We thank the staff of the Second Core Lab, Department of Medical Research, National Taiwan University Hospital for their technical support.

所有的程序遵循本研究中，在视觉与眼科声明协会研究给予了动物在眼科和视觉研究的用途和台大医院的机构动物护理和使用委员会批准。 1.隔离，原代培养制备与牛的CEC的免疫染色<ol><li>从本地屠宰场获得新鲜牛的眼睛。 </li><li>消毒在3分钟10％w / v的聚乙烯吡咯烷酮 - 碘溶液中的眼睛。用磷酸盐缓冲盐水（PBS）溶液洗涤。 </li><li>收获在无菌条件下解剖刀和剪刀角膜按钮。去皮后弹力与在解剖显微镜下用镊子膜（请注意，在这项研究中，牛眼睛被一个本地屠场去核，因此，在第一研究过程是preenucleated眼睛的在实验室消毒）。 </li><li>在1毫升试试孵育后弹力膜PSIN在37℃下30分钟。收集通过离心牛的CEC在112×g离心5分钟。重悬细胞于1ml补充激素上皮培养基（SHEM）含有HEPES缓冲的Dulbecco改进的Eagle培养基和哈姆的F12培养基，补充有5％胎牛血清，0.5％二甲亚砜，2纳克/毫升人表皮的等体积的生长因子，5毫克/毫升的胰岛素，5毫克/毫升转铁蛋白，5纳克/毫升硒，1纳摩尔/升霍乱毒素，50毫克/毫升庆大霉素，和1.25毫克/毫升两性霉素B的</li><li>种子的细胞（每眼约1×10 5个细胞）到一个直径6cm培养皿。培养他们在SHEM。孵育在37℃下的菜在5％CO 2在空气气氛中。改变培养基每3天。 </li><li>当细胞达到汇合时，把它们洗干净的PBS，并在37°C孵育他们在1ml胰蛋白酶的5分钟。离心112×g离心5分钟收集它们。重悬在1ml的闪的细胞沉淀。 <ol&gt; <li>算在血球细胞。种子在盖玻片的细胞在24孔板一个1×10 4每孔的密度，并培养细胞的SHEM。调查马立马司他的EnMT抑制效果，孵育细胞SHEM 10马立马司他的μM加入到培养基中，并改变它的培养基，每3天。 </li></ol></li><li>固定细胞在含有250微升4％多聚甲醛的，pH值7.4所指示的时间点上，在室温下30分钟。用250微升0.5％的Triton X-100的5分钟通透。用10％的牛血清白蛋白阻断30分钟。 </li><li>孵育针对活性β-连环蛋白（ABC），蜗牛和蛞蝓的初级抗体的细胞在4℃下过夜。使用的初级抗体的稀释度为1:200。抗体稀释在10mM PBS中，1％w / v的牛血清白蛋白，和0.09％w / v的叠氮化钠组成的抗体稀释液。 </li><li>用PBS洗涤细胞两次15分钟，并孵育米，所述的Alexa Fluor缀合的二级抗体:1小时在室温下（1 100在抗体稀释液）。 </li><li>染液通过覆盖细胞用2微克/毫升4&#39;​​，6-二脒基-2-苯基的细胞核，洗涤细胞两次，用PBS进行15分钟，并用抗褪色安装溶液装入它们。 </li><li>通过使用激光扫描共聚焦显微镜用20X物镜放大倍率获得在405和488纳米的激发波长免疫荧光图像。 </li></ol> 2.鼠角膜内皮细胞冷冻损伤模型和房内注射<ol><li>麻醉12周大的雄性SD大鼠用2％甲苯噻嗪（5.6毫克/千克）和替来他明以及唑拉西泮（18毫克/千克）的肌内注射。轻轻捏住动物的皮肤，以确认在​​不存在皮肤抽搐适当麻醉。 </li><li>灌输0.5％丙美卡因盐酸一滴对每只大鼠的右眼，以尽量减少眼痛和眨眼反射。灌输四环素软膏到左眼防止角膜干燥。 </li><li>冷却在液氮中的不锈钢探针（直径= 3毫米）。不锈钢探针应用于右眼的角膜中央，持续30秒。在操作过程中频繁地灌输的PBS于右眼防止角膜干燥。 </li><li>灌输0.1％阿托品和0.3％硫酸庆大霉素冷冻损伤后立即和每日一次，以减轻来自睫状肌痉挛造成眼部疼痛和预防感染。 <ol><li>手术后，继续使用热灯老鼠温暖，麻醉后观察其恢复每隔15分钟，直到他们重新获得电机控制。此外，应用四环素软膏右眼以防止在恢复期间角膜干。 </li></ol></li><li>重复角膜冷冻损伤连续3天。 </li><li>用于递送马立马司他或bFGF的到大鼠眼睛的前房，如前所述麻醉大鼠。灌输一滴0.5％丙美卡因盐酸成右眼的，以尽量减少眼痛和眨眼反射。 </li><li>灌溉用无菌PBS眼表面。通过上述和平行的平面中插入在paralimbal透明角膜附着到1ml注射器一个地下30针虹膜的手术显微镜下进行前房穿刺术。 </li><li>打开针头斜面向上，并稍微踩下角膜伤口排出一些房水，降低眼压。注入0.02毫升药物的前房内。轻轻压缩与针撤出期间棉尖端的针道。 </li><li>拍摄外眼在手术显微镜下所指示的时间点。 </li></ol> 3.收获鼠角膜按钮和免疫<ol><li>安乐死的大鼠，将它们放置在一个安乐死腔和以每分钟的腔室容积的10-30％的填充速率注入100％的CO 2。保持二氧化碳注入一个缺乏呼吸和褪色的眼睛的颜色后附加分。 </li><li>穿透眼鼠在用锋利的刀片将角膜缘。切沿角膜缘剪刀眼角膜。拼合幻灯片上的眼角膜。作出额外的放射状切口，如果角膜蜷缩起身体。 </li><li>修复角膜用250微升4％多聚甲醛的，pH为7.4，在室温下30分钟。用250微升0.5％的Triton X-100的5分钟通透，并用10％的牛血清白蛋白阻断30分钟。 </li><li>在抗体稀释液200:为1的稀释孵育针对ABC初级抗体的角膜在4℃下过夜。用PBS洗角膜两次15分钟，并与二级抗体孵育它们:在室温下1小时（1 100在抗体稀释液）。染液通过覆盖细胞用2微克/毫升的4&#39;，6-二脒基-2-苯基细胞核，并用PBS洗涤细胞两次15分钟。 </li><li>安装在防褪色安装解决方案的眼角膜。 OB在20X物镜放大倍率覃在405激发波长的免疫荧光图像和561 nm的激光扫描共聚焦显微镜。 </li></ol>

