Topical cyclosporine inhibits conjunctival epithelial apoptosis in experimental murine keratoconjunctivitis sicca.
OBJECTIVE
Increased apoptosis in the conjunctival epithelium has been observed in experimental murine keratoconjunctivitis sicca (KCS). Topical cyclosporine (CsA) has been noted to reduce conjunctival epithelial apoptosis in chronic canine and human KCS. The purpose of this study is to determine if topical CsA treatment inhibits conjunctival epithelial apoptosis in a murine model of KCS.


METHODS
Dry eye was induced in 3 groups of C57BL6 mice by subcutaneous injection of scopolamine TID and exposure to an air draft and low-humidity environment for 16 hours per day for 12 days. The dry eye control group received no topical treatment; a second group received 1 microL of 0.05% CsA topically TID (dry eye + CsA); and the third group received 1 microL of the castor oil vehicle of CsA topically TID (dry eye + vehicle). Normal mice were used as untreated controls. After 12 days, the mice were killed, and the right eyes and eyelids were excised, frozen, and cryosectioned. Transmission electron microscopy (TEM) was performed on conjunctival and corneal samples taken from the left eyes. Apoptosis was detected in frozen sections with the ApopTag (ISOL) In Situ Oligo Ligation Kit, which specifically detects DNA fragmentation. Immunohistochemical staining was performed to detect activated caspase-3. Conjunctival goblet cell number was counted in tissue sections stained with period acid Schiff (PAS) reagent. These assays were performed on 2 separate sets of mice.


RESULTS
Compared with untreated controls and dry eye mice receiving CsA, the number of ISOL-positive epithelial cells in the bulbar and tarsal conjunctiva was significantly greater in the dry eye control and dry eye mice + vehicle groups (P < 0.01 for both groups). There was no significant difference in the number of ISOL-positive conjunctival epithelial cells between the dry eye control and dry eye + vehicle mice. There was no significant difference in ISOL-positive cells in the corneal epithelium between the untreated controls and the 3 treatment groups. Dry eye + CsA mice showed less activated caspase-3 staining than the dry eye control and the dry eye + vehicle groups. TEM showed loss of superficial differentiated cells and extensive nuclear fragmentation characteristic of apoptosis in the dry eye control and dry eye + vehicle groups but not in the dry eye + CsA group. There was significant loss of goblet cells in the bulbar and tarsal conjunctivae of the dry eye control and the dry eye + vehicle groups compared with untreated controls and the dry eye + CsA group.


CONCLUSIONS
Topical CsA significantly reduced conjunctival epithelial apoptosis and protected against goblet cell loss in experimental murine KCS. Inhibition of apoptosis appears to be a key mechanism for the therapeutic effect of CsA for KCS.
Strong
Bradley
B
Ocular Surface Center, Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas 77030, USA.
Farley
William
W
Stern
Michael E
ME
Pflugfelder
Stephen C
SC
D000287:Administration, Topical; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002452:Cell Count; D003228:Conjunctiva / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D016572:Cyclosporine / Q000494:pharmacology*; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure; D020397:Goblet Cells / Q000187:drug effects / Q000473:pathology; D007124:Immunoenzyme Techniques; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D007638:Keratoconjunctivitis Sicca / Q000201:enzymology / Q000473:pathology / Q000517:prevention & control*; D051379:Mice; D008810:Mice, Inbred C57BL; D009883:Ophthalmic Solutions; D012601:Scopolamine / Q000633:toxicity
D007166:Immunosuppressive Agents; D009883:Ophthalmic Solutions; D016572:Cyclosporine; D012601:Scopolamine; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases