We thank Christine &#214;r&#252;n and Jan A. M. Sochurek for their help with experimental methods.

All procedures involving animal tissue follow the guidelines of the local Animal Care and Ethics Committee.
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. 
	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>
2. Preparation for histology
<ol>
	<li>Prepare Sorensen&#8217;s buffer with a pH of 7.4 containing 19.6 mL of 133 mM KH2PO4 and 80.4 mL of 133 mM Na2HPO4.</li>
	<li>Remove the medium from the cornea-containing well and fixate the tissue for 20 min at room temperature (RT) using methanol-free paraformaldehyde (4%) in Sorensen&#8217;s buffer.</li>
	<li>Place tissue in 20% sucrose in PBS until tissue sinks (1 h) and then in 30% sucrose in PBS overnight at RT. Take care to avoid contact with bubbles and the air surface interface. Embed tissue in optimal cutting temperature (OCT) compound and store at -80 &#176;C.</li>
	<li>Cut sections 10 &#956;m thick using a cryostat at -27 &#176;C. 
	NOTE: A camel hairbrush is useful to help guide the emerging section over the knife blade.</li>
	<li>Transfer the section to a microscope slide by touching the slide to the tissue within 1 min of cutting it to avoid freeze-drying of the tissue. Store the slides at -80 &#176;C.</li>
</ol>
3. Hematoxylin and eosin (H&#38;E) staining
<ol>
	<li>Air dry sections for several minutes to remove moisture.</li>
	<li>Stain with filtered 0.1% Mayers hematoxylin for 10 min in a 50 mL tube.</li>
	<li>In a Coplin jar, rinse in cool running ddH2O for 5 min and dip in 0.5% eosin 10x.</li>
	<li>Dip in ddH2O until the eosin stops streaking and then dip in 50% (10x) as well as 70% (10x) EtOH.</li>
	<li>Equilibrate in 95% EtOH (30 s) and 100% EtOH (60 s) before dipping in xylene several times.</li>
	<li>Finally mount and coverslip the specimen before taking images using a light microscope.</li>
</ol>