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The authors have no competing financial interests to declare.

的CEC是众所周知的倾向中的细胞增殖经受EnMT。制定战略用于抑制EnMT过程用于治疗目的时，EnMT机构的透彻理解是必要的。我们描述了2模型，研究EnMT，即在体 ​​外培养模型大鼠角膜内皮冷冻损伤模型牛CEC。我们的研究结果证明了这两款车型的EnMT过程。此外，马立马司他的EnMT抑制效应再现这两种模式，这表明这些2种型号共享相同的机制。 体外培养模型牛CEC提?...

Corneal endothelial cells (CECs) play a vital role in maintaining corneal clarity and thus visual acuity by regulating the hydration status of the corneal stroma through active pumping1. Because of the limited proliferative potential of human CECs, the cell number decreases with age, and the repair of corneal endothelial wounds following injury is usually achieved through cell enlargement and migration, rather than cell mitosis2. When the CEC count decreases below a threshold of approximately 500 cells/mm2, the dehydration status of the corneal stroma cannot be maintained, leading to bullous keratopathy and vision impairment3,4.
The limited proliferative potential of human CECs has been attributed to several factors, including reduced expression of the epidermal growth factor and its receptor in aging cells5, antiproliferative TGF&#946;2 in the aqueous humor6, and contact inhibition2,7. Although some growth factors, such as basic fibroblast growth factor (bFGF), can increase proliferation in a cultured human corneal endothelium, the culture efficiency remains limited8,9. Furthermore, CECs may undergo a phenotypic change during ex vivo expansion, resembling epithelial-mesenchymal transition (EMT)10-13. Endothelial-mesenchymal transition (EnMT) is characterized by cell junction destabilization, apical-basal polarity loss, cytoskeletal rearrangement, alpha smooth muscle actin expression, and type I collagen secretion14. All of these characteristics may abrogate the normal function of CECs, hampering the use of ex vivo cultured CECs in tissue engineering. Moreover, EnMT has been associated with the pathogenesis of several corneal endothelial diseases, including Fuchs endothelial corneal dystrophy and retrocorneal membrane formation15,16. Therefore, understanding the mechanism of EnMT may aid in manipulating the EnMT process and facilitate the regeneration of CECs to enable competent function. 
In this study, we described a method for isolating bovine CECs from the corneal button. In the primary culture in vitro, the EnMT process, including a phenotypic change, the nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, was observed. We further described a method for demonstrating EnMT in vivo by using a rat corneal endothelium cryoinjury model. Using these 2 models, we demonstrated that marimastat, a broad-spectrum matrix metalloproteinase (MMP) inhibitor, can suppress the EnMT process. The described protocols facilitate the detailed analysis of the EnMT mechanism and the development of strategies for manipulating the EnMT process for further clinical application.

