Assessment of Cornea and Tear Smoothness in an Autoimmune Dry Eye Rat Model

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

1. Preparation of Lacrimal Gland Extract

NOTE: Rats were anesthetized with the intraperitoneal injection of ketamine (75 mg/kg) and xylazine (10 mg/kg). Proper anesthetization was confirmed by toe pinching and tail pinching. Ophthalmic gel was applied to the rat's eyes to prevent dryness after each procedure. The anesthetized rats were placed under far infrared lights to keep the animals warm until they fully recovered. During the procedures and recovery time, animals were closely monitored by researchers. All the materials and surgery tools were sterile before use. At the end of the experiment, rats were euthanized by the intraperitoneal injection of pentobarbital (80 mg/kg). Complete euthanasia was verified by lack of cardiac pulse and no blink reflex by touching the eyeball. Rats were housed in standard conditions: room temperature, 21-23 °C; relative humidity, 30-70%; light-dark cycle, alternating 12 h (7 AM to 7 PM).

1. Place the euthanized female Sprague-Dawley rats (age range from 8 weeks to 16 weeks) flat, with one ear against the table and the other facing up. Make a 10-mm incision superior-inferiorly under the exposed ear with a pair of spring scissors. Remove the lacrimal gland by dissecting it from the surrounding connective tissue and from the drainage duct.
   1. Store the glands at -80 °C until required. Thaw the glands on ice. Mince as finely as possible on ice with scissors. Add 150 µL of phosphate-buffered saline or PBS with 1x protease inhibitor per lacrimal gland.
2. Sonicate the samples on ice for 5 min at 20 kHz with the sonicator set at 10 s on, and 10 s off at 30% amplitude. Centrifuge the sonicated samples for 20 min at 13,000 x g and 4 °C.
3. Pipette the supernatant and transfer it to a new tube. Aliquot the supernatant to 1.5 mL tubes and store them at -80 °C. Measure the protein concentration with a bicinchoninic acid assay, as per the manufacturer's instructions.

2. Preparation of Emulsion and Pertussis Toxin

1. Emulsion
   1. Add 40 mg of heat-killed Mycobacterium tuberculosis H37Ra into 10 mL of complete Freund's adjuvant containing 1 mg/mL Mycobacterium tuberculosis H37Ra and mix well.     
      NOTE: The final complete Freund's adjuvant contains 5 mg/mL Mycobacterium tuberculosis H37Ra.
   2. Weigh the ovalbumin, dissolve it in PBS, and prepare an antigen mixture containing 2 mg/mL ovalbumin and 10 mg/mL lacrimal gland extract. Aiming for an injection volume of 200 µL for each animal, prepare a master mixture based on the animal numbers.
   3. Transfer incomplete Freund's adjuvant or complete Freund's adjuvant to a 50 mL tube, ensuring that the volume of adjuvant to the antigen mixture is in a 1:1 ratio. Add the antigen mixture drop-by-drop to the adjuvant while vortexing with the highest speed so that does not result in spillage. Continue vortexing for 5 min after all the antigen has been added.
   4. Transfer the emulsion to a 5 mL syringe and link it with another 5 mL syringe through a syringe connector. Push the emulsion from one syringe to the other to mix it. 
      NOTE: The emulsion is ready if a single droplet remains as a sphere when placed in water. Only freshly prepared emulsion or emulsion stored overnight at 4 °C should be used.
2. Pertussis toxin
   1. Reconstitute 50 µg of pertussis toxin in 500 µL of water to make a final concentration of 100 ng/µL. Votex the container for 30 seconds to make sure the toxin dissolves completely.
      NOTE: Do not sterilize by filtration, as this will result in a loss of material. Do not freeze. This solution remains active for at least 6 months at 4 °C.
   2. Dilute 100 ng/µL of pertussis toxin to 3 ng/µL using PBS. Transfer 100 µL of the diluted pertussis toxin to a 1 mL Luer-lock injection syringe with a 27 G needle.

3. Immunization of the Lewis Rats

1. On day 0, mix the emulsion a few times. Distribute 200 µL of the emulsion, which contains 1 mg of lacrimal gland extract and 200 µg of ovalbumin in complete Freund's adjuvant, into 1-mL Luer-lock syringes with 27G needles. Inject the emulsion subcutaneously at the base of the rat tails without anesthesia.
   NOTE: The tail does not need to be pre-warmed before the injection. Each rat is immunized with 1 mg of lacrimal gland extract and 200 µg of ovalbumin in complete Freund's adjuvant with 500 µg of Mycobacterium tuberculosis H37Ra.
2. On day 14, inject 200 µL of the emulsion, which contains 1 mg of lacrimal gland extract and 200 µg of ovalbumin in incomplete Freund's adjuvant, in the same manner as on day 0. Inject 300 ng of pertussis toxin in 100 µL of PBS intraperitoneally per rat on the same day.

4. Injection of the Antigen Mixture into the Forniceal Subconjunctiva and Lacrimal Gland to Recruit Antigen-specific Immune Cells and Cause Local Inflammation

1. Calculate the amount of ovalbumin and lacrimal gland extract based on the number of rats in the experiment. Weigh the required amount of ovalbumin and combine it with the defrosted lacrimal gland extract prepared in step 1, making antigen solution containing 1 mg/mL ovalbumin and 1 mg/mL lacrimal gland extract.
2. Anesthetize the rats with ketamine (75 mg/kg) and xylazine (10 mg/kg) by intraperitoneal injection. Pinch the toes of rats to ensure that proper anesthesia has been achieved (i.e., no responsive movement is observed after the pinch). Inject 5 µL of antigen into the forniceal subconjunctiva of the eye. Inject 20 µL of antigen into the lacrimal gland.

5. Assessment of Dry Eye Features

NOTE: The anesthetized rats should be held gently by a gloved hand in an upright position on a flat surface to avoid movement.

1. Measure the tear volume with phenol red thread.
   1. Use one pair of forceps to hold the thread and another to pull the lower eyelid of the rat. Place the thread at the proximal corner of the lower fornix for 1 minute and then remove the thread.     
      NOTE: Tears make the wetted part of the thread red in color.
   2. Take an image of the length of the wetted part of the thread next to a ruler with millimeter markings. Measure the wetted length to the tenth of a millimeter using an image software (e.g., ImageJ).
2. Measure cornea/tear smoothness.
   1. Place the rat under a stereomicroscope equipped with a ring illuminator and a camera. Apply 5 µL of saline to the rat cornea. Passively blink by moving the upper and lower eyelids with gloved fingers ~5 times to spread the saline.
   2. Focus the ring illuminator on the middle of the cornea surface under 1.6x magnification. Acquire photographic images after 10 s.      
      NOTE: The ring illuminator projects two circular rows of dot images on the corneas of the animal. The regular spacing of the undistorted dots suggests a smooth cornea/tear layer.