<ol>
	<li>DelMonte, D. W., Kim, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+physiology+of+the+cornea.">Anatomy and physiology of the cornea.</a> Journal of Cataract and Refractive Surgery. 37 (3), 588-598 (2011).</li><li>Edelhauser, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+balance+between+corneal+transparency+and+edema:+the+Proctor+Lecture.">The balance between corneal transparency and edema: the Proctor Lecture.</a> Investigative Ophthalmology &amp; Visual Science. 47 (5), 1754-1767 (2006).</li><li>Tuft, S. J., Coster, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+corneal+endothelium.">The corneal endothelium.</a> Eye. 4, 389-424 (1990).</li><li>Bourne, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+the+corneal+endothelium+in+health+and+disease.">Biology of the corneal endothelium in health and disease.</a> Eye. 17, 912-918 (2003).</li><li>He, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+map+of+the+human+corneal+endothelial+cell.">3D map of the human corneal endothelial cell.</a> Scientific Reports. 6, 29047 (2016).</li><li>Gain, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+Survey+of+Corneal+Transplantation+and+Eye+Banking.">Global Survey of Corneal Transplantation and Eye Banking.</a> JAMA Ophthalmology. 134 (2), 167-173 (2016).</li><li>Schwartzkopff, J., Bredow, L., Mahlenbrey, S., Boehringer, D., Reinhard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+corneal+endothelium+following+complete+endothelial+cell+loss+in+rat+keratoplasty.">Regeneration of corneal endothelium following complete endothelial cell loss in rat keratoplasty.</a> Molecular Vision. 16, 2368-2375 (2010).</li><li>Bredow, L., Schwartzkopff, J., Reinhard, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+corneal+endothelial+cells+following+keratoplasty+in+rats+with+bullous+keratopathy.">Regeneration of corneal endothelial cells following keratoplasty in rats with bullous keratopathy.</a> Molecular Vision. 20, 683-690 (2014).</li><li>Bartakova, A., Kunzevitzky, N. J., Goldberg, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+Cell+Therapy+for+Corneal+Endothelium.">Regenerative Cell Therapy for Corneal Endothelium.</a> Current Ophthalmology Reports. 2 (3), 81-90 (2014).</li><li>Zhao, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+three-dimensional+organ+culture+model+for+corneal+wound+healing+and+corneal+transplantation.">Development of a three-dimensional organ culture model for corneal wound healing and corneal transplantation.</a> Investigative Ophthalmology &amp; Visual Science. 47 (7), 2840-2846 (2006).</li><li>Aron-Rosa, D., Aron, J. J., Griesemann, M., Thyzel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+neodymium-YAG+laser+to+open+the+posterior+capsule+after+lens+implant+surgery:+a+preliminary+report.">Use of the neodymium-YAG laser to open the posterior capsule after lens implant surgery: a preliminary report.</a> Journal - American Intra-Ocular Implant Society. 6 (4), 352-354 (1980).</li><li>Vogel, A., Hentschel, W., Holzfuss, J., Lauterborn, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cavitation+bubble+dynamics+and+acoustic+transient+generation+in+ocular+surgery+with+pulsed+neodymium:+YAG+lasers.">Cavitation bubble dynamics and acoustic transient generation in ocular surgery with pulsed neodymium: YAG lasers.</a> Ophthalmology. 93 (10), 1259-1269 (1986).</li><li>Vogel, A., Schweiger, P., Frieser, A., Asiyo, M. N., Birngruber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraocular+Nd:YAG+laser+surgery:+laser-tissue+interaction,+damage+range,+and+reduction+of+collateral+effects.">Intraocular Nd:YAG laser surgery: laser-tissue interaction, damage range, and reduction of collateral effects.</a> IEEE Journal of Quantum Electronics. 26 (12), 2240-2260 (1990).</li><li>Zhu, Q., Zhu, Y., Tighe, S., Liu, Y., Hu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+Human+Corneal+Endothelial+Cells+In+Vitro.">Engineering of Human Corneal Endothelial Cells In Vitro.</a> International Journal of Medical Sciences. 16 (4), 507-512 (2019).</li><li>Li, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotinamide+inhibits+corneal+endothelial+mesenchymal+transition+and+accelerates+wound+healing.">Nicotinamide inhibits corneal endothelial mesenchymal transition and accelerates wound healing.</a> Experimental Eye Research. 184, 227-233 (2019).</li><li>Pescina, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+convenient+ex+vivo+model+for+the+study+of+the+transcorneal+permeation+of+drugs:+histological+and+permeability+evaluation.">Development of a convenient ex vivo model for the study of the transcorneal permeation of drugs: histological and permeability evaluation.</a> Journal of Pharmaceutical Sciences. 104 (1), 63-71 (2015).</li><li>Smeringaiova, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+Wound+Repair+of+the+Organ-Cultured+Porcine+Corneas.">Endothelial Wound Repair of the Organ-Cultured Porcine Corneas.</a> Current Eye Research. 43 (7), 856-865 (2018).</li><li>Yamashita, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rabbit+Corneal+Endothelial+Dysfunction+Model+Using+Endothelial-Mesenchymal+Transformed+Cells.">A Rabbit Corneal Endothelial Dysfunction Model Using Endothelial-Mesenchymal Transformed Cells.</a> Scientific Reports. 8 (1), 16868 (2018).</li><li>Schubert, H. D., Trokel, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+repair+following+Nd:YAG+laser+injury.">Endothelial repair following Nd:YAG laser injury.</a> Investigative Ophthalmology &amp; Visual Science. 25 (8), 971-976 (1984).</li><li>Zhang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rabbit+Model+of+Corneal+Endothelial+Injury+Established+Using+the+Nd:+YAG+Laser.">Rabbit Model of Corneal Endothelial Injury Established Using the Nd: YAG Laser.</a> Cornea. 36 (10), 1274-1281 (2017).</li><li>McCally, R. L., Bonney-Ray, J., de la Cruz, Z., Green, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+endothelial+injury+thresholds+for+exposures+to+1.54+micro+m+radiation.">Corneal endothelial injury thresholds for exposures to 1.54 micro m radiation.</a> Health Physics. 92 (3), 205-211 (2007).</li><li>Nash, J. P., Wickham, M. G., Binder, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+damage+following+focal+laser+intervention.">Corneal damage following focal laser intervention.</a> Experimental Eye Research. 26 (6), 641-650 (1978).</li></ol>

The authors have nothing to disclose.

