A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Our method provides the missing reliable animal model of dry eye disease which can be acute or chronic thanks to our method of injecting all lacrimal glands and repeat injections. This technique reliably delivers Concanavalin A to the inferior lacrimal gland using ultrasound guidance to the glands of widely variable sizes and up to superior lacrimal gland making this method a complete approach. This model provides a simple, optimized, nonsurgical means to induce aqueous-deficient dry eye disease.

It is well-suited to study drug efficacy and disease pathophysiology. Identifying posterior incisure and utilizing ultrasound localization can be challenging at first. Removing all the fur is critical to aid in ultrasound localization for both these steps.

Demonstrating the procedure will be Wei Huang, a graduate student from my laboratory. After confirming mild sedation by observing a relaxed head position with the ear lobes no longer fully upright, use a micropipette to apply 25 microliters of preservative-free lidocaine to the first eye and place a flexible wire lid speculum between the eyelids. Using 0.3 forceps, grasp the nictating membrane at its apex and extend it over the cornea.

Using a 26 gauge sharp needle, inject lidocaine 1%with 1:100, 000th epinephrine subconjuctivally into the base of the nictating membrane. A moderate bleb should form over the membrane. Then remove the speculum and make an identical injection into the left nictating membrane.

After approximately five minutes, place the lid speculum back into the fellow eye and use 0.3 forceps to retract the nictating membrane at its apex. Using Westcott scissors, cut the nictating membrane off at its base and remove the speculum. Then place topical antibiotic ointment on the eye.

For palpebral portioning of the superior lacrimal gland injection, after confirming sedation, shear off the fur in the pre-orbital and scalp area and use depilatory cream to completely remove any residual fur. After two minutes remove the cream and apply 25 microliters of 1%preservative-free lidocaine to the appropriate eye. Evert the upper eyelid and apply gentle medial pressure to the posterior orbital rim until the protuberance marking the palpebral portion of the gland is seen.

Using fine-toothed forceps and a tuberculin syringe equipped with a 27 gauge needle, directly penetrate the gland via a transconjunctival approach advancing the needle two millimeters into the tissue and inject 100 microliters of a 500 microgram solution of Concanavalin A or Con A.Immediately after the injection, apply medial pressure to the globe to cause the orbital superior lacrimal gland to protrude from the posterior incisure. Using closed curved forceps, indent the area until the bony opening in the skull is felt and apply further modest pressure with forceps to leave an indentation in the skin to serve as the landmark for the needle placement. Insert a tuberculin syringe equipped with a 27 gauge needle perpendicular to the skin over the indentation mark about a quarter inch into the incision and redirect the needle posteriorly and externally toward the lateral canthus aiming for the midpoint between the injection site and the bony orbital rim.

Once the hub of the needle is reached, slowly inject 0.2 milliliters of a 1, 000 microgram solution of Con A.For ILG injection, view the animal from the side to locate the prominence of the ILG along the lower anterior portion of the orbit. Use a surgical marking pen to draw a vertical line onto the skin where the superficial part of the ILG gland transitions from its superficial resting place on the zygomatic bone to its more medial location in the orbit. Sweep a vertically held ultrasound probe across the line on the skin to identify the end of the zygomatic bone.

The ILG transition occurs where the image of the gland changes from a circumscribed hyperechoic line to one without a recognizable medial border. The relative position of the handpiece to the line drawn on the skin when this change is observed will be the injection site. To place Con A into a gland at a point just medial to the zygomatic arch bone, set the desired depth of injection as the depth of the zygomatic bone hyperechoic signal plus one millimeter minus the known length of the needle.

Insert the needle about 12 millimeters into the gland at the injection site before slowly withdrawing until the length of the exposed needle outside of the body is equal to the calculated difference. Then inject 0.2 milliliters of a 1, 000 microgram of Con A solution and confirm the success of the injection by ultrasound. The ILG should exhibit a characteristic hyperechoic space.

Con A injections induce a strong inflammatory response in the lacrimal gland characterized by a dense lymphocytic infiltrate that is accompanied by a decreased tear production. Tear lactoferrin levels are suppressed resulting in a compromised corneal and conjunctival epithelium and an increased rose bengal staining. The injection of the three orbital LG tissues produces a consistent and uniform dry eye disease state.

A single set of Con A injections produces dry eye disease lasting about one week with all of the clinical parameters normalizing by day 10. Sequential Con A injections about one week apart extend the duration of the dry eye disease accordingly. After approximately five sets of injections, the dry eye disease state often becomes permanent without the need for further injections.

Optimally localizing the lacrimal gland structures is most important. Familiarity with cranial anatomy and attention to fine detail including removal of the fur along with skill using the ultrasound all improve results. As this straightforward technique require use of sharps, investigation should take proper precautions to prevent needle stick injuries.