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

Summary

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.

Abstract

When developing novel antimicrobials, the success of animal trials is dependent on accurate extrapolation of antimicrobial efficacy from in vitro tests to animal infections in vivo. The existing in vitro tests typically overestimate antimicrobial efficacy as the presence of host tissue as a diffusion barrier is not accounted for. To overcome this bottleneck, we have developed an ex vivo porcine corneal model of bacterial keratitis using Pseudomonas aeruginosa as a prototypic organism. This article describes the preparation of the porcine cornea and protocol for establishment of the infection. Bespoke glass molds enable straightforward setup of the cornea for infection studies. The model mimics in vivo infection as bacterial proliferation is dependent on the ability of the bacterium to damage corneal tissue. Establishment of infection is verified as an increase in the number of colony forming units assessed via viable plate counts. The results demonstrate that infection can be established in a highly reproducible fashion in the ex vivo corneas using the method described here. The model can be extended in the future to mimic keratitis caused by microorganisms other than P. aeruginosa. The ultimate aim of the model is to investigate the effect of antimicrobial chemotherapy on the progress of bacterial infection in a scenario more representative of in vivo infections. In so doing, the model described here will reduce the use of animals for testing, improve success rates in clinical trials and ultimately enable rapid translation of novel antimicrobials to the clinic.

Introduction

Corneal infections are important causes of blindness and occur in epidemic proportions in low- and mid-income countries. The etiology of the disease varies from region to region but bacteria account for a large majority of these cases. Pseudomonas aeruginosa is an important pathogen that causes a rapidly progressive disease. In many cases, patients are left with stromal scarring, irregular astigmatism, require transplant or in the worst case scenario, lose an eye^1,2.

Bacterial keratitis caused by P. aeruginosa is a difficult eye infection to treat particularly due to the increasing emergence of antimicrobial resistant strains of P. aeruginosa. Within the last decade, it has become apparent that testing and developing new treatments for corneal infections, in general, and those caused by Pseudomonas sp., in particular, are essential to combat the current trend in antibiotic resistance³.

For testing the efficacy of new treatments for corneal infections, conventional in vitro microbiological methods are a poor surrogate due to the difference in bacterial physiology during laboratory culture and during infections in vivo as well as due to the lack of the host interface^4,5. In vivo animal models, however, are expensive, time-consuming, can only deliver a small number of replicates and raise concerns about animal welfare.

In this article, we demonstrate a simple and reproducible organotypic ex vivo porcine model of keratitis that can be used to test various treatments for acute and chronic infections. We have used P. aeruginosa for this experiment but the model also works well with other bacteria, and organisms such as fungi and yeast which cause keratitis.

Protocol

Albino laboratory rabbits were sacrificed in the laboratory for other planned experimental work under home office approved protocols. The eyes were not required for experimental use in those studies so they were used for this protocol.

1. Sterilization

1. CRITICAL STEP: Disinfect all forceps and scissors by soaking for 1 h in 5% (v/v) solution of Distel in distilled water, clean with a brush, rinse with tap water and sterilize in an oven at 185 °C for a minimum of 2 h.
2. Sterilize all other glassware and reagents by autoclaving at 121 °C for 15 minutes or prepare reagents according to the manufacturer’s instructions. Carry out the following procedures in a class II microbiology safety cabinet.

2. Sample collection

1. Collection of porcine eyes
   1. Large white landrace sows, a cross with a Hampshire boar was used. The animals were stunned with an electric current and the eyes were enucleated 2 h later in the abattoir.
   2. CRITICAL STEP: Once enucleated, transfer the eyes to the lab in a sterile phosphate buffered saline (PBS) solution to prevent them from drying out and process them immediately upon arrival.
2. Collection of rabbit eyes
   1. Excise the corneas and send to the lab in sterile PBS.

