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E. W. Haselhorst, R. Y. Reins, A. M. McDermott; Antimicrobial Peptides and Corneal Epithelial Wound Healing. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3392.
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Antimicrobial peptides such as defensins and cathelicidins have antibacterial activity and modulate immune function and cell behaviours relevant to wound healing. We investigated corneal epithelial healing and antimicrobial peptide expression before and after injury in genetically modified mice deficient in β-defensin-1 (mBD-1 KO) or cathelin-related antimicrobial peptide (CRAMP KO).
Wounding: Mice were anaesthetized then the corneal epithelium was removed in an area marked by a 2mm trephine by alger brush (imaging) or scraping with a sterile blade (RT-PCR). Imaging: the wounded area was stained with fluorescein and digitally imaged under UV illumination every 4-8 hours after injury. The area of the wound was calculated using NIH image software. RT-PCR: corneal epithelial cells were collected by scraping, then re-grown cells were collected 24 or 48hrs after injury and RT-PCR performed to detect the presence of mBD-1, 2, 3, 4, 5, 6 or CRAMP.
There was no significant difference in healing among wild type (WT) and CRAMP KO mice. 36hrs after injury, 5 of 7 WT and 5 of 8 CRAMP KO mice had healed completely. The % area healed was greater in mBD-1 KO mice than C57BL/6 control animals at 16, 20, 24, 32 and 36 hrs after injury, with data at 20, 24 and 32 hrs being significantly different (P</= 0.02, student’s t-test). 40hrs after injury 4 of 6 control and 5 of 5 mBD-1 KO mice were completely healed. In mBD-1 KO mice, mBD-2, 3 and CRAMP were increased (1.5-3 fold) 24 hrs after injury and mBD-2, 3, 4, 5 and CRAMP were increased (4-12 fold) at 48hrs, whereas mBD-6 was decreased (n=2). In heterozygous CRAMP mice mBD-1 and 6 were decreased (1.5 fold) and mBD-2, 3, 4 and 5 were increased (1.5-6 fold) 48hrs after injury (n=2).
Increased defensin and CRAMP expression after injury is in keeping with a role for these peptides as antimicrobial agents and modulators of wound healing. Notably more peptides were expressed at 48hrs when the wounds were closed than at 24 hrs when active cell migration was occurring. Lack of CRAMP did not significantly impair wound healing, although lack of mBD-1 accelerated the process, suggesting that defensins and cathelicidins have different roles in modulating wound healing.
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