A Mouse Model for Corneal Neovascularization by Alkali Burn

All the animal work, including the experimental procedures and euthanasia, was approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine with Protocol Number AN-8790.

1. Preparation of 1 N NaOH

1. Add 4 mL of sterile deionized water to a 15 mL centrifuge tube. Weigh out 400 mg of sodium hydroxide (NaOH), and add to the tube carefully.
2. Dissolve the NaOH by slowly stirring the solution using a glass rod. Make up the volume to 10 mL by adding sterile deionized water to the tube, and mix again by gently inverting the tube up and down. Close the cap tightly, and store the solution at room temperature.
3. Prepare the fresh solution every month because the concentration of the NaOH solution may be reduced by the solution absorbing carbon dioxide in the air.
4. Always gently mix the NaOH solution before use.
   ​CAUTION: Prepare the solution inside a chemical hood, and wear appropriate personal protective equipment (PPE).

2. Preparation of the 4% paraformaldehyde (PFA) solution

1. Add 30 mL of 1x phosphate-buffered saline (PBS) to a glass beaker. Weigh out 4 g of paraformaldehyde (PFA), and add it to the beaker.
2. Keep the beaker on a hot plate at 60 °C with stirring. Add the 1 N NaOH solution dropwise to raise the pH until the solution clears.
3. Check and adjust the pH to 7.4 using 1 N hydrochloric acid (HCl). Adjust the final volume to 50 mL with 1x PBS.
4. Cool and filter the solution. Store the solution at 4 °C.
   ​CAUTION: Prepare the solution in a fume hood while wearing appropriate PPE.

3. Preparing the ketamine/xylazine cocktail

1. Prepare the ketamine/xylazine cocktail by adding 0.8 mL of ketamine (stock concentration: 100 mg/mL) and 0.16 mL of xylazine (stock concentration: 100 mg/mL) to 9.4 mL of saline.
2. Store the cocktail in sterile injection bottles at room temperature (RT).

4. Alkali burn on the mouse cornea

1. Inject meloxicam (4-6 mg/kg of body weight) subcutaneously 30 min prior to the procedure for pain relief. Anesthetize the mice (C57BL/6J, 6-8 weeks of age, male) using an i.p. injection of the ketamine/xylazine cocktail (Ketamine 80 mg/kg and Xylazine 16 mg/kg of body weight).
2. Check the reflex response (pedal withdrawal) by pinching the toes of the mouse, and confirm the absence of the reflex. Apply one drop of topical anesthetic, 0.5% proparacaine, on the corneal surface of one eye, and a drop of artificial tears on the other eye.
3. Using a 2 mm biopsy punch, punch out Whatman filter paper discs.
4. Add 2 µL of 1N NaOH to a clean Petri dish. Put the 2 mm filter paper disc on the 1 N NaOH drop, and allow it to soak for 15 s.
5. Pick up the filter paper with forceps, and apply the filter paper to the proparacaine-treated eye at the center of the cornea for 30 s.
   NOTE: The filter paper must be touching only the center of the cornea, and care must be taken to avoid the movement of the filter paper once placed, as moving the filter paper can cause burns to the adjacent tissues.
6. Wash the eye by flushing with 20 mL of sterile saline solution in a sterile syringe.
   NOTE: Care must be taken to ensure that the cornea, along with the conjunctival sac, is washed thoroughly to ensure no further damage to the cornea or surrounding tissue. Washing the conjunctival sac will further prevent symblepharon.
7. Wipe the excess saline gently from the eyes and the surrounding area using disposable soft wipes. Afterward, keep the mice in a recovery cage on a warm heating pad until ambulatory.
   NOTE: Mice are monitored daily after the alkali burn for 3 days. If symptoms of pain or stress are observed, meloxicam (4-6 mg/kg of body weight) is administered subcutaneously.

