Establishing a Porcine Ex Vivo Cornea Model for Studying Drug Treatments against Bacterial Keratitis

This article describes a step-by-step protocol to set up an ex vivo porcine model of bacterial keratitis. Pseudomonas aeruginosa is used as a prototypic organism. This innovative model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue.

The ex vivo porcine keratitis model presented here provides researchers developing novel antimicrobials with a representative in vitro model to more accurately determine antimicrobial efficacy at the preclinical stages. We have used Pseudomonas aeruginosa for this experiment, but the model also works well with other bacteria and organisms such as fungi and yeast. Use sterile forceps to transfer the eyeball to a Petri dish.

Remove the conjunctiva and muscle tissue around the eyeball using a scalpel blade number 15 and forceps. Then while holding the optic nerve with the forceps, gently lift the eyeball and transfer it to a half liter jar filled with sterile PBS. Once all eyeballs are cleared of surrounding tissue, use sterile forceps to move them to another half liter jar filled with 3%povidone iodine in PBS.

After the eyeballs have been in the jar for one minute, transfer them to a third jar containing sterile PBS. Place an eyeball on a clean Petri dish and use the forceps to hold it steady. With a number 10A scalpel blade, make a cut near the cornea.

Use scissors to excise the cornea leaving about three millimeters of sclera surrounding it. Ensure the sharp end of the scissors is in the suprachoroidal space and does not pierce the iris. Hold the corneoscleral button with forceps and use another pair of pointed end forceps to gently separate the uveal tissue.

Then lift the corneoscleral button to separate it from the remainder of the eyeball. Briefly rinse the button in a 1.5%solution of povidone iodine in PBS. Then place the button in sterile PBS in a 12-well plate.

After processing no more than 40 eyeballs, place each corneoscleral button in its own 34 millimeter Petri dish with the epithelial side up. To each dish, add three milliliters of culture medium pre-warmed to 37 degrees Celsius. Incubate the Petri dishes for 24 hours at 37 degrees Celsius in a humidified tissue culture incubator.

Use aseptic technique to remove the media from the Petri dishes and replace it with three milliliters of fresh pre-warmed culture media containing antibiotics. After incubating the corneas for 48 hours, remove the media again and rinse the corneas with two milliliters of PBS. Then add antibiotic-free media and incubate for two to three days to remove residual antibiotics from the tissue.

Change the media at least one more time within these three days. Discard corneas if any turbidity develops in the antibiotic-free medium. Add 10 milliliters of LB to a 50 milliliter conical flask with a foam stopper.

Inoculate the broth with a colony of P.aeruginosa from a fresh agar plate and incubate at 37 degrees Celsius for three to four hours until the bacteria are in mid log phase. Transfer the bacterial culture to a 50 milliliter tube and centrifuge it at 3, 000 times G for five minutes. Remove the supernatant and resuspend the cell pellet in PBS.

After washing the cells two more times and resuspending them in PBS, adjust the optical density of the suspension at 600 nanometers to approximately 0.6 using sterile PBS as a blank. Remove the media from the Petri dish and rinse the corneas twice with one milliliter of sterile PBS. Hold the cornea with forceps and squeeze gently.

Use a 10A scalpel to make four cuts, two vertical, two horizontal in the central section of the corneoscleral button through the epithelial layer to the underlying stroma. Place a sterile glass mold in a six-well plate with the wide part up. Then place the cornea in the middle of the glass mold with the epithelium side facing down and the wounded part of the cornea centered in the glass mold.

Fill the glass mold completely by adding one milliliter of agar solution. After allowing the agar to set, invert the glass mold so that the corneal epithelium is facing upwards. The glass mold has to be sealed with agar up to the brim to prevent leaking of the inoculum or drug solution.

Pipette 200 microliters of the bacterial culture directly into a cut area. Also for each experiment, set up a control by adding 200 microliters of sterile PBS to one cornea instead of adding the bacterial culture. Next, add 185 microliters of PBS to the top of each cornea to keep the epithelium moist.

Then add one milliliter of DMEM without antibiotics to the bottom of each well. Incubate the six-well plate at 37 degrees Celsius with humidity and 5%carbon dioxide for up to 24 hours. Discard the DMEM from the six-well plate and one milliliter of sterile PBS to each well to rinse it.

Remove the PBS gently without touching the central part of the corneoscleral button. Remove the glass mold using sterile forceps and place it in 5%Distel. Gently rinse the top of the corneoscleral button twice with one milliliter of PBS.

Use fine tip forceps to lift the edge of the corneoscleral button, detach it from the agar underneath and transfer it to a 50 milliliter tube filled with one to two milliliters of ice cold PBS. To help detach bacteria from the corneal epithelium and the cut area, add PBS to the tube and use a fine tip homogenizer to shear the top of the infected cornea. The tissue does not have to be completely liquidized.

Vortex to resuspend settled bacteria and add 20 microliters of the homogenized cornea to 180 microliters of PBS. Then perform serial dilutions of the homogenate in a 96-well plate. Pipette 10 microliters of the diluted homogenate onto a blood agar plate.

After incubating the plate for 16 hours, count the number of colony forming units. Porcine corneas usually swell after a few days in medium. There was no significant difference in colony forming units obtained from corneas with and without addition of dextran, which is usually added to prevent swelling of the cornea.

Corneas are typically wounded to help the bacteria penetrate the epithelium. Although there was no significant difference in the progress of infection between wounded and unwanted corneas, there was more variation among replicates in wounded corneas. Washing the corneas twice with PBS removes excess bacteria that did not attach to the epithelium.

There is a significant difference in CFU between washed and unwashed porcine corneas infected with P.aeruginosa PAO1 for 24 hours. There was no significant difference in CFU counts between porcine and rabbit corneas infected with PA14 and PAO1. The results for both models were reproducible.

After 24 hours, the corneas infected with either Pseudomonas strain always developed opacity and the cut area becomes more visible and open in comparison to the uninfected cornea. Infected and treated corneas that were processed with this method can be further used for waxing, cryopreservation, sectioning, staining with hematoxylin and Olsen, immunostaining, and confocal microscopy. The protocol contributes to greater control over drug design and formulation at the preclinical stages, which would increase success at clinical trials, reduce the use of animals, and result in faster translation of new antimicrobials to the clinic.