This study investigates the induction of dry eye in rats through the administration of an antigen-adjuvant emulsion. The process involves T cell activation and the subsequent immune response leading to inflammation and tissue damage in the ocular surface.
Take a rat previously immunized with an antigen-adjuvant emulsion containing lacrimal gland-derived autoantigens and foreign antigens.
Re-inject the emulsion and intraperitoneally inject pertussis toxin, ensuring maximal T cell activation.
Activated cells differentiate into circulating effector memory T cells, TEMs and tissue-resident memory T cells, TRMs. A percentage of TRMs resides in ocular surface tissues.
After a defined interval, inject the antigens into the forniceal sub-conjunctiva and lacrimal glands, inducing localized inflammation.
Subconjunctival antigen-presenting cells, APCs, present antigens to the TRMs, triggering cytokine release.
Cytokines induce conjunctival epithelial cells to release chemokines, which recruit TEMs.
APCs reactivate the infiltrated TEMs, triggering proinflammatory cytokine release.
Cytokines cause the death of mucin-producing conjunctival goblet cells, decreasing mucin levels, and causing tear film instability.
Cytokines induce lacrimal gland cell death — reducing tear production — and stimulate corneal epithelial cells to produce matrix metalloproteinases, damaging the cornea.
Assess the rat for the clinical features indicating successful dry eye induction.
On day zero, distribute 200 microliters of the emulsion which contains 1 milligram of lacrimal gland extract and 200 micrograms of ovalbumin, and complete Freund's adjuvant into 1-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 and 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 1 milligram per milliliter of ovalbumin and 1 milligram per milliliter of lacrimal gland extract.
After anesthetizing the rats, inject 5 microliters of antigen into the fornicial subconjuctiva of the eye, then, inject 20 microliters of antigen into the lacrimal gland.
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