Name: scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model
Uploaded: 2016-04-03T14:00:00.000+00:00
Duration: 5 min 56 s
Description: Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

作者非常感谢达莉亚塞拉女士和EMI沙龙先生在实验室中他们的专业和优秀的技术工作。

动物按照关于在研究中使用的动物的ARVO分辨率处理。该研究方案经实验动物研究机构委员会（批准号022-4598-2; 021211）。 1.准备手术＃1 <ol><li>权衡和麻醉13日龄新西兰白兔用盐酸氯胺酮100毫克/毫升（100毫克/千克）和盐酸赛拉嗪25毫克/毫升（12.5毫克/千克）的肌肉注射。这些剂量使用，由于手术过程的长度。由于缺乏疼痛反射保证麻醉的适当水平。 </li><li>使用无菌棉签，申请眼药膏的少量非操作眼以防止角膜干燥。在操作过程中应用0.9％生理盐水滴在操作眼睛角膜，以防止角膜干燥。 </li><li>在局部麻醉后每只眼睛执行三个轴向长度测量（oxybuprocain使用超声A-扫描仪Ë盐酸盐0.4％），然后平均的测量。垂直应用探头角膜中央。 </li></ol> 2.手术＃1 - 预交联步骤<ol><li>使用眼科手术镊子和剪刀眼科手术显微镜下，进行手术的眼科镊子和剪刀360°结膜peritomy。 </li><li>使用有角度的钳或肌肉挂钩确定的四个眼外直肌和2-0编织丝非针刺缝合隔离。 注:丝线缝合将与所希望的方向移动的地球帮助。眼球被分成四个直肌肌肉（四象限）之间的象限。 <ol><li>标记用皮肤标记物两个区在每个象限中，一个在赤道巩膜和一个在巩膜。这些是在照射区域。 </li></ol></li><li>准备3毫升水5ml注射器含0.1％右旋糖酐自由，核黄素-5- phosphat即注射器连接到＆G泪道插管或地下25锥形hydrodelineator。 </li><li>制备的照射装置，其包括连接到一个斜面向下定制光纤的UV A（370纳米）的光源。继能源动力和校准测量（见下文说明），将设备设置为57毫瓦/平方厘米。 注意:校准由制造商进行。这种方法提供了57焦耳/ cm 2的总UVA光剂量。 （在0.2 平方厘米57毫瓦/平方厘米2是使用11.4毫瓦/ 0.2 平方厘米200秒11.4毫瓦/ 0.2厘米2的每0.2 2.2Ĵ厘米2点的累积负载） </li></ol> 3.手术＃1 - 交联<ol><li>通过在选定的象限的相反方向拉动丝线移动眼球待处理（ 例如，上拉和鼻治疗下颞象限时）。执行使用2-0编织丝非针刺缝合眼球运动，我solates每个直肌。 </li><li>应用含有0.1％游离葡聚糖核黄素-5-磷酸盐到照射开始前的照射区20秒光敏剂溶液。 </li><li>使用斜面向下定制光纤200秒的照射时间照射的照射区。测量每个区0.2平方厘米以4mm的半径的区域。 </li><li>应用在200秒照射周期含有0.1％游离葡聚糖核黄素-5-磷酸盐到辐照区每20秒光敏剂溶液。 注:执行照射和两名外科医生同时核黄素的下降。 </li><li>重复步骤3.1-3.4为四个象限眼球的。 </li></ol> 4.手术＃1 - 后交联步骤<ol><li>刮眼睛周围的皮毛。采用手术眼科剪修剪眼睑边缘，然后轻轻地缝合上，用4-0丝线编织象牙色下眼睑 - 3/8圈重诗切割 - 13毫米长度C-3针（tarsorrhaphy）。在步骤（氯霉素5％）的端部适用于盖缘眼药膏少量。 </li></ol> 5.手术＃1 - 术后护理<ol><li>保持加热灯下动物恢复过程中。无人看管，直到它已经恢复了足够的意识，以保持胸骨斜卧不要让动物。 </li><li>将动物在干净的笼子新的床上用品，直到其完全恢复。恢复后，将动物将被返回到动物房。返回动物对他的母亲，只有当清醒。 </li><li>在第一个48小时，检查有关的一般情况和疾病或感染的迹象的动物。辖镇痛按部1如果需要观察兔行为的变化辖镇痛。 （安乃近降至50毫克/ kg，每6小时）。 </li></ol> 6.手术＃2 <ol><li>之后一下55天urgery＃1，称重并用氯胺酮盐酸盐100毫克/毫升（100毫克/千克）和赛拉嗪盐酸盐25毫克/毫升（12.5毫克/千克）的肌内注射麻醉兔。 </li><li>使用眼科手术显微镜下手术眼科剪，去掉tarsorrhaphy。在表面麻醉下使用超声A-扫描器（盐酸奥布卡因0.4％）后，每一只眼睛执行三个轴向长度测量，然后平均的测量。垂直应用探头角膜中央。 </li><li>安乐死腹腔苯巴比妥钠（200毫克/ 1.5 kg体重）的兔子。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