3. Preparation of the corneoscleral button

1. Use sterile forceps to hold the tissue surrounding the eyeball and transfer it to a Petri dish. Remove the conjunctiva and muscle tissue around the eyeball on a Petri dish using scalpel blade no. 15 and forceps.
2. Gently lift the eyeball while holding the optic nerve with forceps and transfer to a 0.5 L jar filled with sterile PBS.
3. Once all eyes are cleared of surrounding tissue, move them using sterile forceps to another 0.5 L jar filled with 3% (v/v) povidone iodine in PBS and leave for 1 min.
4. Transfer eyeballs to another 0.5 L jar with sterile PBS.
5. Use forceps to hold the eye still on a Petri dish and make a cut near the cornea with a scalpel blade no 10A.
6. CRITICAL STEP: Hold the edge of the cut and use scissors to excise the cornea leaving about 3 mm of sclera surrounding the cornea. Ensure the sharp end of scissors does not pierce the iris or the choroidal tissue and is in the supra-choroidal space.
7. Hold the corneoscleral button with forceps and use another pair of pointed end forceps to gently separate the uveal tissue.
8. Lift the corneoscleral button from remaining globe and briefly rinse it in 1.5% (v/v) povidone iodine solution in PBS in a 12 well plate.
9. Place the corneoscleral button into another 12 well plate filled with sterile PBS.
10. After processing all eyes (recommended maximum 40 eyes in one batch), place each corneoscleral button to an individual Petri dish (34 mm diameter) epithelial side up and pour in 3 mL of culture medium pre-warmed to 37 °C.
    NOTE: The composition of the culture medium is as follows: Dulbecco’s modified Eagle’s medium (DMEM): Ham’s [1:1] supplemented with 5 μg∙mL^-1 insulin and 10 ng∙mL^-1 epidermal growth factor (EGF), 10% (v/v) foetal calf serum (FCS), 100 U∙mL^-1 penicillin, 100 U∙mL^-1 streptomycin and 2.5 μg∙mL^-1 amphotericin B. As an optional step, the medium can be supplemented with 50 g∙L^-1 dextran to prevent swelling of the excised cornea during the further incubation steps.
11. Incubate at 37 °C in a humidified tissue culture incubator.

4. Maintenance of the corneoscleral buttons

1. After 24 hours, use aseptic technique to remove media and replace with 3 mL of fresh pre-warmed culture media containing antibiotics. Keep the corneoscleral buttons in media with antibiotics for 48 h to disinfect the corneas. Incubate at 37 °C in a humidified tissue culture incubator.
2. CRITICAL STEP: After 48 hours, remove the media and rinse corneas with 2 mL of PBS. Then keep the corneoscleral buttons in antibiotic-free media for a minimum of two or ideally three days before experimental infection, to remove residual antibiotics from the tissue.
3. Incubate at 37 °C in a humidified tissue culture incubator. Change media at least one more time within these three days. Discard corneas if any turbidity develops in the antibiotic-free medium.

5. Preparation of an inoculum

1. Pour 10 mL of LB broth into a 50 mL conical flask with a foam stopper.
2. Transfer a colony of P. aeruginosa strain PAO1 or strain PA14 from a fresh agar plate and incubate at 37 °C for 3-4 h until the bacteria are in mid-log phase.
3. Transfer the culture of bacteria to a 50 mL tube and centrifuge at 3,000 x g for 5 min. Remove the supernatant and re-suspend the cell pellet in PBS.
4. Repeat step 5.3 two more times to wash the cells. Re-suspend the cell pellet in PBS and adjust the optical density at 600 nm to approximately 0.6 using sterile PBS as a blank.

6. Infecting the corneoscleral button

1. Remove media from the Petri dish and rinse corneas twice with 1 mL of sterile PBS.
2. Gently squeeze forceps while holding the cornea in-between. 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.
3. Place a sterile glass mold in a 6-well plate with the wide part up and place the cornea in the middle of the glass mold, epithelium side facing down. Make the cut right in the center of the bottom part of the glass mold.
4. CRITICAL STEP: Pour 1 mL of 1% (w/v) low melting point agar dissolved in DMEM to fill the glass mold with cornea completely.
5. Allow the agar to set and then invert the glass mold so that the corneal epithelium is facing upwards.
6. Pipette 15 μL of the bacterial culture with OD_600nm = 0.6 (for P. aeruginosa this equates to approximately 1 x 10⁷ colony forming units (CFU) in 15 μL) directly into a cut area and then add 85 μL of PBS to the top to keep the corneal epithelium moist. Dilute the remaining bacterial culture and plate on agar to count colony forming units for the inoculum.
7. Add 1 mL of DMEM without antibiotics to the bottom of each well with the glass mold. Incubate the 6-well plate with the infected corneoscleral buttons in a humidified incubator at 37 °C with 5% CO₂ for up to 24 h.
8. Set up uninfected control cornea alongside every experiment. To set up uninfected control, replace the 15 μL of bacterial culture in step 6.6 with sterile PBS.

