Abstract
PurposeThis study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding.MethodsLaboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs.ResultsThe proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining.ConclusionsFollowing wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.
Highlights
The ocular surface is a complex unit comprised of various epithelial and glandular tissues that secrete essential tear film components
The statistical analysis revealed that wounded eyes had significantly increased proMMP-9, MMP-9 and proMMP-2 activity during early (8 to 16 hours) and late (24 to 36 hours) migration of the corneal epithelium (p,0.001, Table 2)
Procedural control eyes exhibited an increase of MMP activity during the stage equivalent to early wound healing, but only proMMP-2 was found to be significantly elevated in tears collected at 8 to 16 hours and 24 to 36 hours (p,0.001, Table 2)
Summary
The ocular surface is a complex unit comprised of various epithelial and glandular tissues (cornea, bulbar and palpebral conjunctiva, lacrimal glands and accessory eyelid glands) that secrete essential tear film components. These tissues are connected by a continuous epithelium as well as the nervous, immune and endocrine systems [1,2]. Changes in the protein composition of collected tears have been associated with ocular wound healing [4,5,6] and ocular surface conditions such as dry eye [7,8,9], meibomian gland dysfunction and keratoconjunctivitis sicca [9,10], as well as systemic conditions including diabetes [11] and breast cancer [12]. Most MMPs are secreted in an enzymatically inactive state, as pro-enzymes, which are activated in a proteolytic manner by direct cleavage of the propeptide by another MMP or a non-proteolytic manner by organomercurials
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