Funding for this work was supported by Trefoil Therapeutics and NIH NCATS TRND CRADA #2016-04. The authors would like to thank Tony Wong for histopathology advice and services, the Nikon Imaging Center at UC San Diego for use of their confocal microscope, and Drs. Natalie Afshari and Marian Macsai for their advice on surgical technique. In addition, the authors extend their gratitude to the donors of eyes and the eye banks for providing corneas.

This work used existing specimens without identification of subjects and is exempt from IRB approval under 45 CFR 46.101(b)(4).
Human donor corneas were obtained from various eye banks across the US (see Table of Materials). Corneas were individually judged dystrophic by the eye banks based on findings such as guttae, pleomorphism, polymegathism, and/or low ECD upon specular evaluation. Figure 1 presents Trypan Blue staining in the cornea during wound creation, immediately following stripping, and after 2 weeks in culture.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63482/63482fig01.jpg" /> 
Figure 1: Diagram representing cornea during wound creation, immediately post-stripping, and after 14 days in culture as visualized by Trypan Blue. The reduction in stained area between these timepoints was measured to determine the level of healing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Wound creation
<ol>
	<li>Using sterile forceps, remove the cornea from media and rinse in 1x PBS to remove residual media and cell debris. After rinsing, place the cornea endothelial side up on the lid of a Petri dish.</li>
	<li>Pipette 30 &#181;L of Trypan Blue into a welled dish. Dip a new 4 mm biopsy punch in Trypan Blue and tap off any excess.</li>
	<li>Using both hands, position the punch above the center of the cornea and lower it straight down onto the endothelial surface, applying minimal pressure.</li>
	<li>Without changing the position or pressure on the cornea, shift the punch to one hand and reach for forceps with the newly freed hand. Use the forceps to hold the cornea in place while gently twisting the biopsy punch about 90&#176; back and forth several times.</li>
	<li>Lift the punch straight up from the cornea and set aside.</li>
	<li>Rinse once more in 1x PBS to remove excess Trypan Blue.</li>
</ol>
2. Descemet&#39;s stripping
<ol>
	<li>Transfer cornea on a Petri dish lid to a dissecting scope. 
	NOTE: Do not leave cornea dry longer than 5 min. If more time is needed to complete the process, rinse again in 1x PBS to rehydrate the endothelium.</li>
	<li>Holding the cornea in place with curved forceps, score Descemet&#39;s membrane by lightly dragging the tip of a sharp 30 G needle along the ring of Trypan Blue left by the biopsy punch. Use minimal pressure to avoid disrupting the underlying stroma.</li>
	<li>With a Sinskey hook, use gentle scooping motions to lift and peel back Descemet&#39;s membrane around the wound edge, working toward the center of the lesion. 
	NOTE: Descemet&#39;s membrane should come up with very little resistance. If difficulty is experienced, one is likely pulling up stroma. If this happens, start again, lifting from a new point along the wound edge.</li>
	<li>Once the majority of Descemet&#39;s membrane has been separated from the stroma, use Gorovoy forceps to remove the membrane and set aside.</li>
	<li>Examine the stripped area for any remaining pieces of membrane and remove with Gorovoy forceps.</li>
</ol>
3. Trypan Blue staining
NOTE: Following Descemet&#39;s stripping, stain the cornea with Trypan Blue to visualize the wound area.
<ol>
	<li>Return the cornea on a Petri dish lid to the biosafety cabinet and rinse in 1x PBS containing 0.01% (w/v) CaCl2 and MgCl2 to remove cell debris and facilitate tight adherence of remaining CECs to the intact Descemet&#39;s membrane.</li>
	<li>Place cornea in a welled dish and pipette 30 &#181;L of Trypan Blue onto the endothelial layer for 30 s.
	<ol>
		<li>Use forceps to gently rock the cornea to ensure that the entire endothelial surface is covered.</li>
	</ol>
	</li>
	<li>Rinse off excess Trypan Blue in 1x PBS with 0.01% (w/v) CaCl2 and MgCl2 and image the stained cornea under a dissecting microscope for the Day 0 timepoint.
	<ol>
		<li>Be sure cornea is balanced such that light is evenly transmitted throughout and positioning is replicable across timepoints. 
		&#8203;NOTE: Forceps can be used to hold cornea in place while imaging if needed.</li>
	</ol>
	</li>
</ol>
4. Culture period
<ol>
	<li>Culture corneas in a six-well plate containing low-serum media consisting of OptiMEM; 1x insulin, transferrin, and selenium; 1x antibiotic/antimycotic; 0.02 mg/mL CaCl2; 0.2 mg/mL ascorbic acid; and 0.8% heat inactivated fetal bovine serum. Culture the left cornea in 8 mL of low-serum media alone, and the right cornea in 8 mL of low-serum media supplemented with 100 ng/mL eFGF1 (NM141).
	<ol>
		<li>Incubate at 37 &#176;C with 6% CO2 for 14 days with daily media changes.</li>
	</ol>
	</li>
	<li>Repeat the Trypan Blue staining procedure on Days 3, 6, 9, 12, and 14, and image each cornea immediately after staining. Be sure to keep camera settings consistent across all timepoints.</li>
	<li>On Day 12, add 10 &#181;M EdU to both control and eFGF1 (NM141)-supplemented media and incubate for 48 h to label proliferating cells. Renew media with EdU added again on Day 13.</li>
	<li>On Day 14, 48 h after the addition of EdU, Trypan stain and image corneas as usual, except instead of 1x PBS with CaCl2 and MgCl2, use plain 1x PBS for rinses to prevent the calcium from interacting with the Alizarin Red stain.</li>
