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>R...

<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 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 border="0" cellpadding="0" cellspacing="0" style="width:933px;" width="932">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:313px;">BARRON VACUUM TREPHINE</td>
			<td style="width:213px;">Katena</td>
			<td style="width:344px;">K20-2058</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cryostat</td>
			<td>Leica</td>
			<td>CM 3050S</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dulbecco&#8217;s Modified Eagle&#8217;s Medium - high glucose</td>
			<td>PAA</td>
			<td>E-15009</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Eye holder</td>
			<td>Self</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Inverted Microscope</td>
			<td>Leica</td>
			<td>DMI 6000 B</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">KH2PO4</td>
			<td>Merck</td>
			<td>529568</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Na2HPO4</td>
			<td>Merck</td>
			<td>1065860500</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nd:YAG laser</td>
			<td>Zeiss Meditec</td>
			<td>visuLAS YAG II plus</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT Tissue Tek</td>
			<td>Sakura Finetechnical</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Phosphate Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>10010056</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Porcine serum</td>
			<td>Sigma-Aldrich</td>
			<td>12736C</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Spectral-domain optical coherence tomograph</td>
			<td>Heidelberg Engineering</td>
			<td>Spectralis</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tissue culture plate 12-well</td>
			<td>Sarstedt</td>
			<td>833921</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Two-Photon Microscope</td>
			<td>JenLab</td>
			<td>DermaInspect</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">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 ...

Current disease models for corneal endothelial cell loss focus on the destruction of the endothelium and more likely represent the final stages of the disease when corneal transplantation is inevitable. New treatments using stem cells or gene therapy, however, are now available that could be more useful for the early stages of the disease, the models for which are lacking. Noninvasive intraocular surgery by photodisruption using an Nd:YAG laser has become a routine procedure for ophthalmologists.After obtaining freshly enucleated porcine eyes from the local abattoir, place the samples in four degrees Celsius full medium. Use scissors to remove the extracellular tissues and soak the eyes in 5%povidone iodine ophthalmic solution for five minutes. Next, place the disinfected samples in sterile PBS at room temperature.Using a spectral domain optical coherence tomography device, screen the eyes for major anterior segment pathologies such as corneal scarring, edema, and other opacities. After screening, position the eyes in front of a slip lamp unit equipped with an Nd:YAG laser with a wavelength of 1, 064 nanometers and a focal spot diameter of 10 micrometers in air. Select the 12X magnification and deflect the illumination to visualize the individual corneal layers.After setting the pulse energy and focus point to the appropriate parameters for the selective ablation of the corneal endothelial cells, apply several laser shots to the tissue. Then under a dissecting microscope, place a clear cornea paracentesis close to the limbus and inject viscoelastic to stabilize the anterior chamber. Then use an eight millimeter trephine to excise the laser treated central cornea and place the isolated cornea into one well of a 12-well plate endothelial side up.When all of the corneas have been collected, add three millimeters of full medium to each sample well and incubate the specimens for up to three days at 37 degrees Celsius. At the end of the incubation, replace the medium in each well with methanol-free 4%paraformaldehyde in Sorensen's buffer for a 20-minute incubation at room temperature. At the end of the incubation, place the fixed samples in 20%sucrose in PBS for about one hour until the corneas sink.Transfer the samples into 30%sucrose in PBS overnight before embedding the tissues in optimal cutting temperature for storage at minus 80 degrees Celsius. Use a cryostat set to minus 27 degrees Celsius to obtain 10 micrometer thick sections of the frozen tissues collecting each section on a microscope slide within one minute of it being acquired. Then store the slides at minus 80 degrees Celsius until staining.For hematoxylin and eosin staining, air dry the frozen sections for several minutes to remove moisture before staining with filtered 0.1%Mayer's hematoxylin for 10 minutes in a 50 milliliter tube. At the end of the incubation, rinse the slides in double distilled water for five minutes in a cuvette. Next, dip the slides in 0.5%eosin 10 times, followed by dip rinsing in double distilled water until the eosin stops streaking.Dip the rinsed slides 10 times in 50%ethanol and 10 times in 70%ethanol. After the last 70%ethanol dip, equilibrate the sections in 95%ethanol for 30 seconds, followed by 60 seconds in 100%ethanol before several dips in xylene. Then mount the specimens with coverslip before obtaining images on a light microscope.Two photon and light microscopy images should be independently evaluated as no damage, too much damage, or correct amount of damage. Based on these assessments, a heat map can then be calculated to select the right constellation of laser parameters for selective ablation of the corneal endothelial cells with minimal damage to the surrounding tissue. Previous analysis has determined that the focal point of the laser must be at least 0.15 millimeters behind the corneal endothelium for the lowest pulse energy tested.For pulse energies higher than 2.9 millijoules, the longest focal distance tested is still too close to the endothelium. Various cytoprotective agents can be tested while the excised specimens are in culture. If proven successful, they can be added to the irrigation solution for treatment.

development-noninvasive-laser-assisted-experimental-model-corneal

Research

JoVE Journal

Medicine

5.2K Views.  University of Lübeck. Gli attuali modelli di malattia per la perdita di cellule endoteliali corneali si concentrano sulla distruzione dell'endotelio e più probabilmente rappresentano le fasi finali della malattia quando il trapianto corneale è inevitabile. Sono ora disponibili nuovi trattamenti con cellule staminali o terapia genica, tuttavia, che potrebbero essere più utili per le prime fasi della malattia, i cui modelli sono carenti. La chirurgia intraoculare non invasiva per fotodisrruzione utilizzando un la...