作者要感谢切斯特菲尔德的艾略特·阿巴特奥提供猪眼。玻璃戒指是谢菲尔德大学化学系的玻璃鼓风机丹·杰克逊根据我们的设计制作的。作者要感谢医学研究理事会（MR/S004688/1）的资助。作者还要感谢沙纳利·迪克韦拉夫人在角膜制备方面的技术帮助。作者要感谢乔纳森·埃默里先生在图片格式化方面所起的帮助。

白化病实验室的兔子被牺牲在实验室里，根据内政部批准的协议进行其他计划中的实验工作。这些研究不需要眼睛进行实验性使用，因此它们用于此协议。
1. 绝育
<ol><li>关键步骤:在蒸馏水中浸泡 1 小时（v/v/v） Distel 溶液，用刷子清洁，用自来水冲洗，并在 185 °C 的烤箱中消毒，至少 2 小时，对所有钳子和剪刀进行消毒。</li>
	<li>通过在 121 °C 下自动拍击 15 分钟或根据制造商的指示准备试剂，对所有其他玻璃器皿和试剂进行消毒。在 II 类微生物安全柜中执行以下程序。</li>
</ol>2. 样本收集
<ol><li>猪眼的集合<ol>
<li>大型白色陆地赛母猪，一个十字架与汉普郡野猪被使用。动物们被电流惊呆了，眼睛在2小时后在屠宰场被细胞核化了。</li>
		<li>关键步骤:一旦电子核化，将眼睛转移到实验室在无菌磷酸盐缓冲盐水（PBS）溶液，以防止他们干涸，并在抵达后立即处理他们。</li>
	</ol>
</li>
	<li>兔子眼的集合<ol>
<li>将角膜消费税化，然后用无菌PBS送到实验室。</li>
	</ol>
</li>
</ol>3. 角膜硬膜按钮的准备
<ol><li>使用无菌钳子握住眼球周围的组织，并将其转移到培养皿中。使用 15 号手术刀刀片和钳子取出培养皿上的眼球周围的结膜和肌肉组织。</li>
	<li>轻轻抬起眼球，同时用钳子握住视神经，转移到一个装满无菌PBS的0.5L罐子里。</li>
	<li>一旦所有的眼睛都清除了周围的组织，用无菌钳子将它们转移到另一个0.5升罐中，里面装满了PBS中3%（v/v）的多维酮碘，然后离开1分钟。</li>
	<li>将眼球转移到另一个带有无菌 PBS 的 0.5 L 罐子上。</li>
	<li>使用钳子将眼睛静止在培养皿上，并用没有 10A 的手术刀刀片在角膜附近切开。</li>
	<li>关键步骤:抓住切口的边缘，用剪刀切除角膜，在角膜周围留下大约3毫米的硬膜。确保剪刀的锐端不会刺穿虹膜或胆囊组织，并处于超胆囊空间。</li>
	<li>用钳子按住角膜硬化按钮，然后使用另一对尖端钳子轻轻地分离子宫组织。</li>
	<li>将角膜硬化按钮从剩余的球体中提起，并在 12 个井板中的 PBS 中短暂冲洗 1.5% （v/v） povidone 碘溶液。</li>
	<li>将角膜硬盘按钮放入另一个充满无菌 PBS 的 12 个井板中。</li>
	<li>处理完所有眼睛（建议一批最多40只眼睛），将每个角膜硬化按钮放在单个培养皿（直径34毫米）上部，倒入3mL的培养介质预热至37°C。 注:培养介质的组成如下:杜尔贝科的改良鹰的介质（DMEM）:哈姆的[1:1]辅以5微克∙mL-1胰岛素和10∙mL-1表皮生长因子（EGF），1 10% （v/v） 胎儿小腿血清 （FCS）， 100 U∙mL-1青霉素， 100 U∙mL- 1链霉素和 2.5 μg∙mL- 1安非他明 B.作为可选步骤，可补充50克∙L-1德克斯特兰，以防止在进一步的孵化步骤中切除角膜肿胀。</li>
	<li>在加湿组织培养孵化器中以37°C孵化。</li>
</ol>4. 角膜硬性按钮的维护
<ol><li>24小时后，使用无菌技术去除介质，代之以含有抗生素的3mL新鲜预热培养物。用抗生素将角膜硬性按钮放在介质中48小时，对角膜进行消毒。在加湿组织培养孵化器中以37°C孵化。</li>