</ol>
5. Alizarin Red staining
<ol>
	<li>On Day 14, prepare 5% Alizarin Red in 0.9% saline solution. 
	NOTE: Alizarin Red will not dissolve completely in saline solution. Upon combining, vortex solution and rock for at least 1 h. Vortex once more and filter before use to remove undissolved particles.</li>
	<li>Once Trypan staining and imaging have been completed, transfer corneas to a welled dish and add 200 &#181;L Alizarin Red to the endothelial surface of each cornea.
	<ol>
		<li>Stain for 2 min, then rinse each cornea in a Petri dish of 1x PBS to remove excess Alizarin Red before placing in a six-well plate pre-filled with 8 mL of 1x PBS for two 5 min washes.</li>
	</ol>
	</li>
	<li>After the second wash, image the stripped area of each cornea under a dissecting microscope. 
	&#8203;NOTE: Unlike with Trypan imaging, corneas can be left in 1x PBS to image Alizarin staining to prevent them from drying out during the process.</li>
</ol>
6. Fixation and permeabilization
<ol>
	<li>Once Alizarin images have been captured, transfer corneas to a 12-well plate and wash in 4 mL of 1x PBS containing 0.05% Tween-20 (PBS-T) for 30 min to remove any particles from tissue prior to fixation.</li>
	<li>Fix corneas in 4 mL of 4% PFA for 30 min at room temperature.</li>
	<li>Permeabilize cells for 5 min in 4 mL of 1x PBS containing 1% Triton X-100, then wash three times in 4 mL of plain 1x PBS for 30 min each.</li>
</ol>
7. Immunohistochemistry
<ol>
	<li>Block corneas for 1 h at 37&#176;C in 4 mL of 1x PBS containing 2% bovine serum albumin and 2% goat serum.</li>
	<li>Incubate in 1 mL of blocking solution containing 2.5 &#181;g/mL primary antibody mouse anti-ZO-1 for 1 h at 37&#176;C, then wash three times in 4 mL of 1x PBS for 30 min each. 
	NOTE: The plate can be tilted during antibody staining to minimize the volume of reagents needed. Just ensure that the entire cornea is completely submerged in antibody solution.</li>
	<li>Perform the EdU Click-iT reaction using a cocktail containing 0.88 mg/mL ascorbic acid, 0.26 mg/mL CuSO4, and 2.5 &#181;M Alexa Fluor 488 azide.
	<ol>
		<li>Prepare reaction components
		<ol>
			<li>Add 500 &#181;L of 1x PBS to 88 mg of ascorbic acid and vortex until dissolved.</li>
			<li>Add 840 &#181;L of deionized water to 44 mg of CuSO4 and vortex until dissolved.</li>
		</ol>
		</li>
		<li>Prepare cocktail by adding the following reagents to 6 mL of 1x PBS in this order, inverting tube to mix after each addition: 30 &#181;L of ascorbic acid solution, 30 &#181;L of CuSO4 solution, and then 15 &#181;L of Alexa Fluor 488 
		NOTE: Once prepared, this solution is light sensitive. For the remainder of the protocol, corneas should be protected from light whenever possible.</li>
		<li>Add 1 mL of EdU reaction cocktail to each cornea and react for 1 h at room temperature.</li>
	</ol>
	</li>
	<li>Add 4 mL of 10 &#181;g/mL Hoechst 33342 to each cornea and react for 2 min at room temperature, then wash three times in 1x PBS-T for 30 min each.</li>
	<li>Incubate in 1 mL of blocking solution containing 2 &#181;g/mL Alexa Fluor 555 labeled goat anti-mouse IgG for 1 h at 37&#176;C, then wash three times in 1x PBS-T for 30 min each.</li>
	<li>Store corneas at 4 &#176;C in 4 mL of 1x PBS with 1% anti/anti, 0.1% ciprofloxacin, and 0.1% Amphotericin B to prevent microbial growth.</li>
	<li>When performing confocal imaging, mount whole corneas in 200 &#181;L of mounting medium on glass slides with coverslip taped down to flatten.</li>
</ol>
8. Trypan Image Analysis
<ol>
	<li>Perform all image analysis using NIH&#39;s open-source software ImageJ. Open an image using File &#62; Open and click Image &#62; Adjust &#62; Color threshold.</li>
	<li>Use the &#34;Hue&#34; sliders to encompass only the blue range and adjust the upper &#34;Brightness&#34; slider all the way to the left to include more of the stained area. 
	NOTE: If this action causes parts of the image that are not Trypan stained to be included, the &#34;Brightness&#34; slider can be returned to its original level.</li>
	<li>Slowly adjust the upper &#34;Saturation&#34; slider until the thresholding accurately outlines the stained area. 
	NOTE: It is helpful to have a copy of the original image open to refer to during this process.</li>
	<li>Click the Select button within the color threshold menu, then use the selection brush tool to de-select areas outside of the wound that were picked up.</li>
	<li>Click Analyze &#62; Measure to report the stained area in pixels.</li>
	<li>De-select all using Edit &#62; Selection &#62; Select None and use the oval selection tool to fit the limbus as closely as possible, then click Analyze &#62; Measure to report the area of the cornea in pixels. 
	NOTE: Brightness can be turned up temporarily to aid in visualization of the limbus if this part of the image is too dark.</li>
	<li>Record area values and divide the stained area by the area of the whole cornea, then multiply by 100 to calculate the percent stained.</li>
	<li>Repeat this process two more times for each image, then take the average of the three measurements.</li>
</ol>
9. Statistical Analysis
<ol>
	<li>Once all images have been analyzed, divide the average percent stained on Day 14 by the percent stained on Day 0 to determine the residual unhealed area. Subtract this amount from 100% to calculate the percentage of the stripped area healed over 14 days.</li>
	<li>Use a paired t-test to compare percent healed values between control and treatment groups.</li>
</ol>

