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, 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.
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.
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
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.
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 rotating. Following the protocol, ex vivo culture of the murine eyeballs can be examined and documented daily within a 48-well culture plate under a stereomicroscope (Figure 3C). A day after debriding the murine corneal epithelium, one circular fluorescein-stained epithelial defect measured 2 mm in diameter can be revealed in digital photographs obtained under Cobalt blue light (Figure 3D). Initial irregularly stained wound margin or negative fluorescein staining means incomplete or failed removal of the corneal epithelium. In the normal process of wound healing, the corneal epithelial defect will heal with reduced fluorescein-stained area in 2-3 days.
In vivo rabbit model of corneal alkali injury:
Before any procedure, intact rabbit corneal epithelium cannot be stained with fluorescein staining. After creating alkali injury to the rabbit corneal epithelium, positive fluorescein staining can be observed with or without Cobalt blue light over the central cornea with a clear and complete circular margin (Figure 4A–B and Figure 5B). Incomplete stain with unfilled area represents residual corneal epithelial tissues or failed staining. During regular follow-up, the corneal epithelial wound re-epithelializes with ingrowth of pannus from the limbus, followed by reduced stained area (Figure 5C). The epithelial defect heals within 3-4 weeks. If corneal ulcer, dellen, large epithelial defect, or massive whitish or mucous discharge develops abruptly, insecure tarsorrhaphy, exposed sutures, a mispositioned nictitating membrane, or a foreign body within palpebral conjunctiva should be considered.
Figure 1: Procedures to set up a mouse model of corneal mechanical injury. (A) Topical anesthesia is applied before the procedure. (B) Gentle indentation is done over the central cornea with a 2 mm skin biopsy trephine. (C) Corneal rust ring remover is used to remove the central corneal epithelium. (D) An epithelial defect is stained with fluorescein to confirm the defect area and compare it with the region marked in B. (E,F) The mouse eyeball is harvested and transferred onto a 48-well plate covered with wax beforehand. Please click here to view a larger version of this figure.
Figure 2: Steps to build up a rabbit model of alkali corneal injury. (A) Topical anesthesia is applied to the ocular surface. (B) A lid speculum is used to open the upper and lower eyelids, without folding or squeezing the nictitating membrane. (C) A NaOH-soaked trephined filter paper (8 mm in diameter) is placed onto the central cornea. (D) Corneal rust ring remover is used to debride the 8 mm central epithelium down to the Bowman's layer. (E) The epithelium defect is stained with fluorescein. (F) After the procedure, tarsorrhaphy is performed to protect the wound from scratches. Please click here to view a larger version of this figure.
Figure 3: Positive and negative results in a mouse model of corneal mechanical injury. (A) Intact mouse corneal epithelium without any staining before the procedure. (B) In vivo positive staining with fluorescein on the murine corneal wound without Cobalt blue light. (C) Ex vivo culture of the mouse eyeball without adding fluorescein stain before adding culture medium. (D) Positive staining with fluorescein on corneal epithelial defect in ex vivo mouse model. The 2 mm epithelial defect generally heals within 2-3 days. Please click here to view a larger version of this figure.
Figure 4: Results of fluorescein staining in a rabbit model of corneal alkali injury. (A) Positive fluorescein staining under Cobalt blue light. The photograph was taken just after the mechanical corneal injury. (B) Positive staining with fluorescein dye could also be observed on rabbit ocular surface without Cobalt blue light. (C) Negative staining on the healed ocular surface. Please click here to view a larger version of this figure.
Figure 5: Time course of wound healing and appearance of re-epithelialization in a rabbit model of corneal alkali injury. (A) Re-epithelialization in mouse and rabbit models takes 2-3 days and 3-4 weeks, respectively. (B) An 8 mm epithelial defect stained with fluorescein after alkali burn in a rabbit model. Cobalt blue light was used as the light source. The photograph was taken just after the alkali injury. (C) A healed epithelial defect in the rabbit eye 3 weeks after alkali injury, showing reduced stain area. Please click here to view a larger version of this figure.
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 small volume. At the same time, surrounding functional units, such as eyelids, lacrimal system, and the conjunctiva, can be evaluated under in vivo conditions, as opposed to cell culture or ex vivo culture conditions. Tarsorrhaphy may be still required in this situation if mice movements may affect the experimental condition. It is easier to observe and quantify a circular wound than a simple linear scratch wound4. However, skin punch to create a demarcation line on the cornea should be done carefully without cutting through the Bowman's membrane, which will otherwise leave a deep corneal injury or a penetrating wound. The mechanical corneal wound could also be created by an 8 mm corneal trephine and a scalpel blade in a rabbit model15, wherein a deeper wound down to anterior stroma rather than the corneal epithelium was presented.
