Name: ex vivo and in vivo animal models for mechanical and chemical injuries of corneal epithelium
Uploaded: 2022-04-06T13:00:00
Description: Watch this Scientific Journal Video about Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium at JoVE.com

The study was funded by the Atomic Energy Council of Taiwan (Grant No. A-IE-01-03-02-02), Ministry of Science and Technology (Grant No. NMRPG3E6202-3), and Chang Gung Medical Research Project (Grant No. CMRPG3H1281).

All of the experimental procedures in animal studies were approved by the Research Ethics Committee at the Chang Gung Memorial Hospital and adhered to the ARVO statement for use of animals in ophthalmic and vision research.
1. Ex vivo wound healing model of the mouse corneal epithelium
<ol>
	<li>Preparation of the mice
	<ol>
		<li>Administer general anesthesia to C57BL/6 mice by intraperitoneal delivery of ketamine hydrochloride (80-100&#160;mg/kg of body weight) an...

<ol>
	<li>Sridhar, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+of+cornea+and+ocular+surface.">Anatomy of cornea and ocular surface.</a> Indian Journal of Ophthalmology. 66 (2), 190-194 (2018).</li><li>Henriksson, J. T., McDermott, A. M., Bergmanson, J. P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dimensions+and+morphology+of+the+cornea+in+three+strains+of+mice.">Dimensions and morphology of the cornea in three strains of mice.</a> Investigative Ophthalmology &amp; Visual Science. 50 (8), 3648-3654 (2009).</li><li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MANF+promotes+diabetic+corneal+epithelial+wound+healing+and+nerve+regeneration+by+attenuating+hyperglycemia-induced+endoplasmic+reticulum+stress.">MANF promotes diabetic corneal epithelial wound healing and nerve regeneration by attenuating hyperglycemia-induced endoplasmic reticulum stress.</a> Diabetes. 69 (6), 1264-1278 (2020).</li><li>Ma, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+epithelial+injury-induced+norepinephrine+promotes+Pseudomonas+aeruginosa+keratitis.">Corneal epithelial injury-induced norepinephrine promotes Pseudomonas aeruginosa keratitis.</a> Experimental Eye Research. 195, 108048 (2020).</li><li>Chan, M. F., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+corneal+injury.">Animal models of corneal injury.</a> Bio Protocol. 5 (13), 1516 (2015).</li><li>Lan, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+and+function+of+mesenchymal+stem+cells+in+corneal+injury.">Kinetics and function of mesenchymal stem cells in corneal injury.</a> Investigative Ophthalmology &amp; Visual Science. 53 (7), 3638-3644 (2012).</li><li>Watanabe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+corneal+epithelial+wound+healing+in+vitro+and+in+vivo+by+annexin+A5.">Promotion of corneal epithelial wound healing in vitro and in vivo by annexin A5.</a> Investigative Ophthalmology &amp; Visual Science. 47 (5), 1862-1868 (2006).</li><li>Murataeva, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+CB2R+receptors+are+upregulated+with+corneal+injury+and+regulate+the+course+of+corneal+wound+healing.">Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing.</a> Experimental Eye Research. 182, 74-84 (2019).</li><li>Carter, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+impact+of+2D+and+3D+culture+conditions+on+the+therapeutic+effects+of+human+mesenchymal+stem+cell+secretome+on+corneal+wound+healing+in+vitro+and+ex+vivo.">Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo.</a> Acta Biomaterialia. 99, 247-257 (2019).</li><li>Sanie-Jahromi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+limbal+stem+cells+on+polycaprolactone+and+polycaprolactone/gelatin+fibrous+scaffolds+and+transplantation+in+animal+model.">Propagation of limbal stem cells on polycaprolactone and polycaprolactone/gelatin fibrous scaffolds and transplantation in animal model.</a> Bioimpacts. 10 (1), 45-54 (2020).</li><li>Sun, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+membrane+protein+(EMP2)+antibody+blockade+reduces+corneal+neovascularization+in+an+In+vivo+model.">Epithelial membrane protein (EMP2) antibody blockade reduces corneal neovascularization in an In vivo model.</a> Investigative Ophthalmology &amp; Visual Science. 60 (1), 245-254 (2019).</li><li>Yang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cannabinoid+receptor+1+suppresses+transient+receptor+potential+vanilloid+1-induced+inflammatory+responses+to+corneal+injury.">Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury.</a> Cell Signal. 25 (2), 501-511 (2013).</li><li>Bai, J. Q., Qin, H. F., Zhao, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+on+mouse+model+of+grade+II+corneal+alkali+burn.">Research on mouse model of grade II corneal alkali burn.</a> International Journal of Ophthalmology. 9 (4), 487-490 (2016).</li><li>Oh, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+protein+TSG-6+reduces+inflammatory+damage+to+the+cornea+following+chemical+and+mechanical+injury.">Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (39), 16875 (2010).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+effects+of+biohcly+in+an+in+vivo+model+of+mechanical+wounds+in+the+rabbit+cornea.">Evaluation of the effects of biohcly in an in vivo model of mechanical wounds in the rabbit cornea.</a> Journal of Ocular Pharmacology and Therapeutics. 35 (3), 189-199 (2019).</li><li>Gong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+nintedanib+thermos-sensitive+hydrogel+on+neovascularization+in+alkali+burn+rat+model.">Effect of nintedanib thermos-sensitive hydrogel on neovascularization in alkali burn rat model.</a> International Journal of Ophthalmology. 13 (6), 879-885 (2020).</li><li>Yao, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+mesenchymal+stem+cells+on+cornea+wound+healing+induced+by+alkali+burn.">Role of mesenchymal stem cells on cornea wound healing induced by alkali burn.</a> PLoS One. 7 (2), 30842 (2012).</li></ol>