我们使用具有核黄素和UVA辐照交联技术中存在的预防轴性近视的体内研究的第一个在兔模型。虽然不同的实验室动物可以在这种类型的研究中使用，我们选择了兔大多是由于眼睛的大小，并在巩膜表面上执行交联的需要。 我们发现，暴露兔子巩膜和缝合上下眼睑是具有挑战性的程序。我们建议修整眼睑边缘，并使用在我们的协议中提到的缝线，以确保眼睑被牢?...

近视是最常见的折射病症。在美国和欧洲近视的患病率被报告为30％左右，而在亚洲国家，它会影响到一般人群1,2的60％。近视的进展出现在多达近视患者的50％，通常在约-0.5屈光度超过两年的时间间隔3的速率。近视实行的医疗费用相当高，包括费用眼镜，隐形眼镜和屈光手术以及与青光眼，白内障，视网膜脱离和视力损伤4-6的增加健康风险的成本。 在近视的动物实验中，视力降低是由眼睑缝合7-10，封堵器放置在离眼睛和角膜纹身11短距离感应。但是，对于在这些研究中，以发生人工近视，遮挡过程必须执行在非常年幼的动物，因为没有视力剥夺实验卡尔灭蝇灯出对成人标本证明是成功的。 之一的高度近视的重要特征是与巩膜的渐进变薄，可能是巩膜的病理变化，由于视觉剥夺12或后正视的干扰反馈机制由于巩膜的一些代谢紊乱，如在Ehlers-当洛综合征13。最终，双方机制导致了拉伸和巩膜，视网膜和脉络膜变薄，由于近视巩膜的结构异常，如胶原减少纤维直径14,15和骚乱在原纤维16。 几项研究已经表明，受损的胶原交联是在近视巩膜17-18的减弱过程的一个重要因素。 Wollensak 等 19-21应用光敏剂核黄素和紫外线A（UVA）照射引起的胶原交联（370纳米）并指出在猪和人巩膜的刚性的显著，157％的增加在体外 19和增加了465％，在兔子巩膜刚性体内 （杨氏模量）20。 交联也对兔子巩膜长期效果体内 :4个月后，3天后的刚性增加了320.4％，277.6％和8个月（杨氏模量）22后502％。 治疗尝试阻止近视进展已经发表23-26但这些方法的成功是有争议的。防止进行性近视的没有有效的手段已被发现的日期。 近视的病因仍有争议，及其治疗提出了挑战。这些发现的基础上，它是假设，巩膜交联剂可以作为近视进展的基础巩膜处理的装置。这项研究的目的是检查巩膜胶原crosslinki纳克通过视轴闭塞诱导的轴性近视的发展的影响。

<table><tbody><tr><td>2-0 braided silk non-needled sutures&amp;nbsp;</td><td>ETHICON</td><td>W193</td><td></td></tr><tr><td>4-0 braided silk ivory color&amp;nbsp;</td><td>ETHICON</td><td>W816</td><td></td></tr><tr><td>0.1% dextran-free riboflavin-5-phosphate 1 mg:1 ml</td><td>Concept for Pharmacy Ltd</td><td>D2-5025</td><td></td></tr><tr><td>UV A (370 nm) light source&amp;nbsp;</td><td>O/E LAND Inc</td><td>NCSU033B</td><td></td></tr><tr><td>Beveled down custom made fiber optic&amp;nbsp;</td><td>Prizmatix Ltd</td><td></td><td></td></tr><tr><td>26 G lacrimal cannula&amp;nbsp;</td><td>Beaver-visitec International Ltd.&amp;nbsp;</td><td>REF581276</td><td></td></tr><tr><td>25 G tapered hydrodelineator [Blumenthal]&amp;nbsp;</td><td>Beaver-visitec International Ltd.&amp;nbsp;</td><td>REF585107</td><td></td></tr><tr><td>13 days old rabbits</td><td>Harlan&amp;nbsp;</td><td>1NZWR40</td><td></td></tr><tr><td>Ultrasonic biometer</td><td>Allergan-Humphrey</td><td>820-519</td><td></td></tr><tr><td>Skin marker</td><td>Devon</td><td>4237101664X</td><td></td></tr></tbody></table>

scleral cross-linking using riboflavin and ultraviolet-a radiation for prevention of axial myopia in a rabbit model

