管这些免疫的大鼠发展为DED，但与对照组相比，眼球组织中效应记忆T细胞没有显着增加。 这种技术的局限性在于实现模型需要70天的时间。效应记忆T细胞是正常大鼠眼中的主要T细胞亚群。在该模型中，自身免疫性DED导致眼球中CD3 +效应记忆T细胞的增加。优先的药物临床上抑制效应记忆T细胞，例如Kv1.3钾通道的选择性抑制剂，因此可以对自身免疫性DED 28具有治疗益处。

干眼症（DED）是眼泪和眼表的多因素疾病，导致不适，视觉障碍和泪膜不稳定症状，这可能导致眼表受损。它伴随着泪膜的渗透压增加和眼表1的炎症。与DED相关的症状是灼热，刺痛，烧伤，异物感，撕裂，眼睛疲劳和干燥2,3 。 DED的两个主要原因是减少泪液分泌腺体的泪液产生和撕裂膜4的过度蒸发。在患有自身免疫性疾病的患者中，例如干燥综合征，系统性红斑狼疮和类风湿关节炎对睑板腺的免疫损伤降低了对于泪液稳定性至关重要的脂质的表达。此外，对眼表的免疫损伤降低了产品对于表面润湿性很重要的粘蛋白。一起，这些过程累积地引起慢性干眼5,6,7 。 泪液替代和抗炎治疗是治疗的主要内容。然而，目前DED（ 即皮质类固醇和环孢菌素）的抗炎疗法具有广泛的免疫抑制作用，导致严重的不良反应8,9,10 。需要合适的动物模型来测试新型免疫调节剂以治疗自身免疫性干眼症。 具有特定遗传缺陷的小鼠11,12,13 ，缺乏特异性基因的小鼠14,15和过表达免疫原性的转基因小鼠ory基因已被用作自身免疫干眼症的模型16,17 。抗原诱导的自身免疫动物模型也在小鼠18 ，兔子19和大鼠20,21 中报道。在这里，我们描述了抗原诱导的慢性自身免疫干眼模型。这个模型是两个早期模型的修改;一个使用泪腺提取物，第二个使用自体抗原（ 即， klk1b22）来自泪腺20,21 。 通过用卵白蛋白，完全弗氏佐剂和含有Sprague-Dawley大鼠的泪腺提取物的乳剂（ 图1 ）皮下免疫6至8周龄的雌性Lewis大鼠诱导该疾病。在不完全弗氏佐剂中用相同抗原进行第二次免疫两周后给药。为了将抗原特异性免疫细胞募集到泪腺和眼表面，第6〜7周将泪腺提取物和卵白蛋白（1mg / mL）的混合物注射入穹隆结膜下和泪腺（ 图1 ）。超过85％的大鼠在第一次免疫后70天开始出现干眼症的特征。这些特征包括减少的泪液生成（ 图2 ），增加的角膜荧光素染色（ 图3 ）和降低的撕裂稳定性（ 图4 ）。通过流式细胞术对正常大鼠眼球中的T细胞的免疫分析显示CD3 +效应记忆T细胞的优势（ 图5和6 ）。具有自身免疫性DED的大鼠显示CD3 +效应记忆T细胞的增加以及初始和中央记忆T细胞相应的减少（ 图6 </stro纳克&gt;）。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P2714-1BTL</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermal Scientific</td><td>23227</td><td></td></tr><tr><td>Mycobacterium tuberculosis H37Ra&amp;nbsp;</td><td>Becton, Dickinson and company</td><td>231141</td><td></td></tr><tr><td>complete Freund&#039;s adjuvant</td><td>Becton, Dickinson and company</td><td>231131</td><td></td></tr><tr><td>ovalbumin</td><td>Sigma-Aldrich</td><td>A5503-10G</td><td></td></tr><tr><td>incomplete Freund&#039;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5506-6X10ML</td><td></td></tr><tr><td>pertussis toxin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7208-50UG</td><td></td></tr><tr><td>fluorescein sodium solution</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sonicator</td><td>Sonics&amp;nbsp;</td><td>Vibra-Cell</td><td></td></tr><tr><td>phenol red thread</td><td>Tianjin Jingming New Technological development Co. LTD.</td><td>NA</td><td></td></tr><tr><td>Stereo microscope with ring light illuminator and camera</td><td>Carl Zeiss</td><td>NA</td><td></td></tr><tr><td>Micro IV microscope&amp;nbsp;</td><td>Phoenix Research Labs</td><td>NA</td><td></td></tr></tbody></table>

a chronic autoimmune dry eye rat model with increase in effector memory t cells in eyeball tissue

