The authors thank Alcon for providing financial support for these studies.

1. Human corneal epithelial cell culture
<ol>
	<li>Grow immortalized HCEC19 in collagen-coated 75 cm2 flasks with 20 mL of Dulbecco&#39;s modified Eagle medium/nutrient mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 &#176;C with 5% CO2. 
	NOTE: Shaking is not required.</li>
	<li>Change the media every 2&#8722;3 days.</li>
</ol>
2. Preparation of cells for testing
<ol>
	<li>After the cells are almost confluent remove culture media.</li>
	<li>Add 4&#8722;6 mL of cell disassociation solution to each 75 cm2 flasks. Incubate the cells at 37 &#176;C until the cells detach (approximately 20&#8722;30 min), checking under the microscope periodically.</li>
	<li>Add 2&#8722;6 mL of DMEM/F12 containing 10% FBS to each 75 cm2 flask.</li>
	<li>Transfer the contents of the flask to a 50 mL centrifuge tube.</li>
	<li>Centrifuge the cells at 450&#8722;500 x g for 5 min. Pipet out the supernatant and resuspend the cells in pre-warmed media.</li>
	<li>Count the cells using a hemocytometer. Calculate the volume of media that contains a total of 1 x 105 cells.</li>
	<li>Add 1 x 105 cells to each well of a 48 well collagen-1 coated culture plate. Add enough media to the well so that the final volume of media in the well is 0.5 mL of culture DMEM/F12 with 10% FBS. 
	NOTE: An artificial tear formulation may cause cytotoxicity that can be measured by a decrease in the metabolic activity of HCEC. The consistency of the data is dependent on adding the same number of HCEC to each well when seeding. The culture concentration that is recommended for 48 well plates is 1 x 105. Because mammalian cells can settle in the centrifuge tube after resuspending, it is recommended to resuspend the cells by agitation frequently while seeding the plates with cells so that the cell suspension has the HCEC equally distributed.</li>
	<li>Incubate the cells at 37 &#176;C and 5% CO2 for 24 h.</li>
</ol>
3. No desiccation protocol
<ol>
	<li>Control procedure
	<ol>
		<li>After the 24 h incubation remove the culture media from the plate.</li>
		<li>Immediately treat with 150 &#181;L of a test formulation and media control solution.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 30 min.</li>
		<li>Remove the test solutions from the cells.</li>
		<li>Add 0.5 mL of 10% metabolic dye solution to test for metabolic activity. Incubate the cells for another 4 h at 37 &#176;C and 5% CO2.</li>
		<li>After the 4 h incubation period, remove 100 &#181;L of metabolic dye solution from each well and place each 100 &#181;L into a well of a 96 well plate.</li>
		<li>Measure the fluorescence of each well using a fluorescent plate reader (Table of Materials). Set the excitation wavelength at 540 nm and the emission wavelength at 590 nm.</li>
	</ol>
	</li>
	<li>No desiccation protocol (recovery procedure)
	<ol>