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DDE, SP, GD, and JW are employees of and hold equity in Trefoil Therapeutics, Inc. DDE is inventor on patents covering eFGF1 (NM141).

Many ophthalmologists have concerns about recommending DSO to their patients for two main reasons: 1) the lengthy healing process, and 2) lack of data (DSO is a new concept in the field of ophthalmic surgery). The research we have presented would be of great utility for easing both of these concerns. Based on data from this study and others, the FDA has approved a Phase 2 clinical trial where eFGF1 (NM141) will be administered at varying dosing schedules to patients undergoing DSO17.
The method described above was modeled after a study performed by Soh et al., where corneal endothelial healing was evaluated with and without the ROCK inhibitor Y-27632 in both scratched and peeled wounds18. While Y-27632 accelerated endothelial regeneration with Descemet&#39;s membrane still intact, no substantial healing was found in stripped wounds even with treatment. Using a similar stripping technique followed by treatment with or without eFGF1 (NM141), the observations we found were not consistent with those of Soh and colleagues. The absence of Trypan Blue staining in many treated corneas at Day 14, and the presence of ZO-1 positive tight junctions within the reformed endothelial layer argues that an intact barrier, part of the CEC&#39;s natural function, was restored in both normal and dystrophic corneas. Though not quantified in this study, the presence of EdU-positive cells in and around the stripped area also suggests proliferation as a healing mechanism, one which we have previously established can be stimulated by eFGF1 (NM141) in injured corneas16. Statistical analysis showed treatment with eFGF1 (NM141) resulted in significantly greater healing from DSO, on average over twice as high as control corneas at the 14 day timepoint. Though healing rates vary moderately between individuals-a typical characteristic of donor corneas-the results&#39; replicability over large sample sizes also evidences a highly measurable method. To our knowledge, there are no other examples of an ex vivo Descemet&#39;s stripping model in the literature.
Key components of the protocol itself that would serve valuable to other researchers studying DSO are the use of Trypan Blue to detect bare stroma, and the image processing technique used to measure the stained area. Trypan Blue is commonly used in ophthalmic surgery, particularly when working with Descemet&#39;s membrane to detect non-viable cells and assist in visibility of the tissue. The staining timepoints included in this protocol allowed for effective repeat staining without over-exposing corneas to Trypan Blue, as it has shown to be toxic to CECs at high concentrations19. The reduction of stained area over 14 days in all corneas, confirmed by Alizarin Red and immunohistochemistry to be the result of migrated CECs, demonstrates a simple and reproducible method to measure healing. Using ImageJ&#39;s color threshold menu, multiple analysts collected data with standard deviations consistently below 1% (data not shown). Though alternative programs may perform similarly, ImageJ is an open-source software capable of producing accurate area measurements to track healing.
There is, however, one aspect of the stripping protocol that we found to be both necessary for wound creation, yet obstructive to the overall healing process. Using a sharp, 30 G needle to score Descemet&#39;s membrane along the mark left by the biopsy punch enables the creation of a smooth, circular wound which is noted by clinicians to support faster healing10. At the same time, this step is damaging to the cornea, as it can create tears in the stromal fibers causing stromal cell death, impeding migration of endothelial cells across the wound edge, and inducing the formation of nodules that result in more persistent post-operative edema20. Clinicians performing DSO typically use a reverse Sinskey hook to initiate the wound, but without any intraocular pressure keeping the cornea taut, this tool is less effective in the ex vivo model. An alternative tool capable of tearing Descemet&#39;s membrane without damaging the underlying stroma would improve upon the protocol, for example the irrigation and aspiration handpiece recommended by Macsai and Shiloach10. Further experimentation will be needed to determine whether this technique is compatible with the ex vivo model.
A challenge which appears inherent to the ex vivo model is the frequent occurrence of CEC death in the area peripheral to the wound, particularly in dystrophic corneas. This occasionally obscured quantitation of the wound area, since the accuracy of the color thresholding tool becomes more limited as the stained area extends beyond the center of the cornea where its curvature results in uneven light distribution. However, these variable measurements primarily occurred at earlier timepoints before peripheral staining gradually receded as damaged CECs cleared and neighboring cells stretched, migrated, or proliferated to replace them. By the final 14 day timepoint, the stained area had localized back to the center of the cornea, and all images were measurable. A similar observation was made with comparable frequency by Soh et al., where five of 14 normal corneas presented what they termed &#39;Premature Culture Failure&#39; (PCF) early in the culture period18. While the damage reversed itself over time in our corneas and yet was permanent in their case, this may be attributed to the fact that their method required wounding a larger area of the cornea. The observation of more prevalent peripheral Trypan staining in dystrophic corneas may indicate that dystrophic corneas are more susceptible to endothelial cell death than healthy corneas. While the exact cause of this cell death has yet to be elucidated, we believe it is unlikely that this issue will be relevant to human corneas in vivo. Damage to the peripheral endothelium has not been reported in any clinical case studies of DSO to our knowledge, suggesting that this phenomenon is unique to donor corneas cultured ex vivo6,8,10,11,21. Aside from two instances where only control corneas stained, all observations of peripheral staining were paired, so it is unlikely that the cause was exposure to eFGF1 (NM141). However, it is possible that in those instances treatment may have provided a protective effect against the damage that otherwise would have induced peripheral staining in both corneas. Further investigation on this hypothesis is needed.
Another limitation to this method is sourcing donor corneas representative of the FECD phenotype for which DSO is intended. Donor corneas of any kind are scarce, hence the need for a surgery that avoids the use of donor tissue. For our purposes, the only corneas available are those rejected from transplant for various reasons. Eye banks further classify these corneas as normal or dystrophic based on criteria including presence of guttae, low or un-measurable ECD, and irregular CEC morphology. Confirming a dystrophic diagnosis prior to accepting tissue from an eye bank is also nearly impossible, since most donors&#39; medical history does not include past ocular history and the only information provided is the ECD value, the technician&#39;s notes, and in some cases a representative specular image. The dystrophic corneas obtained for this study did not show confluent central guttae upon confocal microscopy performed after the culture period was complete, suggesting that they may represent &#34;early&#34; stages of FECD. We do not expect this to have a significant impact on the implications of the study, as the purpose of DSO is to remove confluent areas of guttae, allowing healthy peripheral cells to migrate inward.
This method provides a highly applicable and reproducible technique to evaluate agents that might impact CEC proliferation and migration. The model has several features that make it more physiologically relevant than in vitro models that involve cultured CECs, even when seeded onto human corneal tissue graft22,23,24. First, the CECs to be stimulated are in a monolayer exactly as they exist in the patient&#39;s eye, and are migrating across the corneal stroma as they would after clinical DSO. The stroma in question is from the same patient as the CECs, thus controlling for potential FECD-related stromal differences. There is no need to explant, dissociate, and expand cultures of CECs with the associated challenges and potential for Endothelial to Mesenchymal Transition (EnMT) during the culturing process. The protocol described is itself very similar to the clinical DSO procedure. While it bypasses the culture and expansion steps, this method does have the limitation that the duration of study is restricted by corneal swelling, as the epithelial layer is not maintained. This inhibits us from investigating the morphology of CECs that have migrated to cover the stripped area, leaving it unclear whether they will eventually rearrange into a hexagonal array in this model. Garcin et al. have developed one potential solution with their active storage machine (ASM), a device shown to keep corneas in culture for up to 3 months with significantly less edema than corneas maintained in traditional organ culture25. Such a device may be helpful in replicating and expanding upon this work.
This model has potential utility in testing other wound healing therapies (e.g., ROCK inhibitors), evaluating modifications to surgical technique and comparing healing across different donor populations or stages of disease. We hope this research, in conjunction with clinical trial data as it comes out, encourages clinicians to consider DSO as a valuable treatment option for their eligible FECD patients.