Corneal rust ring removal is another pivotal issue that is worth mentioning. Since the mouse eyeball is small, over-removal or under-removal of the corneal epithelium can occur thus affecting the accuracy of the research. Marking cornea with a skin biopsy punch and fluorescein-guided operation will help reduce these mistakes. Although cautery and scalpel blades have been proposed as tools to remove corneal epithelium in animal models6,7, the damage over the ocular surface may not be easily controlled and reproduced in the same way, which potentially leads to inconsistent results in further experiments.
Compared to the in vivo condition, ex vivo cultured mouse eyeballs in 48-well plates are easier to manipulate due to a larger working space on the plates and can be used to test complex agents in various culture mediums at the same time, such as drug-eluting contact lenses and cell therapies. When mouse eyeballs are being harvested and transferred onto a 48-well plate, meticulous protection of the cornea is important to avoid additional artificial damage to the ocular surface and rupture of the eyeballs. For the following study, the eyeball can be fixed within the paraffin-coated well with the cornea facing upward and immersed within a culture medium. Floating or rotating eyeballs or dehydrated cornea will hinder the results. Since this ex vivo mouse model focuses on changes over the ocular surface, other functional units, such as lacrimal gland and eyelids, are not discussed in this ex vivo model. Ex vivo mouse model also reduces the cost of breeding and housing mice and saves experimental space, compared to in vivo animal model. This model is suitable for a short-term study, rather than a long term one since potential tissue infection and organ failure may develop in the long run.
Although the mouse model costs less and can be scaled up in the laboratory, a small surface area potentially limits the observation of detailed changes of the cornea such as lipid deposition and neovascularization by stereomicroscopes. Instead, a rabbit model of corneal injury with a larger diameter of the eyeball can generally compensate for this disadvantage. By adjusting the concentration of NaOH and the soaking time, different extents of the severity of corneal alkali burn can be created. In rat chemical burn models, 1 N NaOH was used to soak the cornea with 3 mm filter paper for 40 s and 4 mm filter paper for 20 s, providing a similar but smaller area for observation16,17. To keep the alkali burn consistent in size and concentration, a brand new and sharp punch is suggested in preparation of 8 mm filter paper to avoid any fibre or unfilled corner at the paper margin. Sufficient irrigation to wash out chemical agents from the ocular surface and conjunctival sac is required to reduce continuous damage outside the wound. Since rabbit nictitating membrane may interfere with experimental procedure and induce pain when corroded by alkali agents, it must be carefully protected and put back to physiological position after the procedure to reduce additional inflammation over the ocular surface. After the procedure of alkali burn, rabbit corneal wound can be protected by tarsorrhaphy or other material such as contact lens to secure quality and consistence of experiments.
The authors have nothing to disclose.
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).
6/0 Ethicon vicryl suture | Ethicon | 6/0VICRYL | tarsorrhaphy |
Barraquer lid speculum | katena | K1-5355 | 15 mm |
Barraquer needle holder | Katena | K6-3310 | without lock |
Barron Vacuum Punch 8.0 mm | katena | K20-2108 | for cutting filter paper |
C57BL/6 mice | National Laboratory Animal Center | RMRC11005 | mouse strain |
Castroviejo forceps 0.12 mm | katena | K5-2500 | |
Corneal rust ring remover with 0.5 mm burr | Algerbrush IITM; Alger Equipment Co., Inc. Lago Vista, TX | CHI-675 | for debridement of the corneal epithelium |
Filter paper | Toyo Roshi Kaisha,Ltd. | 1.11 | |
Fluorescein sodum ophthalmic strips U.S.P | OPTITECH | OPTFL100 | staining for corneal epithelial defect |
Ketamine hydrochloride | Sigma-Aldrich | 61763-23-3 | intraperitoneal or intramuscular anesthetics |
New Zealand White Rabbits | Livestock Research Institute, Council of Agriculture,Executive Yuan | Rabbit models | |
Normal saline | TAIWAN BIOTECH CO., LTD. | 100-120-1101 | |
Proparacaine | Alcon | ALC2UD09 | topical anesthetics |
Skin biopsy punch 2mm | STIEFEL | 22650 | |
Sodium chloride (NaOH) | Sigma-Aldrich | 1310-73-2 | a chemical agent for alkali burn |
Stereomicroscope | Carl Zeiss Meditec, Dublin, CA | SV11 | microscope for surgery |
Westcott Tenotomy Scissors Medium | katena | K4-3004 | |
Xylazine hydrochloride 23.32 mg/10 mL | Elanco animal health Korea Co., LTD. | 047-956 | intraperitoneal or intramuscular anesthetics |