The authors would like to thank Ms. Tin Min Qi and Dr. Veluchamy Amutha Barathi and her team for their help with handling the animals. This work was supported by NHIC-I2D-1409007, SingHealth Foundation SHF/FG586P/2014, and NMRC/CSA/045/2012.

Animals were handled according to institutional guidelines and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The study protocol was approved by the Institutional Animal Care and Use Committee of SingHealth.
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 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&#160;blink reflex by touching the eyeball. Rats were housed in standard conditions: room temperature, 21-23 &#176;C; relative humidity, 30-70%; light-dark cycle, alternating 12 h (7 AM to 7 PM).
<ol>
	<li>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.
	<ol>
		<li>Store the glands at -80 &#176;C until required. Thaw the glands on ice. Mince as finely as possible on ice with scissors. Add 150 &#181;L of PBS with 1x protease inhibitor per lacrimal gland.</li>
	</ol>
	</li>
	<li>Sonicate the samples on ice for 5 min at 20 kHz with the sonicator set at 10 s on, 10 s off at 30% amplitude. Centrifuge the sonicated samples for 20 min at 13,000 x g and 4 &#176;C.</li>
	<li>Pipette the supernatant and transfer it to a new tube. Aliquot the supernatant to 1.5 mL tubes and store them at -80 &#176;C. Measure the protein concentration with a bicinchoninic acid assay22, as per the manufacturer&#39;s instructions.</li>
</ol>
2. Preparation of Emulsion and Pertussis Toxin
<ol>
	<li>Emulsion
	<ol>
		<li>Add 40 mg of heat-killed Mycobacterium tuberculosis H37Ra into 10 mL of complete Freund&#39;s adjuvant containing 1 mg/mL Mycobacterium tuberculosis H37Ra and mix well. 
		NOTE: The final complete Freund&#39;s adjuvant contains 5 mg/mL Mycobacterium tuberculosis H37Ra.</li>
		<li>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 &#181;L for each animal, prepare a master mixture based on the animal numbers.</li>
		<li>Transfer incomplete Freund&#39;s adjuvant or complete Freund&#39;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 that does not result in spillage. Continue vortexing for 5 min after all the antigen has been added.</li>
		<li>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 &#176;C should be used.</li>
	</ol>
	</li>
	<li>Pertussis toxin
	<ol>
		<li>Reconstitute 50 &#181;g of pertussis toxin in 500 &#181;L of water to make a final concentration of 100 ng/&#181;L. Votex the container for 30 s 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 &#176;C.</li>
		<li>Dilute 100 ng/&#181;L of pertussis toxin to 3 ng/&#181;L using PBS. Transfer 100 &#181;L of the diluted pertussis toxin to a 1 mL Luer-lock injection syringe with a 27 G needle.</li>
	</ol>
	</li>
</ol>
3. Immunization of the Lewis Rats
<ol>
	<li>On day 0, mix the emulsion a few times. Distribute 200 &#181;L of the emulsion, which contains 1 mg of lacrimal gland extract and 200 &#181;g of ovalbumin in complete Freund&#39;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 &#181;g of ovalbumin in complete Freund&#39;s adjuvant with 500 &#181;g of Mycobacterium tuberculosis H37Ra.</li>
	<li>On day 14, inject 200 &#181;L of the emulsion, which contains 1 mg of lacrimal gland extract and 200 &#181;g of ovalbumin in incomplete Freund&#39;s adjuvant, in the same manner as on day 0. Inject 300 ng of pertussis toxin in 100 &#181;L of PBS intraperitoneally per rat on the same day.</li>
</ol>
4. Injection of the Antigen Mixture into the Forniceal Subconjunctiva and Lacrimal Gland to Recruit Antigen-specific Immune Cells and Cause Local Inflammation
<ol>
	<li>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.</li>
	<li>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 &#181;L of antigen into the forniceal subconjunctiva of the eye. Inject 20 &#181;L of antigen into the lacrimal gland.</li>
</ol>
5. Assessment of Dry Eye Features
NOTE: For steps 5.1-5.3, the anesthetized rats should be held gently by a gloved hand in an upright position on a flat surface to avoid movement.
<ol>
	<li>Measure the tear volume with phenol red thread.
	<ol>
		<li>Use one pair of forceps to hold the thread and another to pull the lower eyelid of rat. Place the thread at the proximal corner of the lower fornix for 1 min and then remove the thread. 
		NOTE: Tears make the wetted part of the thread red in color.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Measure cornea/tear smoothness.
	<ol>
		<li>Place the rat under a stereomicroscope equipped with a ring illuminator and a camera. Apply 5 &#181;L of saline to the rat cornea. Passively blink by moving the upper and lower eyelids with gloved fingers ~5 times to spread the saline.</li>
		<li>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. Regular spacing of the undistorted dots suggests a smooth cornea/tear layer.</li>
	</ol>
	</li>
	<li>Measure corneal damage with fluorescein staining.
	<ol>
		<li>Add 2 &#181;L of 0.2% fluorescein to the rat cornea. Passively open and close the rat eyelids 3 times with a gloved finger to spread the fluorescein dye on the surface of the eye.</li>