Fuchs Endothelial Corneal Dystrophy (FECD) is a disease characterized by lost pump function in corneal endothelial cells (CECs) and the excessive buildup of collagen and other extracellular matrix proteins on the surface of Descemet&#39;s membrane, forming corneal guttae1. The only known treatment for FECD is endothelial keratoplasty in varying forms, all of which come with risk of rejection and endothelial cell loss2. While advancements in ophthalmic surgery have enabled these procedures to become less invasive over time, any form of transplantation comes with the risk of rejection and possibility of lifetime steroid use, a treatment with its own concomitant adverse events. Moreover, the global donor tissue shortage is such that only one donor cornea is available for every 70 patients in need3. Given these challenges, researchers and clinicians are exploring surgical methods that avoid the need for donor tissue altogether. One of these experimental techniques is Descemet&#39;s Stripping Only (DSO) or Descemetorhexis without Endothelial Keratoplasty (DWEK), in which FECD patients with guttae localized to the center of the cornea have a central 4 mm circle of Descemet&#39;s membrane stripped without graft placement. The removal of guttae encourages healthy peripheral cells to migrate inward and reform the endothelial monolayer, eventually reversing stromal edema and improving vision. The concept was originally described in a series of case studies in which patients underwent surgery that was complicated by detachment of Descemet&#39;s membrane, but CEC repopulation still occurred4,5,6,7. Though there are many advantages to this method, the healing process is lengthy and inconsistent, as some patients require rescue transplantation if no healing is seen in the months following surgery8. For these reasons, a drug that stimulates faster migration and proliferation of CECs may be beneficial in the recovery process of FECD patients that have undergone DSO.
Several recent studies have evaluated ROCK inhibitors as a supplemental treatment for patients undergoing DSO, and found that treated patients recovered faster and had higher central endothelial cell densities (ECD) than those in the DSO only group9,10,11. However, due to small sample sizes and differences between dosing regimens, more data is needed to better understand the efficacy of ROCK inhibitors in this setting.
Fibroblast Growth Factors have also been shown to stimulate regeneration of the corneal endothelium both in vitro with bovine CECs, and in vivo in feline corneas12,13. eFGF1 (NM141) is an engineered version of FGF-1 containing several amino acid substitutions to stabilize the molecule, as opposed to the native FGF-1, which has a much shorter half-life14,15. We have previously demonstrated the ability of eFGF1 (NM141) to stimulate proliferation of CECs ex vivo in quartered human corneas16. This study sought to improve upon that work by establishing the first successful ex vivo model of DSO in both normal and dystrophic corneas to determine whether adjunctive treatments such as eFGF1 (NM141) accelerate healing in this application.

