Name: development of a noninvasive, laser-assisted experimental model of corneal endothelial cell loss
Uploaded: 2020-04-24T14:30:00
Description: Watch this Scientific Journal Video about Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss at JoVE.com

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.

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Research

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Medicine

Orthotopic Aortic Transplantation: A Rat Model to Study the Development of Chronic Vasculopathy

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant vasculopathy (TVP).
The commonly used animal model for cardiovascular chronic rejection studies is the heterotopic heart transplant model performed in laboratory rodents. This model is used widely in experiments since Ono and Lindsey (3) published their technique. To analyze the findings in the blood vessels, the heart has to be sectioned and all vessels have to be measured.
Another method to investigate chronic rejection in cardiovascular questionings is the aortic transplant model (1, 2). In the orthotopic aortic transplant model, the aorta can easily be histologically evaluated (2). The PVG-to-ACI model is especially useful for CAV studies, since acute vascular rejection is not a major confounding factor and Cyclosporin A (CsA) treatment does not prevent the development of CAV, similar to what we find in the clinical setting (4). A7-day period of CsA is required in this model to prevent acute rejection and to achieve long-term survival with the development of TVP.
This model can also be used to investigate acute cellular rejection and media necrosis in xenogeneic models (5).

This video demonstrates the orthotopic aortic transplant model as a simple model to study the development of transplant vasculopathy (TVP) in rats.

Research models of chronic rejection are essential to investigate pathobiological and pathophysiological processes during the development of transplant ...

Corneal Donor Tissue Preparation for Endothelial Keratoplasty

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full thickness corneal transplantation has been the treatment to restore sight in those limited by corneal disease. Some disadvantages to this approach include a high degree of post-operative astigmatism, lack of predictable refractive outcome, and disturbance to the ocular surface. The development of Descemet's stripping endothelial keratoplasty (DSEK), transplanting only the posterior corneal stroma, Descemet's membrane, and endothelium, has dramatically changed treatment of corneal endothelial disease. DSEK is performed through a smaller incision; this technique avoids 'open sky' surgery with its risk of hemorrhage or expulsion, decreases the incidence of postoperative wound dehiscence, reduces unpredictable refractive outcomes, and may decrease the rate of transplant rejection3-6.
Initially, cornea donor posterior lamellar dissection for DSEK was performed manually1 resulting in variable graft thickness and damage to the delicate corneal endothelial tissue during tissue processing. Automated lamellar dissection (Descemet's stripping automated endothelial keratoplasty, DSAEK) was developed to address these issues. Automated dissection utilizes the same technology as LASIK corneal flap creation with a mechanical microkeratome blade that helps to create uniform and thin tissue grafts for DSAEK surgery with minimal corneal endothelial cell loss in tissue processing.
Eye banks have been providing full thickness corneas for surgical transplantation for many years. In 2006, eye banks began to develop methodologies for supplying precut corneal tissue for endothelial keratoplasty. With the input of corneal surgeons, eye banks have developed thorough protocols to safely and effectively prepare posterior lamellar tissue for DSAEK surgery. This can be performed preoperatively at the eye bank. Research shows no significant difference in terms of the quality of the tissue7 or patient outcomes8,9 using eye bank precut tissue versus surgeon-prepared tissue for DSAEK surgery. For most corneal surgeons, the availability of precut DSAEK corneal tissue saves time and money10, and reduces the stress of performing the donor corneal dissection in the operating room. In part because of the ability of the eye banks to provide high quality posterior lamellar corneal in a timely manner, DSAEK has become the standard of care for surgical management of corneal endothelial disease.
The procedure that we are describing is the preparation of the posterior lamellar cornea at the eye bank for transplantation in DSAEK surgery (Figure 1).

Endothelial corneal transplantation is a surgical technique for treatment of posterior corneal diseases. Mechanical microkeratome dissection to prepare tissue results in thinner, more symmetric grafts with less endothelial cell loss and improved outcomes. Dissections can be performed at the eye bank prior to corneal transplantation surgery.

Over the past ten years, corneal transplantation surgical techniques have undergone revolutionary changes1,2. Since its inception, traditional full ...

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 ...

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.

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

Corneal Donor Tissue Preparation for Descemet's Membrane Endothelial Keratoplasty

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, along with its basement membrane (Descemet&#39;s membrane) is introduced onto the recipient&#39;s posterior stroma3. Unlike Descemet&#8217;s Stripping Automated Endothelial Keratoplasty (DSAEK), where additional donor stroma is introduced, no unnatural stroma-to-stroma interface is created. As a result, the natural anatomy of the cornea is preserved as much as possible allowing for improved recovery time and visual acuity4. Endothelial Keratoplasty (EK) is the procedure of choice for treatment of endothelial dysfunction. The advantages of EK include rapid recovery of vision, preservation of ocular integrity and minimal refractive change due to use of a small, peripheral incision1. DSAEK utilizes donor tissue prepared with partial thickness stroma and endothelium. The rapid success and utilization of this procedure can be attributed to availability of eye-bank prepared precut tissue. The benefits of eye-bank preparation of donor tissue include elimination of need for specialized equipment in the operating room and availability of back up donor tissue in case of tissue perforation during preparation. In addition, high volume preparation of donor tissue by eye-bank technicians may provide improved quality of donor tissue. DSAEK may have limited best corrected visual acuity due to creation of a stromal interface between the donor and recipient cornea. Elimination of this interface with transplantation of only donor Descemet&#39;s membrane and endothelium in DMEK may improve visual outcomes and reduce complications after EK5. Similar to DSAEK, long term success and acceptance of DMEK is dependent on ease of availability of precut, eye-bank prepared donor tissue. Here we present a stepwise approach to donor tissue preparation which may reduce some barriers eye-banks face in providing DMEK grafts.

