Name: use of rabbit eyes in pharmacokinetic studies of intraocular drugs
Uploaded: 2016-07-23T13:00:00.000+00:00
Duration: 10 min 2 s
Description: Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

We would like to thank Ms. Ji Hyun Park and Ji Yeon Park for their technical assistance in the animal experiments. This work was supported by a grant from the Seoul National University Bundang Hospital Research Fund (grant number: Grant No. 14-2014-022) and from a grant (CCP-13-02-KIST) from the Convergence Commercialization Project of the National Research Council of Science and Technology, Seoul, Korea.

我们的协议如下首尔国立大学盆唐医院的机构动物护理和使用委员会（IACUC），其批准的所有在该协议提出的动物的程序及动物护理方法的指导方针。该IACUC是完全符合指南的实验室动物的护理和使用（2011）第八版。所有的程序都用坚持协会的研究在视觉与眼科声明对动物眼科和视觉研究使用动物的指导进行。单独的笼子被用于房屋的兔子。另外的手术或制剂在进行该实验前（ 即 ，灭菌）可以不需要。 1.药物眼内注射到兔眼<ol><li>麻醉健康新西兰白兔体重1.5-2公斤，盐酸替来他明和唑拉西泮的混合肌肉注射盐酸盐（15毫克/千克）和赛拉嗪盐酸盐（5毫克/千克）。通过监控眼睑（闪烁）反射或夹捏耳廓验证麻醉。 注:如果眼内药物结合眼部色素，结合度可能会导致药物的药代动力学眼部修饰。例如，在有色兔的玻璃体和房水的颜料结合药的半衰期可以增加相比，在白兔14。在这种情况下，人的眼睛具有不同程度的色素沉着和白化兔眼可能不代表人类对应从着色兔眼所获得的数据可以更相似的，适用于人眼。因此，考虑药物相互作用色素，使用色素或白兔应慎重考虑，其结果应与兔的应变（色素）来解释。然而，白兔被推荐用于比较药动学性质如对d研究地毯颜料相互作用可能在比较的混杂因素。 <ol><li>申请用1％盐酸丙美卡因眼科眼药水局部麻醉剂。使用兽医软膏（2％羟丙基甲基纤维素），防止眼睛干涩，直到从麻醉中恢复。 注意:切勿将无人看管的兔子，而麻醉。 </li></ol></li><li>扩张瞳孔与盐酸去氧肾上腺素和托的一至两滴。 </li><li>对于眼内注射前手术准备，应用5-10％碘伏到用棉签5眼周皮肤。放置5-10％碘伏一滴在眼睛的结膜。 </li><li>辖眼压的药物无论是玻璃体或使用房内无菌技术5。 注:在全身循环吸收的药物也可能会渗透到对侧眼。当药物施用于双眼，一只眼睛的药物浓度可通过药物注射到受影响另一只眼睛。如果它被确认对侧注射的影响可以忽视，因为在循环的药物不能渗透另一只眼睛，使用双眼为眼内注射的，可以认为，因为它是经济的并最小化牺牲动物的数量在药代动力学研究。对于下面的眼内注射全身浓度药代动力学研究，只使用一只眼睛是适当的。 <ol><li>用于玻璃体内注射，使用地下30针和任一商业1毫升注射器，胰岛素注射器，或玻璃注射器垂直于巩膜表面5注入药物后面的颞象限手术缘玻璃体内1毫米。 注:药物用于前房和玻璃体内注射的体积变化取决于药物进行调查。有关使用兔眼抗VEGF剂的药物动力学实验中，先前的研究中所用0.025，0.05（最常见的），或者0.1米升贝伐单抗或兰尼单抗的前房或玻璃体内注射的。