		<li>Repeat steps 3.1.1&#8722;3.1.4.</li>
		<li>Add 0.5 mL of DMEM/F12 media to each well.</li>
		<li>Incubate one set of cultures for 18 h at 37 &#176;C and 5% CO2.</li>
		<li>Remove the media and add 0.5 mL of 10% metabolic dye solution. Repeat steps 3.1.6 and 3.1.7.</li>
	</ol>
	</li>
</ol>
4. Desiccation protocol
<ol>
	<li>Control procedure
	<ol>
		<li>After the 24 h incubation (step 2.8), remove the culture media from the plate.</li>
		<li>Immediately treat with 150 &#181;L of a test formulation and media control solution.</li>
		<li>Incubate the cells at 37 &#176;C and 5% CO2 for 30 min.</li>
		<li>Remove the test solutions from the cells.</li>
		<li>Place the cells in a 37 &#176;C and 45% RH chamber for 5 min to desiccate the cells.</li>
		<li>Add 0.5 mL of 10% metabolic dye solution to test metabolic activity. Incubate the cells for another 4 h at 37 &#176;C and 5% CO2.</li>
		<li>After the 4 h incubation period, remove 100 &#181;L of metabolic dye solution from each well and place each 100 &#181;L into a well of a 96 well plate.</li>
		<li>Measure the fluorescence of each well using a fluorescent plate reader. Set the excitation wavelength at 540 nm and the emission wavelength at 590 nm.</li>
	</ol>
	</li>
	<li>Recovery procedure
	<ol>
		<li>Repeat steps 4.1.1-4.1.5.</li>
		<li>Add 0.5 mL of DMEM/F12 media to each well.</li>
		<li>Incubate one set of cultures for 18 h at 37 &#176;C and 5% CO2.</li>
		<li>Remove the media and add 0.5 mL of 10% metabolic dye solution. Repeat steps 4.1.7 and 4.1.8.</li>
	</ol>
	</li>
</ol>
5. Data analysis
<ol>
	<li>Calculate the average fluorescence of the media control. Calculate the percent viability of the sample using the follow formula: 
	Percent viability of the sample = test sample fluorescence/media control fluorescence x 100 
	NOTE: The media control average is 100% viability. The metabolic dye by itself has a small amount of fluorescence associated with it. The fluorescence of the metabolic dye reagent can be subtracted from the control and test sample fluorescence prior to calculating % viability of the test samples to the media control.</li>
	<li>Perform a normality test and a test for equal variances on the control and test samples.</li>
	<li>If the normality test passes and the variances are equal then perform a one-way ANOVA. To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Tukey or Dunnett&#39;s multiple comparisons tests).</li>
	<li>If the normality test passes and the variances are different then perform the Welch&#39;s ANOVA. To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Dunnett&#39;s or Games-Howell multiple comparisons tests).</li>
	<li>If the normality test fails use a nonparametric test (i.e., Kruskal-Wallis test). To identify significant differences between specific groups perform a pairwise comparisons post hoc test (i.e., Dunn&#39;s test).</li>
</ol>