	<li>关键步骤: 48 小时后， 取出介质， 用 2 ml 的 Pbs 冲洗角膜。然后在无抗生素介质中将角膜硬化按钮保存至少两天或理想情况下，在实验感染前三天，从组织中取出残留的抗生素。</li>
	<li>在加湿组织培养孵化器中以37°C孵化。在这三天内至少再更换一次媒体。如果无抗生素介质中出现任何浊度，请丢弃角膜。</li>
</ol>5. 准备接种
<ol><li>用泡沫塞将 10 毫升 LB 汤倒入 50 毫升圆锥形烧瓶中。</li>
	<li>将一群 P.阿鲁吉诺萨 菌株PAO1或菌株PA14从新鲜的醋板中转移，在37°C下孵育3-4小时，直到细菌处于中日志阶段。</li>
	<li>将细菌培养转移到 50 mL 管和离心机中，3，000 x g，5 分钟。取出超常剂并重新悬浮 PBS 中的细胞颗粒。</li>
	<li>重复步骤 5.3 两次来清洗细胞。重新悬挂PBS中的细胞颗粒，并将光学密度调整为600nm至0.6左右，使用无菌PBS作为空白。</li>
</ol>6. 感染角膜硬膜按钮
<ol><li>从培养皿中取出介质，用 1 mL 无菌 PBS 冲洗角膜两次。</li>
	<li>轻轻挤压钳子，同时夹住中间的角膜。使用 10A 手术刀在角膜硬化按钮的中心部分（通过上皮层到底层频闪）进行四次切割（两次垂直，两次水平）。</li>
	<li>将无菌玻璃模具放在一个6井板中，宽部分向上，并将角膜放在玻璃模具中间，上皮侧朝下。在玻璃模具底部的中心进行切割。</li>
	<li>关键步骤: 倒 1 毫升 1% （w/v） 低熔点琼脂溶解在 DMEM 中， 以完全填充玻璃模具角膜。</li>
	<li>让琼脂设置，然后倒置玻璃模具，使角膜上皮朝上。</li>
	<li>带 OD600nm = 0.6 的细菌培养物的移液器 15 μL（对于 P. aeruginosa 来说，这相当于大约 1 x 107 菌落形成单位 （CFU） 在 15 μL 中直接进入切割区域，然后将 85 μL 的 PBS 添加到顶部以保持角膜上皮湿润。稀释阿加上剩余的细菌培养和板，以计数菌落形成单位的培养。</li>
	<li>加入1毫升的DMEM没有抗生素到每个井的底部与玻璃模具。在 37 °C 的加湿孵化器中用受感染的角膜硬化按钮孵化 6 井板，5% CO2 可长达 24 小时。</li>
	<li>在每个实验旁边设置未受感染的对照角膜。要建立未受感染的控制，在步骤 6.6 中用无菌 PBS 替换 15 μL 的细菌培养。</li>
</ol>7. 角膜的同质化以收获细菌
<ol><li>从 6 井板底部丢弃 DMEM 介质，并添加 1 mL 的无菌 PBS 冲洗井底。</li>
	<li>通过管道轻轻取出 PBS，而无需触摸角膜硬性按钮的中心部分。使用无菌钳子取出玻璃环，并将其放置在 5% Distel 中。</li>
	<li>用 1 mL 的 PBS 轻轻冲洗角膜硬化按钮的顶部两次 [可选] 。</li>
	<li>用细尖钳按住角膜硬化按钮的边缘，并将其从下面的琼脂中分离。</li>
	<li>将角膜转移到一个50 mL管充满了冰冷1-2 mL的PBS。</li>
	<li>使用精细的尖端同质化剂来纯粹的受感染角膜的顶部。组织不必完全液化。同质化剂有助于将细菌从角膜上皮和切口区域分离。</li>
	<li>在PBS中将角膜漩涡几秒钟以混合内容。</li>
	<li>将 20μL 的同质物添加到 180μL 的 PBS 中，并在 96 井板中执行串行稀释。</li>
	<li>连续稀释悬架至10-4 和10-5 稀释和移液器10μL的稀释同质细菌到血糖板。孵化板8小时，并计算CFU的数量。在测试抗菌素的效果时，必须通过实验得出适当的稀释因子。</li>
</ol>

