Desiccating Stress-Induced MMP Production and Activity Worsens Wound Healing in Alkali-Burned Corneas

Abstract

Purpose: To evaluate the effects of dry eye on ocular surface protease activity and sight threatening corneal complications following ocular surface chemical injury.

Methods: C57BL/6 mice were subjected to unilateral alkali burn (AB) with or without concomitant dry eye for 2 or 5 days. Mice were observed daily for appearance of corneal perforation. Whole corneas were harvested and lysed for RNA extraction. Quantitative real-time PCR was performed to measure expression of inflammation cytokines, matrix metalloproteinases (MMP). Matrix metalloproteinase-9 activity, gelatinase activity, and myeloperoxidase (MPO) activity were evaluated in corneal lysates. Presence of infiltrating neutrophils was evaluated by immunohistochemistry and flow cytometry.

Results: Eyes subjected to the combined model of AB and dry eye (CM) had 20% sterile corneal perforation rate as soon as 1 day after the initial injury, which increased to 35% by 5 days, delayed wound closure and increased corneal opacity. Increased levels of IL-1β, -6, and MMPs-1, -3, -8, -9, and -13, and chemokine (C-X-C motif) ligand 1 (CSCL1) transcripts were found after 2 days in CM compared with AB corneas. Increased MMP-1, -3, -9, and -13 immunoreactivity and gelatinolytic activity were seen in CM corneas compared with AB. Increased neutrophil infiltration and MPO activity was noted in the CM group compared with AB 2 days post injury.

Conclusions: Desiccating stress worsens outcome of ocular AB, creating a cytokine and protease storm with greater neutrophil infiltration, increasing the risk of corneal perforation.

Figure 1

Dry eye jeopardized cornea integrity after AB injury. (A) Schematic of experimental design. A unilateral AB in the right cornea was performed as described in the Materials and Methods. Mice were then kept in a normal vivarium room (resulting in AB and UT corneas) or subjected to DS in a specially designed room (resulting in DS or combined model [CM = AB + DS] corneas). (B) Digital images of bright field pictures from control, AB, CM, and DS 5 days post injury. Note: perforation in the combined group. (C) Rate of ocular perforation in eyes subjected to AB and CM. (D) Representative H&E staining of cryosections from control, AB, CM, and 5 days post injury. Perforated CM corneas developed massive inflammatory infiltration forming waves of inflammatory cells within the cornea stroma. Cornea perforation is sealed by lens and iris. Alkali-burned corneas had loose epithelium and moderate inflammatory cells in the cornea stroma and anterior chamber. Original magnification: ×10. (E) Representative digital images of corneas 1 hour after creation of AB lesion and representative pictures of open and closed wounds 2 and 5 days after initial lesion used to generate the survival curve of corneal re-epithelization seen in (F). (F) Survival rate of wound closure in AB and CM groups. (G) Representative bright field digital images of corneas showing opacification scale used to generate the graph seen in ([H]; corneal opacity score). (H) Corneal opacity score in AB and CM groups. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 2

Early increase cytokine and MMP storm in combined model corneas. Relative fold of expression of IL-6 and MMPs-1, -2, -3, -8, -9, -12, and -13, in whole corneas subjected to AB or CM. Bar graphs show means ± SEM of one representative experiment with five samples per group/time point (experiment was repeated thrice with similar results). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Increased MMP protein expression in combined model of AB and dry eye. (A) Representative merged digital images of corneas cryosections immunostained for MMP-1, -3, -13, and -9 (all in green) with propidium iodide nuclei counterstaining (red) in corneas subjected to AB, DS, or CM. Scale bars: 50 μm. (B) Matrix metalloproteinase–9 activity in whole-cornea lysates in AB, CM, and untreated group. (C) Representative zymogram showing MMP-9 bands whole-cornea lysates in the treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

Increased neutrophil infiltration in CM groups. (A) Relative fold of expression of IL-1β and CXCL1 in whole corneas subjected to AB, or CM (AB + dry eye). Bar graphs show means ± SEM of one representative experiment with five samples per group/time point (experiment was repeated twice with similar results). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (B) Representative merged digital images of corneas cryosections immunostained for IL-1β (in green) with propidium iodide nuclei counterstaining (red) in corneas subjected to AB or CM. Scale bars: 50 μm. (C) Representative pictures of Gr-1⁺ cells (red) of central cornea cryosections among the AB, CM, and control groups used to generate the bar graph showing counts in (D). Insets are representative pictures of limbal area. (D) Bar graphs (mean ± SEM) of Gr-1⁺ cell counts in whole cornea/groups. (E) Representative histogram (top panel) and dot plot (bottom panel) of CD45⁺ and Gr-1⁺ cells evaluated by flow cytometry. Numbers in the squares indicate percentage of positive cells. (F) Bar graphs (mean ± SEM) of frequency of CD45⁺ and CD45⁺Gr-1⁺ and CD45⁺F4/80⁺ cells quantified by flow cytometry in whole-cornea, single-cell suspension. (G) Myeloperoxidase activity in whole-corneas lysates 2 days after injury in untreated, AB and CM groups (mean ± SEM). *P < 0.05, ***P < 0.001, ****P < 0.0001. FSC-A, forward side scatter area.