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Science</td><td>470134-946</td><td></td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP9700-100</td><td></td></tr><tr><td>Calcium chloride (CaCl)</td><td>Amresco</td><td>1B1110-500G</td><td></td></tr><tr><td>Chex-all II sterilzation pouches</td><td>Propperman&amp;nbsp;</td><td>24008</td><td></td></tr><tr><td>Cirpofloxacin hydrochloride</td><td>Alfa Aesar</td><td>J61970</td><td></td></tr><tr><td>Copper (II) sulfate pentahydrate (CuSO4)</td><td>Sigma</td><td>469130-50g</td><td></td></tr><tr><td>Dissecting microscope</td><td>Nikon</td><td>SMZ1270</td><td></td></tr><tr><td>Dry vacuum pump</td><td>Welch</td><td>2019B-01</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Thermo Scientific</td><td>A31606-01</td><td></td></tr><tr><td>Frosted micro slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Galaxy miniStar microcentrifuge</td><td>VWR</td><td>C1413, VWR</td><td></td></tr><tr><td>Goat anti-mouse IgG (H+L) secondary antibody, Alexa Fluor 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(ITS)</td><td>Corning</td><td>41400-045</td><td></td></tr><tr><td>Iris scissors, 11 cm</td><td>World Precision Instruments</td><td>501264-G</td><td></td></tr><tr><td>L- Ascorbic acid</td><td>Sigma</td><td>A4544-25G</td><td></td></tr><tr><td>Manual single channel pipet</td><td>Rainin</td><td>&amp;nbsp;17014-392, -391, -382</td><td></td></tr><tr><td>Needle PrecisionGlide 30G</td><td>Becton Dickinson</td><td>305106</td><td></td></tr><tr><td>N-Met141 TTHX1114</td><td>Biopharmaceutical Development Program</td><td>NA</td><td></td></tr><tr><td>Opti-Mem I + GlutaMAX-1 (Opti-MEM)</td><td>Thermo Scientific</td><td>51985-034</td><td></td></tr><tr><td>Orion Star A211 pH meter</td><td>Thermo Scientific</td><td>STARA211</td><td></td></tr><tr><td>Petri dishes</td><td>VWR</td><td>89107-632</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>BDH</td><td>BDH9258-500G</td><td></td></tr><tr><td>Potassium phosphate monobasic (KH2PO4)</td><td>VWR</td><td>0781-500G</td><td></td></tr><tr><td>Powerpette plus pipet controller</td><td>VWR</td><td>75856-456</td><td></td></tr><tr><td>Precision water bath 188</td><td>Precision Scientific Incorporated</td><td>WB05</td><td>Set to 37&amp;deg;C</td></tr><tr><td>Purifier Class II model biosafety cabinet</td><td>Labconco</td><td>36213043726</td><td></td></tr><tr><td>Safe-Lock tubes, 1.5 mL</td><td>Eppendorf</td><td>22363212</td><td></td></tr><tr><td>Scalpel size 22 stainless steel&amp;nbsp;</td><td>Sklar</td><td>446479</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>VWR</td><td>2041-2.5K</td><td></td></tr><tr><td>Sodium hosphate dibasic (Na2HPO4)</td><td>VWR</td><td>0404-1KG</td><td></td></tr><tr><td>Standard shaker</td><td>VWR</td><td>89032-092</td><td></td></tr><tr><td>Standard solid refrigerator</td><td>VWR</td><td>10820-402</td><td>Set to 4&amp;deg;C</td></tr><tr><td>Sterilmatic autoclave</td><td>Market Forge</td><td>STM-EL</td><td></td></tr><tr><td>Syringe filters</td><td>VWR</td><td>28145-477</td><td></td></tr><tr><td>Test tube rocker</td><td>Thermo Scientific</td><td>M48725Q</td><td></td></tr><tr><td>Tru-Punch disposable biopsy punch, 4 mm</td><td>Sklar</td><td>96-1146</td><td></td></tr><tr><td>Trypan Blue</td><td>Thermo Scientific</td><td>15250-061</td><td></td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P7949-100mL</td><td></td></tr><tr><td>Vibrance antifade mounting medium with DAPI</td><td>Vector Laboratories Inc.</td><td>H-1800</td><td></td></tr><tr><td>VistaVision cover glasses, no. 1</td><td>VWR</td><td>16004-098</td><td></td></tr><tr><td>Vortex Genie 2</td><td>Fisher Scientific</td><td>G-560</td><td></td></tr><tr><td>ZO-1 monoclonal antibody (ZO1-1A12)</td><td>Thermo Scientific</td><td>33-9100</td><td></td></tr></tbody></table>

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.