Mouse and rabbit models of corneal injury provide a useful ex vivo and in vivo platform for monitoring wound healing, testing new therapeutics, and studying underlying mechanisms of wound healing and treatment pathways. Different animal models can be used for a short-term or long-term experiment, depending on the purpose of the research. For instance, after creating an epithelial defect on mouse cornea in vivo, a confined epithelial defect could be used to monitor liquid therapeutic agents in a...

The human cornea consists of five major layers and plays a pivotal role in ocular refraction to maintain visual acuity and structural integrity for protecting intraocular tissues1. The outermost part of the cornea is the corneal epithelium, composed of five to six layers of cells that sequentially differentiate from the basal cells and move upward to shed from the ocular surface1. Compared to the cornea in humans and New Zealand rabbits, mouse cornea has a similar corneal structure, but thinner periphery than the central part due to a reduced thickness in the epithelium and the stroma2. Because of its unique position in the ocular optic system, many external insults such as mechanical injury, bacterial inoculation, and chemical agents may easily endanger epithelial integrity and further lead to vision-threatening epithelium defect, infectious keratitis, corneal melting, and even corneal perforation.
Although various therapeutic agents, such as lubricants, antibiotics, anti-inflammatory agents, auto-serum products, and amniotic membrane have already been used to improve re-epithelialization and reduce scarring, other potential treatment modalities that can enable wound healing, reduce inflammation, and suppress scar formation are still being developed and tested on different platforms. Various animal models for corneal epithelial wound healing have been proposed, including corneal epithelium removal with a corneal rust ring remover in diabetic mouse3, linear scratches over mouse corneal epithelium by a sterile 25 G needle for bacterial inoculation4, trephine-assisted removal of the corneal epithelium by corneal rust ring remover5, epithelial cautery over half of the cornea and limbus6, trephine-facilitated rabbit corneal abrasion by a dulled scalpel blade7, and bovine cornea injury by flash freezing in liquid nitrogen8.
Other than mechanical injury to the corneal epithelium, chemical agents are also common insults to the ocular surface, especially acidic and alkali agents. Sodium hydroxide (NaOH, 0.1-1 N for 30-60 s) is one of the commonly used chemicals in murine and rabbit models of corneal chemical burn9,10,11,12,13. 100% ethanol had also been applied to the cornea in the rat chemical burn model, followed by additional mechanical scrapping using a surgical blade14. Since maintenance of a healthy ocular surface relies on functional units, including the eyelids, Meibomian glands, lacrimal system, the conjunctiva, and the cornea, in vivo animal models have some merits over ex vivo cultured cornea epithelial cells or corneal tissues. In this article, the mouse model of corneal abrasion wound, and the rabbit model of corneal alkali burn are demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6/0 Ethicon vicryl suture</td>
			<td>Ethicon</td>
			<td>6/0VICRYL</td>
			<td>tarsorrhaphy</td>
		</tr>
		<tr>
			<td>Barraquer lid speculum</td>
			<td>katena</td>
