The overall goal of this procedure is to induce clinical features of tear dysfunction and lacrimal gland inflammation in rats using subcutaneous immunization and subconjunctival and intralacrimal gland injection of lacrimal gland extracts for assessment of therapies in autoimmune dry eye. This method can help to answer key questions in dry eye, such as whether drugs that suppress T cells in autoimmune disease are efficacious. The main advantage of this technique, it produces signs of ocular disease similar to human dry eye and also quantifies the T cells infiltrating the lacrimal gland and the eye.
The implications of this animal model extend towards understanding lymphocyte trafficking because spleen and lymph nodes can be studied. Generally, individual new to this method will struggle because of the lack of suitable instrumentation for documenting changes in the small rat eye. Visual demonstration of this method is critical as certain steps are difficult to learn because of the rapid drying of the rat eye and the lack of blinks after anesthesia.
After euthanizing a female Sprague Dawley rat according to the text protocol, place the animal flat with one ear against the table and the other facing up. With a pair of scissors, make a 10 millimeter incision superior-inferiorly under the exposed ear. Then, remove the lacrimal gland by dissecting it from the surrounding connective tissue and from the drainage duct.
Next, using scissors, mince the tissue as finely as possible on ice. Then, add 150 microliters of PBS with 1X protease inhibitor per lacrimal gland. With the sonicator set at 10 seconds on, 10 seconds off, and 30%amplitude, sonicate the samples on ice at 20 kilohertz for five minutes.
Then, centrifuge the sonicated samples at 13, 000 times g and four degrees Celsius for 20 minutes. Transfer the supernatant to a new tube. Then, aliquot the supernatant to 1.5 milliliter tubes, and store them at minus 80 degrees Celsius.
Transfer complete Freund's adjuvant containing five milligrams per milliliter of Mycobacterium tuberculosis H37Ra or incomplete Freund's adjuvant to a 50 milliliter tube, ensuring that the volume of adjuvant to the antigen mixture is in a one-to-one ratio. Add the antigen mixture containing two milligrams per milliliter ovalbumin and 10 milligrams per milliliter lacrimal gland extract drop by drop to the adjuvant solution while vortexing with the highest speed that does not result in spillage. Continue vortexing for five minutes after all antigen has been added.
Transfer the emulsion to a five milliliter syringe, and use a syringe connector to link it with another five milliliter syringe. Then, push the emulsion from one syringe to the other to mix it. On day zero, distribute 200 microliters of the emulsion, which contains one milligram of lacrimal gland extract and 200 micrograms of ovalbumin in complete Freund's adjuvant, into one milliliter Luer lock syringes, and equip the syringes with 27 gauge needles.
Without using anesthesia, subcutaneously inject the emulsion at the base of the rat tails. Then, on day 14, in a similar manner, inject 200 microliters of the same antigen in incomplete Freund's adjuvant. On the same day, intraperitoneally inject 300 nanograms of pertussis toxin in 100 microliters of PBS.
On day 48, weigh the required amount of ovalbumin and dissolve it in PBS. Then, combine ovalbumin with the defrosted lacrimal gland extract prepared earlier in this video, making antigen solution containing one milligram per milliliter of ovalbumin and one milligram per milliliter of lacrimal gland extract. After anesthetizing the rat, inject five microliters of antigen into the forniceal subconjunctiva of the eye.
Then, inject 20 microliters of antigen into the lacrimal gland. While holding an anesthetized rat according to the text protocol, to measure the tear volume with phenol red thread, 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 one minute, and then remove the thread.
Then, place the thread next to a ruler with millimeter markings, and take an image of the length of the wetted part of the thread. To measure the cornea tear smoothness, place the rat under a stereo microscope equipped with a ring illuminator and a camera. Then, apply five microliters of saline to the rat cornea.
With gloved fingers, passively blink by moving the upper and lower eyelids approximately five times to spread the saline. Under 1.6 times magnification, focus the ring illuminator on the middle of the cornea surface. Then, after 10 seconds, acquire photographic images.
Use fluorescein scanning to measure corneal damage by adding two microliters of 0.2%fluorescein to the rat cornea. With a gloved finger, passively open and close the rat eyelids three times to spread the fluorescein dye on the surface of the eye. After one minute, use a three milliliter syringe to draw one milliliter of saline.
Then, position the syringe about two to three millimeters anterior to the cornea and gently apply saline onto the eye. With the background light switched off, acquire images under the cobalt blue filter of an ocular imaging microscope. After euthanizing the rat according to the text protocol, use scissors to remove the upper and lower eyelids.
Free the globe by severing the extraocular muscles, the optic nerve, and the forniceal conjunctiva. Make an incision to open up the eyeball. Remove the lens in vitreous.
Then, put the dissected eyeballs into 1.5 milliliter tubes on ice. Finally, collect the lacrimal glands as described earlier in this video. This figure shows representative images of phenol red threads from control and DED rat.
The length of the phenol red threads in the DED group is shorter than the control group, indicating less tear volume. Fluorescein binds to damaged corneal epithelium and thus is used to measure the extent of damage. In this experiment, fluorescein spots on the corneal surface of DED rats were graded from zero to two and compared to control rats.
Rats with DED have more fluorescein staining than control rats, suggesting corneal damage. As seen here, if the corneal surface is smooth with high tear stability, the image of the illuminator ring on the ocular surface is round and perfect. Distortion of the image indicates reduced corneal smoothness and an unstable tear film, as was observed to a higher distortion degree in the DED group.
This flow cytometry analysis shows that the predominant T cell subset in normal rat eyeball tissues are effector memory T cells. In the eyeballs of DED rats, approximately 70%of the CD3-positive T cells are effector memory T cells while in control rats this number is approximately 50%Eyeballs of DED rats have significantly higher effector memory T cells than those of control rats. Following this procedure, staining for other kinds of immune cells can be performed to answer additional questions.
For example, you can stain for innate immune cells or B lymphocytes. After its development, this technique paved the way for researchers to understand ocular surface immunology and, further, to study dry eye in humans and Sjogren's syndrome. After watching this video, you should have a good understanding of how to induce autoimmune dry eye in rats and to conduct robust documentation of the outcomes.
