1. Glaucoma Medications and Dry Eye
Margaret Zhang
COSMOS at UC Davis, July 2015
Cluster 1: Biotechnology

2. What is Dry Eye?
Also known as keraconjunctivitis sicca and Sjögren’s syndrome
Disease of the tear film and ocular surface that results in ocular discomfort and visual disturbance
People either do not produce enough tears or have a poor quality of tears
A common and often chronic problem, particularly in older adults
14.5% of population affected in the US
17.9% women; 10.5% men
~30% of population age 50 and older
Chronic-long term disease

3. Ocular Surface and Tear Film
Ocular surface is covered by the tear film
Tear film has three layers: oil, water, and mucin
Mucin layer plays a critical role in:
maintaining the normal function of the tear film
preventing dry eye

4. MUC5AC
Mucins are a group of glycoproteins secreted from ocular surface tissues to the tear film
Three types of mucins are present at the ocular surface
Large gel-forming mucins
Small soluble mucins
Membrane associated mucins
MUC5AC
A large gel-forming mucin
Expressed by conjunctival goblet cells

5. MUC5AC Expression is Reduced in Dry Eye Patients
Using Real-Time PCR, MUC5AC mRNA detection was significantly lower in dry eye patients in comparison to normal eye patients.
Using ELISA, MUC5AC protein levels were significantly reduced in the tear fluid of dry eye patients in comparison to normal eye patients.
Argüeso P, et al., Invest Ophthalmol Vis Sci. 2002;43:

6. Topical Ocular Medications and Dry Eye
Many factors are associated with induction of dry eye, including topical ocular medications
Glaucoma is another common and chronic ocular disease, leading to blindness without treatment
Caused by intraocular pressure (IOP)
Long-term use of topical glaucoma medications has been reported to induce or worsen dry eye disease
Timolol
Widely used glaucoma medication
Used to lower ocular pressure
Available with preservative BAK and preservative- free
Active chemical compound: Timolol maleate

7. Researchable Question: Does Timolol, a topical glaucoma medication, decrease MUC5AC expression, thus causing dry eye?

8. Overview of Procedure
Treatment of Mice with Timolol /-BAK
Measurement of MUC5AC Expression (RT-PCR)
Dry Eye Test (CFS and Tear Production)
Normal and dry eye mice will be used in this study.

9. Methods Animals: 8-week old C57BL/6 mice. Materials:
0.25% Timolol with preservative (Betimol®, Vistakon, Jacksonville, FL)
0.25% Timolol preservative-free (Timoptic®, Aton Pharma, Lawrenceville, NJ)
Cyclosporine A (Restasis®, Allergan, Irvine, CA)
A topical ocular medication approved by FDA for increasing tear production in patients with dry eye
Positive control in this study

10. Induction of Dry Eye
Place mice in a Controlled Environment Chamber (CEC)
Allows continuous regulation of:
Relatively low humidity below 30%
Constant temperature of
21–23oC
Airflow of 15 L/minute for 24 hours a day
(A) Overview of the system with air pump (1), temperature–humidity recorder (2), water separator (3), desiccators (4), and flowmeter with valve (5).
(B) Detail of the modified cage with temperature–humidity probe (6) and space available for desiccants (7)
Barabino S et al., Invest Ophthalmol Vis Sci. 2005; 46:

11. Methods: Treatment Regimen
Treatment regimen for normal or dry eye mice (n=8 mice/group)
Treatment
Dose (a drop)
Frequency
Control
Saline
Twice daily for 2 weeks
BAK
(Benzalkonium chloride)
0.01% BAK in saline
Timolol Maleate - BAK
0.25% ophthalmic solution
(Preservative-free)
Timolol Maleate + BAK
Cyclosporine A
0.05% ophthalmic emulsion

12. Measurement of MUC5AC Expression
Real Time PCR:
Isolate total RNA from the conjunctival tissues using the RNeasy microkit and store RNA at −80°C in Trizol until future use
Synthesize the first strand of cDNA with random hexamers using SuperScriptTM III Reverse Trancriptase
Perform Real-Time PCR using FAM-MGB dye and predesigned primers for GAPDH and MUC5AC
Load cDNA in each well and perform assays in duplicates
Use GAPDH gene as the endogenous reference for each reaction
Normalize the results using the cycle threshold (CT) of GAPDH
Use the relative mRNA level in the untreated group as the normalized control for the others groups

