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

Overview

In this video, we demonstrate the inoculation of Concanavalin A, a lymphocyte mitogen, into the inferior lacrimal gland of the eye of a rabbit to induce dry eye disease. Ultrasound or sonogram images are used to guide the precise location of the injection site and also to confirm the success of inoculation.

Protocol

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).

Representative Results

Figure 1: Preparation of rabbit for concanavalin A injections. (A) Small shears are used to remove fur, allowing easier visualization of landmarks to identify the orbital superior lacrimal gland. (B) Nair is used to remove hair that remains after shearing.

Figure 2: Localization of the inferior lacrimal gland. (A) The prominence of the superficial portion of the ILG seen through the lower lid. The curvilinear pen mark denotes the lower position of the gland. The vertical line, under the nasal limbus, denotes the approximate position where the ILG transitions to a deeper position within the orbit and serves as a visual reference for the US. (B) US hand-piece sweeping across the area of the vertical line; the US monitor will show where the zygomatic bone ends, where the ILG transitions and where the ConA injection should be given ("injection site").

Figure 3: Ultrasound image of the inferior lacrimal gland. Upper Panel: The ILG as it moves deeper in orbit to lie beneath the zygomatic arch. The dashed line represents the line on skin across which the US probe is swept. Middle Panels: As the hand-piece is swept across this line, the examiner looks for loss of the zygomatic bone echo that is present in the left image (arrow) and disappears in the right. Lower Panels: Images of the ILG taken before (left) and after (right) injection of ConA. Development of a large cystic space within the gland confirms proper delivery.

Figure 4: Injection of the inferior lacrimal gland. Injection of the ILG is done at the location identified by US. The depth of injection is calculated as described in the text (step 2.4.6). Calipers (seen behind the needle) ensure that the needle is placed at the proper depth before injection.

Materials

Name	Company	Catalog Number	Comments
Rabbit, New Zealand White	Charles River Labs, Waltham, MA		Research animals, 2-3 kg
Aceproinj (acepromazine)	Henry Schein Animal Health, Dublin, OH	NDC11695-0079-8	0.1ml/kg subcutaneously injection for rabbit sedation
Ultrasound probe	VisualSonics Toronto, Ont	MX 550 S	Untrasonography-guide ConA injection for inferior lacrimal gland
Caliper	Bausch and Lomb (Storz), Bridgewater, NJ	E-2404	Caliper used to measure length of needle during ConA injection
Concanavalin A	Sigma, St. Louis, MO	C2010	Make 5mg/ml in PBS for injection into rabbit lacrimal glands
Isoflurane	Henry Schein, Melville, NY	29405
Anesthesia vaporizer	VetEquip, Pleasanton, CA	Item #911103
26-gauge needles (5/8)	Becton Dickinson and Company, Franklin Lakes, NJ	305115	Needles for injecting ConA into the lacrimal glands
27-gauge needles (5/8)	Becton Dickinson and Company, Franklin Lakes, NJ	305921	Needles for injecting ConA into the lacrimal glands