eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of organ culture and laser treatment
<ol>
	<li>Obtain freshly enucleated porcine eyes from the local abattoir. Keep them cool (4 &#176;C) in Dulbecco&#39;s modified Eagle medium (DMEM) with high glucose, supplemented with L-glutamine, sodium pyruvate, penicillin/streptomycin (1%), and porcine serum (10%), henceforth referred to in this article as full medium.</li>
	<li>Remove extracellular tissues with scissors and soak the eyes in 5% povidone-iodine ophthalmic solution for 5 min before placing them in sterilized phosphate-buffered saline (PBS) until use.</li>
	<li>Screen eyes for major anterior segment pathologies, such as corneal scarring, edema, and other opacities with a spectral-domain optical coherence tomography device (Table of Materials).</li>
	<li>Position the eyes in front of a slit-lamp unit equipped with a Nd:YAG laser (Table of Materials), which has a wavelength of 1,064 nm and a focal spot diameter of 10 &#181;m in air.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: For optimal positioning a 3D-printed holding apparatus is used, which was designed to hold the eye firm, without putting too much pressure on it (Figure 1).</li>
	<li>Use a magnification of 12x and deflect the illumination to visualize the individual corneal layers.</li>
	<li>Set the pulse energy (e.g., 1.6 mJ) and focus point (e.g., 0.16 mm) for selective ablation of endothelial cells.</li>
	<li>Place a clear cornea paracentesis close to the limbus and inject viscoelastic (Table of Materials) to stabilize the anterior chamber.</li>
	<li>Excise the laser-treated central cornea using an 8 mm trephine.</li>
	<li>Place the excised cornea in a well of a 12-well plate with the endothelial site facing upwards and incubate the specimen in 3 mL of full medium at 37 &#176;C for up to 3 days. 
	NOTE: Potential cytoprotective agents can be added to the medium during this step.</li>
</ol>

<table><tbody><tr><td>Barron vacuum trephine&amp;nbsp;</td><td>Katena</td><td>K20-2058</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM 3050S</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium - high glucose</td><td>PAA</td><td>E-15009</td><td></td></tr><tr><td>Eye holder</td><td>Self</td><td>N/A</td><td></td></tr><tr><td>Inverted Microscope</td><td>Leica</td><td>DMI 6000 B</td><td></td></tr><tr><td>KH2PO4</td><td>Merck</td><td>529568</td><td></td></tr><tr><td>Na2HPO4</td><td>Merck</td><td>1065860500</td><td></td></tr><tr><td>Nd:YAG laser</td><td>Zeiss Meditec</td><td>visuLAS YAG II plus</td><td></td></tr><tr><td>OCT Tissue Tek</td><td>Sakura Finetechnical</td><td>4583</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>10010056</td><td></td></tr><tr><td>Porcine serum</td><td>Sigma-Aldrich</td><td>12736C</td><td></td></tr><tr><td>Spectral-domain optical coherence tomograph</td><td>Heidelberg Engineering</td><td>Spectralis</td><td></td></tr><tr><td>Tissue culture plate 12-well</td><td>Sarstedt</td><td>833921</td><td></td></tr><tr><td>Viscoelastic</td><td>OmniVision</td><td>Methocel</td><td></td></tr></tbody></table>

laser-assisted selective ablation of porcine corneal endothelial cells: a technique to induce focused porcine corneal endothelial injury for ex vivo studies

Source: Holzhey, A. et al., <a href="https://www.jove.com/v/60542">Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss.</a> J. Vis. Exp. (2020)
This video demonstrates a non-invasive laser-assisted procedure to selectively damage the corneal endothelial cells in the porcine eye. The developed ex vivo model of endothelial cell damage can be used to study diseases of the corneal endothelium.