		<li>After 1 min, draw 1 mL of saline with a 3 mL syringe (without any needle attachment), position the syringe about 2-3 mm anterior to the cornea, and gently propel saline onto the eye.</li>
		<li>Acquire images under the cobalt blue filter (i.e., ~400 nm) of an ocular-imaging microscope with the background lights switched off. 
		NOTE: Images were subsequently analyzed by a grading system modified from previous publications23. Images were then analyzed by subjectively grading the number, area, and intensity of the green spots from 0 to 2, where 0 indicates their absent, 1 indicates punctate staining of less than 50 spots, and 2 indicates punctate staining of more than 50 spots.</li>
	</ol>
	</li>
	<li>Euthanize the rats through the intraperitoneal injection of pentobarbital (80 mg/kg). Remove the upper and lower eyelids using scissors. Fix and prolapse the eyeballs by pushing down on the periocular tissues with forceps. Free the globe by severing the extraocular muscles, the optic nerve, and the forniceal conjunctiva.</li>
	<li>Fix the position of the rat eyeball with forceps and open it with a circumferential incision along the equator. Remove the lens and vitreous. Put the dissected eyeballs into 1.5 mL tubes on ice.</li>
	<li>Collect the lacrimal glands, as described in step 1.1. Immediately transfer the dissected eyeball tissues and lacrimal glands to the laboratory to prepare for flow cytometry analysis.</li>
	<li>Isolated the immune cells with collagenase and dispase II digestion methods24,25. Proceed to use flow cytometry to profile the T-cell subpopulation in the eyeball tissues26. 
	NOTE: The panel of antibodies are: anti-CD45APC-Cyanine7 (OX-1), anti-CD3 BV421 (1F4), anti-CD4 PE-Cyanine7 (OX-35), anti-CD45RC Alexa647 (OX-22), anti-CD62 PE (HRL1), anti-CD44 FITC (OX-50), and viability cell dye 7-AAD. Among the CD45+CD3+7AAD- population, na&#239;ve (CD3+CD45RC+), effector memory (TEM, CD3+CD45RC-CD44+CD62L-), and central memory (TCM, CD3+CD45RC-CD44+CD62L+) T cells were gated.</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+definition+and+classification+of+dry+eye+disease:+report+of+the+Definition+and+Classification+Subcommittee+of+the+International+Dry+Eye+WorkShop+(2007).">The definition and classification of dry eye disease: report of the Definition and Classification Subcommittee of the International Dry Eye WorkShop (2007).</a> Ocul Surf. 5 (2), 75-92 (2007).</li><li>Gayton, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Etiology,+prevalence,+and+treatment+of+dry+eye+disease.">Etiology, prevalence, and treatment of dry eye disease.</a> Clin Ophthalmol. 3, 405-412 (2009).</li><li>Tong, L., Tan, J., Thumboo, J., Seow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dry+eye.">The dry eye.</a> Praxis (Bern 1994). 102 (13), 803-805 (2013).</li><li>Messmer, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology,+diagnosis,+and+treatment+of+dry+eye+disease.">The pathophysiology, diagnosis, and treatment of dry eye disease.</a> Dtsch Arztebl Int. 112 (5), 71-81 (2015).</li><li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Curr Allergy Asthma Rep. 14 (1), 403 (2014).</li><li>Stevenson, W., Chauhan, S. K., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+disease:+an+immune-mediated+ocular+surface+disorder.">Dry eye disease: an immune-mediated ocular surface disorder.</a> Arch Ophthalmol. 130 (1), 90-100 (2012).</li><li>Tong, L., Thumboo, J., Tan, Y. K., Wong, T. Y., Albani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+eye:+a+window+of+opportunity+in+rheumatoid+arthritis.">The eye: a window of opportunity in rheumatoid arthritis.</a> Nat Rev Rheumatol. 10 (9), 552-560 (2014).</li><li>Carnahan, M. C., Goldstein, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+complications+of+topical,+peri-ocular,+and+systemic+corticosteroids.">Ocular complications of topical, peri-ocular, and systemic corticosteroids.</a> Curr Opin Ophthalmol. 11 (6), 478-483 (2000).</li><li>Johannsdottir, S., Jansook, P., Stefansson, E., Loftsson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+cyclodextrin-based+aqueous+cyclosporin+A+eye+drop+formulations.">Development of a cyclodextrin-based aqueous cyclosporin A eye drop formulations.</a> Int J Pharm. 493 (1-2), 86-95 (2015).</li><li>Prabhasawat, P., Tesavibul, N., Karnchanachetanee, C., Kasemson, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+cyclosporine+0.05%+eye+drops+in+Stevens+Johnson+syndrome+with+chronic+dry+eye.">Efficacy of cyclosporine 0.05% eye drops in Stevens Johnson syndrome with chronic dry eye.</a> J Ocul Pharmacol Ther. 29 (3), 372-377 (2013).</li><li>Ishimaru, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Severe+destructive+autoimmune+lesions+with+aging+in+murine+Sjogren's+syndrome+through+Fas-mediated+apoptosis.">Severe destructive autoimmune lesions with aging in murine Sjogren's syndrome through Fas-mediated apoptosis.</a> Am J Pathol. 156 (5), 1557-1564 (2000).