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作者没有什么可透露的。

使用外体猪角膜开发这种角膜模型的主要驱动力是为开发新型抗菌素的研究人员提供具有代表性的体外模型，以更准确地确定前脑阶段的抗菌功效。这将为参与开发新的抗菌药物的研究人员提供在临床前阶段对药物设计和配方的更大控制，提高临床试验的成功率，通过有针对性的研究减少动物的使用，并加快将新的抗菌素转化为临床。
多项研究已调查了感染各种动物的外生角膜的影响，例如:兔子6、狗7、山羊8和猪9、10、11。这些研究大多侧重于建立6和可视化感染9的方法，但到目前为止，只有少数出版物侧重于药物测试和准确量化细菌6，7，8，12。
我们模型的主要优点是作为食物链一部分的猪角膜的可用性。因此，使用前体猪角膜符合3R原则，即在研究中取代、改进和减少动物的使用，同时提供宿主接口的代表性模型。如果严格遵守协议，我们观察到角膜外泄的污染没有问题。玻璃模具非常简单、快速、简单，无需任何专用设备。顶部的窄环使少量测试药物（100μL）或细菌的添加变得方便。玻璃模具的环允许PBS与细菌或药物溶液保留在角膜的中心部分，并防止细菌进入角膜下面。环易于清洁和消毒，并允许观察感染期间角膜顶部发生的变化。荧光标记细菌菌株可用于使用荧光共生显微镜可视化感染或量化感染在组织中的传播。整个角膜可以进一步处理组织学或电子显微镜成像。
关键步骤在协议中标记。在执行协议时，必须特别注意这些步骤，以确保成功感染。协议内最关键的步骤是确保角膜在制备过程中用足够的抗生素治疗，以防止感染，然后在引入感染生物体之前充分消除抗生素，在这种情况下 ，P. aeruginosa。在使用此协议进行实验时，在某些情况下，在无抗生素介质的孵化过程中出现浊度。这种浊度表明无抗生素介质中微生物的生长。这可能是由于使用抗生素对角膜的治疗不完整，或者由于处理过程中的污染。这些角膜没有进行进一步实验，被丢弃了。在无抗生素介质中孵育角膜时，通过在孵化器中使用频繁的绝育运行，使用带过滤器的一次性移液器提示，以及在消毒用于从猪眼中切除角膜的工具时适当小心，避免了浊度的发展。另一个关键步骤是在感染前将角膜放入玻璃模具中。玻璃模具使一个人能够保持角膜的凸形。角膜的凸起是角膜表面保留感染剂量或治疗剂的挑战。因此，必须确保角膜和玻璃模具之间有足够的密封。当角膜和玻璃模具之间有足够的密封时，霉菌上方的环形结构创建一个储层，以保留感染剂量或治疗剂。通过将 DMEM agar 完全填充玻璃模具的宽段，确保密封充足。
与任何模型一样，描述的前活体猪角膜模型也有限制。此处描述的模型不模仿整个角膜撕裂膜的组成、流动和补充。闪烁提供的机械动作也未纳入模型。文献中一致认为，泪膜的组成和动力学，和闪烁是重要的防御机制，删除外国粒子和微生物从眼睛13。事实上，该模型也缺乏在体内感染期间触发的免疫反应。在这些防御机制的存在下，体内感染的进展很可能与此处描述的前体内模型所观察到的不同。尽管有这些局限性，但前体猪角膜模型对于测试现有和新兴抗菌素的有效性是相关的，主要有两个原因:1） 前体内模型中的细菌的生理学模仿体内条件，因为细菌增殖取决于它们破坏角膜组织的能力:2） 该模型将三维组织作为治疗的扩散屏障，就像在体内一样。因此，前体内模型比传统的抗菌易感性测试技术具有优势。
这里描述的前活猪角膜模型也可用于研究导致角膜炎的不同菌株、真菌和酵母菌。此前体角膜模型是可重复的，允许一个人在短时间内生成复制，不像在体内模型。理论上可以添加人工眼泪或宿主免疫防御细胞来模拟实时场景，而不是PBS。角膜是从同一品种的猪身上获得的，屠宰时大约21-23周大。因此，与从人类尸体中获得的复制品相比，复制品之间的变异性较小。在过去几年中，使用猪外腔角膜模型进行生物医学应用的概念越来越受欢迎，因为它与人眼在生物学上相似，这使得这个模型更容易比较14。人们越来越有兴趣使用猪角膜移植15，16或作为干眼17或伤口愈合18的模型。