在我们以前的上的抗血管内皮细胞生长因子将0.05ml贝伐单抗15，0.025毫升兰尼单抗，或0.03毫升VEGF-陷阱16的眼内药代动力学研究中使用。无穿刺最大的安全的音量被认为是0.1毫升，尽管很少有证据支持这一17。如果过度体积被注入或者前房内或玻璃体内，眼内压大大增加。除了视神经损害由增加的眼内压（IIOP）直接造成的，即青光眼视神经损伤，在极端情况下，IIOP导致受损眼灌注和视网膜中央动脉阻塞，它类似于在大脑中风。 </li><li>对于前房内注射剂，通过与锥角巩膜缘插入地下30针上和推进它每年注射药物进入前房rallel虹膜平面以减少外伤的虹膜或透镜的危​​险。 注意:根据药物制剂，针大于30 G能够被使用。需要较大号针头的条件包括含有药物的微球，大蛋白质药物，和高粘度的制剂。 </li><li>注射后，压缩注射部位用无菌棉尖涂药以促进伤口愈合。 注意:通常情况下，30秒就足够了伤口愈合，当一个地下30针被用于眼内注射。然而，如果是用于眼内注射较大号针头，更长的时间将是适当的用于伤口的密封，以减少从巩膜创口的泄漏。要检查巩膜创口泄漏是很重要的，以确保在眼内注射后立即使用的眼内药物的量是相同的注射量，特别是如果使用较大号针头。 </li></ol></li><li>每天观察处理后的兔子一周，每周事后一次，重症眼内炎症（结膜充血和积脓）的任何迹象，直到安乐死。 注:镇痛药注射后使用地下30针是不伴随手术后疼痛微创过程不需要如眼内注射。不要返回已动过手术，以该公司的其他动物，直到完全康复的动物。 </li></ol> 2.样品制备<ol><li>对摘除术，安乐死眼内药物注射后的不同时间点的兔子（ 例如 ，1小时或1，2，5，9，14，或30天）。 注意:这些时间点取决于所关注的药物和已知的药物动力学曲线。对于每个时间点，使用至少两个眼球13。对于可靠的数据的质量，采样时间应仔细均衡采样选择，至少四个时间点的药物13的两个半生命中的至少一个时间范围</SUP&gt;。我们认为，6-8时间点与均衡采样可能是适当的。对于小分子（≤1,000达）18，特别是大于1的第一个24小时的过程中的时间点是至关重要的。因此，对于大分子（&gt; 1000道尔顿）18，一个采样周期，如1小时和1，2，5，9，14，30天19可以是一个很好的选择。对于小分子（≤1,000道尔顿），1，2，4和8小时，1，3和7天可以是一个选项20。根据不同的分子量，采样周期可进一步修饰。 <ol><li>安乐死，辖10毫升15％静脉氯化钾与替来他明盐酸盐，唑拉西泮盐酸盐（15毫克/千克），和盐酸赛拉嗪（5mg / kg的）的混合物中的肌肉内注射麻醉家兔后迅速。 </li></ol></li><li>打开与眼睑拉钩睑裂。做一个360°结膜切口2-3毫米后角膜缘和向后通过解剖的C扩展它onjunctiva和来自世界各地眼球筋膜囊。 </li><li>切开眼外肌接近其插入到全球。钳与弯钳视神经然后剪切镊子和地球之间的神经。取出眼球本身同时使周围组织完好无​​损。眼球摘除后，立即冻结了眼球，并将它们存储在-80℃。 注意:有可能是立即冻结两个选项。如果药物浓度在非常早的阶段测量（ 即小于1小时），液氮可能更适合，以保证眼球的更好及时冻结。然而，对于以后期间，冰可用于立即冷却该眼球，然后冰柜可以用于眼球储存在-80℃。 </li><li>获得眼球的所有时间点后，将冷冻的眼球分离成三个室，玻璃体，房水和视网膜/脉络膜。分离defros之前，这些车厢婷。 注意:用于分离成三个隔间的最重要的一点是该过程的速度。眼球应非常迅速分离除霜之前，可以在冰上进行的过程延迟解冻。 <ol><li>要打开全球，做一个切口在角膜缘（300℃以上）用手术刀刀片。获得在虹膜，房水前的冷冻室。 </li><li>通过拉动和使用组织钳取出谴责某个冰冻的虹膜和晶状体。当玻璃体访问时，从剩余的组织（视网膜/脉络膜/巩膜）分离出来获得冷冻玻璃。 </li><li>用15号解剖刀刀片，从底层巩膜分开视网膜/脉络膜组织。 </li></ol></li><li>用于免疫测定，除霜房水样本，并测量各样品的体积。通过从含管的t减去空管的重量测量冷冻样品的重量他冷冻样本。 注意:由于冷冻样品的比重大致为1，各样品的重量可用于计算样品的体积。 </li><li>称量玻璃体样品，除霜的样品中，并在4℃下过夜溶解它们于1.0ml含有旋转器上的1％牛血清白蛋白的磷酸盐缓冲盐水。然后，离心将样品在387×g离心10分钟21。 </li><li>称取冷冻视网膜/脉络膜样品同质化。添加蛋白抽提试剂与组织的1:10（1克组织/ 10 mL试剂的）试剂的比率。用预先冷却microhomogenizer均质化组织。离心10分钟，裂解的样品在12,000-20,000 XG并将上清液转移到一个冷却的EPP管。 </li></ol> 3.免疫注:若干分析方法可用于蛋白质浓度的测定。选择一个合适的定量分析方法，Depending该检测范围。简言之，高效液相色谱法的选择离子监测模式可检测分子的皮克水平，而LC-MS / MS可以检测蛋白质的纳克和皮克水平与MRM / PRM模式分析，分别。 ELISA法的检测极限被认为是在皮克水平。 <ol><li>对于间接酶联免疫吸附测定（ELISA）来测量抗VEGF剂的浓度，使用ELISA试剂盒在96孔板检测感兴趣的药物和生成的已知的药物浓度的标准曲线。 <ol><li>稀玻璃体，房水，并用在1×磷酸盐缓冲盐水（PBS）的0.1％牛血清白蛋白浓度的线性范围内，该视网膜/脉络膜样品，并使用这些用于测定。 </li></ol></li><li>划分在板上的试样成等分试样以100μl/孔。在4℃孵育过夜，然后用200微升0洗涤液的三次洗涤板。05％吐温20在1×PBS中。 注:抗VEGF存在于样品充当用于ELISA的一次抗体;因此，额外使用一个初级抗体是没有必要的。 </li><li>稀释二级抗体，以1:1×PBS中20,000在0.1％BSA中，并加入100微升每孔的稀释的溶液。在4℃下用稀释的第二抗体温育该板过夜后，测量在450nm波长的光密度。减去平均每个标准和样品重复读数的平均值为零的标准光密度。 </li><li>通过使用能够生成四参数逻辑（4-PL）曲线拟合，如SOFTMAX临的计算机软件减少数据创建基于从已知浓度的药物溶液的相对光信号的标准曲线。 [拟合] - 用于创建标准曲线，4-PL曲线拟合可以通过在[标准曲​​线]点击[4-参数]来获得。 </li><li>计算出样品fr处的药物浓度嗡标准曲线。 注:检测限（LOD）的抗VEGF药物的局限性在我们的实验进行了研究。贝伐单抗的最低检测限为0.024-3.125纳克/毫升，并且aflibercept的是0.039-10纳克/毫升。 </li></ol> 4.药代动力学分析方法注:对于PK分析，可以使用任一隔室或者非房室分析。在房室分析，分子的处置行为可以通过公式（模型）进行说明。因此，隔室PK分析可以在任何时间t预测的浓度，而非房室模型不能想像或预测浓度 - 时间曲线为其他给药方案。然而，房室模型拟合可能是一个复杂而漫长的过程。与此相反，假设在非房室模型的限制较少。的非房室方法简单，通常用于计算药代动力学参数如半寿期，清除率和分布容积。我们选择房室模型上的抗VEGF药物药代动力学研究。 <ol><li>分析使用建模软件，如凤凰城的WinNonlin软件房室模型的药物浓度数据。 <ol><li>单击[WNL5经典造型] [PK模式]并映射观察时间，给药量和药物浓度[设置]菜单内</li><li>在[模型选择]标签室模型（ 例如 ，车厢号），并单击[执行]按钮来计算模型参数。 </li></ol></li><li>在分析后，选择最能描述基于以下标准的药物浓度数据的最终室模型:（ 例如 ，标准误差）（1）赤池信息准则，（2）参数估计的精度，以及（3）图形分析（ 例如 ，拟合曲线的善良）。 </li><li>计算感兴趣的药代动力学参数，例如半衰期（T 1/2）和区域的时concentra下化曲线（AUC），从由隔室模型驱动的模型参数和方程。 </li></ol>

