Corneal Repair Models in Mice: Epithelial/Mechanical Versus Stromal/Chemical Injuries

Abstract

The tissue response to injury is a complex process. The cornea is an excellent model for studying wound repair processes because of its simple anatomy, easy accessibility, and normal avascular state. Here, we describe two corneal repair models in mice: an epithelial/mechanical injury model and a stromal/chemical injury model. The two models induce different repair responses, and consequently enable the study of independent repair processes. Here, we describe how these two wound models may be used to study basic cellular and molecular mechanisms of corneal repair.

1. Introduction

The cornea is the front part of the eye and has a critical role in the visual processing system by refracting light through the pupil and lens onto the retina. It also functions as an important protective barrier for the internal contents of the eye against various environmental challenges.

The cornea has a relatively simple anatomy and consists of three specialized cellular layers: epithelium, stroma, and endothelium. Distinct cell types allow each layer to have its own characteristic structure and function. The epithelium is the outermost cellular layer of the cornea and forms the first line of defense against pathogen invasion and external insults. It consists of 5–7 layers of stratified squamous nonkeratinized epithelial cells [1, 2]. The stroma is the thickest layer of the cornea and accounts for approximately 90% of the total corneal thickness [3]. It is mainly composed of water and has a sparse population of keratocytes within a highly ordered collagen-rich extracellular matrix (ECM). The stroma functions to provide mechanical strength and corneal transparency [2, 3]. The endothelium is the innermost, single-cell layer of cells. The endothelial cells contain water channels whose roles are to maintain corneal water homeostasis [4].

Corneal integrity and transparency are critically important for good visual acuity. However, as the external surface of the eye, the cornea is susceptible to a variety of physical, chemical, and infectious insults. Injuries to the cornea may disrupt its normal function and anatomy and lead to visual impairment. Ocular morbidity as a result of corneal injuries can vary considerably, ranging from insignificant superficial wounds to more severe deeper injuries, which can lead to irreversible blindness. Depending on the extent and depth of injuries, the cornea wound-healing processes can vary largely and result in dramatically different repair outcomes.

Corneal wound healing is a complex and dynamic process that leads to the restoration of corneal tissue homeostasis. It involves a highly coordinated interaction of different types of cells, chemokines, growth factors, and ECM components [5–8]. In order to gain deeper insights into these interactions and processes, we have established two corneal repair models in mice: epithelial/mechanical and stromal/chemical injuries.

The epithelial/mechanical wound model injures the corneal epithelial layer and leaves the underlying corneal layers intact. This is a good injury model to study the repair processes of epithelial cell migration, proliferation, and reepithelialization. In this model, a defined area of the central cornea is marked with a trephine and an algerbrush burr is used to remove the epithelial cells within the mark. Fluorescein staining can be used to demarcate the area of the abraded epithelium and measure the rate of reepithelialization over time. We have used the epithelial/mechanical injury model to compare reepithelialization rates and inflammation between injured corneas of wild-type and Mmp12^−/− mice [9]. This injury model allowed us to determine the role of MMP12 in promoting early repair processes following corneal epithelial injury by enhancing epithelial cell migration and neutrophil infiltration [9] (Fig. 1).

Fig. 1. Illustration of a corneal wound shows the cellular and molecular events after epithelial/mechanical injury.

Corneal abrasion leads to increased expression of MMPs, in particular MMP12, which plays an essential role in promoting early corneal epithelial repair by increasing epithelial cell re-epithelializatlon and neutrophil infiltration. In addition, damaged epithelial cells produce growth factors which modulate keratinocyte phenotype and mechanical activity during epithelial cell migration

The stromal/chemical wound model injures the corneal epithelial and stromal layers. This deeper injury model induces an intense immune response and can be used to study the repair processes of corneal inflammation, neovascularization, and fibrosis. In this model, a circular filter paper is soaked in a chemical (e.g., 0.1 N NaOH) and then applied to the central cornea for a defined period of time. The strong immune response induces corneal neovascularization and the expression of fibrotic markers within a few days after injury. We have used the stromal/chemical wound model to compare the inflammatory, neovascular, and fibrotic responses between corneas of injured wild-type and Mmp12^−/− mice [6, 7]. This deeper injury model allowed us to determine that MMP12 has a protective effect on corneal fibrosis during wound repair through its regulation of immune cell infiltration, angiogenesis, and CCL2/CCR2 signaling [6, 7] (Fig. 2).

Fig. 2. Illustration of a corneal wound shows the cellular and molecular events after stromal/chemical injury.

MMP12 expression is increased during corneal wound healing following chemical burns, which exerts a protective effect by inhibiting corneal fibrosis, neovascularization and inflammation through its direct downregulation of CCL2 and its primary receptor CCR2. VEGF, vascular endothelial growth factor

Thus, we have established two corneal injury models in mice that enable the study of different corneal repair processes. We have used these models to determine how MMP12 regulates basic mechanisms involved in corneal reepithelialization, inflammation, neovascularization, and fibrosis. These corneal injury models can be used to further investigate and uncover additional basic repair mechanisms and are also suitable for clinical applications (e.g. preclinical drug safety and efficacy testing).

