Minocycline inhibits alkali burn-induced corneal neovascularization in mice

Abstract

The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1β, 6 (IL-1α, IL-1β, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1β and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1β, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1β, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.

Figure 1. Inhibitory effect of minocycline on corneal neovascularization (CNV).

(A) Representative images of CNV on day 4,7,10 and 14 after alkali burns treated with intraperitoneal injection of PBS, minocycline (30 mg/kg b.i.d) or minocycline (60 mg/kg b.i.d). (B) The percentages of CNV area in the three groups at different checkpoints. The percentage of CNV area in the high-dosage group reduced significantly compared with the control group. There was no statistical difference in the percentage of CNV area between the low-dosage group and control group after injury, except on the fourth day. (n = 32, **p<0.01).

Figure 2. Minocycline promoted corneal epithelial recovery after alkali burns.

(A) Representative images of the mice corneas with fluorescein staining with intraperitoneal injection of minocycline or PBS after alkali burns. (B) The area of epithelial defect in the minocycline-treated groups was significantly smaller than that in the control group. The percentages of corneal epithelial defect in the control group, the low-dosage group, and the high-dosage group were 20.92%±8.77%, 18.62%±5.83% and 11.19%±7.14% on day 4; 16.13%±3.63%, 11.41%±4.47% and 6.02%±3.56% on day 7; 10.40%±4.09%, 6.42%±3.50% and 1.11%±1.24% on day 10; 4.84%±2.58%, 1.85%±1.80% and 0% on day 14, respectively. (*p<0.05,**p<0.01, ***p<0.001).

Figure 3. Anti-inflammatory effects of minocycline on the burned corneas at day 14.

(A–C) H–E staining of the mice cornea. (Magnification, ×400) The corneal thickness was increased in the control group and the low-dosage group than that in the high-dosage group. The number of infiltrated inflammatory cells in one field was lower in the corneal stroma of the high-dosage group (13.8±5.7) than that in the control group (34.8±14.3) (G). (D–F) Immunohistochemistrical staining of PMNs. The PMNs were labeled in brown and the endothelium of corneal vessels was labeled with red arrows. The number of infiltrated PMNs (black arrows) in one field was lower in the corneal stroma of the high-dosage group (6.2±3.7) than that of the control group (15.2±5.0). There were no statistical differences of inflammatory cells or PMNs between low-dosage group and control group (H). (*p<0.05).

Figure 4. The mRNA expression of angiogenesis-related genes detected by real-time RT-PCR.

All the data represented the relative fold change of mRNA expression of the genes of interest. Gene expression in the mice corneas 14 days after alkali burn was significantly lower in the high-dosage drug group: the expression levels were VEGFR1 38.46%, VEGFR2 45%, bFGF 47.97%, IL-1β 12%, IL-612%, MMP-2 41%, IL-1β 48%, MMP-9 14.94%, and MMP-13 4.37% of those in the control group.(n = 7, *p<0.05; **p<0.01).

Figure 5. Gelatin zymography for MMP-2 and -9.

(A) Representative image of gelatin zymography for MMP-2 and -9 of the corneas 14 days after alkali burns, treated with intraperitoneal injection of PBS, minocycline (30 mg/kg b.i.d) and minocycline (60 mg/kg b.i.d). (B) Densitometry analysis showed intraperitoneal injection of minocycline (60 mg/kg b.i.d) significantly reduced the protein expression of MMP-2 and MMP-9 proenzymes and their activated forms in mouse corneas than that in the low-dosage group and the control group (except for the activated MMP-2 protein). (n = 5,*p<0.05; **p<0.01).

Figure 6. The expression of VEGFR1, VEGFR2, IL-1β and IL-6 proteins detected by ELISA.

The expression of VEGFR2 protein in the mice corneas of the control group, the low-dosage group, and the high-dosage group were 7688.31±1274.27, 5956.76±1579.41 and 4691.27±776.67 pg/mg, IL-1β protein was 6285.41±1246.17, 5107.15±694.55 and 3295.47±631.90 pg/mg, and IL-6 protein was 464.06±94.55, 397.90±51.64, and 214.98±67.90 pg/mg, respectively. The protein levels of VEGFR2, IL-1β and IL-6 were lower in the high-dosage group than that in the control group. There were no statistical differences in the expression of VEGFR1 protein among the three groups. (n = 5, *p<0.05; **p<0.01).