Defensin Levels in Tears After an Experimental Corneal Wound in the Rabbit Are Correlated With Wound Healing

Abstract

Abstract: : Purpose: Wound healing is a complex process, prior to regeneration of the epithelium the cornea is vulnerable to entry of pathogens. Upregulation of defensins during this time would be an advantage for the restoration of visual function. Little is known about the changing levels of tear proteins in corneal wound healing. The aim of this study was to use proteomic approaches to examine the levels of defensins in tear protein profiles following corneal wounding. Methods: Tears were collected from New Zealand White rabbits prior to and daily for five days following a standard unilateral 8mm dia. corneal epithelial abrasion. Tear protein profiles obtained from wounded and control eyes were compared using proteomic approaches, SELDI ProteinChip technology and LC–MS (ABI Q–STAR XL). Peptides and proteins of interest were further characterized by nanoESI–MS/MS. Results: Tear protein profiles changed rapidly after a corneal abrasion. The general pattern of the tear protein profile was also correlated with the progress of re–epithelialization. An important finding was that the levels of some of rabbit defensins (NP–1 and NP–2) were elevated after wounding and returning to normal levels when cornea epithelium healing was completed. Specifically, NP–1 and NP–2 levels in rabbit tears were found to be 5∼20 times and 4∼12 times higher than basal levels, respectively, after corneal wounding estimated by SELDI; however, NP–3B and NP–5 were not found to be upregulated. The identity of the rabbit defensins was confirmed by high–resolution mass spectrometry. Conclusions: The marked increase of the levels of selected defensins in tear fluids after corneal wounding suggests these peptides play an important role in protecting the cornea from microbial attack and may also modulate wound healing processes as well.

Keywords: cornea: tears/tear film/dry eye • proteomics • wound healing