近视的个体，特别是那些有严重的近视，是在白内障，青光眼，视网膜脱离和脉络膜视网膜异常高于正常危险。此外，病理性近视是视力障碍和失明1-3常见的不可逆的原因。我们的研究表明巩膜交联使用核黄素和紫外线的辐射对轴性近视的兔模型发展的影响。眼球的眼轴长度是由新西兰13岁天（男性和女性）白兔A扫描超声检测。眼睛然后行360°结膜peritomy巩膜交，其次是tarsorrhaphy。轴向伸长被缝合的右眼眼睑（tarsorrhaphy）13日龄新西兰兔诱导。眼睛被分成象限，每个象限有两个巩膜照射区域，每个区域具有0.2平方厘米的面积和4毫米的半径。交联是通过滴加0.1％进行葡聚糖 - 自由核黄素-5-磷酸盐到紫外线-A照射前照射区20秒，并且在200秒的照射时间每20秒。 UVA辐射（370纳米），在57毫瓦/平方厘米（总UVA光剂量，57焦耳/平方厘米）垂直于巩膜应用。 Tarsorrhaphies进行55天遭到移除，随后重复轴向长度的测量。这项研究表明，核黄素和紫外线的辐射交联巩膜有效防止阻塞引起的轴向伸长在兔模型。

图1和图 2图示表明两组的轴向长度的测量。组1家兔接受巩膜交联和tarsorrhaphy上右眼，而左眼上未（ 图1）进行操作。而左眼上未（ 图2）操作的第2组兔的右眼接受仅peritomy和tarsorrhaphy。 在第1组，其接受巩膜交联和tarsorrhaphy，平均在右眼轴向长度测量眼睑缝合前10....

Watch this Scientific Journal Video about Scleral Cross-linking Using Riboflavin and Ultraviolet-A Radiation for Prevention of Axial Myopia in a Rabbit Model at JoVE.com

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

我们证明与核黄素和UVA巩膜交联的上一个轴向伸长兔眼的影响。轴向伸长率13日龄新西兰兔缝合的右眼眼睑（tarsorrhaphy）（男性和女性）引起的。

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 ...

scleral-cross-linking-using-riboflavin-ultraviolet-radiation-for

Research

JoVE Journal

Medicine

8.8K 次观看.  Rabin Medical Center, Beilinson Campus. 我们证明与核黄素和UVA巩膜交联的上一个轴向伸长兔眼的影响。轴向伸长率13日龄新西兰兔缝合的右眼眼睑（tarsorrhaphy）（男性和女性）引起的。 

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

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

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

视网膜脱离模型鼠害由透明质酸钠的视网膜下注射

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

医学

Three Different Protocols of Corneal Collagen Crosslinking in Keratoconus: Conventional, Accelerated and Iontophoresis

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been introduced as an efficient treatment option. In biological and chemical sciences, crosslinking refers to new chemical bonds formed between reactive molecules. Hence, the aim of corneal collagen CXL is to synthetically increase the formation of crosslinks between collagen fibrils in the corneal stroma. Despite the fact that the efficiency of the conventional CXL (C-CXL) protocol has already been shown in several clinical studies, it might benefit from improvements in duration of the procedure and removal of corneal epithelium. Hence, in order to provide a coherent evaluation of two new and optimized CXL protocols, we studied keratoconus patients who had undergone one of the three CXL treatments: iontophoresis (I-CXL), accelerated CXL (A-CXL), and conventional CXL (C-CXL). A-CXL is a 6 time faster CXL procedure using a ten time higher UVA irradiance but still including an epithelium removal. Iontophoresis is a transepithelial non-invasive technique in which a small electric current is applied to improve riboflavin penetration throughout the cornea. Using anterior segment optical coherence tomography (AS OCT) and in vivo confocal microscopy (IVCM), we conclude that regarding the depth of treatment penetration, conventional CXL protocol remains the standard for treating progressive keratoconus. Accelerated CXL seems to be a quick, effective and safe alternative to treat thin corneas. The use of iontophoresis is still being investigated and should be considered with greater caution.