The authors have no conflicts of interest to disclose.

With the increasing use of intraocular drugs, such as anti-vascular endothelial growth factor (VEGF) agents, for the treatment of diverse ocular diseases, knowledge of the tissue distribution and clearance of the drug after the intraocular injection is important. Understanding the pharmacokinetics of intraocular drugs is important for understanding the efficacy and safety of drugs, determining the optimal dosage of the drugs, and minimizing systemic or intraocular complications. However, detailed pharmacokinetic studies ...

眼内药物递送的出现之前，药物治疗的眼内疾病的主要关注的是与该药物能渗透到眼睛的效率。血眼屏障防止许多物质，包括药物，扩散进入眼睛。因此，可能不容易获得的高于治疗水平的药物的浓度。眼内药物施用方法，包括前房内和玻璃体内注射，能够直接绕过血眼屏障1-3，使药物治疗浓度可在眼4,5来实现。 因此，玻璃体内给药已成为治疗几种眼疾病5,6-一种流行的方法。例如，玻璃体内注射被广泛用于与年龄有关的黄斑变性，糖尿病性视网膜病变，视网膜静脉阻塞，和眼内感染7-10进行。特别是，由于引进抗VEGF药物，玻璃体内注射的次数已显着地增加了对视网膜疾病的治疗。因此，了解这些药物的眼内药物动力学，用于评估药物治疗的有效性和安全性是重要的。 虽然药物的眼内施用被认为是眼病药物治疗的重大突破，监控眼球内的药物浓度是技术要求高。因为人眼仅含有少量的水状液（约200微升）和玻璃体（约4.5毫升， 表1）的，它在技术上是难以得到足够量的眼内流体的测量药物浓度。此外，被用于获得眼部流体的方法，如玻璃体攻丝或前房穿刺放，可能会损坏眼组织，并导致严重的并发症，例如白内障，眼内炎，或视网膜脱离11,12。因此，动物模型中的常用的眼内药物13的药代动力学研究中使用。在这些动物模型中，兔或猴是最常用的动物。 兔子，这是在家庭兔科的顺序兔形目的小型哺乳动物，在世界一些地区被发现。因为兔子是没有攻击性，他们很容易处理，在实验中使用，并观察。成本较低，动物，类似于眼睛大小，以人类随时可用性和大型数据库的信息进行比较青睐执行使用兔眼药代动力学研究。在本文中，对于眼内药物在兔眼药代动力学研究的协议描述。

<table><tbody><tr><td>Zoletil</td><td>Virbac Laboratories, Carros Cedex, France</td><td></td><td></td></tr><tr><td>Xylazine hydrochloride&amp;nbsp;</td><td>Fort Dodge Laboratories, Fort Dodge, IA</td><td></td><td></td></tr><tr><td>Proparacaine hydrochloride (Alcaine)</td><td>Alcon laboratories, Fort Worth, TX</td><td></td><td></td></tr><tr><td>Phenylephrine hydrochloride and tropicamide</td><td>Santen Pharmaceutical, Co., Osaka, Japan</td><td></td><td></td></tr><tr><td>Recombinant Human VEGF 165</td><td>R&amp;amp;D systems</td><td>293-VE-050</td><td></td></tr><tr><td>Carbobate-Bicarbonate buffer</td><td>SIGMA</td><td>C3041-50CAP</td><td></td></tr><tr><td>Nunc Microwell 96F w/lid 
Nunclon D Si</td><td>Thermo SCIENTIFIC</td><td>167008</td><td>96 well plate</td></tr><tr><td>Bovine Serum Albumin (BSA) 25 g(Net)</td><td>BOVOGEN</td><td>BSA025</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4 (1x), 500 ml</td><td>gibco</td><td>10010-023</td><td></td></tr><tr><td>Sheep anti-Human IgG Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-28652</td><td></td></tr><tr><td>Goat Anti-Human IgG Fc(HRP)</td><td>abcam</td><td>ab97225</td><td></td></tr><tr><td>Goat anti-Human IgG, Fab&#039;2 Secondary Antibody, HRP conjugate</td><td>Thermo SCIENTIFIC</td><td>PA1-85183</td><td></td></tr><tr><td>CelLytic MT&amp;nbsp; Cell Lysis Reagent</td><td>SIGMA</td><td>C3228-50ML</td><td>lysis buffer</td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #15</td><td></td></tr><tr><td>100 Scalpel Blades</td><td>nopa instruments</td><td>BLADE #10</td><td></td></tr><tr><td>Feather surgical blade stainless steel</td><td>FEATHER</td><td>11</td><td></td></tr><tr><td>1-StepTM TMB-Blotting substrate solution, 250 ml</td><td>Thermo SCIENTIFIC</td><td>34018</td><td></td></tr><tr><td>Stable Peroxide Substrate Buffer (10x), 100 ml</td><td>Thermo SCIENTIFIC</td><td>34062</td><td></td></tr><tr><td>Softmax Pro</td><td>Molecular Devices</td><td>v.5.4.1</td><td>software for generating standard curve</td></tr><tr><td>SAAM II&amp;nbsp;</td><td>Saam Institute, Seattle, WA</td><td></td><td>software for pharmacokinetic modeling</td></tr><tr><td>Phoenix WinNonlin</td><td>Pharsight, Cary, NC</td><td>v. 6.3</td><td>software for pharmacokinetic modeling</td></tr><tr><td>Avastin (bevacizumab)</td><td>Genentech</td><td></td><td></td></tr></tbody></table>