This experiment was initially performed in 10 pairs of normal research corneas, as they are most readily available. Once the method was shown to be successful, the study was replicated in 11 pairs of dystrophic corneas-labeled as such by eye banks based on specular evaluation-to study the effects of eFGF1 (NM141) in corneas representative of the FECD patient population. For greater clinical relevance, the figures included in the present work represent data collected from dystrophic corneas unless otherwise noted.
After simulating the surgical outcome of DSO, Trypan Blue staining was performed and repeated throughout the 14 day culture period, and the reduction in positive staining quantified to evaluate wound healing (Figure 2). Alizarin Red staining was also performed on Day 14 to delineate cell borders. Dark red areas with no clear pattern visible (referred to hereafter as negative staining) appeared consistently within the unhealed section of the lesion and in other areas of the cornea where cells were presumed to be damaged or missing. Areas that stained positive for Trypan Blue on Day 14 corresponded well with negative staining by Alizarin Red (Figure 2). All dystrophic corneas treated with eFGF1 (NM141) showed greater healing on Day 14 compared to the untreated mate. On average, treated corneas demonstrated 91% healing as opposed to 38% in control corneas, and this difference was statistically significant (p &#60; 0.001) (Figure 3A). This is comparable to results obtained from 20 normal corneas, where controls showed an average of 32% healing and treated corneas reached 81%, which again was statistically significant (p &#60; 0.001) (Supplemental Figure 1). The percent healed was compared between normal and dystrophic corneas using two sample t-tests assuming equal variance, and no significant difference was seen in either the control or treatment groups (data not shown).
Donor information provided by eye banks (Table 1) for both normal and dystrophic corneas was analyzed to determine whether characteristics including age, days stored in Optisol, death to collection interval, cell density, and sex correlate with healing. Pearson&#39;s correlation coefficient (r) was used to investigate all factors except sex, which was evaluated using an unpaired t-test to compare healing between males and females. The absolute value of r was below 0.25 in all cases, indicating that any correlations between healing and age, days stored in Optisol, death to collection interval, or cell density were considered negligible. The difference in healing between males and females was not statistically significant (p = 0.53).
Similar to the normal data set, 10 of the 22 dystrophic corneas presented with notable Trypan staining outside of the stripped area connected to the stained area within the lesion in at least one timepoint (usually Day 3)-an observation we termed peripheral staining (Figure 4A). Of those 10, only two were control corneas whose treated counterparts did not exhibit the same effect. The remaining eight were all matched pairs and presented with similar severity between corneas from the same individual. Although staining patterns were comparable between normal and dystrophic corneas, the frequency of peripheral staining was higher in the dystrophic data set with 45% of corneas positive, compared to 25% in the normal group. Figure 4A shows a pair that was considered positive for peripheral staining, as the stained area outside of the lesion met the wound edge in the Day 6 images and gradually receded. In the corresponding Alizarin Red images, cells in areas with peripheral Trypan staining appeared enlarged and abnormally shaped, much like the cells that migrated into the stripped area, and negative staining correlated with the areas where Trypan Blue was present on Day 14. Despite the large portion of the cornea affected throughout the culture period, partial or complete clearing of the stained periphery occurred in all 10 corneas. Additionally, the final percent healed at Day 14 was within the normal range, respective to treatment group, for all but one cornea. The outlier (0811L, pictured in Figure 4A) returned a negative percent healed value due to the remnants of peripheral staining that still connected to the lesion area at Day 14 (Figure 3A and Figure 4A). A second staining pattern, referred to as far peripheral staining, was captured in the Alizarin Red images from Figure 4B, but was also apparent in Trypan Blue images throughout the culture period in many corneas (Figure 4B). This observation is characterized by a ring of dark staining around the limbus, indicating compromised endothelium. Despite the term, this pattern was so common in both normal and dystrophic corneas that they were not counted as positive for peripheral staining. In these images, the control cornea showed more vibrant and extensive staining on Day 14, despite both corneas possessing similar severity and pattern of staining early in the culture period. Also noted in the treated cornea was a seemingly larger number of cells, which appeared more compact and hexagonal compared to control, though these observations were not quantitatively measured. Due to the curvature in the periphery, only Trypan staining within and immediately surrounding the stripped area was included in quantitative analysis. When percent-stained values were averaged across all dystrophic corneas, the emergence of peripheral staining resulted in an initial increase from that of Day 0, as opposed to the steady decrease seen in normal corneas where peripheral staining was less prevalent (Figure 3B).
Immunohistochemistry visualized by confocal imaging reveals semi-organized expression of ZO-1, a functional marker of CEC tight junctions, in and around the lesion edge (Figure 5), a pattern similarly evidenced by the Alizarin Red staining (Figure 2 and Figure 4). Pleomorphism and polymegathism are visible by ZO-1 in most dystrophic corneas; however, this observation can be attributed to their diseased state and was noted by the eye bank upon specular examination of the corneas prior to culturing. Disorganized ZO-1 expression is observed within the lesion area of treated corneas where cells had migrated inward, also consistent with the Alizarin Red patterns. EdU-incorporating cells can be seen around the border of the lesion and inside of the lesioned area in control and treated corneas (Figure 5). The pattern of migrated CECs also reflects the Trypan Blue results, where in control corneas most cells were found within two fields of the lesion edge, while CECs in treated corneas can be seen throughout the stripped area (Figure 2).
EdU labeling was performed as a qualitative measure in this study to confirm that proliferation contributed to healing. However, quantification of cells incorporating EdU could not be performed systematically due to corneal swelling preventing consistent, replicable images from being captured in and around the lesion. As a surrogate, quantitative analysis performed in a study conducted prior to establishment of the DSO model has been included to demonstrate the effects of eFGF-1 (NM141) on proliferation (Figure 6). Normal human donor corneas were cut into quarters using a scalpel and cultured in the presence of EdU, with or without eFGF1 (NM141) for 48 h as described previously (Eveleth, 2020)16. Imaging and subsequent analysis of Hoechst 33342 and EdU-labeled cells was performed separately in the undisturbed mid zone of the quarters and in the edge zone, within three fields from where the cornea was cut. In the mid zone, the percentage of cells incorporating EdU was low and highly variable across the 10 corneas analyzed. On average, quarters treated with eFGF1 (NM141) had higher levels of EdU incorporation in the mid zone (4.1% &#177; 7.9%) compared to controls (1.8% &#177; 2.9%), though this difference was not statistically significant (p = 0.239). At the wound edge, treated quarters stimulated with eFGF1 (NM141) showed significantly higher rates of EdU incorporation when averaged (18.1% &#177; 11.5%) compared to control quarters (10% &#177; 9.6%, p = 0.012).
Table 1: Donor information for 22&#160;dystrophic and 20 normal corneas used in this study, as well as 10 normal corneas used previously for EdU quantitation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/Table 1_Donor info.zip" target="_blank">Please click here to download this Table.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63482/63482fig02.jpg" /> 
Figure 2: Vital dye staining of corneas post-Descemet&#39;s stripping. Dystrophic human corneas were stripped of the central 4 mm of Descemet&#39;s membrane and incubated with or without eFGF1 (NM141) (100 ng/mL) for 14 days. Lesions were visualized by Trypan Blue staining on days 0, 3, 6, 9, 12, and 14. CEC borders were visualized by Alizarin Red staining on day 14. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63482/63482fig03v2.jpg" /> 
Figure 3: Dystrophic human corneas show significantly greater healing from DSO when cultured with eFGF1 (NM141). (A) Percent healing was determined by measuring the stained area at Day 14 using ImageJ and comparing that to the percent stained at Day 0. (B) The average percent stained graphed over time shows a consistent decrease in normal corneas while peaking at Day 3 in dystrophic corneas due to the higher prevalence of peripheral staining. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63482/63482fig04.jpg" /> 
Figure 4: Vital dye staining of dystrophic corneas with peripheral staining. (A) Trypan staining of the intact Descemet&#39;s membrane can be seen advancing toward the center, then clearing over time in both control and treated corneas. (B) Alizarin patterns around the limbus represent a typical observation of peripheral damage and disorganized re-establishment of the endothelium seen in many donor corneas-in this case dystrophic. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/63482/63482fig05.jpg" /> 
Figure 5: Stimulation of migration and proliferation of CECs by eFGF1 (NM141) in stripped dystrophic corneas. Confocal micrographs of Descemet&#39;s stripped corneas stained for ZO-1 (red), EdU (green), and Hoechst 33342 (blue). The dotted line represents the lesion edge with the stripped area on the left side of the image and the intact Descemet&#39;s membrane on the right. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/63482/63482fig06v2.jpg" /> 
Figure 6: Quantitation of proliferating cells from normal corneas quartered using a scalpel and incubated in media containing EdU with or without eFGF-1 (NM141) for 48 h. Quarters were imaged in triplicate at the undisturbed mid zone and along the cut edge, and counts averaged to determine the percentage of cells incorporating EdU in the mid and edge zones. Figure modified from Journal of Ocular Pharmacology and Therapeutics, used with permission16. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/63482fig06largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Figure 1: Normal human corneas show significantly greater healing from DSO when cultured with eFGF1 (NM141). ImageJ was used to measure the stained areas and determine % healing. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63482/Supplemental Figure 1.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1 at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Medicine