干眼病是一种非常常见的病症，导致发病率和医疗负担，降低生活质量。需要合适的干眼动物模型来测试新型治疗剂以治疗自身免疫性干眼症状。该方案描述了慢性自身免疫性干眼鼠模型。用含有泪腺提取物，卵清蛋白和完全弗氏佐剂的乳剂免疫Lewis大鼠。两周后给予不完全弗氏佐剂中具有相同抗原的第​​二次免疫。这些免疫接种在尾部的基部皮下施用。为了增强眼表面和泪腺的免疫反应，首次免疫6周后​​，将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺。大鼠发展为干眼特征，包括泪液产生减少，撕裂稳定性降低，角膜损伤增加。免疫专业流式细胞分析显示，CD3 +效应记忆T细胞在眼球中占优势。

图1说明了实验设计。在第48天和第70天，在免疫的大鼠中评估干眼临床特征。撕裂体积由酚红线的湿部分的长度表示。 图2显示了来自对照和DED大鼠的酚红线的代表性图像。 DED组苯酚红线长度比对照组短，表明泪液体积较小。 荧光素与受损的角膜上皮结合。因此，通过角膜荧光素染色来测量角膜损伤。 DED大鼠角膜表面的荧光斑点从0级分为2级，与对照组比较。 DED的大鼠比对照大鼠具有更多的荧光素染色（ 图3 ），表明角膜损伤。 角膜光滑在DED和对照大鼠中通过环形照明器进行评估。如果角膜表面光滑，撕裂稳定性高，眼表上的照明环的图像是圆形和完美的。图像变形表示角膜平滑度降低和泪液不稳定。环的变形程度从0到2。在DED组中，注意到更高的环形变形水平（ 图4 ），表明较少的撕裂稳定性。 当干眼症的至少两个临床特征异常时，大鼠被定义为具有干眼症。在24只免疫的大鼠中，21只大鼠在第48天开发了DED。当在第70天评价时，结果是一致的。 流式细胞分析显示正常大鼠眼球组织中主要的T细胞亚群是效应记忆T细胞（ 图5 ）。在DED大鼠的眼球中，〜70％的CD3+ T细胞是效应记忆T细胞，而在对照大鼠中，这个数目是〜50％。 DED大鼠的眼球具有比对照大鼠显着更高的效应细胞记忆T细胞（ 图6 ）。 <img alt="图1" src="/files/ftp_upload/55592/55592fig1.jpg" /> 图1:实验设计示意图。 LG:泪腺DED:干眼病; CFA:完全弗氏佐剂; IFA:不完全弗氏佐剂。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" src="/files/ftp_upload/55592/55592fig2.jpg" /> 图2:酚红线测量撕裂体积。将苯酚红线放置在两只老鼠眼睛的近端拐角处1分钟，然后取出。代表Source显示了来自对照组和DED组的酚红线与标尺的图像。使用ImageJ来测量酚红线的湿部分的长度。比例尺= 1 mm。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" src="/files/ftp_upload/55592/55592fig3.jpg" /> 图3:荧光素染色测量的角膜上皮损伤的代表性图像。每个大鼠角膜用0.2％荧光素染色1分钟，并用至少1mL盐水冲洗。在具有钴蓝光的眼睛成像显微镜下拍摄图像。第一列显示控制角膜的代表性图像。第二列包含具有DED特征的大鼠的角膜染色的代表性图像。绿色荧光斑点表示角膜上皮<html> l伤害。所有图像都以相同的颜色尺度生产。荧光素染色的定量根据绿点的面积和密度进行。比例尺= 1 mm。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" src="/files/ftp_upload/55592/55592fig4.jpg" /> 图4:显示环形照明器反射的代表性角膜图像。通过环形照明器测量大鼠角膜/撕裂平滑度。捕获图像中的环的变形程度是相对撕裂稳定性的量度。左栏显示对照动物的代表性图像，右列在诱导干眼后显示代表性的图像。比例尺= 1 mm。请点击此处查看此图的较大版本。 <img alt="图5" src="/files/ftp_upload/55592/55592fig5.jpg" /> 图5:流式细胞仪分析得到的点图。从眼球组织分离的T细胞用一组抗体染色。在CD45 + CD3 + 7AAD-群体中，选择CD3 + 7-AAD - T细胞。在CD3 + 7-AAD - T细胞中，确定了初始的中枢记忆和效应记忆T细胞群。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" src="/files/ftp_upload/55592/55592fig6.jpg" /> 图6:眼球中的T细胞亚群概况。 CD3 + CD45RC+初始T细胞，CD3 + CD45RC - CD44 + CD62L -效应记忆T（T EM ）细胞和CD3 + CD45RC - CD44 + CD62L +中央记忆T（T CM ）细胞以CD3 + T细胞的百分比表示。结果来自3只对照大鼠和6只DED大鼠。从分离的泪腺的T细胞的分析中获得了类似的结果（数据未显示）。未配对的Student&#39;s t检验用于统计学比较。误差条代表SD。 * p &lt;0.05，** p &lt;0.01。 <a href="http://ecsource.jove.com/files/ftp_upload/55592/55592fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a>