</li><li>Jonsson, R., Tarkowski, A., Backman, K., Holmdahl, R., Klareskog, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sialadenitis+in+the+MRL-l+mouse:+morphological+and+immunohistochemical+characterization+of+resident+and+infiltrating+cells.">Sialadenitis in the MRL-l mouse: morphological and immunohistochemical characterization of resident and infiltrating cells.</a> Immunology. 60 (4), 611-616 (1987).</li><li>Jonsson, R., Tarkowski, A., Backman, K., Klareskog, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+characterization+of+sialadenitis+in+NZB+X+NZW+F1+mice.">Immunohistochemical characterization of sialadenitis in NZB X NZW F1 mice.</a> Clin Immunol Immunopathol. 42 (1), 93-101 (1987).</li><li>Li, H., Dai, M., Zhuang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+T+cell+intrinsic+role+of+Id3+in+a+mouse+model+for+primary+Sjogren's+syndrome.">A T cell intrinsic role of Id3 in a mouse model for primary Sjogren's syndrome.</a> Immunity. 21 (4), 551-560 (2004).</li><li>Shull, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+disruption+of+the+mouse+transforming+growth+factor-beta+1+gene+results+in+multifocal+inflammatory+disease.">Targeted disruption of the mouse transforming growth factor-beta 1 gene results in multifocal inflammatory disease.</a> Nature. 359 (6397), 693-699 (1992).</li><li>Green, J. E., Hinrichs, S. H., Vogel, J., Jay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocrinopathy+resembling+Sjogren's+syndrome+in+HTLV-1+tax+transgenic+mice.">Exocrinopathy resembling Sjogren's syndrome in HTLV-1 tax transgenic mice.</a> Nature. 341 (6237), 72-74 (1989).</li><li>Shen, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+autoimmunity+in+IL-14alpha-transgenic+mice.">Development of autoimmunity in IL-14alpha-transgenic mice.</a> J Immunol. 177 (8), 5676-5686 (2006).</li><li>Hayashi, Y., Hirokawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunopathology+of+experimental+autoallergic+sialadenitis+in+C3H/He+mice.">Immunopathology of experimental autoallergic sialadenitis in C3H/He mice.</a> Clin Exp Immunol. 75 (3), 471-476 (1989).</li><li>Liu, S. H., Zhou, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+dacryoadenitis:+purification+and+characterization+of+a+lacrimal+gland+antigen.">Experimental autoimmune dacryoadenitis: purification and characterization of a lacrimal gland antigen.</a> Invest Ophthalmol Vis Sci. 33 (6), 2029-2036 (1992).</li><li>Liu, S. H., Prendergast, R. A., Silverstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+dacryoadenitis.+I.+Lacrimal+gland+disease+in+the+rat.">Experimental autoimmune dacryoadenitis. I. Lacrimal gland disease in the rat.</a> Invest Ophthalmol Vis Sci. 28 (2), 270-275 (1987).</li><li>Jiang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+experimental+autoimmune+keratoconjunctivitis+sicca+(KCS)+induced+in+Lewis+rat+by+the+autoantigen+Klk1b22.">A new model of experimental autoimmune keratoconjunctivitis sicca (KCS) induced in Lewis rat by the autoantigen Klk1b22.</a> Invest Ophthalmol Vis Sci. 50 (5), 2245-2254 (2009).</li><li>Walker, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bicinchoninic+acid+(BCA)+assay+for+protein+quantitation.">The bicinchoninic acid (BCA) assay for protein quantitation.</a> Methods Mol Biol. 32, 5-8 (1994).</li><li>Lin, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mouse+dry+eye+model+induced+by+topical+administration+of+benzalkonium+chloride.">A mouse dry eye model induced by topical administration of benzalkonium chloride.</a> Mol Vis. 17, 257-264 (2011).</li><li>Cousins, S. W., Streilein, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+detection+of+lymphocyte+proliferation+in+eyes+with+immunogenic+inflammation.">Flow cytometric detection of lymphocyte proliferation in eyes with immunogenic inflammation.</a> Invest Ophthalmol Vis Sci. 31 (10), 2111-2122 (1990).</li><li>Yawata, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+change+in+natural+killer+cell+type+in+the+human+ocular+mucosa+in+situ+as+means+of+immune+evasion+by+adenovirus+infection.">Dynamic change in natural killer cell type in the human ocular mucosa in situ as means of immune evasion by adenovirus infection.</a> Mucosal Immunol. 9 (1), 159-170 (2016).</li><li>Chen, Y., Chauhan, S. K., Lee, H. S., Saban, D. R., Dana, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+dry+eye+disease+is+principally+mediated+by+effector+memory+Th17+cells.">Chronic dry eye disease is principally mediated by effector memory Th17 cells.</a> Mucosal Immunol. 7 (1), 38-45 (2014).</li><li>Goldmuntz, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+autoimmune+disease-resistant+LER+rat:+an+outcross+between+the+buffalo+and+autoimmune+disease-prone+Lewis+inbred+rat+strains.">The origin of the autoimmune disease-resistant LER rat: an outcross between the buffalo and autoimmune disease-prone Lewis inbred rat strains.</a> J Neuroimmunol. 44 (2), 215-219 (1993).</li><li>Cahalan, M. D., Chandy, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+network+of+ion+channels+in+T+lymphocytes.">The functional network of ion channels in T lymphocytes.</a> Immunol Rev. 231 (1), 59-87 (2009).</li></ol>