角膜感染是致盲的重要原因，在低收入和中等收入国家流行。这种疾病的病因因地区而异，但细菌占这些病例的绝大多数。伪多莫纳斯阿鲁吉诺萨是导致一种快速进步的疾病的重要病原体。在许多情况下，患者留下频闪疤痕，不规则的散光，需要移植或在最坏的情况下，失去一只眼睛1，2。
由 P.aeruginosa 引起的细菌性角膜炎是一种难以治疗的眼睛感染，特别是由于抗微生物药物耐药菌 株的日益出现。在过去十年中，很明显，测试和开发新的治疗角膜感染的方法，一般来说，特别是那些由 伪多莫纳斯 sp.引起的，对于对抗抗生素耐药性3的当前趋势至关重要。
为了测试角膜感染新疗法的疗效，传统的体外微生物方法是一种糟糕的替代方法，因为在实验室培养期间和在体内感染期间细菌生理学的差异，以及由于缺乏宿主接口4，5。然而，体内动物模型昂贵、耗时，只能提供少量的复制品，并引起人们对动物福利的担忧。
在本文中，我们展示了一个简单和可重复的角膜炎体外猪模型，可用于测试急性和慢性感染的各种治疗方法。我们已经使用 P.aeruginosa 进行这个实验，但模型也与其他细菌，以及生物体，如真菌和酵母，导致角膜炎运作良好。

<table><tbody><tr><td>50 mL Falcon tube</td><td>SLS</td><td>352070</td><td></td></tr><tr><td>Amphotericin B</td><td>Sigma</td><td>A2942</td><td></td></tr><tr><td>Cellstar 12 well plate</td><td>Greiner Bio-One</td><td>665180</td><td></td></tr><tr><td>Dextran</td><td>Sigma</td><td>31425-100mg-F</td><td></td></tr><tr><td>Distel</td><td>Fisher Scientific</td><td>12899357</td><td></td></tr><tr><td>DMEM + glutamax</td><td>SLS</td><td>D0819</td><td></td></tr><tr><td>Dual Oven Incubator</td><td>SLS</td><td>OVe1020</td><td>Sterilising oven</td></tr><tr><td>Epidermal growth factor</td><td>SLS</td><td>E5036-200UG</td><td></td></tr><tr><td>F12 HAM</td><td>Sigma</td><td>N4888</td><td></td></tr><tr><td>Foetal calf serum</td><td>Labtech International</td><td>CA-115/500</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>15307805</td><td></td></tr><tr><td>Handheld homogeniser 220</td><td>Fisher Scientific</td><td>15575809</td><td>Homogeniser</td></tr><tr><td>Heracell VIOS 160i</td><td>Thermo Scientific</td><td>15373212</td><td>Tissue culture incubator</td></tr><tr><td>Heraeus Megafuge 16R</td><td>VWR</td><td>521-2242</td><td>Centrifuge</td></tr><tr><td>Insulin, recombinant Human</td><td>SLS</td><td>91077C-1G</td><td></td></tr><tr><td>LB agar</td><td>Sigma</td><td>L2897</td><td></td></tr><tr><td>Multitron</td><td>Infors</td><td>Not appplicable</td><td>Bacterial incubator</td></tr><tr><td>PBS</td><td>SLS</td><td>P4417</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>SLS</td><td>P0781</td><td></td></tr><tr><td>Petri dish</td><td>Fisher Scientific</td><td>12664785</td><td></td></tr><tr><td>Petri dish 35x10mm CytoOne</td><td>Starlab</td><td>CC7672-3340</td><td></td></tr><tr><td>Povidone iodine</td><td>Weldricks pharmacy</td><td>2122828</td><td></td></tr><tr><td>Safe 2020</td><td>Fisher Scientific</td><td>1284804</td><td>Class II microbiology safety cabinet</td></tr><tr><td>Scalpel blade number 15</td><td>Fisher Scientific</td><td>O305</td><td></td></tr><tr><td>Scalpel Swann Morton</td><td>Fisher Scientific</td><td>11849002</td><td></td></tr></tbody></table>

establishing a porcine ex vivo cornea model for studying drug treatments against bacterial keratitis