<table><tbody><tr><td>trypsin</td><td>ThermoFisher Scientific</td><td>12604-013</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle medium and Ham&#039;s F12 medium</td><td>ThermoFisher Scientific</td><td>11330</td><td></td></tr><tr><td>fetal bovine serum</td><td>ThermoFisher Scientific</td><td>26140-079</td><td></td></tr><tr><td>dimethyl sulfoxide</td><td>Sigma</td><td>D2650</td><td></td></tr><tr><td>human epidermal growth factor</td><td>ThermoFisher Scientific</td><td>PHG0311</td><td></td></tr><tr><td>insulin, transferrin, selenium&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>41400-045</td><td></td></tr><tr><td>cholera toxin</td><td>Sigma</td><td>C8052-1MG</td><td></td></tr><tr><td>gentamicin</td><td>ThermoFisher Scientific</td><td>15750-060</td><td></td></tr><tr><td>amphotericin B</td><td>ThermoFisher Scientific</td><td>15290-026</td><td></td></tr><tr><td>paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>111219</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787&amp;nbsp;</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>marimastat</td><td>Sigma</td><td>M2699-25MG</td><td></td></tr><tr><td>anti-active beta-catenin antibody</td><td>Millpore</td><td>05-665</td><td></td></tr><tr><td>anti-snail antibody</td><td>Santa cruz</td><td>sc28199</td><td></td></tr><tr><td>anti-slug antibody</td><td>Santa cruz</td><td>sc15391</td><td></td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11001</td><td>for staining of ABC of bovine CECs</td></tr><tr><td>goat anti-mouse IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11003</td><td>for staining of ABC of rat corneal endothelium</td></tr><tr><td>goat anti-rabbit IgG (H+L) secondary antibody</td><td>ThermoFisher Scientific</td><td>A-11008</td><td>for staining of snail and slug of bovine CECs</td></tr><tr><td>antibody diluent</td><td>Genemed Biotechnologies</td><td>10-0001</td><td></td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole</td><td>ThermoFisher Scientific</td><td>D1306</td><td></td></tr><tr><td>mounting medium</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>laser scanning confocal microscope</td><td>ZEISS</td><td>LSM510</td><td></td></tr><tr><td>xylazine&amp;nbsp;</td><td>Bayer</td><td>N/A</td><td></td></tr><tr><td>tiletamine plus zolazepam</td><td>Virbac</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>proparacaine hydrochloride ophthalmic solution</td><td>Alcon</td><td>N/A</td><td>veterinary drug</td></tr><tr><td>0.1% atropine</td><td>Wu-Fu Laboratories Co., Ltd</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>0.3% gentamicin sulfate</td><td>Sinphar Group</td><td>N/A</td><td>clinical drug&amp;nbsp;</td></tr><tr><td>basic fibroblast growth factor</td><td>ThermoFisher Scientific</td><td>PHG0024</td><td>clinical drug&amp;nbsp;</td></tr></tbody></table>

in vitro and in vivo models to study corneal endothelial-mesenchymal transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

牛的CEC的分离后，将细胞在体外培养， 图1给出了牛的CEC的相位对比图像。细胞在汇合的六边形表示细胞没有通过细胞分离过程中角膜基质成纤维细胞的污染。 图2描绘了使用针对ABC，蜗牛和蛞蝓的抗体在一个指定的时间点进行的免疫染色。除了 ​​在体外培养的表型变化，观察到的ABC和EMT调节的相应核易位。 图3示出马...