The authors have no conflicts of interest. L.T. received prior funding and/or gifts from Alcon, Allergan, Santen, Bausch and Lomb, Eyelens, and Eyedetec.

A critical step of this protocol is ensuring the homogeneity of the emulsion. In well-prepared emulsions, the antigens are completely coated with oil, ensuring the slow release of the injected antigen and continuous immune stimulation. Another critical feature of this protocol is the use of Lewis rats. Lewis rats are more sensitive to the development of autoimmune disease than other strains27.
This protocol has been modified from two previously published protocols, which either used lacrimal gland extract only or recombinant Klk1b2220,21. In the current protocol, ovalbumin plus lacrimal gland extract are used as the antigen, and antigen-specific immune cells are attracted to the ocular surface and the lacrimal gland, inducing local tissue damage. Dry eye develops slowly, reaching ~85% by day 48 after the initial immunization. Antigenic challenge to the eye and lacrimal gland on day 48 exacerbates dry eye and ensures its chronicity up to day 70.
Compared to the recombinant Klk1b22 in the Klk-induced DED model, the lacrimal gland extract and ovalbumin used in the current model are cheaper and easier to obtain. The lacrimal gland extract also contains other proteins, apart from Klk, that may induce autoimmunity, so this extract is theoretically more potent than the Klk method at inducing DED. We have also tried immunizing rats with lacrimal gland extract only; although these immunized rats developed DED, there was no significant increase of effector memory T cells in eyeball tissues compared to controls.
The limitation of this technique is that it takes 70 days to achieve the model. Effector memory T cells are the main T-cell subsets in the normal rat eye. In this model, autoimmune DED results in an increase in CD3+ effector memory T cells in the eyeball. Drugs that preferentially suppress effector memory T cells, such as selective inhibitors of the Kv1.3 potassium channel, may therefore have a therapeutic benefit on autoimmune DED28.