use of rabbit eyes in pharmacokinetic studies of intraocular drugs

给药的途径眼压使高浓度的治疗药物的交付，同时尽量减少他们的全身吸收。几种药物给药进入前房或玻璃体和眼内注射已有效地治疗各种眼内疾病。兔眼已广泛用于眼科研究相比，其他哺乳动物的动物是易于处理和经济，兔眼的大小是相似的人眼。使用地下30针，药物可注入兔眼的前房和玻璃体内的空间。然后眼球冷冻直至分析，并且可分为房水，玻璃体和视网膜/脉络膜。玻璃体和视网膜/脉络膜样品可以匀浆并分析前溶解。然后，可以执行免疫测定以测量在每个隔室的眼内药物浓度。合适的药代动力学模型可以是用于计算多个参数，诸如半衰期和药物的最大浓度。兔眼可用于眼内药物的药代动力学研究的良好模式。

用来进行的兔眼用无菌技术感兴趣的药物的玻璃体内注射的过程示于图1。将处理过的眼睛在预定的时间去核并储存在-80℃。对于分析，三个室，房水，玻璃体和视网膜/脉络膜，从冷冻兔眼分离，如在图2中表现出来。所述隔室的样品用于ELISA制备。与第二抗体温育后，光密度在96孔板，其中包含了玻璃体内注射后收集在多个时间点是从三个隔为标?...

Watch this Scientific Journal Video about Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs at JoVE.com

Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

眼内药物的药代动力学研究采用兔眼

The intraocular route of drug administration enables the delivery of high concentrations of therapeutic drugs, while minimizing their systemic absorption. ...

use-of-rabbit-eyes-in-pharmacokinetic-studies-of-intraocular-drugs

Research

JoVE Journal

Medicine

31.7K 次观看.  Seoul National University Bundang Hospital. Rabbits are widely used to study the pharmacokinetics of intraocular drugs. We describe a method for conducting pharmacokinetic studies of intraocular drugs using rabbit eyes.

视频: Use of Rabbit Eyes in Pharmacokinetic Studies of Intraocular Drugs

Novel Photoacoustic Microscopy and Optical Coherence Tomography Dual-modality Chorioretinal Imaging in Living Rabbit Eyes

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into sound waves and is currently under intensive investigation. However, most reported work to date is focused on the imaging of the posterior segment of the eyes of small animals, such as rats and mice, which poses challenges for clinical human translation due to small eyeball sizes. This manuscript describes a novel photoacoustic microscopy (PAM) and optical coherence tomography (OCT) dual-modality system for posterior segment imaging of the eyes of larger animals, such as rabbits. The system configuration, system alignment, animal preparation, and dual-modality experimental protocols for in vivo, noninvasive, label-free chorioretinal imaging in rabbits are detailed. The effectiveness of the method is demonstrated through representative experimental results, including retinal and choroidal vasculature obtained by the PAM and OCT. This manuscript provides a practical guide to reproducing the imaging results in rabbits and advancing photoacoustic ocular imaging in larger animals.

This manuscript describes the novel setup and operating procedure of a photoacoustic microscopy and optical coherence tomography dual-modality system for noninvasive, label-free chorioretinal imaging of larger animals, such as rabbits.

