Therapeutic effects of topical netrin-4 in a corneal acute inflammatory model

Abstract

Aim: To evaluate the therapeutic effect of netrin-4 on the early acute phase of inflammation in the alkali-burned eye.

Methods: Eye drops containing netrin-4 or phosphate buffered saline (PBS) were administered to a alkali-burn-induced corneal acute inflammatory model four times daily. The clinical evaluations, including fluorescein staining and inflammatory index, were performed on day 1, 4 and 7 using slit lamp microscopy. Global specimens were collected on day 7 and processed for immunofluorescent staining. The levels of inflammatory mediators in the corneas were determined by real-time polymerase chain reaction (PCR).

Results: Exogenous netrin-4 administered on rat ocular surfaces showed more improvements in decreasing fluorescein staining on day 4 and 7, and resolved alkali burn-induced corneal inflammation index on day 7 (P<0.01). The levels of IL-1β, IL-6, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) in corneas were decreased in netrin-4-treated groups (P<0.05). In addition, netrin-4 significantly reduced the expression of leukocyte common antigen 45 (CD45) in the alkali-burn cornea (P<0.001).

Conclusion: Topical netrin-4 accelerated wound healing and reduced the inflammation on alkali-burn rat model, suggesting a potential as an anti-inflammatory agent in the clinical to treat the acute inflammation.

Keywords: alkali burn; cornea; inflammation; netrin-4.

Figure 1. Netrin-4 promotes corneal epithelial wound healing after alkali burns

A: Fluorescein staining showed central corneal epithelial defects at day 1 after the alkali burns, and there was gradual decrease of the epithelial defect areas at day 4 and 7 in both groups. The epithelial defects were completely healed on day 7, if the corneas were treated with netrin-4, while the corneas treated with PBS did not heal. Images at different time points are sequential pictures from the same rat eye treated with either PBS or netrin-4. B: The inflammatory index of the ocular surface declined from day 1 to 7 in both groups. However, it was significantly lower in eyes treated with netrin-4 at day 7 (^bP<0.01). The figure is a summary of three experiments with n=3 corneas per time point per group per experiment.

Figure 2. Netrin-4 inhibits cornea inflammation, adhesion and chemotactic factors induced by alkali-burn

Corneal inflammation evaluated by real-time PCR for the mRNA expression of IL-1β (A), IL-6 (B), MIP-1 (C), MCP-1 (D), ICAM-1 (E) and VACM-1 (F) in the corneas of each group. The figure is a summary of three experiments with n=3 corneas per time point per group per experiment. Data are shown as mean±SD. ^aP<0.05, ^bP<0.01, ^cP<0.001. Scale bar: 20 mm.

Figure 3. Netrin-4 inhibits cornea inflammatory cell infiltration after corneal alkali burns

Corneal inflammation evaluated by immunofluorescent staining of CD45 for infiltrating macrophages in the corneal stroma (as red indicated) of the normal c57 (A), control group (C) and netrin-4-treated (E) groups. G: Six representative slices from homologous positions of each sample were selected (three samples for each group). Data are shown as mean±SD. ^cP<0.001. Scale bar: 20 µm.