2. Materials

2.1. Animals and Reagents

2.1.1. Animals

It is important to age- and sex-match the comparative groups of mice because age and sex can influence the rates of corneal repair (see Note 1).

2.1.2. Reagents

All reagents may be prepared and stored at room temperature unless otherwise specified.

1. Sterile ultrapure water (H₂O).

2. Ethanol: 70% solution in water.

3. 1× sterile phosphate-buffered saline solution.

4. Sodium hydroxide (NaOH): 1.0 N (normal) solution. Prepare fresh 0.1 N NaOH by mixing 50 μl 1 N NaOH and 450 μl sterile H₂O to obtain a total volume of 500 μl.

5. Carprofen: Stock 50 mg/ml in PBS. Store at 4 °C. Prepare fresh on the day of the procedure by diluting with sterile 1× PBS into a final concentration of 0.5 mg/ml (final dosage 0.5 mg/kg body weight).

6. Isoflurane: Administered as the inhalant anesthesia using a nose cone system with a properly calibrated vaporizer (see below).

7. Proparacaine hydrochloride ophthalmic solution (0.5%; Bausch & Lomb, NDC: 24208-730-06).

8. Fluorescein sodium and benoxinate hydrochloride ophthalmic solution (0.25%/ 0.4%; Akorn, NDC: 17478-640-10). Prepare fresh on the day of the procedure by diluting (1:40) fluorescein solution in a 1.5 ml tube. Mix 2 μl of the fluorescein stock solution with 78 μl sterile 1 x PBS to obtain a total volume of 80 μl.

2.2. Supplies and Equipment

1. Reusable space gel heating pad (Fisher scientific, Cat. No. 14-370-223).

2. Weck-cel cellulose eye spears (Medtronic, Cat. No. 0008680).

3. Algerbrush II with 0.5 mm Burr (Katena, Cat. No. K2-4900).

4. Trephine with handle (1.5 mm; Beaver-visitec, Cat. No. 9748).

5. Stereomicroscope (for scratch/epithelial injury, need GFP filter; for both injuries, camera attachment is optional) (Leica, Cat. No. MZ16F).

6. Anesthesia machine with an induction chamber, nose cone attachment, and calibrated vaporizer (Summit Anesthesia Solutions, Cat. No. AS-01-0007).

7. Sterile, 1 ml, disposable syringe with needle (VWR, Cat. No. 76290-412).

8. Pipette (1 ml) with sterile aerosol tips (Fisher Scientific, Cat. No. 12111005).

9. Alcohol swabs—isopropyl alcohol 70% (BD, Cat. No. 326895).

10. Filter paper (Thermo Fisher Scientific, Cat. No. 09-795AA). Prepunched 2 mm filter paper discs using an ear punch into a sterile 1.5 ml Eppendorf tube.

11. Ear punch (Roboz, Cat. No. 65-9902).

12. Forceps (Dumont #5).

13. 15 ml conical sterile polypropylene centrifuge tube (Fisher Scientific, Cat. No. 1495970C)

14. 1.5 ml sterile Eppendorf tubes (Fisher Scientific, Cat. No. AM12400)

15. Timer (VWR, Cat. No. 62344-641).

3. Methods

Carry out all procedures at room temperature unless otherwise specified.

Ethical Statement:

All procedures should be conducted in accordance with the tenets of the Declaration of Helsinki.

3.1. Procedure for Epithelial/Mechanical Injury

1. Bench station set-up for surgical procedures (Fig. 3):

   1. Use 70% ethanol to clean the bench station for the surgical procedures.
   2. Place the anesthesia platform under the microscope objective and position the heating pad beneath it.
   3. Affix the nose cone attachment durably on top of the anesthesia platform and connect the induction chamber to the isoflurane vaporizer.
   4. Place the proparacaine bottle, fluorescein solution, Algerbrush, trephine, and Weck-cel spears on the bench station.
   5. Add isoflurane to the anesthesia machine and connect it to the oxygen tank so that the system delivers a blended mixture of oxygen and isoflurane into the induction chamber for mouse anesthesia.
   6. Connect the induction chamber to a charcoal filter canister to capture waste anesthetic gases.
   7. Clean the Algerbrush burr using a 70% isopropyl alcohol swab prior to use.

2. Inject Carprofen intraperitoneally into each mouse based on its body weight to provide postsurgical pain control. Use the following equation to calculate the injection volume (see Note 2).

3. Open the gas supply to the induction chamber. Anesthetize a single mouse by placing it in the isoflurane induction chamber, closing the lid, and then adjusting the oxygen flow to 0.91/min and the isoflurane vaporizer to 1–2%. Once shallow breathing is noted, quickly transfer the mouse to the anesthesia platform under the microscope and fit the mouse into the nose cone. Divert the gas supply from the induction chamber to the nose cone. Check for proper mouse anesthesia using paw pinches. Once the mouse is fully anesthetized, position the mouse’s head on the platform so that the eye to be injured is facing up toward the microscope objective.