4.5K Views.  Trefoil Therapeutics, Inc.. 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). 

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

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene therapy in organ-cultured corneas. The diabetic corneal disease is a complication of diabetes with frequent abnormalities of corneal nerves and epithelial wound healing. We have also documented significantly altered expression of several putative epithelial stem cell markers in human diabetic corneas. To alleviate these changes, adenoviral gene therapy was successfully implemented using the upregulation of c-met proto-oncogene expression and/or the downregulation of proteinases matrix metalloproteinase-10 (MMP-10) and cathepsin F. This therapy accelerated wound healing in diabetic corneas even when only the limbal stem cell compartment was transduced. The best results were obtained with combined treatment. For possible patient transplantation of normalized stem cells, an example is also presented of the optimization of gene transduction in stem cell-enriched cultures using polycationic enhancers. This approach may be useful not only for the selected genes but also for the other mediators of corneal epithelial wound healing and stem cell function.

An example of adenoviral gene therapy in the human diabetic organ-cultured corneas is presented towards the normalization of delayed wound healing and markedly reduced epithelial stem cell marker expression in these corneas. It also describes the optimization of this process in stem cell-enriched limbal epithelial cultures.

The goal of this protocol is to describe molecular alterations in human diabetic corneas and demonstrate how they can be alleviated by adenoviral gene ...