			<td>K1-5355</td>
			<td>15 mm</td>
		</tr>
		<tr>
			<td>Barraquer needle holder</td>
			<td>Katena</td>
			<td>K6-3310</td>
			<td>without lock</td>
		</tr>
		<tr>
			<td>Barron Vacuum Punch 8.0 mm</td>
			<td>katena</td>
			<td>K20-2108</td>
			<td>for cutting filter paper</td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>National Laboratory Animal Center</td>
			<td>RMRC11005</td>
			<td>mouse strain</td>
		</tr>
		<tr>
			<td>Castroviejo forceps 0.12 mm</td>
			<td>katena</td>
			<td>K5-2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Corneal rust ring remover with 0.5 mm burr</td>
			<td>Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX</td>
			<td>CHI-675</td>
			<td>for debridement of the corneal epithelium</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Toyo Roshi Kaisha,Ltd.</td>
			<td>1.11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein sodum ophthalmic strips U.S.P</td>
			<td>OPTITECH</td>
			<td>OPTFL100</td>
			<td>staining for corneal epithelial defect</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>61763-23-3</td>
			<td>intraperitoneal or intramuscular anesthetics</td>
		</tr>
		<tr>
			<td>New Zealand White Rabbits</td>
			<td>Livestock Research Institute, Council of Agriculture,Executive Yuan</td>
			<td></td>
			<td>Rabbit models</td>
		</tr>
		<tr>
			<td>Normal saline</td>
			<td>TAIWAN BIOTECH CO., LTD.</td>
			<td>100-120-1101</td>
			<td></td>
		</tr>
		<tr>
			<td>Proparacaine</td>
			<td>Alcon</td>
			<td>ALC2UD09</td>
			<td>topical anesthetics</td>
		</tr>
		<tr>
			<td>Skin biopsy punch 2mm</td>
			<td>STIEFEL</td>
			<td>22650</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaOH)</td>
			<td>Sigma-Aldrich</td>
			<td>1310-73-2</td>
			<td>a chemical agent for alkali burn</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Carl Zeiss Meditec, Dublin, CA</td>
			<td>SV11</td>
			<td>microscope for surgery</td>
		</tr>
		<tr>
			<td>Westcott Tenotomy Scissors Medium</td>
			<td>katena</td>
			<td>K4-3004</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride 23.32 mg/10 mL</td>
			<td>Elanco animal health Korea Co., LTD.</td>
			<td>047-956</td>
			<td>intraperitoneal or intramuscular anesthetics</td>
		</tr>
	</tbody>
</table>

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 wound healing model of the mouse corneal epithelium: 
After in vivo debridement of mouse corneal epithelium with hand-held corneal rust ring remover, a mildly depressed central corneal area with positive fluorescein stain can be found in the central 2 mm area (Figure 3A-B). After harvesting the mouse eyeball, it was easily fixed onto a wax-coated 48-well culture plate without significant rotati...

Watch this Scientific Journal Video about Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium at JoVE.com

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.

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, ...

Research

JoVE Journal

Medicine

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

Procedure for Human Saphenous Veins Ex Vivo Perfusion and External Reinforcement

The mainstay of contemporary therapies for extensive occlusive arterial disease is venous bypass graft. However, its durability is threatened by intimal ...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence (NIRF) xenograft imaging experiments in mice ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...