Effect of topical fluoroquinolones on the expression of matrix metalloproteinases in the cornea

Abstract

Background: Matrix metalloproteinases play an important role in extracellular matrix deposition and degradation. Based on previous clinical observations of corneal perforations during topical fluoroquinolone treatment, we decided to evaluate the comparative effects of various fluoroquinolone eye drops on the expression of matrix metalloproteinases (MMPs) in cornea.

Methods: Eighty female Lewis rats were divided into two experimental groups: intact and wounded corneal epithelium. Uniform corneal epithelial defects were created in the right eye with application of 75% alcohol in the center of the tissue for 6 seconds. The treatment groups were tested as follows: 1) Tear drops: carboxymethylcellulose sodium 0.5 % (Refresh, Allergan); 2) Ciprofloxacin 0.3% (Ciloxan, Alcon); 3) Ofloxacin 0.3%(Ocuflox, Allergan); 4) Levofloxacin 0.5%(Quixin, Santen). Eye drops were administered 6 times a day for 48 hours. Rats were sacrificed at 48 hours. Immunohistochemical analysis and zymography were conducted using antibodies specific to MMPs-1, 2, 8 and 9.

Results: MMP-1, MMP-2, MMP-8 and MMP-9 expression were detected at 48 hrs in undebrided corneal epithelium groups treated with the topical fluoroquinolones. No statistical difference was observed in quantitative expression of MMPs among ciprofloxacin 0.3%, ofloxacin 0.3%, levofloxacin 0.5%. When the artificial tear group and the fluoroquinolone groups with corneal epithelial defect were compared, increased expression of MMPs was observed as a result of the wound healing process. However, the fluoroquinolone treated group exhibited high statistically significantly levels of MMPs expression.

Conclusions: Our study provides preliminary evidence that topical application of fluoroquinolone drugs can induce the expression of MMP-1, MMP-2, MMP-8 and MMP-9 in the undebrided corneal epithelium compared to artificial tear eye drops.

Figure 1

Immunolocalization of MMP-1 in intact corneal epithelium at 48 hours is shown for each treatment group. Panel a: artificial tears; panel b: Ciprofloxacin 0.3%; panel c: Ofloxacin 0.3%; panel d: Levofloxacin 0.5%. A photomicrograph of the artificial tear group shows negative corneal staining for MMP-1. However, intense corneal epithelial and superficial stromal expression of MMP-1 was identified in intact corneal epithelium groups treated with fluoroquinolone eye drops (panel's b-d). The presence of MMP-1 was detected mostly in the corneal epithelial cells (arrows). (Immunohistochemistry, ×400 magnification).

Figure 2

A representative western blot of MMP-1 and MMP-8 expression of supernatant samples from control (artificial tear) and the fluoroquinolone test groups. Corneas with debrided (OD) and intact (OS) epithelium were analyzed. 1. MW; 2. Tears OS; 3. Tears OD; 4. Ciprofloxacin OS; 5. Ciprofloxacin OD; 6. Ofloxacin OS; 7. Ofloxacin OD; 8. Levofloxacin OS; 9. Levofloxacin OD The molecular sizes of both MMPs were calculated based on a molecular weight standard and recorded in kilodaltons (kDa). The fluoroquinolone eye drops increased the expression of both metalloproteinases, MMP-1 (42 kDa) and of MMP-8 (64 kDa) in intact corneas; whereas, the expression of these MMPs was negative in the controls treated with artificial tears.

Figure 3

Typical gelatin zymography analyses of gelatinase A (MMP-2) and gelatinase B (MMP-9) expression in artificial tear group rat and in the test groups were performed. Corneal conditioned medium samples from debrided (OD) and intact (OS) corneal epithelium of each group were analyzed. 1. Tears OD; 2. Tears OS; 3. Levofloxacin 0.5% OD; 4. Levofloxacin 0.5% OS; 5. Ciprofloxacin OD; 6. Ciprofloxacin OS; 7. Ofloxacin OD; 8. Ofloxacin OS. MMP-9 (92 kDa) and MMP-2 (72 kDa) expression were up regulated in fluoroquinolone treated corneas and were present at basal level in unwounded corneas receiving artificial tears. Corneal epithelium debridement up regulated the expression of MMP-9 and MMP-2 in all test groups.

Figure 4

Effects of fluoroquinolone eye drops on the corneal rat's metalloproteinases expression. The bands densities of western immunoblots for MMP-1 (A) and MMP-8 (B) and zymograms for MMP-2 (C), MMP-9 (D) were subjected to densitometry analysis, the statistical significance was determined by one-way ANOVA test (values are means, error bars). P < 0.05 was considered statistically significant when compared fluoroquinolones groups vs. artificial tear group. Note the highest values of MMPs levels from topical fluoroquinolone groups compared to artificial tear group in unwounded corneas.