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Three authors (Rekha Rangarajan, Howard A. Ketelson, Lakshman Subbaraman) are employees of Alcon. Alcon is the sole source of funding and sponsor of this project. Lyndon W. Jones, over the past 3 years, has received research support or lectureship honoraria from the following companies: Alcon, Allergan, Contamac, CooperVision, GL Chemtec, Inflamax Research, J&#38;J Vision, Menicon, Nature&#39;s Way, Novartis, PS Therapy, Santen, Shire, SightGlass and Visioneering. Lyndon Jones is also a consultant and/or serves on an advisory board for Alcon, CooperVision, J&#38;J Vision, Novartis and Ophtecs. The following list of authors have nothing to disclose: Richard Do, David J. McCanna, Adeline Suko, Daryl Enstone, Jaya Dantam.

This protocol is designed to determine the protective effects of artificial tear solutions against cell desiccation. Initially, the cells are exposed to the dry eye drops to coat the cells. Next, the cells are dried, and the metabolic activity is monitored to assess whether the formulations can mitigate the detrimental effects of desiccation stress. These steps are critical for understanding the overall impact the dry eye formulations could have on the overall viability of corneal epithelial cells.
After most of the dry eye formulations are removed from the culture wells, the chemistry of the remaining molecules that are in contact with the cells will impact cell desiccation. Some products contain micro-emulsions of oils that minimize cell evaporation20. A study that evaluated friction analysis after rinsing showed maintenance of low friction for some dry eye formulations which may be an indicator of the enhanced residence time of the dry eye formulations21. One of the limitations of this assay is that although desiccation protection can be evaluated in comparison to other eye drop formulations the duration of this protection cannot be determined due to the short drying time assessed. Longer drying times in this in vitro assay may require the use of multilayer cultures that have overall more moisture content than a cell monolayer.
The specific incubation times for the formulations on the cells and the duration of drying time may need to be adjusted if temperature and humidity values are changed. Selecting a temperature, RH and air flow for simulating environmental stress conditions that can contribute to dry eye can be difficult as the seasonal and local environmental conditions are quite variable21. Modern-day environment chambers for human studies can vary the temperature between 5 &#176;C and 35 &#176;C, and RH between 5% and 95%22. Using an evaporimeter and tightly fitted goggles it was determined that at RH of 40&#8722;45% the evaporation rate from the cornea was 0.037 &#181;L/cm2/min and at RH of 20&#8722;25% the evaporation rate was 0.065 &#181;L/cm2/min23. If the in vitro model is looking at the protective effect of formulations under extremely low RH conditions such as in airplanes15, deserts, or dry indoor environments16, incubation times may need to be reduced to accommodate the enhanced evaporation rates from the cells. In the human tear fluid, a variety of lipids derived from meibomian and other glands are present that protect the tears from evaporation. These lipids have different melting points24. Temperature changes could impact the fluidity of the tear fluid so tests performed at ambient temperature could have significantly different evaporation rates than tests conducted at 37 &#176;C. Since the average ocular surface temperature range is from 32.9 to 36 &#176;C25, performing the test near this temperature range is recommended.
In addition to the metabolic dye (alamarBlue) used in this protocol, there are other chemical reagents that can be used to assess the effects of product formulations on cell metabolic activity. The tetrazolium salts (MTT, XTT, MTS, WST) are alternative chemicals that can be used. The difference in the tetrazolium salts is that MTT is a positively charged molecule so it can enter the cytoplasm of living cells26. MTT becomes insoluble after its reduction by NAD(P)H27. It must be solubilized prior to reading absorbance on a plate reader. The other tetrazolium salts are negatively charged28. They must be reduced by secondary molecules outside the living cell because they cannot enter the cell and interact directly with intracellular molecules29. For the XTT, MTS, and WST-1 assays, the addition of an electron coupling agent 1-methoxy phenazine methosulfate (PMS) can improve the performance of the assays30. In contrast to the metabolic assays that use tetrazolium salts, alamarBlue does not require an additional solubilizing or electron coupling agents. Also, unlike XTT, MTS or WST-1, alamarBlue penetrates cell membranes and can be reduced directly by cellular enzymes27,29. Direct comparison of alamarBlue to tetrazolium salts have shown that alamarBlue is more sensitive for evaluating the metabolic activity of various cell lines including HCEC3,27,31,32.
Other biochemical endpoints can also be considered for evaluating the protection of human corneal epithelial cells by desiccation. Fluorescent dyes can be used to detect cell membrane permeability and cell esterase activity1. Also, apoptosis can be detected by staining for apoptotic markers on the cell surface or by measuring for caspase activity1,33. Other assays have determined the effects of ophthalmic products on HCEC tight junctions34. These assays could be incorporated in future assessments utilizing the desiccation procedures described in this article.
There are many causes of dry eye. Dry eye can be caused by decreased secretion of tears, increased ocular surface inflammation or increased dehydration at the ocular surface. For the individuals that have evaporative dry eye use of a dry eye drop that prevents their cells from drying during desiccation conditions may alleviate the symptoms of dry eye. One of the problems in some dry eye formulations is the presence of preservatives that may harm the ocular surface. Injuring cells is not helpful to dry eye patients as it can cause more cells to die as injured cells are more susceptible to desiccation stress35.
A recent review article by Garrigue et al.36 provides an assessment of the current knowledge of how lipid-based products act to alleviate evaporative dry eye. Adding lipids to lipid deficient tears can prevent evaporation, thus protecting the cells from desiccation. In addition, humectants are molecules that attract and retain water at the ocular surface37. Both glycerin in solution #1 and propylene glycol in solution #3 are humectants. Solution #3 showed a greater protective effect of the HCEC that may have been due to the lipid and humectant properties of the formulation ingredients.
This protocol is designed to evaluate the metabolic activity of HCEC after exposure to the dry eye formulations and to desiccation stress. By using the methods described in this article, new nonirritating and protective eye drops can be developed for people that suffer from evaporative dry eye.