<img alt="Figure 1" src="/files/ftp_upload/20908/20908_Figure1.jpg" /> 
Figure 1: Experimental setup.&#160;(A) Eyes were fixed in a partially 3D-printed holding apparatus, which allowed precise alignment with respect to the laser beam. (B) Before laser treatment, the tissue was evaluated with an anterior segment optical coherence tomography device to check for major anterior segment pathologies. Positions of focal laser points are in...

Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

Source: Holzhey, A. et al., Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss. J. Vis. Exp. (2020)
This ...

The innermost layer of the porcine cornea - the transparent avascular front eye portion - consists of corneal endothelial cells, or CECs, supported by the basement membrane - the Descemet&#39;s membrane, or DM. CECs maintain corneal transparency and optimal vision. Any damage to them could affect vision.
For selective CEC damage, begin with a freshly harvested porcine eye in a suitable medium. Remove the surrounding tissues. Treat the eye with an appropriate disinfectant to eliminate surface microbial contamination. Following saline wash, optically examine the anterior eye to rule out any abnormalities before laser irradiation.
Secure the eye in a customized holding apparatus in front of a slit-lamp unit equipped with a laser of a specific wavelength. Visualize the individual corneal layers. With appropriate laser energy and focus point position, direct the laser pulses to the corneal endothelial layer. The focused short-duration, high-power pulses, cause selective CEC rupture from the exposed surface while avoiding heat diffusion, thereby protecting the DM or stromal tissue.
Creating an entry port close to the limbus - the corneoscleral junction, inject a viscoelastic solution into the anterior chamber containing the cornea to stabilize it and protect the remaining CECs. Excise and transfer the laser-treated corneal segment into a media-containing multi-well plate, CEC side facing upward. The ex vivo corneal endothelium injury model can be used for further studies.

laser-assisted-selective-ablation-porcine-corneal-endothelial-cells

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Porcine

973 次观看. 来源:Holzhey， A. et al.， 角膜内皮细胞损失的无创激光辅助实验模型的开发。J. Vis. Exp. （2020 年） 该视频演示了一种选择性损伤猪眼角膜内皮细胞的无创激光辅助手术。开发的内皮细胞损伤离体模型可用于研究角膜内皮疾病。 

视频: 猪角膜内皮细胞的激光辅助选择性消融:一种诱导离体研究聚焦猪角膜内皮损伤的技术 - 概念

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.

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

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

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 ...

科研

JoVE Journal

发育生物学

An &#8220;All-laser&#8221; Endothelial Transplant

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an intended depth, and a near infrared diode laser to weld the corneal tissue. The proposed technique enables to reach the three main goals in endothelial keratoplasty: a precise control in the thickness of the donor tissue; its easy insertion in the recipient bed and a reduced risk of donor lenticule dislocation. The donor cornea thickness is measured in the surgery room with optical coherence tomography (OCT), in order to correctly design the donor tissue dimensions. A femtosecond laser is used to cut the donor cornea. The recipient eye is prepared by manual stripping of the descemetic membrane. The donor endothelium is inserted into a Busin-injector, the peripheral inner side is stained with a proper chromophore (a water solution of Indocyanine Green) and then it is pulled in the anterior chamber. The transplanted tissue is placed in the final and correct location and then diode laser welding is induced from outside the eyeball. The procedure has been performed on more than 15 patients evidencing an improvement in surgery performances, with a good recovery of visual acuity and a reduced donor lenticule dislocation event.

An all laser procedure is proposed in the endothelial transplant. The surgical technique is based on the use of a femtosecond laser to prepare the donor tissue. Laser welding technique is then used to secure the donor endothelium in the correct position.

"全激光"内皮移植

The &#8220;all laser&#8221; assisted endothelial keratoplasty is a procedure that is performed with a femtosecond laser used to cut the donor tissue at an ...

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 ...

Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies

成績單

Laser-assisted Selective Ablation of Porcine Corneal Endothelial Cells: A Technique to Induce Focused Porcine Corneal Endothelial Injury for Ex Vivo Studies