Dry eye disease (DED) is a multifactorial disease of the tears and the ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability, which can lead to damage to the ocular surface. It is accompanied by increased osmolarity of the tear film and by inflammation of the ocular surface1. Symptoms associated with DED are burning, stinging, grittiness, foreign body sensation, tearing, ocular fatigue, and dryness2,3. The two main causes of DED are reduced tear production by the tear secretion gland and the excessive evaporation of the tear film4. In patients with autoimmune diseases, such as Sjogren syndrome, systemic lupus erythematosus, and rheumatoid arthritis immune damage to the meibomian glands reduces the expression of lipids essential for tear stability. Also, immune damage to the ocular surface decreases the production of mucins important for surface wettability. Together, these processes cumulatively cause chronic dry eye5,6,7.
Tear replacement and anti-inflammatory therapy are the mainstays of therapy. However, current anti-inflammatory therapies for DED (i.e., corticosteroids and cyclosporine) are broadly immunosuppressive, leading to serious adverse effects8,9,10. There is need for a suitable animal model to test novel immunomodulatory agents to treat autoimmune dry eye.
Mice with specific genetic defects11,12,13, mice lacking specific genes14,15, and transgenic mice overexpressing immunoregulatory genes have been used as models of autoimmune dry eye16,17. Antigen-induced autoimmune animal models have also been reported in mice18, rabbits19, and rats20,21. Here, we describe an antigen-induced model of chronic autoimmune dry eye. This model is a modification of two earlier models; one used lacrimal gland extracts, and the second used an autoantigen (i.e., klk1b22) from the lacrimal glands20,21.
The disease was induced by the subcutaneous immunization of 6 to 8 week-old female Lewis rats with ovalbumin, complete Freund&#39;s adjuvant, and an emulsion containing lacrimal gland extracts from Sprague-Dawley rats (Figure 1). A second immunization with the same antigen in incomplete Freund&#39;s adjuvant was administered two weeks later. To recruit antigen-specific immune cells to the lacrimal gland and ocular surface, mixture of lacrimal gland extract and ovalbumin (1 mg/mL) was injected into the forniceal subconjunctiva and lacrimal glands at the 6th-7th week (Figure 1). More than 85% of the rats developed characteristic features of dry eye 70 days after the first immunization. These features include reduced tear production (Figure 2), increased corneal fluorescein staining (Figure 3), and decreased tear stability (Figure 4). Immune profiling of the T cells in the eyeballs of normal rats by flow cytometry reveals a preponderance of CD3+ effector memory T cells (Figures 5 and 6). Rats with autoimmune DED show an increase in CD3+ effector memory T cells and a corresponding decreases in na&#239;ve and central memory T cells (Figure 6).