13. Dry Eye Test Measurement of Tear Production:
Tear production was measured with the phenol red thread test as a diagnostic tool to study the effect of treatment on dry eye
Remove excess tears for a standard time of 4 seconds
Under a magnifying fluorescent lamp, hold threads with jeweler forceps and place threads in the lateral cantus of the conjunctival fornix of the right eye for 30 seconds
Using hemacytometer scale, read the tear distance (in millimeters) under a microscope

14. Dry Eye Test Corneal Fluorescein Staining:
Fluorescein staining of the corneal epithelium is used as a diagnostic tool to study the effect of treatment on the ocular surface
Perform CFS:
before placing the mice into the Controlled Environment Chamber (Day 0)
before the instillation of topical medications (Day 2)
after two weeks (Day 14)
Apply fluorescein into the inferior conjunctival sac of the eye using a micropipette
Wait three minutes
Evaluate punctate staining on the corneal surface using a slit lamp biomicroscope under cobalt blue light
Assess the corneal surface staining using the National Eye Institute grading system

15. Estimated Results
In normal mice, Timolol decreased MUC5AC mRNA expression and tear production, and increased CFS.
In dry eye mice, Timolol further decreased MUC5AC mRNA expression and tear production, and further increased CFS.
No difference between two Timolol formulations (with or without BAK), suggesting that these above changes were not due to BAK, the preservative agent.

16. Dry Eye Related Measurements
Increased Corneal Fluorescein Staining (CFS)
Decreased MUC5AC Expression
Decreased Tear Production

17. Corneal Fluorescein Staining (CFS) in Normal and Dry Eye Mouse Models

18. MUC5AC Expression in Conjunctival Epithelial Cells in Normal and Dry Eye Mouse Models
MUC5AC mRNA Level

19. Tear Production in Normal and Dry Eye Mouse Models
Tear Volume

20. What To Do If You Have Glaucoma
Low risk for dry eye
Timolol
Other glaucoma medications
High risk for dry eye
Surgery?
The findings from this study may help develop new glaucoma drugs which do not interfere with mucin production and cause dry eye in patients with high risk for development of dry eye disease.

21. Potential Issues
Because only one glaucoma medication was tested, is it accurate to say glaucoma medications induce dry eye?
Is glaucoma medication in general or just this agent associated with the relationship between glaucoma medications and dry eye?
Since this study was performed in mice, will the same findings be repeated in humans?

22. Potential Future Studies
Test agents from different class of glaucoma medications
Similar study done on humans
Supplement data with more tests
Impression cytology (measure MUC5AC expression)
Schirmer’s test (measure tear production)

23. Acknowledgements
Thanks to the authors of the articles that I have cited to generate this research proposal.
Special thanks to Paul Feldstein, Adam Telleen, LeAnn Linsay, Dana de Farcy, Ann Moriarty, and all of my fellow Biotechnology cluster students.

24. Bibliography
Gipson IK, Hori Y, Argüeso P. Character of ocular surface mucins and their alteration in dry eye disease. Ocul Surf. 2004;2(2):
Argüeso P, Balaram M, Spurr-Michaud S, Keutmann HT, Dana MR, Gipson IK. Decreased levels of mucin MUC5AC in tears of patients with Sjögren syndrome. Invest Ophthalmol Vis Sci the goblet cell Apr;43(4):
Gipson IK, Argüeso P. Role of mucins in the function of the corneal and conjunctival epithelia. Int Rev Cytol. 2003;231:1-49.
Barabino S, Shen L, Chen L, Rashid S, Rolando M, Dana MR. The controlled- environment chamber: a new mouse model of dry eye. Invest Ophthalmol Vis Sci ;46(8):
Fraunfelder FT, Sciubba JJ, Mathers WD. The role of medications in causing dry eye. J Ophthalmol. 2012;2012:
Tokuda N, Kitaoka Y, Matsuzawa A, Miyamoto J, Sakae S, Munemasa Y, Takagi H. The Effect of Rebamipide on Ocular Surface Disorders Induced by Latanoprost and Timolol in Glaucoma Patients. J Ophthalmol. 2015; 2015:

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