在开发新型抗菌药物时，动物试验的成功取决于从体外试验到体内动物感染的抗菌效果的准确推断。现有的体外测试通常高估了抗菌效能，因为主机组织作为扩散屏障的存在没有被考虑在内。为了克服这一瓶颈，我们开发了一种细菌性角膜炎的前活体猪角膜模型，使用 伪多莫纳斯阿鲁吉诺萨 作为原型生物体。本文描述了猪角膜的准备和建立感染的协议。定制玻璃模具可直接设置角膜用于感染研究。该模型模拟体内感染，因为细菌增殖取决于细菌损害角膜组织的能力。感染的建立被证实为通过可行的板数评估的殖民地形成单位数量的增加。结果表明，使用此处描述的方法，可以在前体角膜中以高度可重复的方式建立感染。该模型可以在未来扩展，以模拟角膜炎引起的微生物以外的 P.阿鲁吉诺萨。该模型的最终目标是研究抗菌化疗对细菌感染进展的影响，在更能代表体内感染的情况下。通过这样做，这里描述的模型将减少动物用于测试，提高临床试验的成功率，并最终使新型抗菌素快速翻译到诊所。

玻璃模具的设计是一个创新和原创的想法，其使用使我们能够建立模型在一致的方式与最小的/没有污染的问题。这些模具是由谢菲尔德大学的玻璃鼓风机根据设计（图1A）制备的。实验设置保持角膜的凸形，并将细菌保存在发生感染的上皮上部（图1B）上。
猪角膜通常在中等的几天后膨胀。这是正常的，我们发现角膜之间没有显著的区别，没有添加德克斯特兰，这通常是为防止角膜肿胀（图1H）而添加的。角膜通常受伤，以帮助细菌穿透上皮。虽然受伤（切口）和未切割（未切割）角膜之间的感染进展没有显著差异，但我们注意到未切割角膜（图1C）的复制品之间存在更多差异。用PBS洗两次角膜可以去除不附着在上皮上的多余的细菌。24小时（图1D）中感染P.阿鲁吉诺萨PAO1的洗过和未洗过的猪角膜在CFU方面有显著差异。感染 PA14 和 PAO1 的猪角膜和兔角膜的 CFU 计数没有显著差异（图 1E，1F）。两种型号的结果都是可重复的。24小时后，感染假角膜菌株的角膜总是发育不透明，与未受感染的角膜（图1G）相比，切口区域变得更加明显和开放。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61156/61156fig01.jpg"> 图1:感染 伪多莫纳斯阿鲁吉诺萨的外体角膜。（A） 用于维持角膜形状和促进细菌引入和治疗的玻璃模具的示意图图片。玻璃模具的厚度为 1.5 mm，与用牛酸盐玻璃制成的试管的厚度相同。（B） 实验设置的示意图。（C） 测试同质化后伤人对最终CFU计数的影响。未切割 （n = 16） 和切口 （n = 28） 角膜感染了 P. 阿鲁吉诺萨 PAO1 和 P. 阿鲁吉诺萨 PA14 24 小时。角膜在均质化前用 1 mL 的 PBS 洗净。错误条表示标准偏差。（D） 测试用2×1 mL的PBS（n = 6）清洗角膜的效果，并在感染 P.aeruginosa PAO1后24小时内不清洗（n =6）。错误条表示标准偏差。（E） 感染 P. 阿鲁吉诺萨 PAO1 和 P. 阿鲁吉诺萨 PA14 的猪角膜的最终 CFU 计数为 24 小时 （n = 10）。角膜被洗净和切开。错误条表示标准偏差。（F） 感染 P.阿鲁吉诺萨 PAO1和 P.阿鲁吉诺萨 PA14的兔子角膜的最终CFU计数为24小时（n =6）。角膜被洗净和切开。错误条表示标准偏差。（G） 感染 P.阿鲁吉诺萨 PAO1的外活猪角膜图片24小时。控制受伤，但没有添加细菌。受感染的角膜受伤，切口侧增加了10 7 个CFU。没有从对照角膜中恢复CFU。（H） 最终 CFU 在感染 P. aeruginosa PAO1 24 小时后从用德克斯特兰 （n = 2） 治疗的角膜和那些没有德克斯特兰 （n = 9） 的角膜中恢复。角膜被洗净和切开。错误条表示标准偏差。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61156/61156fig01large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>

Watch this Scientific Journal Video about Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis at JoVE.com

Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

本文描述了建立细菌性角膜炎前活体猪模型的分步协议。 伪多莫纳斯阿鲁吉诺萨 被用作原型生物体。这种创新模式模仿体内感染，因为细菌的增殖取决于细菌破坏角膜组织的能力。

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to ...