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>Sigma-Aldrich</td><td>P2714-1BTL</td><td></td></tr><tr><td>Pierce BCA Protein Assay Kit</td><td>Thermal Scientific</td><td>23227</td><td></td></tr><tr><td>Mycobacterium tuberculosis H37Ra&amp;nbsp;</td><td>Becton, Dickinson and company</td><td>231141</td><td></td></tr><tr><td>complete Freund&#039;s adjuvant</td><td>Becton, Dickinson and company</td><td>231131</td><td></td></tr><tr><td>ovalbumin</td><td>Sigma-Aldrich</td><td>A5503-10G</td><td></td></tr><tr><td>incomplete Freund&#039;s adjuvant&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F5506-6X10ML</td><td></td></tr><tr><td>pertussis toxin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P7208-50UG</td><td></td></tr><tr><td>fluorescein sodium solution</td><td>Bausch &amp;amp; Lomb U.K Limited</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sonicator</td><td>Sonics&amp;nbsp;</td><td>Vibra-Cell</td><td></td></tr><tr><td>phenol red thread</td><td>Tianjin Jingming New Technological development Co. LTD.</td><td>NA</td><td></td></tr><tr><td>Stereo microscope with ring light illuminator and camera</td><td>Carl Zeiss</td><td>NA</td><td></td></tr><tr><td>Micro IV microscope&amp;nbsp;</td><td>Phoenix Research Labs</td><td>NA</td><td></td></tr></tbody></table>

a chronic autoimmune dry eye rat model with increase in effector memory t cells in eyeball tissue

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable dry eye animal model to test novel therapeutics to treat autoimmune dry eye conditions. This protocol describes a chronic autoimmune dry eye rat model. Lewis rats were immunized with an emulsion containing lacrimal gland extract, ovalbumin, and complete Freund&#39;s adjuvant. A second immunization with the same antigens in incomplete Freund&#39;s adjuvant was administered two weeks later. These immunizations were administered subcutaneously at the base of the tail. To boost the immune response at the ocular surface and lacrimal glands, lacrimal gland extract and ovalbumin were injected into the forniceal subconjunctiva and lacrimal glands 6 weeks after the first immunization. The rats developed dry eye features, including reduced tear production, decreased tear stability, and increased corneal damage. Immune profiling by flow cytometry showed a preponderance of CD3+ effector memory T cells in the eyeball.

Figure 1 illustrates the experiment design. On both day 48 and day 70, dry eye clinical features are assessed in the immunized rats. The tear volume is represented by the length of the wet part of the phenol red thread. Figure 2 shows representative images of phenol red threads from control and DED rats. 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. Thus, corneal damage is measured by corneal fluorescein staining. Fluorescein spots on the corneal surface of DED rats were graded from 0 to 2 and compared to control rats. Rats with DED have more fluorescein staining than control rats (Figure 3), suggesting corneal damage.
The corneal smoothness in DED and control rats was assessed by the ring illuminator. 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. The distortion degree of the ring was graded from 0 to 2. A higher ring distortion level was noted in the DED group (Figure 4), indicating less tear stability.
Rats are defined as having dry eye when at least two clinical features of dry eye are abnormal. Among the 24 immunized rats, 21 rats developed DED on day 48. The results were consistent when evaluated on day 70.
The flow cytometry analysis shows that the predominant T-cell subset in normal rat eyeball tissues are effector memory T cells (Figure 5). In the eyeballs of DED rats, ~70% of the CD3+ T cells are effector memory T cells, while in control rats, this number is ~50%. Eyeballs of DED rats have significantly higher effector memory T cells than those of control rats (Figure 6).
<img alt="Figure 1" src="/files/ftp_upload/55592/55592fig1.jpg" /> 
Figure 1: Schematic of the Experimental Design. LG: lacrimal gland; DED: dry eye disease; CFA: complete Freund&#39;s adjuvant; IFA: incomplete Freund&#39;s adjuvant. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55592/55592fig2.jpg" /> 
Figure 2: Phenol Red Thread Measures the Tear Volume. Phenol red thread is placed at the proximal corner of both rat eyes for 1 min and is then removed. Representative images of the phenol red thread, together with a ruler, from both control and DED groups are shown. ImageJ was used to measure the length of the wet part of the phenol red threads. Scale bar = 1 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55592/55592fig3.jpg" /> 
Figure 3: Representative Images of the Corneal Epithelial Damage Measured by Fluorescein Staining. Each rat cornea was stained with 0.2% fluorescein for 1 min and flushed with at least 1 mL of saline. Images were taken under an eye imaging microscope with cobalt blue light. The first column shows representative images of control corneas. The second column contains representative images of corneal staining from rats with DED features. The green fluorescent spots indicate corneal epithelial damage. All images were produced on the same color scale. The quantification of fluorescein staining was performed according to the area and density of the green spots. Scale bar = 1 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55592/55592fig4.jpg" /> 
Figure 4: Representative Cornea Images Showing the Reflection of the Ring Illuminator. Rat corneal/tear smoothness was measured by a ring illuminator. The distortion degree of the ring in the captured images is a measure of relative tear stability. The left column shows the representative images in control animals, and the right column shows representative images after the induction of dry eye. Scale bar = 1 mm. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55592/55592fig5.jpg" /> 
Figure 5: Dot Plots Derived from Flow Cytometry Analysis. T cells isolated from eyeball tissues were stained with a panel of antibodies. In the CD45+CD3+7AAD- population, CD3+7-AAD- T cells were gated. Among the CD3+7-AAD- T cells, the na&#239;ve, central memory, and effector memory T cell populations were determined. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55592/55592fig6.jpg" /> 
Figure 6: T-cell Subpopulation Profile in the Eyeballs. CD3+CD45RC+ na&#239;ve T cells, CD3+CD45RC-CD44+CD62L- effector memory T (TEM) cells, and CD3+CD45RC-CD44+CD62L+ central memory T (TCM) cells are presented as the percentage of CD3+ T cells. Results are from 3 control rats and 6 DED rats. Similar results were obtained from the analysis of T cells from isolated lacrimal glands (data not shown). The unpaired Student&#39;s t-test was used for statistical comparison. The error bars represent the SD. * p &#60;0.05, ** p &#60;0.01. <a href="http://cloudflare.jove.com/files/ftp_upload/55592/55592fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue at JoVE.com

