Minocycline and Diacetyl Minocycline Eye Drops Reduce Ocular Neovascularization in Mice

Abstract

Purpose: To evaluate the efficacy of minocycline and a novel, modified minocycline analogue that lacks antimicrobial action, diacetyl minocycline (DAM), on choroidal neovascularization (CNV) in mice of both sexes.

Methods: CNV was induced via laser injury in female and male C57BL/6J mice. Minocycline, DAM, or saline was administered via topical eye drops twice a day for 2 weeks starting the day after laser injury. CNV volume was measured using immunohistochemistry labeling and confocal microscopy.

Results: Minocycline reduced lesion volume by 79% (P ≤ 0.0004) in female and male mice. DAM reduced lesion volume by 73% (P ≤ 0.001) in female and male mice. There was no significant difference in lesion volume between minocycline and DAM treatment groups or between female and male mice.

Conclusions: Both minocycline and DAM eye drops significantly reduced laser-induced CNV lesion volume in female and male mice. While oral tetracyclines have been shown to mitigate pathologic neovascularization in both preclinical studies and clinical trials, the present data are the first to suggest that tetracycline derivatives may be effective to reduce pathologic CNV when administered via topical eye drops. However, the action is unrelated to antimicrobial action. Targeted delivery of these medications via eye drops may reduce the potential for systemic side effects.

Translational relevance: Topical administration of minocycline and/or DAM via eye drops may represent a novel therapeutic strategy for disorders involving pathologic CNV.

Figure 1.

Experimental timeline and methods. (A) Timeline of ocular laser surgeries, drug treatments, dissections, immunohistochemistry, and confocal microscopy. (B) Laser disruption of Bruch's membrane to induce CNV formation. (C) Topical eye drop administration technique. (D) Enucleation and dissection of eyes: eyes were hemisected to separate anterior and posterior halves, retina was separated from underlying choroid, and radial cuts were made to facilitate flattening of samples for staining, flat mounting, and microscopy.

Figure 2.

Minocycline and DAM eye drops reduce CNV volume in female and male mice. (A) Representative images of CNV lesions in mice treated with saline (n = 4 females and 4 males), minocycline (n = 4 females and 4 males), or DAM (n = 4 females and 4 males) for 2 weeks. Lesions were labeled with fluorescent markers for isolectin IB4 (red; blood vessels), phalloidin (green; RPE), and DAPI (blue; nuclei). All settings were kept constant across all images, for both confocal microscopy and image analysis. Scale bar: 100 µm. (B) Plot of CNV lesion volumes in saline, minocycline, and DAM treatment groups. Each point corresponds to the average volume of CNV lesions in both left and right eyes of a single mouse. A one-way ANOVA with Bonferroni's multiple comparisons test was used to analyze differences between control, minocycline, and DAM treatment groups with data from male and female mice combined. *P < 0.0005, **P < 0.001. Error bars represent standard error of the mean.

Figure 3.

Representative three-dimensional reconstructions of CNV lesions in female mice. Mice were treated with saline (n = 4 females and 4 males), minocycline (n = 4 females and 4 males), or DAM (n = 4 females and 4 males) eye drops for 2 weeks. All settings were kept constant across all images, for both confocal microscopy and image analysis. Scale bar: 100 µm. Three channels: green (RPE), red (blood vessels), and blue (nuclei) channels superimposed. Green channel: phalloidin-labeled RPE. Red channel: isolectin IB4-labeled blood vessels. Blue channel: DAPI-labeled nuclei.

Figure 4.

Representative three-dimensional reconstructions of CNV lesions in male mice. Mice were treated with saline (n = 4 females and 4 males), minocycline (n = 4 females and 4 males), or DAM (n = 4 females and 4 males) eye drops for 2 weeks. All settings were kept constant across all images, for both confocal microscopy and image analysis. Scale bar: 100 µm. Three channels: green (RPE), red (blood vessels), and blue (nuclei) channels superimposed. Green channel: phalloidin-labeled RPE. Red channel: isolectin IB4-labeled blood vessels. Blue channel: DAPI-labeled nuclei.

Figure 5.

Antimicrobial activity of minocycline and DAM. (A) CFU assays to determine antibacterial activity of minocycline and DAM. DAM exhibited no antibacterial activity against E. coli at concentrations up to 10 µg/mL. (B) Zone of inhibition assays to determine antibacterial activity of minocycline and DAM. DAM exhibited no antibacterial activity against E. coli at concentrations up to 10 µg/mL. (C) CFU assays to determine antifungal activity of minocycline and DAM. DAM exhibited no antifungal activity against C. albicans at concentrations up to 100 µg/mL. All experiments were repeated in triplicate. Error bars represent standard error of the mean.