Many different ingredients in multiple-dose ophthalmic solutions are needed to maintain stability, pH, osmolarity, and efficacy. However, some chemicals such as preservatives or surfactants in ocular solutions can cause damage to the corneal epithelium1,2. Cell viability tests using human corneal epithelial cells (HCEC) can be performed to assess the potential damage caused by these various ingredients in ophthalmic formulations. One of the in vitro assays that is particularly useful for assessing cell damage is the alamarBlue assay3. In this assay, the metabolic dye (i.e., alamarBlue) contains resazurin, which functions as a cell viability indicator. During cellular respiration, resazurin is reduced to resorufin by accepting electrons4. The reduction of resorufin causes a change from a non-fluorescent molecule to a fluorescent molecule that can be detected using a spectrofluorometer5.
This investigation uses two different treatments of the corneal epithelial cells to determine how dry eye products may perform in an in vitro model. In the first evaluation, the cells are treated with these products for 30 min. After treatment, the cells are then evaluated for metabolic activity immediately after exposure to the dry eye products and after a recovery time interval. This assessment determines the effect of these products on cells prior to desiccation in a humidity chamber. Solution preservatives, surfactants or buffers in these solutions may have some cytotoxic effects on the cells that can be detected using the metabolic dye.
The second evaluation determines the metabolic activity of the cells after exposure to the dry eye products and desiccation. In this assessment, if the product treatment offers protection to the cells from desiccation damage, the metabolic activity of the cells will be maintained. Cell protection from the harmful effects of desiccation may occur if the dry eye product can coat the cells with lipids or the product may absorb liquid from the surroundings adding moisture to the cells.
This protocol is designed to simulate severe conditions of environmental stress on cells. Other studies have used various models to create this external stress. Some studies have dried the cells in laminar flow hoods6,7,8, whereas others have used room temperature and various relative humidity (RH) values9,10,11. A method of simulating dry eye conditions is to increase the osmolarity of the incubating solution12,13. Another in vitro method for simulating adverse environmental conditions is to add cytokines to the cell media to simulate the tear fluid of dry eye patients14. Although these tests are interesting models that can evaluate how chemicals reduce osmotic stress or stimulate the immune system, these models do not directly show how chemical formulations can protect cells from desiccation stress.
The desiccation model uses a temperature/humidity chamber 37 &#176;C and 45% RH. It simulates environmental conditions that can cause evaporation from the ocular surface. Simulations of other extreme environmental conditions such as the low humidity found in airplane cabins15 or in indoor environments during the winter months16 can also be performed using this method.
Cell culture modeling is used to simulate the effect of desiccation stress on the human cornea. In order to detect cell stress, cell viability testing is undertaken to determine the impact on cell health. Several different physiological tests can be performed on cells to determine if they are stressed17. The analysis performed in this test procedure uses the commercially available metabolic dye (Table of Materials). It has the advantage over many other cell stress tests in that it is non-toxic, soluble in growth media and can evaluate cells without cell fixation18. In this procedure the cells are tested for metabolic activity immediately after each treatment and a second set of cells are placed back into growth media and evaluated after 18 h recovery. The reason for testing immediately and then after a recovery period is to identify cell damage that causes immediate effects on metabolic activity and cell damage that causes delayed effects. Also, it provides an opportunity to evaluate the impact of these formulations on short-term versus long-term damage and the ability of the formulations to recover/not recover from the initial insult.
This investigation is used to compare various lipid-containing dry eye products for their effect on HCEC metabolic activity with and without desiccation. The ingredients in the dry eye products tested are described in Table of Materials. The ingredients in solution #1 are carboxymethylcellulose sodium (0.5%), glycerin (1%), and polysorbate 80 (0.5%), in solution #2 they are light mineral oil (1.0%) and mineral oil (4.5%), and in solution #3 the lipid is mineral oil and propylene glycol is the demulcent. These lipids are the formulation components that are expected to provide protection of the HCEC from desiccation.