A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Dry eye disease is a very common condition that causes morbidity and healthcare burden and decreases the quality of life. There is a need for a suitable ...

a-chronic-autoimmune-dry-eye-rat-model-with-increase-effector-memory

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

11.4K Views.  Singapore Eye Research Insitute, A Member of SingHealth. This report describes a method to induce chronic experimental autoimmune dry eye in Lewis rats through immunization with an emulsion of rat lacrimal gland extract, ovalbumin, and complete Freund's adjuvant, followed by the injection of lacrimal gland extract and ovalbumin into the forniceal subconjunctiva and lacrimal glands six weeks later.

Video: A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular ...

Induction of Ocular Surface Inflammation and Collection of Involved Tissues

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular surface gland network. Meibomian glands (MG) secrete lipids that create a covering layer that prevents the evaporation of the aqueous part of the tear film. Neutrophils and extracellular DNA traps populate MG and the ocular surface in a mouse model of allergic eye disease. Aggregated neutrophil extracellular traps (aggNETs) formulate a mesh-like matrix composed of extracellular chromatin that occludes MG outlets and conditions MG dysfunction. Here, a method for inducing ocular surface inflammation and MG dysfunction is presented. The procedures for collecting organs related to the ocular surface, such as the cornea, conjunctiva, and eyelids, are described in detail. Using established techniques for processing each organ, the major morphological and histopathological features of MG dysfunction are also shown. Ocular exudates offer the opportunity to assess the inflammatory state of the ocular surface. These procedures enable the investigation of topical and systemic anti-inflammatory interventions at the preclinical level.

Ocular surface inflammation harms the ocular surface tissues and compromises vital functions of the eye. The present protocol describes a method to induce ocular inflammation and collect compromised tissues in a mouse model of Meibomian gland dysfunction (MGD).

Ocular surface diseases include a range of disorders that disturb the functions and structures of the cornea, conjunctiva, and the associated ocular ...

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer&#39;s tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

This article describes the development of a method to induce acute or chronic dry eye disease in rabbits by injecting concanavalin A to all portions of the orbital lacrimal gland system. This method, superior to those already reported, generates a reproducible, stable model of dry eye suitable for the study of pharmacological agents.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for ...