Corneal Donor Tissue Preparation for Descemet&#039;s Membrane Endothelial Keratoplasty

Endothelial keratoplasty is a surgical technique continuously evolving as advancements result in transplantation of thinner grafts with each succession1. Here we present a technique for controlled manual tissue dissection to allow safe and repeatable separation of endothelium and Descemet&#39;s membranes from donor corneoscleral buttons for transplantation2.

Descemet&#8217;s Membrane Endothelial Keratoplasty (DMEK) is a form of corneal transplantation in which only a single cell layer, the corneal endothelium, ...

Evaluation of a Novel Laser-assisted Coronary Anastomotic Connector - the Trinity Clip - in a Porcine Off-pump Bypass Model

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the Trinity Clip, is developed based on the excimer laser-assisted nonocclusive anastomosis technique. The Trinity Clip connector enables simplified, sutureless, and nonocclusive connection of the graft to the coronary artery, and an excimer laser catheter laser-punches the opening of the anastomosis. Consequently, owing to the complete nonocclusive anastomosis construction, coronary conditioning (i.e., occluding or shunting) is not necessary, in contrast to the conventional anastomotic technique, hence simplifying the off-pump bypass procedure. Prior to clinical application in coronary artery bypass grafting, the safety and quality of this novel connector will be evaluated in a long-term experimental porcine off-pump coronary artery bypass (OPCAB) study. In this paper, we describe how to evaluate the coronary anastomosis in the porcine OPCAB model using various techniques to assess its quality. Representative results are summarized and visually demonstrated.

This paper describes a novel nonocclusive coronary anastomotic connector in a porcine off-pump coronary artery bypass (OPCAB) model. This easy-to-use coronary connector has intrinsic potential to facilitate minimally invasive OPCAB surgery.

To simplify and facilitate beating heart (i.e., off-pump), minimally invasive coronary artery bypass surgery, a new coronary anastomotic connector, the ...

Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can ...

A Neonatal Mouse Spinal Cord Compression Injury Model

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal cord. A major current area of research is focused on the mechanisms of adaptive plasticity that underlie spontaneous or induced functional recovery following SCI. Spontaneous functional recovery is reported to be greater early in life, raising interesting questions about how adaptive plasticity changes as the spinal cord develops. To facilitate investigation of this dynamic, we have developed a SCI model in the neonatal mouse. The model has relevance for pediatric SCI, which is too little studied. Because neural plasticity in the adult involves some of the same mechanisms as neural plasticity in early life1, this model may potentially have some relevance also for adult SCI. Here we describe the entire procedure for generating a reproducible spinal cord compression (SCC) injury in the neonatal mouse as early as postnatal (P) day 1. SCC is achieved by performing a laminectomy at a given spinal level (here described at thoracic levels 9-11) and then using a modified Yasargil aneurysm mini-clip to rapidly compress and decompress the spinal cord. As previously described, the injured neonatal mice can be tested for behavioral deficits or sacrificed for ex vivo physiological analysis of synaptic connectivity using electrophysiological and high-throughput optical recording techniques1. Earlier and ongoing studies using behavioral and physiological assessment have demonstrated a dramatic, acute impairment of hindlimb motility followed by a complete functional recovery within 2 weeks, and the first evidence of changes in functional circuitry at the level of identified descending synaptic connections1.

This article describes a method for generating a reproducible spinal cord compression injury (SCI) in the neonatal mouse. The model provides an advantageous platform for studying mechanisms of adaptive plasticity that underlie spontaneous functional recovery.

Spinal cord injury (SCI) typically causes devastating neurological deficits, particularly through damage to fibers descending from the brain to the spinal ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

An Experimental Model of Diet-Induced Metabolic Syndrome in Rabbit: Methodological Considerations, Development, and Assessment

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased prevalence due to the rise of sedentary lifestyles and unhealthy eating habits. Thanks to animal models, basic research can investigate the mechanisms underlying pathological processes such as MetS. Here, we describe the methods used to develop an experimental rabbit model of diet-induced MetS and its assessment. After a period of acclimation, animals are fed a high-fat (10% hydrogenated coconut oil and 5% lard), high-sucrose (15% sucrose dissolved in water) diet for 28 weeks. During this period, several experimental procedures were performed to evaluate the different components of MetS: morphological and blood pressure measurements, glucose tolerance determination, and the analysis of several plasma markers. At the end of the experimental period, animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia with low HDL, high LDL, and an increase of triglyceride (TG) levels, thus reproducing the main components of human MetS. This chronic model allows new perspectives for understanding the underlying mechanisms in the progression of the disease, the detection of preclinical and clinical markers that allow the identification of patients at risk, or even the testing of new therapeutic approaches for the treatment of this complex pathology.

We describe methods to develop an experimental model of diet-induced metabolic syndrome (MetS) in rabbits using a high-fat, high-sucrose diet. Animals developed central obesity, mild hypertension, pre-diabetes, and dyslipidemia, thus reproducing the main components of human MetS. This chronic model will allow acquisition of knowledge underlying mechanisms of disease progression.

In recent years, obesity and metabolic syndrome (MetS) have become a growing problem for public health and clinical practice, given their increased ...