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) and xylazine (5-10 mg/kg of body weight).&#160;</li>
		<li>Ensure that general anesthesia is working by confirming loss of movement to a noxious stimulus and loss of righting reflex in mice.</li>
		<li>Fix the mice head by hand and apply topical anesthesia on both eyes with one drop of 0.5% proparacaine hydrochloride ophthalmic solution (Figure 1A). Disinfect the ocular surface and lids three times with 5% betadine.&#160;</li>
	</ol>
	</li>
	<li>Creation of murine corneal epithelial wound model 
	NOTE: Perform the below procedures under a stereomicroscope. The corneal wound was created in vivo, not ex vivo, to manipulate the eyeball better and close to the real-world situation. This is a terminal procedure; therefore, only clean instruments (not sterile technique) are&#160;required.&#160;
	<ol>
		<li>Mark the central cornea of the mouse using a skin biopsy punch (2 mm in diameter) to confirm a well-circumscribed and well-measurable area of the wound.</li>
		<li>Gently indent the punch over the central cornea to leave a circular mark (Figure 1B). Utilizing a hand-held corneal rust ring remover with a 0.5 mm burr, debride the corneal epithelium down to the Bowman&#39;s layer ensuring not to damage the latter (Figure 1C). Remove the residual, loose tissues inner to the wound margin with corneal forceps.</li>
		<li>Confirm the area of debridement with fluorescein staining (Figure 1D). To perform fluorescein staining, put a drop of normal saline onto a fluorescein paper to dissolve fluorescein, and then place the fluorescein-containing drop onto murine epithelial defect for visualizing it under Cobalt blue light.</li>
	</ol>
	</li>
	<li>Ex vivo culture of the murine corneal abrasion wound model.
	<ol>
		<li>For harvesting the murine eyeballs, proceed as follows.</li>
		<li>Sacrifice the mice by cervical dislocation after inducing anesthesia with 5% isoflurane in an induction chamber. Ensure that anesthesia is working by confirming the loss of movement to a noxious stimulus and loss of righting reflex in mice.</li>
		<li>Gently press with the tip of forceps at the superior and inferior orbital rims to push the eyeball out. Introduce the tip of the closed corneal scissors into the retrobulbar space along the inferior orbital wall, ensuring not to penetrate the eyeball.</li>
		<li>Hold the eyeball steady with 0.3 mm corneal forceps, and then cut off the optic nerve and periorbital soft tissue with corneal scissors to isolate the eyeball.</li>
		<li>For ex vivo culturing of murine eyeballs, proceed as follows.</li>
		<li>Prepare a 48-well plate with melted wax inside the well and wait for solidification. With the tip of conjunctiva forceps, create a round hole on the surface of solidified wax for accommodating the eyeballs.</li>
		<li>Place the harvested eyeballs directly onto the 48-well plate (Figure 1E) with wax-covered bottoms and sidewalls to establish stabilization (Figure 1F).</li>
		<li>Culture the eyeballs with Dulbecco&#39;s modified eagle medium (DMEM) containing 1% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37 &#176;C with or without antibiotics, depending on the purpose of the study. 
		NOTE: If the model is used for studying corneal epithelial wound healing, antibiotics would be required to prevent infection. However, if this model is used to evaluate the efficacy of antibiotics or mixed agents, prophylactic antibiotics would not be necessary.</li>
		<li>Immerse the ocular surface with the culture medium without causing the eyeball to float.</li>
		<li>Document the course of wound healing by fluorescein staining (step 1.2.3) and collecting photographs with a digital camera under Cobalt blue light. 
		&#8203;NOTE: In prospective experiments with mice models of mechanical corneal injury, those receiving corneal abrasion and tested further for the efficacy of therapeutic agents are viewed as an experimental group, and those receiving corneal abrasion without further treatment are regarded as a negative control group.</li>
	</ol>
	</li>
</ol>
2. In vivo rabbit model of corneal alkali injury