Watch this Scientific Journal Video about In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition at JoVE.com

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

牛角膜内皮细胞的原代培养物用于研究角膜内皮 - 间充质转换的机制。此外，大鼠角膜内皮冷冻损伤模型被用来证明在体内角膜内皮-间充质转换。

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

in-vitro-vivo-models-to-study-corneal-endothelial-mesenchymal

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

7.9K 次观看.  Far Eastern Memorial Hospital. 牛角膜内皮细胞的原代培养物用于研究角膜内皮 - 间充质转换的机制。此外，大鼠角膜内皮冷冻损伤模型被用来证明在体内角膜内皮-间充质转换。 

视频: In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide suffering from corneal vision loss1. To address the demand for viable human corneas, significant progress in three-dimensional (3D) tissue engineering has been made2,3,4. These cornea models range from simple monolayer systems to multilayered models, leading to 3D full-thickness corneal equivalents2.
However, the use of a 3D tissue-engineered cornea in the context of in vitro disease models studied to date lacks resemblance to the multilayered 3D corneal tissue structure, function, and the networking of different cell types (i.e., nerve, epithelium, stroma, and endothelium)2,3. In addition, the demand for in vitro cornea tissue models has increased in an attempt to reduce animal testing for pharmaceutical products. Thus, more sophisticated models are required to better match systems to human physiological requirements, and the development of a model that is more relevant to the patient population is absolutely necessary. Given that multiple cell types in the cornea are affected by diseases and dystrophies, such as Keratoconus, Diabetic Keratopathy, and Fuchs, this model includes a 3D co-culture model of primary human corneal fibroblasts (HCFs) from healthy donors and neurons from the SH-SY5Y cell line. This allows us for the first time to investigate the interactions between the two cell types within the human corneal tissue. We believe that this model could potentially dissect the underlying mechanisms associated with the stromal-nerve interactions of corneal diseases that exhibit nerve damages. This 3D model mirrors the basic anatomical and physiological nature of the corneal tissue in vivo and can be used in the future as a tool for investigating corneal defects as well as screening the efficacy of various agents before animal testing.

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

This protocol describes a novel three-dimensional in vitro model, where corneal stromal cells and differentiated neuronal cells are cultured together to assist in the examination and understanding of the interactions of the two cell types.

角膜组织工程: 人角膜间质神经相互作用的体外模型

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

科研

发育生物学

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

小鼠角膜移植模型，研究实体器官移植的最常见形式

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

医学

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

角膜微囊含量：血管生成在小鼠眼模型

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

神经科学

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

生物工程

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 ...

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, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

生物学

体内心脏移植研究内皮到间充质转化

A Neonatal Heterotopic Rat Heart Transplantation Model for the Study of Endothelial-to-Mesenchymal Transition

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and precludes patients with congenital critical aortic stenosis and hypoplastic left heart syndrome (HLHS) from curative anatomical biventricular surgical repair. Surgical resection is currently the only available therapeutic option, but EFE often recurs, sometimes with an even more infiltrative growth pattern into the adjacent myocardium.
To better understand the underlying mechanisms of EFE and to explore therapeutic strategies, an animal model suitable for preclinical testing was developed. The animal model takes into consideration that EFE is a disease of the immature heart and is associated with flow disturbances, as supported by clinical observations. Thus, the heterotopic heart transplantation of neonatal rat donor hearts is the basis for this model.
A neonatal rat heart is transplanted into an adolescent rat&#39;s abdomen and connected to the recipient&#39;s infrarenal aorta and inferior vena cava. While perfusion of the coronary arteries preserves the viability of the donor heart, flow stagnation within the LV induces EFE growth in the very immature heart. The underlying mechanism of EFE formation is the transition of endocardial endothelial cells to mesenchymal cells (EndMT), which is a well-described mechanism of early embryonic development of the valves and septa but also the leading cause of fibrosis in heart failure. EFE formation can be macroscopically observed within days after transplantation. Transabdominal echocardiography is used to monitor the graft viability, contractility, and the patency of the anastomoses. Following euthanasia, the EFE tissue is harvested, and it shows the same histopathological characteristics as human EFE tissue from HLHS patients.
This in vivo model allows for studying the mechanisms of EFE development in the heart and testing treatment options to prevent this pathological tissue formation and provides the opportunity for a more generalized examination of EndMT-induced fibrosis.

作者聚焦:用于研究内皮-间充质转化的新生儿异位大鼠心脏移植模型  

This work presents an animal model of endothelial-to-mesenchymal transition-induced fibrosis, as seen in congenital cardiac defects such as critical aortic stenosis or hypoplastic left heart syndrome, which allows for detailed histological tissue evaluation, the identification of regulatory signaling pathways, and the testing of treatment options.

新生儿异位大鼠心脏移植模型在内皮间充质转化研究中的应用

Endocardial fibroelastosis (EFE), defined by subendocardial tissue accumulation, has major impacts on the development of the left ventricle (LV) and ...