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

Photoacoustic ocular imaging is an emerging ophthalmic imaging technology that can noninvasively visualize ocular tissue by converting light energy into ...

科研

工程学

Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal abnormalities. In addition, pathological myopia is a common irreversible cause of visual impairment and blindness1-3. Our study demonstrates the effect of scleral crosslinking using riboflavin and ultraviolet-A radiation on the development of axial myopia in a rabbit model. The axial length of the eyeball was measured by A-scan ultrasound in New Zealand white rabbits aged 13 days (male and female). The eye then underwent 360&#176; conjunctival peritomy with scleral crosslinking, followed by tarsorrhaphy. Axial elongation was induced in 13 day-old New Zealand rabbits by suturing their right eye eyelids (tarsorrhaphy). The eyes were divided into quadrants, and every quadrant had two scleral irradiation zones, each with an area of 0.2 cm&#178; and a radius of 4 mm. Crosslinking was performed by dropping 0.1% dextran-free riboflavin-5-phosphate onto the irradiation zones 20 sec before ultraviolet-A irradiation and every 20 sec during the 200 sec irradiation time. UVA radiation (370 nm) was applied perpendicular to the sclera at 57 mW/cm&#178; (total UVA light dose, 57 J/cm&#178;). Tarsorrhaphies were removed on day 55, followed by repeated axial length measurements. This study demonstrates that scleral crosslinking with riboflavin and ultraviolet-A radiation effectively prevents occlusion-induced axial elongation in a rabbit model.

We demonstrate the effect of scleral crosslinking with riboflavin and UVA on an axial elongation rabbit eye. Axial elongation was induced in 13 day-old New Zealand rabbits (male and female) by suturing their right eye eyelids (tarsorrhaphy).

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

Myopic individuals, especially those with severe myopia, are at higher-than-normal risk of cataract, glaucoma, retinal detachment and chorioretinal ...

医学

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

一种用于兔视网膜下手术分步协议

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such as the cornea and conjunctiva. While therapeutic effects and pharmacokinetics of the drugs upon subconjunctival injection have been described in some studies, very few assess the ocular distribution of drugs or drug delivery systems (DDS). The latter is critical for the optimization of intraocular DDS design and drug bioavailability to achieve the desired ocular localization and duration of action (e.g., acute versus. prolonged). This study establishes the use of fiberoptic confocal laser microendoscopy (CLM) to qualitatively study the ocular distribution of fluorescent liposomes in real-time in live mice after sub-conjunctival injection. Being designed for in vivo visual inspection of tissues at the microscopic level, this is also the first full description of the CLM imaging method to study spatio-temporal distribution of injectables in the eye after subconjunctival injection.

Spatio-Temporal In Vivo Imaging of Ocular Drug Delivery Systems using Fiberoptic Confocal Laser Microendoscopy

We present a protocol for the use of fiberoptic confocal laser microendoscopy (CLM) to non-invasively study the spatio-temporal distribution of liposomes in the eye after subconjunctival injection.

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

Subconjunctival injection is an attractive route to administer ocular drugs due to easy trans-scleral access that bypasses anterior ocular barriers, such ...

生物工程

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 ...

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 ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

神经科学

用于免疫组织化学研究的兔眼解剖和冷冻切片

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

作者聚焦：优化兔模型中免疫组织化学的视网膜组织处理

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

获得全兔眼的高质量冷冻切片 

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

眼内药物的药代动力学研究采用兔眼

摘要

探索更多视频

此视频中的章节

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

一种用于兔视网膜下手术分步协议

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

获得全兔眼的高质量冷冻切片

获得全兔眼的高质量冷冻切片

获得全兔眼的高质量冷冻切片

眼内药物的药代动力学研究采用兔眼

摘要

探索更多视频

此视频中的章节

新型光声显微镜与光学相干层析成像在活兔眼中的双模脉络成像

巩膜交联兔模型使用核黄素和紫外辐射的轴性近视防治

一种用于兔视网膜下手术分步协议

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空 体内 成像

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

角膜缘血管丛全圆烧灼治疗啮齿动物手术诱发的青光眼

获得全兔眼的高质量冷冻切片

获得全兔眼的高质量冷冻切片

获得全兔眼的高质量冷冻切片

使用光纤共聚焦激光微内窥镜检查的眼部药物输送系统的时空体内成像