NOTE: In this model, an alkali burn injury is induced followed by mechanical debridement of the corneal epithelium to generate a well-defined and even wound area for subsequent quantification. Sterilize all instruments before use.
<ol>
	<li>Preparation of the rabbit with pre-operative analgesia, including intramuscular injection of systemic analgesics and topical eye drops.
	<ol>
		<li>Administer general anesthesia to New Zealand white rabbits by intramuscular injection of ketamine hydrochloride (35-44 mg/kg of body weight), mixed with xylazine (5-10 mg/kg of body weight) at the hind leg.</li>
		<li>After positioning the rabbit and covering it with a towel, apply topical anesthesia over the right eye with a drop of 0.5% proparacaine hydrochloride ophthalmic solution (Figure 2A) under a stereomicroscope.&#160;Disinfect the ocular surface and lids three times with 5% betadine.</li>
	</ol>
	</li>
	<li>Inducing alkali burn injury over the cornea
	<ol>
		<li>Place circular filter papers with a diameter of 8 mm (cut using an 8 mm punch) in a Petri dish. Using a dropper, add 0.5 N sodium hydroxide (NaOH) into the Petri dish to soak the filter papers. Drain excess NaOH solution from the filter papers before placing them onto the rabbit cornea. 
		CAUTION: 0.5 N NaOH may cause severe erosive injury to human tissues. Wear gloves when handling. If the skin or the eyes come in contact with NaOH droplets, irrigation with copious amounts of normal saline and medical help are required to reduce further damage.</li>
		<li>After opening the eyelids with a lid speculum and confirming that the rabbit nictitating membrane is not interfering with the insertion of filter paper (Figure 2B), place the circular filter paper soaked in 0.5 N NaOH onto the central cornea for 30 s, and then remove it with forceps (Figure 2C).</li>
		<li>After removing the filter paper, rinse the ocular surface with 10 mL of normal saline to wash out alkali material.</li>
	</ol>
	</li>
	<li>Completing corneal epithelial defect
	<ol>
		<li>Debride the corneal epithelium within the opacified area down to the Bowman&#39;s membrane using a corneal rust ring remover with a 0.5 mm burr (Figure 2D).</li>
		<li>Confirm the area of debridement with fluorescein staining under the Cobalt blue light and remove residual corneal epithelium using corneal forceps (Figure 2E).</li>
	</ol>
	</li>
	<li>Secure wound condition with tarsorrhaphy
	<ol>
		<li>Confirm that the nictitating membrane smoothly covers the ocular surface and corneal epithelial defect at the nasal side. Ensure that the nictitating membrane is not folded or distorted too much to interfere with the process of wound healing and the experiment.</li>
		<li>Perform a temporary tarsorrhaphy with or without topical agents using a 6-0 suture to protect the ocular surface and prevent the rabbit from scratching it (Figure 2F). Ensure that the suture for tarsorrhaphy is at 3-4 mm from upper and lower lid margins with 4-5 ties and longer knots to prevent the rabbit from breaking the sutures. 
		NOTE: If the experiment is not involved in an antibiotics study, topical agents with antibiotics could be considered.</li>
		<li>In this rabbit model, those receiving alkali burn and removal of corneal epithelium were regarded as the control group. 
		NOTE: In prospective experiments, the rabbits receiving alkali burn corneal injury and further treated with therapeutic agents is viewed as an experimental group. The rabbits receiving alkali burn treatment only, without further treatment, are regarded as a negative control group.</li>
	</ol>
	</li>
	<li>Post-operative analgesia and pain control
	<ol>
		<li>Assess the physiological condition and USDA pain levels for 7 days after the procedure, by monitoring pain and distress in the animals. Consider the use of tobramycin ointment and one drop of 0.5% proparacaine hydrochloride ophthalmic solution according to the result of the assessment. Administer Buprenorphine HCl (0.03 mg/kg) every 6-8 hours for&#160;3 days. 
		NOTE: For daily measurement of defect area and observation after surgery, the procedure belongs to USDA category D.</li>
	</ol>
	</li>
</ol>