establishing-porcine-ex-vivo-cornea-model-for-studying-drug

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

5.1K 次观看.  University of Sheffield. 本文描述了建立细菌性角膜炎前活体猪模型的分步协议。 伪多莫纳斯阿鲁吉诺萨 被用作原型生物体。这种创新模式模仿体内感染，因为细菌的增殖取决于细菌破坏角膜组织的能力。

视频: Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1). Herpes keratitis is currently the leading cause of both cornea-derived and infection-associated blindness in the developed world. Typical presentation of herpes keratitis includes infection of the corneal epithelium and sometimes the deeper corneal stroma and endothelium, leading to such permanent corneal pathologies as scarring, thinning, and opacity 1.
Corneal HSV-1 infection is traditionally studied in two types of experimental models. The in vitro model, in which cultured monolayers of corneal epithelial cells are infected in a Petri dish, offers simplicity, high level of replicability, fast experiments, and relatively low costs. On the other hand, the in vivo model, in which animals such as rabbits or mice are inoculated directly in the cornea, offers a highly sophisticated physiological system, but has higher costs, longer experiments, necessary animal care, and a greater degree of variability. 
In this video article, we provide a detailed demonstration of a new ex vivo model of corneal epithelial HSV-1 infection, which combines the strengths of both the in vitro and the in vivo models. The ex vivo model utilizes intact corneas organotypically maintained in culture and infected with HSV-1. The use of the ex vivo model allows for highly physiologically-based conclusions, yet it is rather inexpensive and requires time commitment comparable to that of the in vitro model.

Ex Vivo Organotypic Corneal Model of Acute Epithelial Herpes Simplex Virus Type I Infection

In this video article we describe the use of a new ex vivo model of acute herpes simplex virus type I corneal epithelial infection.

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

Herpes keratitis is one of the most severe pathologies associated with the herpes simplex virus-type 1 (HSV-1).  Herpes keratitis is currently the leading ...

科研

神经科学

Porcine Corneal Tissue Explant to Study the Efficacy of Herpes Simplex Virus-1 Antivirals

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a seroprevalence that ranges from 60-90% worldwide, the Herpes Simplex Virus type-1 (HSV-1) commonly causes mucocutaneous lesions of the orofacial region which also manifest as lesions and infection-associated blindness. While current antiviral drugs are effective, emergence of resistance and persistence of toxic side-effects necessitates development of novel antivirals against this ubiquitous pathogen. Although in vitro assessment provides some functional data regarding an emerging antiviral, they do not demonstrate the complexity of ocular tissue in vivo. However, in vivo studies are expensive and require trained personnel, especially when working with viral agents. Hence ex vivo models are efficient yet inexpensive steps for antiviral testing. Here we discuss a protocol to study infection by HSV-1 using porcine corneas ex vivo and a method to treat them topically using existing and novel antiviral drugs. We also demonstrate the method to perform a plaque assay using HSV-1. The methods detailed may be used to conduct similar experiments to study infections that resemble the HSV-1 pathogen.

We describe the use of a porcine cornea to test the antiviral efficacy of experimental drugs.

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

Viruses and bacteria can cause a variety of ocular surface defects and degeneration such as wounds and ulcers through corneal infection. With a ...

免疫与感染

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

创面愈合研究的前活体角膜器官培养模型

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

医学

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

使用分裂的角质按钮的孔膜内皮器官培养模型

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

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, ...

<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 ...

Corneoscleral Button Isolation: A Surgical Procedure to Obtain Corneoscleral Button from Porcine Eye

Source: Okurowska, K., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/61156">Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis.</a> J. Vis. Exp. (2020).
In this video, we show a procedure to surgically isolate the corneoscleral button to generate an ex vivo porcine cornea model. The model is helpful for histological, pathological, and pharmacological studies due to its similarity to the human eye.

角膜巩膜纽扣分离术:一种从猪眼获取角膜巩膜纽扣的外科手术

Source: Okurowska, K., et al. Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis. J. Vis. Exp. (2020).
...

建立猪外维沃角膜模型研究药物治疗细菌性角膜炎

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此视频中的章节

体外器官角膜模型急性上皮疱疹，单纯疱疹病毒I型感染

猪角膜组织外植研究单纯疱疹病毒-1抗病毒药物的功效

创面愈合研究的前活体角膜器官培养模型

使用分裂的角质按钮的孔膜内皮器官培养模型

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

角膜上皮机械和化学损伤的离体和体内动物模型

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

角膜巩膜纽扣分离术:一种从猪眼获取角膜巩膜纽扣的外科手术