Medicine

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Scopolamine-Induced Lacrimal Gland Dysfunction in Rats

A Rat Dry Eye Model with Lacrimal Gland Dysfunction Induced by Scopolamine

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal tear production can lead to a disturbance in the ocular surface environment, including corneal damage and inflammation. In severe cases, ADDE can cause vision loss or even blindness. Currently, dry eye treatment is limited to eye drops or physical therapy, which can only alleviate eye discomfort symptoms and cannot fundamentally cure dry eye syndrome. To restore the function of the lacrimal gland in dry eye, we have created an animal model of lacrimal gland dysfunction in rats induced by scopolamine. Through the comprehensive evaluation of the lacrimal gland, corneas, conjunctivas, and other factors, we aim to provide a full understanding of the pathological changes of ADDE. Compared with the current dry eye mouse model, this ADDE animal model includes a functional evaluation of the lacrimal gland, providing a better platform for studying lacrimal gland dysfunction in ADDE.

Author Spotlight: Challenges in Developing Dry Eye Animal Models and Future Research Directions

Here, we establish a rat model of lacrimal gland dysfunction to provide a basis for the study of aqueous-deficient dry eye.

Aqueous-deficient dry eye (ADDE) is a type of dry eye disease that can result in the reduction of tear secretion quantity and quality. Prolonged abnormal ...

Establishment of A Mouse Model of Aqueous Deficiency Dry Eye

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its underlying mechanisms. Therefore, we developed a mouse model of dry eye disease by surgically removing both the extraorbital lacrimal glands (ELG) and intraorbital lacrimal glands (ILG) to investigate the ocular surface pathology in the context of aqueous deficiency dry eye. Two weeks post operation, the mice exhibited severe dry eye manifestations, including reduced tear secretion, corneal epithelial irregularities, positive fluorescein sodium staining, and neovascularization. Histological examination via hematoxylin and eosin staining revealed inflammatory cell infiltration and corneal epithelium dysplasia. Immunofluorescence staining and quantitative reverse-transcription polymerase chain reaction revealed decreased expression of the normal corneal epithelial biomarkers K12 and Pax6 and increased expression of Sprr1b in the corneal epithelium. These ocular manifestations indicated abnormal corneal epithelial differentiation. Furthermore, immunofluorescence staining of Ki67 revealed the increasing cell proliferation. In conclusion, the ELG plus ILG excision model proved suitable for studying changes in the ocular surface and elucidating the mechanisms underlying aqueous deficiency dry eye.

Here, we present a method to establish a mouse model of aqueous-deficient dry eye by excising the extraorbital and infraorbital lacrimal glands and evaluate the changes in the ocular surface in aqueous deficiency dry eye.

Dry eye disease is a prevalent condition affecting 5%-50% of the global population. Animal model investigations play a crucial role in understanding its ...

Quantification of Tear Volume in an Autoimmune Dry Eye Rat Model using the Phenol Red Thread

Source: Hou, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a> J. Vis. Exp. (2017)
This video demonstrates the phenol red thread test to determine tear volume in an autoimmune dry eye rat model. The pH-sensitive acidic phenol red thread changes color when it comes in contact with the alkaline tears. The shorter length of color change in the phenol red thread corresponds to reduced tear volume.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Immunology

Immunopathology

Assay and Analysis

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

Source: Hou, A. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for assessing corneal smoothness and tear film stability in an autoimmune rat model. It highlights the impact of gland dysfunction on tear film stability, crucial for understanding dry eye disease.

Fluorescein Staining in an Autoimmune Dry Eye Rat Model to Examine Corneal Damage

Source: Hou Ahiua, et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a> J.Vis. Exp. (2017)
This video demonstrates the staining of the damaged cornea using fluorescein dye to evaluate the extent of autoimmune dry eye disease in a rat model. The green fluorescence spots of the stained collagen fibers indicate corneal epithelial damaged cells.

A Technique to Induce Chronic Experimental Autoimmune Dry Eye in Rats

Source: Hou, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55592">A Chronic Autoimmune Dry Eye Rat Model with Increase in Effector Memory T Cells in Eyeball Tissue.</a> J. Vis. Exp. (2017).
This video demonstrates a technique to induce chronic experimental autoimmune dry eye disease in rats. Upon injecting an antigen-adjuvant emulsion containing autoantigens and foreign antigens into a rat at predefined intervals, chronic eye inflammation is induced in the immunized rat by injecting the same antigens into the ocular surface tissues.