<table><tbody><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Vented Flask</td><td>Corning</td><td>354485</td><td>This is the BioCoat brand collagen coated</td></tr><tr><td>96 well plate</td><td>costar</td><td>3370</td><td></td></tr><tr><td>alamarBlue</td><td>Fisher Scientific</td><td>dal 1025</td><td></td></tr><tr><td>Corning 48 Well plates</td><td>Corning</td><td>354505</td><td>This is the BioCoat brand collagen coated</td></tr><tr><td>Cytation 5</td><td>BioTek</td><td>CYT5MPV</td><td>Can read fluorescence from 280 - 700 nm (for assay 540/590)</td></tr><tr><td>DMEM/F12 with L-glutamine and 15 mM HEPES</td><td>Gibco</td><td>11039-021</td><td>There is No Phenol Red in this media</td></tr><tr><td>Fetal Bovine Serum</td><td>Hycone</td><td>SH30396.03</td><td></td></tr><tr><td>Human Corneal Epithelial Cells</td><td>University of Ottawa</td><td>N/A</td><td>SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169&amp;ndash;72.</td></tr><tr><td>Humidity/Temperature Chamber</td><td>Associated Environmental Systems</td><td>LH-1.5</td><td>Economy Line Humidity Chamber</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>100X concentration so add 1ml to each 99ml of media</td></tr><tr><td>Refresh Optive Advanced (solution #1)</td><td>Allergan, Inc</td><td></td><td>Carboxymethylcellulose Sodium (0.5%),Glycerin (1%),polysorbate 80 (0.5%),-Lubricants- Inactive ingredients-boric acid; carbomer copolymer type A; castor oil; erythritol; levocarnitine; purified water; Purite (stablized oxychloro complex)and may contain sodium hydroxide to adjust pH.</td></tr><tr><td>Soothe XP (solution #2)</td><td>Bausch &amp;amp; Lomb</td><td></td><td>Light mineral oil (1.0%), Mineral Oil (4.5%)-emolients-Inactive ingredients-boric acid, edetate disodium, octoxynol-40, polyquaternium-1 (preservative), polysorbate 80, purified water, sodium borate decahydrate. May contain hydrochloric acid and/or sodium hydroxide (to adjust pH).</td></tr><tr><td>Systane Complete (solution #3)</td><td>Alcon Inc.</td><td></td><td>Propylene Glycol 0.6% Lubricant Inactive ingredients- boric acid, dimyristoyl phosphatidylglycerol, edatate disodium, hydroxypropyl guar, mineral oil, polyoxl 40 stearate, POLYQUAD&amp;reg; (polyquaternium-1) 0.001% preservative, sorbitan tristearate, sorbitol and purified water. May contain hydrochloric acid and/or sodium hydroxide to adjust pH.</td></tr><tr><td>TrypLE Express (cell disassociation solution)</td><td>Fisher Scientific</td><td>&lt;a href=&quot;https://www.thermofisher.com/order/catalog/product/12605036&quot; target=&quot;_blank&quot;&gt;12605036&lt;/a&gt;</td><td></td></tr></tbody></table>

effect of artificial tear formulations on the metabolic activity of human corneal epithelial cells after exposure to desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