Developmental Biology

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

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

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

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

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

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

Bioengineering

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

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

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

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

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

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

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

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

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

An Epithelial Abrasion Model for Studying Corneal Wound Healing

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its transparency. However, due to its external position, the cornea is highly susceptible to a wide variety of injuries that can lead to the loss of corneal transparency and eventual blindness. Efficient corneal wound healing in response to these injuries is pivotal for maintaining corneal homeostasis and preservation of corneal transparency and refractive capabilities. In events of compromised corneal wound healing, the cornea becomes vulnerable to infections, ulcerations, and scarring. Given the fundamental importance of corneal wound healing to the preservation of corneal transparency and vision, a better understanding of the normal corneal wound healing process is a prerequisite to understanding impaired corneal wound healing associated with infection and disease. Toward this goal, murine models of corneal wounding have proven useful in furthering our understanding of the corneal wound healing mechanisms operating under normal physiological conditions. Here, a protocol for creating a central corneal epithelial abrasion in mouse using a trephine and a blunt golf club spud is described. In this model, a 2 mm diameter circular trephine, centered over the cornea, is used to demarcate the wound area. The golf club spud is used with care to debride the epithelium and create a circular wound without damaging the corneal epithelial basement membrane. The resulting inflammatory response proceeds as a well-characterized cascade of cellular and molecular events that are critical for efficient wound healing. This simple corneal wound healing model is highly reproducible and well-published and is now being used to evaluate compromised corneal wound healing in the context of disease.

Here, a protocol for creating a central corneal epithelial abrasion wound in the mouse using a trephine and a blunt golf club spud is described. This corneal wound healing model is highly reproducible and is now being used to evaluate compromised corneal wound healing in the context of diseases.

The cornea is critical for vision, accounting for about two-thirds of the refractive power of the eye. Crucial to the role of the cornea in vision is its ...

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. Constructing appropriate animal models can help us focus on the effects of severe inflammation on corneal limbal stem cells. A 2 mm rust remover&#160;was&#160;used to remove the central corneal epithelium of Sprague Dawley (SD) rats to create an injury. Then, the central stroma of the cornea was sutured with nylon sutures to induce persistent inflammation. In this way, a corneal inflammation model with central corneal epithelium abrasion and central stroma suturing was constructed, which induced severe corneal inflammation. The changes in corneal inflammation and the condition of the limbal stem cells at 1, 3, and 7 days post-modeling were observed. On the 3rd day after modeling, the rats&#39; corneal limbus was severely edematous, with obvious neovascularization and local hyperplasia, which are typical signs of limbal stem cell deficiency. By the 7th day, the corneal edema gradually worsened, and the neovascularization continued to increase. Through quantitative reverse transcription polymerase chain reaction (RT-qPCR) and immunofluorescence staining, we found that the corneal epithelial inflammatory factors were significantly upregulated, the corneal epithelial differentiation was abnormal, the corneal epithelial stem cells were significantly reduced, and the cell proliferation and stemness had also decreased. Therefore, this model demonstrates that severe inflammation can induce limbal stem cell damage without directly damaging the limbal stem cells. The model is beneficial for observing the effects of severe inflammation on the biological mechanisms of stem cells and provides an ideal platform for studying the mechanisms of corneal epithelial stem cell dysfunction induced by inflammation.

We developed a rat model of severe corneal inflammation through corneal epithelium curettage combined with corneal sutures. The study evaluated corneal inflammation patterns, epithelial proliferation, and changes in limbal stem cells under inflammatory conditions.

Corneal inflammation, especially severe corneal inflammation, plays a significant role in the development of corneal limbal stem cell dysfunction. ...

Ex Vivo Porcine Corneal Culture System for Toxicity Testing and Wound Healing

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular toxicity assessment. An air/liquid corneal culture system using porcine eyes was developed, and ex vivo epithelial wound healing was utilized as a critical parameter for these studies. Fresh pig corneas were processed for organ culture, with or without epithelial wounding. The corneas were cultured in a humidified 5% CO2 incubator at 37 &#176;C in MEM, with or without testing agents. Corneal permeability and wound healing rates were measured, and epithelial cells and/or whole corneas can be processed for immunohistochemistry, western blotting, and qPCR for molecular and cellular analyses. This study describes a detailed protocol and presents two studies using this ex vivo system. The data show that porcine corneal organ culture, combined with epithelial wound healing, is a suitable ex vivo model for chemical toxicity testing, studying diabetic keratopathy, and identifying potential therapies.

Author Spotlight: Exploring Corneal Innate Immunity and Delayed Wound Healing in Diabetic Patients

Porcine corneal ex vivo organ culture and epithelial wound healing provide an economical, ethical, reproducible, and quantitative means for testing the ocular toxicity of chemicals. They also aid in elucidating mechanisms underlying the regulation of epithelialization and tissue repair, and in evaluating therapeutics for treating diabetic keratopathy and delayed wound healing.

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

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Chapters in this video

Adenoviral Gene Therapy for Diabetic Keratopathy: Effects on Wound Healing and Stem Cell Marker Expression in Human Organ-cultured Corneas and Limbal Epithelial Cells

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

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

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

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

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

An Epithelial Abrasion Model for Studying Corneal Wound Healing

Establishing a Severe Corneal Inflammation Model in Rats Based on Corneal Epithelium Curettage Combined with Corneal Sutures

Evaluating Therapeutic and Chemical Toxicity Using Organ-Cultured Porcine Corneas and Epithelial Wound Healing