5. Examination and assessment of neovascularization and opacity

1. In anesthetized mice, examine the eyes under a dissection microscope on day 10 after the burn, and obtain images using a camera attached to the dissection scope to score the opacity and neovascularization.
   NOTE: A regular dissection scope with a camera attached is sufficient.
2. While observing the cornea through the dissection microscope, score the opacity after the burn based on the following scale¹²:
   0 = No opacity; clear cornea
   1 = Mild opacity; slight haziness in the iris and pupil areas; iris and pupil easily visible
   2 = Moderate opacity; iris and pupil barely visible
   3 = Severe opacity; iris or pupil not visible
   4 = Opaque cornea; iris and pupil not visible
3. While observing the cornea through the dissection microscope, score the CoNV based on the following scale¹²:
   0 = No neovascularization; no new vessels from the limbus
   1 = Mild neovascularization; new vessels originating from the limbus
   2 = Moderate neovascularization; blood vessels originated from the limbus and growing toward the center of the cornea
   3 = Severe neovascularization; blood vessels originating from the limbus and reaching and/or crossing the center of the cornea
4. Use a Student's t-test to statistically compare the opacity and neovascularization scores between the alkali burn and healthy eye groups.
5. Euthanize the mice on day 10 by isoflurane exposure at 5% until 1 min after breathing stops, followed by cervical dislocation, and collect the corneas for flat-mount imaging.

6. Optical coherence tomography (OCT) imaging

1. Take OCT images of the anterior segment of the eyes in anesthetized mice on day 10 after the burn. Perform OCT image acquisition as a volume scan using IR + OCT mode with a 30° field of view and 100% IR intensity.
2. Quantify the thickness of the corneas using ImageJ software.
3. To measure the thickness, use the Line Selection tool in ImageJ software to create a straight line between the anterior and posterior surfaces at the central cornea.
4. Click on Analyze > Measure in the software tools to transfer the values to the data window.
5. Copy the values to a spreadsheet file, and statistically compare the corneal thickness between the alkali burn and healthy eye groups using a Student's t-test.
   ​NOTE: The thickness of the cornea is the distance from a point on the anterior corneal surface to the closest point on the posterior corneal surface at the corneal center.

7. Immunostaining for CoNV on flat-mount corneas

1. Euthanize the mice on day 10 post alkali burn, and enucleate the eyes by blunt dissection.
2. Pull the eyelids apart using the thumb and index fingers, and place forceps under the eye globe. Close the forceps, and gently pull the eyeball off from the orbit.
3. Place the eyeballs in 1x PBS. For each eyeball, remove the cornea from the eye globe by first making an incision using a 30 G needle below the limbus area.
4. Cut around the limbus area using corneal micro scissors, with the incision as the starting point, and slowly separate the cornea and limbus from the globe.
5. Clean the corneas gently using a fine paintbrush to remove the iris. Fix the corneas in 4% paraformaldehyde for 1 h.
6. Wash the corneas three times for 20 min each in 1x PBS at room temperature (RT).
7. Incubate in a blocking buffer (1x PBS supplemented with 0.1% Triton-X 100 and 5% bovine serum albumin [BSA]) for 1 h at RT.
8. Transfer the corneas to an antibody solution containing primary antibodies. Prepare the antibody solution in 1x PBS supplemented with 1% BSA, 0.1% Triton-X 100, Dylight550-conjugated anti-CD31 mAb (1:100), and Alexa Fluor488-conjugated anti-LYVE-1 mAb (1:100).
9. Incubate for 3 days at 4 °C. Wash the corneas in 1x PBS three times for 20 min each.
10. Stain the nuclei using Hoechst stain solution (1:1,000) for 5 min in the dark.
11. Flatten the corneas with radial cuts, and mount them on a pre-cleaned glass slide using mounting medium and coverslips. Seal the coverslips with clear nail polish, and dry the slides overnight in the dark before analysis by confocal microscopy.
12. Image the flat-mounted corneas using confocal microscopy by stitching individual Z-stack images; use a 10x objective, 488 nm and 561 nm lasers, and a resolution of 512 pixels x 512 pixels per slice on non-resonant galvano scanners.
13. Quantify the density of CD31⁺ blood and LYVE-1⁺ lymph vessels using ImageJ software.
14. To determine the vascular density, convert the confocal images to an 8-bit image.
15. Choose Vascular Density from the Plugins.
16. Choose the region of interest on the image, and click on OK. The measurements will open in a new data window.
17. Copy the values to a spreadsheet file, and statistically compare the vascular density between the alkali burn and healthy eye groups using a Student's t-test.
    NOTE: CD31, also called platelet endothelial cell adhesion molecule-1 (PECAM-1), is a cell adhesion molecule involved in angiogenesis and is highly expressed in the endothelial cells of early and mature blood vessels¹³. LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1) is a cell surface marker on lymphatic endothelial cells and can be used as a lymphangiogenesis marker¹⁴.