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The authors have no competing financial interests.

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><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, ...

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3.3K Views.  Chang-Gung Memorial Hospital. Ex vivo en in vivo diermodellen werden opgezet voor het bestuderen van mechanisch en chemisch hoornvliesletsel. De techniek biedt een eenvoudig op te bouwen platform voor onderzoekers om mechanische en chemische schade aan het hoornvlies te bestuderen. Om een murine cornea-epitheelwond te creëren, markeert u het centrale hoornvlies van de verdoofde muis met behulp van een huidbiopsiestoot om een goed afgebakend en goed meetbaar gebied van de wond te bevestigen.Laat de pons voorzichtig...

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

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

Developmental Biology

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

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 ...

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called &lsquo;angiogenic privilege' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.

Neovascularization (NV) of the cornea can complicate multiple visual pathologies. Utilizing a controlled, alkali-burn injury model, a quantifiable level of corneal NV can be produced for mechanistic study of corneal NV and evaluation of potential therapies for neovascular disorders.

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the ...

Corneal Epithelial Abrasion with Ocular Burr As a Model for Cornea Wound Healing

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to injury. In fact, the most common type of eye injury found in clinic is a corneal abrasion. Here, we utilize an ocular burr to induce an abrasion resulting in removal of the corneal epithelium in vivo on anesthetized mice. This method allows for targeted and reproducible epithelial disruption, leaving other areas intact. In addition, we describe the visualization of the abraded epithelium with fluorescein staining and provide concrete advice on how to visualize the abraded cornea. Then, we follow the timeline of wound healing 0, 18, and 72 h after abrasion, until the wound is re-epithelialized. The epithelial abrasion model of corneal injury is ideal for studies on epithelial cell proliferation, migration and re-epithelialization of the corneal layers. However, this method is not optimal to study stromal activation during wound healing, because the ocular burr does not penetrate to the stromal cell layers. This method is also suitable for clinical applications, for example, pre-clinical test of drug effectiveness.

This protocol describes a method to inflict an abrasion to the ocular surface of the mouse, and to follow the wound healing process thereafter. The protocol takes advantage of an ocular burr to partially remove the surface epithelium of the eye in anaesthetized mice.

The murine cornea provides an excellent model to study wound healing. The cornea is the outermost layer of the eye, and thus is the first defense to ...

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 ...

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 ...

A Modified Biopsy Punch Method for Precise Alkali Burns in Mice

Corneal and Limbal Alkali Injury Induction Using a Punch-Trephine Technique in a Mouse Model

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of corneal injury models, researchers aim to enhance their understanding of how the cornea heals and develop strategies to prevent and manage corneal opacities. Chemical injury is one of the most popular injury models that has extensively been studied on mice. Most previous investigators have used a flat paper soaked in sodium hydroxide to induce corneal injury. However, inducing corneal and limbal injury using flat filter paper is unreliable, since the mouse cornea is highly curved. Here, we present a new instrument, a modified biopsy punch, that enables the researchers to create a well-circumscribed, localized, and evenly distributed alkali injury to the murine cornea and limbus. This punch-trephine method enables researchers to induce an accurate and reproducible chemical burn to the entire murine cornea and limbus while leaving other structures, such as the eyelids, unaffected by the chemical. Moreover, this study introduces an enucleation technique that preserves the medial caruncle as a landmark for identifying the nasal side of the globe. The bulbar and palpebral conjunctiva, and lacrimal gland are also kept intact using this technique. Ophthalmologic examinations were performed via slit lamp biomicroscope and fluorescein staining on days 0, 1, 2, 6, 8, and 14 post-injury. Clinical, histological, and immunohistochemical findings confirmed limbal stem cell deficiency and ocular surface regeneration failure in all experimental mice. The presented alkali corneal injury model is ideal for studying limbal stem cell deficiency, corneal inflammation, and fibrosis. This method is also suitable for investigating pre-clinical and clinical efficacies of topical ophthalmologic medications on the murine corneal surface.

Author Spotlight: Advancing Research in Corneal Opacity Treatment and Regeneration 

This protocol describes a method to induce an accurate and reproducible corneal and limbal alkali injury in a mouse model. The protocol is advantageous as it allows for an evenly distributed injury to the highly curved mouse cornea and limbus.

The cornea is critical for vision, and corneal healing after trauma is fundamental in maintaining its transparency and function. Through the study of ...

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.

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

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