Corneal collagen cross-linking (CXL) is the only conservative treatment currently available to halt keratoconus progression by improving the biomechanical rigidity of the corneal stroma. The aim of this manuscript is to highlight the methods of three different protocols of CXL: conventional CXL (C-CXL), accelerated CXL (A-CXL), and iontophoresis CXL (I-CXL).

角膜胶原交联的圆锥角膜的三种不同的协议:常规，加速和离子导入

Keratoconus is a bilateral and progressive corneal ectasia. In order to slow down its progression, corneal collagen cross-linking (CXL) has recently been ...

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 ...

In vivo Structural Assessments of Ocular Disease in Rodent Models using Optical Coherence Tomography

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable tool to evaluate and characterize changes in a variety of retinal and ocular disease and injury models. In light induced retinal degeneration models, SD-OCT can be used to track thinning of the photoreceptor layer over time. In glaucoma models, SD-OCT can be used to monitor decreased retinal nerve fiber layer and total retinal thickness and to observe optic nerve cupping after inducing ocular hypertension. In diabetic rodents, SD-OCT has helped researchers observe decreased total retinal thickness as well as decreased thickness of specific retinal layers, particularly the retinal nerve fiber layer with disease progression. In mouse models of myopia, SD-OCT can be used to evaluate axial parameters, such as axial length changes. Advantages of SD-OCT include in vivo imaging of ocular structures, the ability to quantitatively track changes in ocular dimensions over time, and its rapid scanning speed and high resolution. Here, we detail the methods of SD-OCT and show examples of its use in our laboratory in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia. Methods include anesthesia, SD-OCT imaging, and processing of the images for thickness measurements.

Here, we describe the use of spectral-domain optical coherence tomography (SD-OCT) to visualize retinal and ocular structures in vivo in models of retinal degeneration, glaucoma, diabetic retinopathy, and myopia.

使用光学相干断层扫描对啮齿动物模型中眼部疾病的体内结构评估

Spectral-domain optical coherence tomography (SD-OCT) is useful for visualizing retinal and ocular structures in vivo. In research, SD-OCT is a valuable ...

生物工程

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

兔巩膜二次谐波信号作为治疗组织交叉连接 (txl-) 评价近视的工具

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

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, ...

Subretinal Transplantation of Human Embryonic Stem Cell Derived-retinal Pigment Epithelial Cells into a Large-eyed Model of Geographic Atrophy

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, which leads to subsequent degeneration of vital retinal structures (e.g., photoreceptors) causing severe vision impairment. Similarly, RPE-loss and decrease in visual acuity is seen in long-term follow up of patients with advanced wet age-related macular degeneration (AMD) receiving intravitreal anti-vascular endothelial growth factor (VEGF) treatment. Therefore, on the one hand, it is fundamental to efficiently derive RPE cells from an unlimited source that could serve as replacement therapy. On the other hand, it is important to assess the behavior and integration of the derived cells in a model of the disease entailing surgical and imaging methods as close as possible to those applied in humans. Here, we provide a detailed protocol based on our previous publications that describes the generation of a preclinical model of GA using the albino rabbit eye, for evaluation of the human embryonic stem cell derived retinal pigment epithelial cells (hESC-RPE) in a clinically relevant setting. Differentiated hESC-RPE are transplanted into naive eyes or eyes with NaIO3-induced GA-like retinal degeneration using a 25 G transvitreal pars plana technique. Evaluation of degenerated and transplanted areas is performed by multimodal high-resolution non-invasive real-time imaging.

Retinal pigment epithelial cells could serve as a cell-replacement therapy for the advanced form of dry age-related macular degeneration. This protocol describes the generation of a large-eyed model of geographic atrophy and the subretinal transplantation of human embryonic stem cell-derived retinal pigment epithelial cells into this model of disease.

人胚胎干细胞源性视网膜色素上皮细胞移植视网膜大眼模型的地理萎缩

Geographic atrophy (GA), the late stage of dry age-related macular degeneration is characterized by loss of the retinal pigment epithelial (RPE) layer, ...

生物学

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.