Differential Effects of Dexamethasone and Doxycycline on Inflammation and MMP Production in Murine Alkali-Burned Corneas Associated with Dry Eye

Abstract

Alkali burns to the cornea are among the most devastating injuries to the eye. The purpose of this study was to evaluate the effects of dexamethasone (Dex) or doxycycline (Doxy) on protease activity and corneal complications in a combined model (CM) of alkali burn and dry eye. C57BL/6 mice were subjected to the CM for 2 or 5 days (D). Mice were topically treated either with Dex (0.1%), Dox (0.025%) or vehicle QID and observed daily for appearance of corneal perforation. Quantitative real time PCR was performed to measure expression of inflammation cytokines and matrix metalloproteinases (MMPs) in whole cornea lysates. No perforations were observed in the Dex-treated corneas. All wounds in Doxy-treated corneas were closed 2D post-injury, and they had significantly lower corneal opacity scores at days 4 and 5 post-injury compared to BSS treatment. Dex-treated corneas had the lowest corneal opacity scores. Dex treatment significantly decreased expression of IL-1β, IL-6, MMPs -1, -9, -13, and TIMP-1 after 2 days but increased levels of MMP-8, while Doxy treatment significantly decreased IL-1β, IL-6, MMP-8, and -9, compared to vehicle. Decreased MMP-1, -9 and -13 immunoreactivity and gelatinolytic activity were seen in corneas treated with Doxy and Dex compared to vehicle. Increased neutrophil infiltration and myeloperoxidase activity was noted in the vehicle group compared to Dex 2 days post-injury. These findings demonstrate that early initiation of anti-inflammatory therapy is very efficacious in preserving corneal clarity and facilitating wound healing, while modulating MMP production and suppressing neutrophil infiltration.

Keywords: MMPs; alkali injury; corticoid; dexamethasone; doxycycline; dry eye; neutrophils; ocular perforation.

Figure 1

Anti-inflammatory therapy improves clinical parameters in a combined model (CM) of alkali burn and dry eye. A. Schematic of experimental design of the combined model of alkali burn and dry eye. A unilateral alkali burn (AB) was created on the right cornea as described in materials and methods. Mice were then subjected to desiccating stress and topically treated with either balanced salt solution (BSS), dexamethasone (Dex) or doxycycline (Doxy). Control mice were kept in a normal vivarium room (untreated animals, UT). B. Bright field digital images of alkali burn and dry eye combined model treated with either balanced salt solution (BSS), dexamethasone (Dex) or doxycycline (Doxy) 5 days (5D) post-injury. Scale bar=1000 μm. C. Representative digital images of corneas post-injury stained with 0.1% sodium fluorescein after creation of alkali burn lesion and induction of dry eye topically treated with BSS, Dex or Doxy for 5 days and then topically treated for 16 days in normal environment. Scale bar=1000 μm. D. Corneal opacity score in CM corneas topically treated with BSS, Dex, or Doxy. Asterisks show significant p value compared to vehicle. E. Survival rate of wound closure in CM corneas topically treated with BSS, Dex, or Doxy. F. Rate of ocular perforation in eyes subjected to CM topically treated with BSS, Dex, or Doxy. *p<.05, **p<.01; ***p<.001, ****p<.0001. BSS=corneas subjected to alkali burn and dry eye and treated topically with balanced salt solution, Dex=corneas subjected to alkali burn and dry eye and treated topically with dexamethasone, Doxy=corneas subjected to alkali burn and dry eye and treated topically with doxycycline, D=days.

Figure 2

Inflammatory cytokines and MMPs decrease after anti-inflammatory therapy in a combined model (CM) of alkali burn and dry eye. Relative fold expression of IL-6, collagenases (MMPs -1, -8 and 13), the stromelysin MMP-3, the gelatinase MMP-9 and TIMP-1 in whole corneas subjected to a combined model of alkali burn and dry eye topically treated with BSS, Dex, or Doxy. Bar graphs show means ± SEM of one representative experiment with four samples per group/time point (experiment was repeated three times with similar results). UT=untreated cornea, BSS=corneas subjected to alkali burn and dry eye and treated topically with balanced salt solution, Dex=corneas subjected to alkali burn and dry eye and treated topically with dexamethasone, Doxy=corneas subjected to alkali burn and dry eye and treated topically with doxycycline, D=days. *p<.05; **p<.01; ***p<.001, ****p<.0001.

Figure 3

Anti-inflammatory therapy decreases MMP protein expression and gelatinolytic activity. A. Representative merged digital images of cornea cryosections immunostained for MMP-1, MMP-9, MMP-13 and NGAL (all in green) with propidium iodide nuclei counterstaining (red) subjected to a combined model of alkali burn and dry eye topically treated with BSS, Dex, or Doxy for 2 or 5 days (2D or 5D, respectively). Scale bar=50 μm. B. Representative merged digital images of in situ zymography of central and peripheral cornea (green) with propidium iodide nuclei counterstaining (red) in all treatment groups. Doxy 2D was incubated with inhibitor (inh) provided in the kit and used as negative control. Scale bar=50μm. C. Representative gelatin zymogram showing MMP-9 and NGAL-MMP-9 bands in whole corneal lysates in the treatment groups. D. Representative digital images of cornea cryosections immunostained for Integrin β4 (in green) in all treatment groups. Nuclear counterstaining was omitted to facilitate visualization of immunofluorescent staining. Scale bar=50 μm. UT=untreated cornea, BSS=corneas subjected to alkali burn and dry eye and treated topically with balanced salt solution, Dex=corneas subjected to alkali burn and dry eye and treated topically with dexamethasone, Doxy=corneas subjected to alkali burn and dry eye and treated topically with doxycycline, D=days, NGAL=Neutrophil-gelatinase associated lipocalin, NC=negative control.

Figure 4

Anti-inflammatory therapy decreases neutrophil infiltration. A. Representative merged digital images of corneal cryosections immunostained for MMP-8 (in green) with propidium iodide nuclei counterstaining (red) in corneas subjected to the combined model topically treated with BSS, Dex or Doxy. NC= negative control. Scale bar=100μm. B. Relative fold expression of IL-1β and CXCL1 in whole corneas subjected to alkali burn and dry eye topically treated with BSS, Dex, or Doxy. Bar graphs show means ± SEM of one representative experiment with five samples per group/time point (experiment was repeated three times with similar results). UT=untreated cornea. *p<.05; **p<.01; ***p<.001, ****p<.0001. C. Representative merged digital images of central cornea cryosections immunostained for IL-1β (in green) with propidium iodide nuclei counterstaining (red) in corneas subjected to combined model topically treated with BSS, Dex, or Doxy. NC=negative control. Scale bar=50μm. D. Representative images of Gr-1⁺ cells (red) of central cornea cryosections from animals subjected to a combined model of alkali burn and dry eye topically treated with BSS, Dex or Doxy used to generate the bar graph showing counts in D. E. Bar graphs (mean±SEM) of Gr-1⁺ cell counts in experimental groups. F. Myeloperoxidase (MPO) activity in whole corneas lysates from animals subjected to a combined model of alkali burn and dry eye topically treated with BSS, Dex or Doxy 2 days after injury (mean±SEM). *p<.05; ***p<.001; ****p<.0001. UT=untreated cornea, BSS=corneas subjected to alkali burn and dry eye and treated topically with balanced salt solution, Dex=corneas subjected to alkali burn and dry eye and treated topically with dexamethasone, Doxy=corneas subjected to alkali burn and dry eye and treated topically with doxycycline, D=days. NC=negative control