Ultrasound-Guided Concanavalin A Injection Into Inferior Lacrimal Gland: A Technique to Inoculate Inferior Lacrimal Gland With Concanavalin A to Induce Dry Eye Disease in Rabbit Model

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Animals and Housing

1. Acquire New Zealand White (NZW) rabbits weighing 2-3 kg.
2. House the rabbits singly in cages with strict temperature (65 ± 5 °F) and humidity (45 ± 5%) control. Lighting should have a 12 h on/off cycle.
3. Provide unlimited access to water and standard rabbit chow. Eliminate dietary enrichments as they may contain vitamin A that affects the eye.
4. Acclimate the animals for at least 2 weeks prior to induction of dry eye.

2. Induction and Treatment of Dry Eye

1. Sedate the rabbits with acepromazine 0.2 mg/kg subcutaneously.
2. Shear off the fur in the periorbital and scalp area and completely remove any residual fur using Nair. Leave the skin entirely smooth for better visualization of the anatomical hallmarks and US-guided injection of concanavalin A (Figure 1).
3. Induce moderate sedation by giving isoflurane using a gas mask with O₂ flow set at 1 L/min and isoflurane delivery set to 5%.
4. Injection of the inferior lacrimal gland
   1. View the animal from the side. The prominence of the ILG can be seen along the lower anterior portion of the orbit (Figure 2A).
   2. Draw a vertical line using a surgical marking pen or suitable permanent marker on the skin where the superficial part of the ILG gland transitions from its superficial (more external) resting place on the zygomatic bone to its more medial location in the orbit. This is typically inferior to the anterior limbus (Figure 2A).
   3. Identify the end of the zygomatic bone by sweeping the vertically-held US probe across this line on the skin. The ILG transition occurs where the image of the gland changes from clearly circumscribed (hyperechoic line of the zygomatic bone is seen along the lower edge of the gland in the image) to one without a recognizable medial border (the zygomatic bone echo is no longer present, Figure 3).
   4. Observe the relative position of the hand-piece to the line drawn on the skin when the US screen shows this change. This is the "injection site" where Concanavalin A (ConA) should be given.
   5. Control the depth of injection so as to place ConA into the gland at a point just medial to the zygomatic arch bone.
   6. Determine the depth of injection as follows: Set the desired depth of injection as the depth of the zygomatic bone (hyperechoic signal) plus 1 mm. Subtract this value from the known length of the needle (15 mm in this example).
   7. Insert the needle into the gland at the "injection site" ~12 mm, then slowly withdraw it until the length of the exposed needle outside the body (measured with surgical calipers) is equal to the difference calculated in step 2.4.6 (Figure 4). Inject 1000 µg of ConA in 0.2 mL.
      NOTE: To ensure that the capsule of the gland is pierced and not simply pushed by the needle, the needle should be inserted ~12 mm or almost to the hub before its withdrawal begins.
   8. Repeat the US to confirm the success of the injection. The ILG should show a characteristic hypoechoic space (Figure 3).