The results of this pilot study indicate that a Nd:YAG laser can be used to selectively ablate corneal endothelial cells when appropriate parameters for energy dose and focus point position are chosen.
As the endothelial function is important for corneal transparency and safeguarding the cornea from stromal edema, models of endothelial dysfunction play an important role in the development of anti-edematous drugs or surgical procedures. There are several established in vitro models for mimickin...

Transparency of the cornea is essential for the transmission of light to the retina and its photoreceptors1. In this regard, a relative state of dehydration is critical to keep the collagen fibers within the corneal stroma correctly aligned. This homeostasis is maintained by corneal endothelial cells (CEC) located on the Descemet&#8217;s membrane (DM)2. The endothelium is the innermost corneal layer. It has an important barrier and pump function, which is crucial for corneal transparency3. In contrast to the epithelium, the endothelium is not able to self-renew4. Therefore, any cell damage caused by disease or trauma stimulates the remaining endothelial cells to enlarge and migrate, to cover resulting defects and to maintain corneal functionality5. However, if the CEC density falls below a critical threshold, decompensation of the endothelium leads to an edema, resulting in blurred vision and discomfort or even severe pain4. Despite the availability of drugs to relieve symptoms, currently the only definitive treatment in these cases is corneal transplantation, which can be performed in the form of a full-thickness graft or a lamellar endothelial transplantation. The latter procedure is available as Descemet&#39;s membrane endothelial keratoplasty (DMEK) as well as Descemet&#39;s stripping automated endothelial keratoplasty (DSAEK)6. However, the protection of remaining CEC and enhancing their survival could be an alternative target, which needs an adequate disease model to test potential therapeutic drugs.
Current CEC loss disease models focus on the destruction of the endothelium through the injection of toxic agents (e.g., benzalkonium chloride) into the anterior chamber or by mechanical abrasion of the cells using an invasive descemetorhexis technique7,8. While these models are well established, disadvantages such as general inflammatory response and imprecise collateral damage exist. Therefore, these models are more likely to represent final stages of the disease, when the above-mentioned surgical options are inevitable.
With advances in cellular treatment strategies such as stem cells and gene therapy, the application of these cellular therapies could be useful in early stages of CEC loss9. Subsequently, we need a model that represents these earlier stages of the disease more adequately. In this regard, cell culture models have improved over the last decade but are still limited in their validity, as cells in vitro cannot come close to replicating the complex interactions that occur between the different cell types within the cornea10. Therefore, ex vivo and in vivo disease models are still in high demand and improving the existing ones is of utmost interest.
Noninvasive, intraocular surgery by photodisruption using a neodymium:YAG (Nd:YAG) laser has become a routine procedure for ophthalmologists worldwide since its introduction in the late 1970s11. Photodisruption relies on nonlinear light absorption leading to the formation of plasma, generation of acoustic shock waves, and creation of cavitation bubbles, whenever the application site is located in a liquid environment12. In general, these processes contribute to the intended effect of precise tissue cutting. However, they can also be the source of unnecessary collateral damage limiting the local confinement of laser surgery13.
The prediction of resulting mechanical effects has significantly improved through characterization of the shock wave propagation and cavitation course. It is our goal to target CEC with as little damage to surrounding tissue as possible to provide a noninvasive, laser-assisted experimental disease model for the early stages of CEC loss. For this purpose, it is necessary to determine the optimal pulse energies and positions of the focal spots of the laser.

<table><tbody><tr><td>BARRON VACUUM TREPHINE</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&amp;rsquo;s Modified Eagle&amp;rsquo;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>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Merck</td><td>529568</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</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>Two-Photon Microscope</td><td>JenLab</td><td>DermaInspect</td><td></td></tr><tr><td>Viscoelastic</td><td>OmniVision</td><td>Methocel</td><td></td></tr></tbody></table>

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.

Using the procedure presented here, we treated eyes with a Nd:YAG laser, evaluating different pulse energies (1.0&#8722;4.6 mJ) and positions of focal points (distance from the posterior surface of the cornea: 0.0&#8722;0.2 mm) to find the optimal parameters. Multiple replicates (n = 3) were evaluated for each constellation of the laser parameters (12 x 21).
In addition to the above-mentioned protocol, specimen was analyzed with a two-photon microscope before fixation and H&#38;E staining. The...

Watch this Scientific Journal Video about Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss at JoVE.com

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

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).