7. Homogenization of the cornea to harvest the bacteria

1. Discard the DMEM medium from the bottom of the 6 well plate and add 1 mL of sterile PBS to rinse the bottom of the well.
2. Remove PBS gently by pipetting without touching the central part of the corneoscleral button. Remove the glass ring using sterile forceps and place it in the 5% Distel.
3. Gently rinse the top of the corneoscleral button with 1 mL of PBS twice [optional].
4. Hold the edge of the corneoscleral button with fine tip forceps and detach it from the agar underneath.
5. Transfer the cornea to a 50 mL tube filled with ice cold 1-2 mL of PBS.
6. Use a fine tip homogenizer to sheer the top of the infected cornea. The tissue does not have to be completely liquidized. The homogenizer helps to detach bacteria from the corneal epithelium and the cut area.
7. Vortex the cornea in PBS for a few seconds to mix the contents.
8. Add 20 μL of the homogenate to 180 μL of PBS and perform serial dilutions in a 96 well plate.
9. Serially dilute the suspension to 10^-4 and 10^-5 dilution and pipette 10 μL of the diluted homogenate with bacteria onto a blood agar plate. Incubate the plate for 8 hours and count the number of CFU. When testing the effect of antimicrobials, the appropriate dilution factor must be arrived at experimentally.

Results

The design of the glass molds are an innovative and original idea, the use of which allowed us to set up the model in a consistent fashion with minimal/no issues with contamination. The molds were prepared by a glass blower at the University of Sheffield based on a design (Figure 1A). The experimental setup maintains the convex shape of the cornea and holds bacteria on the top of the epithelium where infection takes place (Figure 1B).

Discussion

The main driver behind the development of this keratitis model using ex vivo porcine cornea is to provide researchers developing novel antimicrobials with a representative in vitro model to more accurately determine antimicrobial efficacy at the preclinical stages. This will provide researchers involved in developing new antimicrobials greater control over drug design and formulation at the pre-clinical stages, increase success at clinical trials, reduce use of animals by enabling targeted studies and result in faster tr...

Materials

Name	Company	Catalog Number	Comments
50 mL Falcon tube	SLS	352070
Amphotericin B	Sigma	A2942
Cellstar 12 well plate	Greiner Bio-One	665180
Dextran	Sigma	31425-100mg-F
Distel	Fisher Scientific	12899357
DMEM + glutamax	SLS	D0819
Dual Oven Incubator	SLS	OVe1020	Sterilising oven
Epidermal growth factor	SLS	E5036-200UG
F12 HAM	Sigma	N4888
Foetal calf serum	Labtech International	CA-115/500
Forceps	Fisher Scientific	15307805
Handheld homogeniser 220	Fisher Scientific	15575809	Homogeniser
Heracell VIOS 160i	Thermo Scientific	15373212	Tissue culture incubator
Heraeus Megafuge 16R	VWR	521-2242	Centrifuge
Insulin, recombinant Human	SLS	91077C-1G
LB agar	Sigma	L2897
Multitron	Infors	Not appplicable	Bacterial incubator
PBS	SLS	P4417
Penicillin-Streptomycin	SLS	P0781
Petri dish	Fisher Scientific	12664785
Petri dish 35x10mm CytoOne	Starlab	CC7672-3340
Povidone iodine	Weldricks pharmacy	2122828
Safe 2020	Fisher Scientific	1284804	Class II microbiology safety cabinet
Scalpel blade number 15	Fisher Scientific	O305
Scalpel Swann Morton	Fisher Scientific	11849002