Results from a study comparing three dry eye formulations are shown in Figure 1. This figure shows that there are differences in the effect the three products, solution #1, solution #2 and solution #3 (Table of Materials) have on the viability of HCEC. Solution #1 and solution #2 had a significant effect on the metabolic activity of HCEC before desiccation. This means that there are formulation components in these products that can disrupt the metabolic activity of the HCEC. The additional drop in cell metabolic activity that occurred after 18 h recovery of cells exposed to solution #1 shows that HCEC were injured initially and after 18 h the injury was not repaired. In comparison, solution #3 had only a mild effect on the metabolic activity of the HCEC.
Figure 2 shows the effect of these lipid-containing dry eye formulations on cell protection. With a near 0% HCEC metabolic activity at the 18 h recovery interval, solution #1 and solution #2 did not protect cells from desiccation stress. Solution #3, however, offered some protection against desiccation stress.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60812/60812fig1.jpg" /> 
Figure 1: Effect of dry eye lipid enhanced products on cell viability. Relative viability measured by HCEC metabolic activity after treatment with lipid-containing dry eye products. * = statistical significance (p &#60; 0.05) relative to solution #3, &#916; = statistical significance (p &#60; 0.05) relative to the media control for the appropriate recovery period. The height of the bar represents the mean value, and the error bars are the SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60812/60812fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60812/60812fig2.jpg" /> 
Figure 2: Effect of dry eye lipid enhanced products on desiccation protection. Effect of lipid-containing dry eye formulations on cell protection. Solution #1 and solution #2 did not protect cells from desiccation stress, and they did not recover from desiccation effects over time. In contrast, solution #3 offered some protection against desiccation stress. * = p &#60; 0.05 relative to solution #3, and &#916; = p &#60; 0.05 relative to untreated (control) media for the appropriate recovery time. The height of the bar represents the mean value, and the error bars are the SD. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60812/60812fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation at JoVE.com

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

effect-artificial-tear-formulations-on-metabolic-activity-human

Nanyang Technological University

Research

JoVE Journal

Medicine

3.8K Views.  Alcon Vision, LLC. Het doel van dit protocol is om te evalueren of verschillende kunstmatige traanformuleringen menselijke hoornvliesepitheelcellen kunnen beschermen tegen uitdroging met behulp van een in vitro model. Na blootstelling aan kunstmatige traanformuleringen en uitdroging worden menselijke hoornvliesepitheelcellen beoordeeld op metabole activiteit.

Video: Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

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 ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

Immunology and Infection

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

Determining the Toxicity of UV Radiation and Chemicals on Primary and Immortalized Human Corneal Epithelial Cells

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) human corneal epithelial cell cultures. Cells were exposed to UV radiation and toxic doses of benzalkonium chloride (BAK), hydrogen peroxide (H2O2), and sodium dodecyl sulfate (SDS). Metabolic activity was measured using a metabolic assay. The release of inflammatory cytokines was measured using a multi-plex interleukin (IL)-1&#946;, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-&#945;) assay, and cells were evaluated for viability using fluorescent dyes. 
The damaging effects of UV on cell metabolic activity and cytokine release occurred at 5 min of UV exposure for iHCEC and 20 min for pHCEC. Similar percent drops in metabolic activity of the iHCEC and pHCEC occurred after exposure to BAK, H2O2, or SDS, and the most significant changes in cytokine release occurred for IL-6 and IL-8. Microscopy of fluorescently stained iHCEC and pHCEC BAK-exposed cells showed cell death at 0.005% BAK exposure, although the degree of ethidium staining was greater in the iHCECs than pHCECs. Utilizing multiple methods of assessing toxic effects using microscopy, assessments of metabolic activity, and cytokine production, the toxicity of UV radiation and chemical toxins could be determined for both primary and immortalized cell lines.

This article describes the procedures used to evaluate the toxicity of UV radiation and chemical toxins on a primary and immortalized cell line.

This article describes the methods of measuring the toxicity of ultraviolet (UV) radiation and ocular toxins on primary (pHCEC) and immortalized (iHCEC) ...

Biology

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding 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.

Due to its anatomical and physiological similarities to the human eye, the porcine eye serves as a robust model for biomedical research and ocular ...

Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model

Source: Hou, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for assessing corneal smoothness and tear film stability in an autoimmune rat model. It highlights the impact of gland dysfunction on tear film stability, crucial for understanding dry eye disease.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

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