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

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Research

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Medicine

5.3K Views.  University of Lübeck. De huidige ziektemodellen voor hoornvlies endotheelcelverlies richten zich op de vernietiging van het endotheel en vertegenwoordigen waarschijnlijk de laatste stadia van de ziekte wanneer hoornvliestransplantatie onvermijdelijk is. Nieuwe behandelingen met behulp van stamcellen of gentherapie, echter, zijn nu beschikbaar die nuttiger zou kunnen zijn voor de vroege stadia van de ziekte, de modellen waarvoor ontbreken. Niet-invasieve intraoculaire chirurgie door fotodisruptie met behulp van een...

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

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.

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

Developmental Biology

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

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.

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

Neuroscience

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.

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 ...

A Simple Mechanical Procedure to Create Limbal Stem Cell Deficiency in Mouse

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with conjunctiva. Patients suffer from recurrent corneal defects, pain, inflammation, and loss of vision.
Previously, a murine model of LSCD was described and compared to two other models. The goal was to produce a consistent mouse model of LSCD that both mimics the phenotype in humans and lasts long enough to make it possible to study the disease pathophysiology and to evaluate new treatments. Here, the technique is described in more detail.
A motorized tool with a rotating burr has been designed to remove the rust rings from the corneal surface or to smooth the pterygium bed in patients. It is a suitable device to create the desired LSCD model. It is a readily available, easy-to-use tool with a fine tip that makes it appropriate for working on small eyes, as in mice. Its application prevents unnecessary trauma to the eye and it does not result in unwanted injuries, as often is the case with chemical injury models. As opposed to a blunt scraper, it removes the epithelium with the basement membrane. In this protocol, the limbal area was abraded two times, and then the whole corneal epithelium was shaved from limbus to limbus. To avoid stroma injury, care was taken not to brush the corneal surface once the epithelium was already removed.

The following article provides an easy, reproducible technique to effectively create a sustainable mouse model of limbal stem cell deficiency (LSCD). This animal model is useful in testing and comparing the efficacy of treatments for limbal stem cell diseases.

Limbal stem cell deficiency (LSCD) is a state of malfunction or loss of limbal epithelial stem cells, after which the corneal epithelium is replaced with ...

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this protocol, we aim to guide the reader not simply through the technical considerations of generating laser-induced lesions to trigger neovascular processes, but rather focus on the powerful information that can be obtained from multimodal longitudinal in vivo imaging throughout the follow-up period.
The laser-induced mouse CNV model was generated by a diode laser administration. Multimodal in vivo imaging techniques were used to monitor CNV induction, progression and regression. First, spectral domain optical coherence tomography (SD-OCT) was performed immediately after the lasering to verify a break of Bruch&#39;s membrane. Subsequent in vivo imaging using fluorescein angiography (FA) confirmed successful damage of Bruch&#39;s membrane from serial images acquired at the choroidal level. Longitudinal follow-up of CNV proliferation and regression on days 5, 10, and 14 after the lasering was performed using both SD-OCT and FA. Simple and reliable grading of leaky CNV leasions from FA images is&#160;presented. Automated segmentation for measurement of total retinal thickness, combined with manual caliber application for measurement of retinal thickness at CNV sites, allow unbiased evaluation of the presence of edema. Finally, histological verification of CNV is performed using isolectin GS-IB4 staining on choroidal flatmounts. The staining is thresholded, and the isolectin-positive area is calculated with ImageJ.
This protocol is especially useful in therapeutics studies requiring high-throughput-like screening of CNV pathology as it allows fast, multimodal, and reliable classification of CNV pathology and retinal edema. In addition, high resolution SD-OCT enables the recording of other pathological hallmarks, such as the accumulation of subretinal or intraretinal fluid. However, this method does not provide a possibility to automate CNV volume analysis from SD-OCT images, which has to be performed manually.

In Vivo Multimodal Imaging and Analysis of Mouse Laser-Induced Choroidal Neovascularization Model

Here, we present the usefulness of longitudinal in vivo imaging in the follow-up of morphological changes of laser-induced choroidal neovascularization in mice.

Laser-induced choroidal neovascularization (CNV) is a well-established model to mimic the wet form of age-related macular degeneration (AMD). In this ...

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.

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

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

Corneal Sensory Fiber Damage Protocols for Axon Regeneration Studies

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

Author Spotlight: Advancing Corneal Innervation Research Through Innovative Models 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

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

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