Watch this Scientific Journal Video about A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue at JoVE.com

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

该报告描述了通过用大鼠泪腺提取物，卵清蛋白和完全弗氏佐剂的乳液免疫在Lewis大鼠中诱导慢性实验性自身免疫性干眼的方法，随后将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺六周后。

慢性自身免疫性干眼鼠模型增加效应记忆T细胞在眼球组织

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

a-chronic-autoimmune-dry-eye-rat-model-with-increase-effector-memory

Research

JoVE Journal

Immunology and Infection

11.4K 次观看.  Singapore Eye Research Insitute, A Member of SingHealth. 该报告描述了通过用大鼠泪腺提取物，卵清蛋白和完全弗氏佐剂的乳液免疫在Lewis大鼠中诱导慢性实验性自身免疫性干眼的方法，随后将泪腺提取物和卵清蛋白注射入穹隆结膜下和泪腺六周后。 

视频: A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

实验性自身免疫性葡萄膜炎:眼内炎症小鼠模型

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

科研

免疫与感染

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 ...

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 ...

东莨菪碱诱导的大鼠泪腺功能障碍

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

作者聚焦：开发干眼动物模型的挑战和未来的研究方向

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

东莨菪碱诱导泪腺功能障碍的大鼠干眼模型

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

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 ...

Quantification of Tear Volume in an Autoimmune Dry Eye Rat Model using the Phenol Red Thread

Source: Hou, A. et al., <a href="https://app.jove.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a> J. Vis. Exp. (2017)
This video demonstrates the phenol red thread test to determine tear volume in an autoimmune dry eye rat model. The pH-sensitive acidic phenol red thread changes color when it comes in contact with the alkaline tears. The shorter length of color change in the phenol red thread corresponds to reduced tear volume.

使用 Phenol Red 线定量自身免疫性干眼大鼠模型中的泪液量

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Assay and Analysis

Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model

Source: Hou, A. et al., <a href="https://app.jove.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for assessing corneal smoothness and tear film stability in an autoimmune rat model. It highlights the impact of gland dysfunction on tear film stability, crucial for understanding dry eye disease.

慢性自身免疫性干眼鼠模型增加效应记忆T细胞在眼球组织

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Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

使用 Phenol Red 线定量自身免疫性干眼大鼠模型中的泪液量

自身免疫性干眼症大鼠模型中角膜和泪液光滑度的评估