The overall goal of this experiment is to investigate the pharmacokinetic properties of intraocular drugs using rabbit eyes. This method can help answer key questions in the ocular pharmacology field, such as the pharmacokinetics of intravitreally injected drugs. The main advantage of this technique is the rabbit eyeballs are relatively large, and thus intravitreal injection techniques and intraocular distribution of injected drugs can be comparable to humans.
In addition, rabbits are easier to handle and less expensive than primates. The implications of this technique stands toward intraocular drug delivery, because use of drug delivery greatly affect the drug concentration and duration of action in ocular compartment. Thus, drug concentrations over time should be investigated in each compartment.
Demonstrating the procedure will be Hye Kyoung Hong, a post-doc from my laboratory. After anesthetizing a healthy New Zealand white rabbit, according to the text protocol, apply a topical anesthetic eyedrop, then apply wetting ointment or gel onto the cornea to prevent eye dryness until recovery from anesthesia. To dilate the pupil, apply one or two drops of phenylephrine hydrochloride, and trapicamide.
Use a cotton swab to apply 10 percent povidone iodine to the periocular skin. Then place one drop of five percent povidone iodine on the conjunctiva of the eye. Next, using a 30 gauge needle attached to a syringe, inject the drug of interest intravitreally, one millimeter behind the surgical limbus in the supratemporal quadrant, perpendicularly to the scleral surface.
Minimizing leakage from the sclera is important, as leakage can be a source of bias. To prevent leakage from the vitreous cavity, a cotton tip should be applied following the needle removal, and compression should be applied for at least 30 seconds. After allowing enough time for wound healing, observe the treated rabbits daily for one week, and once a week afterwards for any sign of severe intraocular inflammation.
After euthanizing the rabbits according to the text protocol, use an eyelid retractor to open the palpebral fissure. Two to three millimeters posterior to the limbus make a 360 degree conjunctival incision and extended posteriorally by dissecting the conjunctiva and tenon's capsule from the globe. Cut the extraocular muscles close to their insertion into the globe.
Then, with curved forceps, clamp the optic nerve and cut the nerve between the forceps and the globe. Remove the eyeball itself while leaving the surrounding tissues intact. Then, using liquid nitrogen, immediately freeze the eyeballs and store them at 80 degrees Celsius.
After isolating the eyeballs and freezing them for all of the time points, before defrosting, quickly separate the frozen eyeballs into three compartments. The vitreous, the aqueous humor, and the retina choroid. Separation of the frozen eyeball into three compartments is not an easy task if the eyeball is thawed.
Quick separation of the three compartments while keeping the tissue frozen is the key step for success for partition. Next, to open the globe, use a scalpel blade to make an incision in the corneal limbus, and incise and separate the anterior segment from the posterior segment, then isolate the aqueous humor, which is the frozen compartment in front of the iris. With tissue forceps, pull and excoriate the frozen iris and lens to remove the tissues.
Then when the vitreous becomes accessible, separate it from the remaining retina, choroid and sclera. Now, using a number 15 scalpel blade, separate the retinochoroid tissue from the underlying sclera. For immunoassays, defrost the aqueous humor samples and measure the volume of each sample.
Determine the weight of the frozen samples by subtracting the weight of an empty tube from that of the tube containing the frozen sample. After weighing the vitreous samples, use PBS with one percent BSA to defrost and solubilize the tissue. Then place on a rotator at four degrees Celsius overnight.
Then, centrifuge the tissue at 387 times G for 10 minutes. To homogenize the retinochoroid samples after weighing the frozen tissue, add protein extraction reagent at a one to 10 ratio of tissue to reagent. Then using a pre-chilled tissue microhomogenizer, homogenize the tissue.
Centrifuge the lysed sample for 10 minutes at 12, 000 to 20, 000 times G, and transfer the supernatant to a chilled EPP tube. To carry out an ELISA, use 0.1 percent BSA in one XPBS to dilute the vitreous, aqueous humor, and retinochoroid samples to concentrations that are within the limit of detection, and use these for the assay. Aliquot 100 microliters per well of the samples and known concentrations of the drug of interest to the 96 well plate to construct a standard curve.
Then incubate the plate overnight at four degrees celsius, before using 200 microliters of washing solution to wash the plate three times. Dilute the secondary antibody one to 20, 000 in PBS with 0.1 percent BSA, and add 100 microliters to each well of the assay plate. Following the incubation of the plate at four degrees Celsius overnight, measure the OD 450.
After creating a standard curve and calculating drug concentrations in the samples according to the text protocol, analyze the drug concentration data with compartmental models using modeling software such as Phoenix Winnonlin software. In WNL5 classic modeling, click PK model, and in the setup menu, map the observation time, administered dose, and drug concentration. Select the final compartment model and calculate pharmecokinetic parameters of interest according to the text protocol.
Following intravitreal injections of a drug of interest, and preparation of tissue samples as demonstrated in this video, ELISA was carried out to determine the concentration of the drug in the vitreous, the aqueous humor, and the retinochoroid. As shown here, the optical density of the color correlates with the concentration of the drug of interest. The concentration data that is calculated from the standard curve can be fitted to the pharmecokinetic model, and the pharmecokinetic parameters can be determined from the fitted line.
In this experiment, the pharmecokinetics of intravitreally injected Bevacizumab, in vitrectomized, and to non-vitrectomized eyes for multiple timepoints were evaluated and compared using the following equation. As shown here, the estimated concentration according to the fitted model, matched quite well to the actual measured values. There were no significant differences in vitreous concentration or half-life of Bevacizumab, suggesting that the role of vitreous in the distribution and clearance of Bevacizumab is insignificant.
Once mastered, the critical eyeball separation technique can be done in 10 minutes if it is performed properly. While attempting this procedure, it is important to remember that the time points for sacrificing rabbits and inoculating their eyeballs should be individualized to the drug of interest, as pharmacokinetic parameters such as molecular weight can vary widely. After its development, this technique paved the way for researchers in the field of ocular pharmacology to explore intraocular pharmacokinetics in a rabbit eye.
After watching this video, you should have a good understanding of how to perform an intraocular pharmacokinetic study using rabbit eyes. Don't forget that the differences between rabbit and human eyes should be carefully considered when extrapolating the draw pharmacokinetic properties that are obtained from animal studies to humans.