4. Place one drop of Proparacaine on the mouse cornea and wait for 30 s.

5. Use a Weck-cel eye spear to gently dry off the cornea by dabbing both corners of the eye.

6. Use one hand to gently apply periocular pressure to proptose the eye. (Optional step: Take a bright-field image of the cornea prior to injury).

7. While looking through the stereo microscope, use the other hand to gently mark the central cornea with a trephine and turn it to approximately three clock hours to make a ring-shaped circle. Attempt to make a single mark.

8. Turn on the Algerbrush and make an epithelial defect in the center cornea. The remaining epithelium within the trephine mark can be further removed using the rotating burr. Alternatively, the remaining epithelium can be manually removed with the Algerbrush off and using its dull blades to extend the epithelial defect to the trephine mark.

9. Apply 20 μl of fluorescein solution to the injured cornea. Change to the GFP filter on the microscope to confirm the epithelial injury. (Optional step: take a picture of the epithelial defect using the GFP filter; Fig. 4).

10. Place another drop of Proparacaine solution onto the injured cornea to ease postsurgical pain.

11. Remove mouse from the nose cone and place it onto a heating pad in a recovery cage. Allow the mouse to awaken unperturbed. Monitor the mouse for signs of postsurgical pain and eye infections.

Fig. 3.

Setup of the operating table for epithelial/mechanical and stromal/chemical injuries

Fig. 4.

Fluorescein staining of cornea reveal the epithelial defects of the center cornea after epithelial/mechanical and stromal/chemical injuries in WT and Mmp12^−/− mice

3.2. Procedure for Stromal/Chemical Injury

1. Bench station set-up for surgical procedures:

   1. Use 70% ethanol to clean the bench station for the surgical procedures.
   2. Place the anesthesia platform under the microscope objective and position the heating pad beneath it.
   3. Affix the nose cone attachment durably on top of the anesthesia platform and connect the induction chamber to the isoflurane vaporizer.
   4. Place the Proparacaine bottle, fluorescein solution, prepunched 2 mm filter discs, 0.1 N NaOH solution, forceps, and Weck-cel spears on the bench station. Have 500 μl 1× PBS ready in a pipette.
   5. Add isoflurane to the anesthesia machine and connect it to the oxygen tank so that the system delivers a blended mixture of oxygen and isoflurane into the induction chamber for mouse anesthesia.
   6. Connect the induction chamber to a charcoal filter canister to capture waste anesthetic gases.
   7. Clean the forceps tips using a 70% isopropyl alcohol swab prior to use.
   8. Set the timer for both 10 s and 30 s.

2. Inject Carprofen intraperitoneally into each mouse based on its body weight to provide postsurgical pain control. Use the following equation to calculate the injection volume (see the Note).

3. Open the gas supply to the induction chamber. Anesthetize a single mouse by placing it in the isoflurane induction chamber, closing the lid, and then adjusting the oxygen flow to 0.91/min and the isoflurane vaporizer to 1–2%. Once shallow breathing is noted, quickly transfer the mouse to the anesthesia platform under the microscope and fit the mouse into the nose cone. Divert the gas supply from the induction chamber to the nose cone. Check for proper mouse anesthesia using paw pinches. Once the mouse is fully anesthetized, position the mouse’s head on the platform so that the eye to be injured is facing up toward the microscope objective.

4. Place one drop of Proparacaine on the mouse cornea and wait for 30 s.

5. Use a Weck-cel eye spear to gently dry off the cornea by dabbing both corners of the eye.

6. Use the forceps to obtain and hold the edge of a filter paper. Fully immerse the filter paper into the 0.1 N NaOH solution for 10 s.

7. Use one hand to proptose the mouse eye by gently applying periocular pressure (Optional step: Take a bright-field image of the cornea prior to injury).

8. While looking through the stereo microscope, use forceps in the other hand to place the NaOH-soaked filter paper onto the center cornea for exactly 30 s. Use the forceps to gently press down on the filter paper to ensure that the entire disc makes contact with the cornea.

9. After 30 s, use the forceps to remove the filter paper disc from the cornea.

10. Quickly flush the eye with 500 μl 1× PBS to remove residual NaOH.

11. Gently dry off the ocular surface with Weck-cel spears.

12. Apply 20 μl of the fluorescein solution to the injured cornea. Change to the GFP filter on the microscope to confirm and assess the corneal injury. (Optional step: take a picture of the epithelial defect using the GFP filter; Fig. 4).

13. Place another drop of Proparacaine solution onto the injured cornea to ease postsurgical pain.

14. Remove the mouse from the nose cone and place it onto a heating pad in a recovery cage. Allow the mouse to awaken unperturbed. Monitor the mouse for signs of postsurgical pain and eye infections.