Elsevier

Experimental Eye Research

Volume 83, Issue 4, October 2006, Pages 728-735
Experimental Eye Research

Wound healing following refractive surgery in hens

https://doi.org/10.1016/j.exer.2006.02.017Get rights and content

Abstract

The wound-healing response is critical to the outcome of refractive surgery and studying wound healing contributes to an understanding of the pathophysiology of other corneal injuries. Animal models allow research to be conducted with sufficient samples and under controlled parameters. We studied the hen to determine the healing process from clinical, biophysical, and biological standpoints after photorefractive keratectomy (PRK). PRK (−6.0 diopters) was performed in hen eyes. At 3, 6, 12, 24, 48, and 72 h and 5, 7, 15, 30, and 60 days postoperatively, we studied the clinical follow-up, objective measurements of light transmission (direct transmittance), apoptosis by TUNEL assay, proliferation by immunocytochemical analysis of 5-bromo-2′-deoxyuridine, and expression of alpha smooth muscle actin (SMA) in myofibroblasts in the corneas. Hen corneas reepithelialize quickly. Haze developed from 5 to 60 days after surgery and was correlated with the appearance and finalization of the expression of SMA. The direct transmittance of light was low during the first 15 days and improved at 30 and 60 days. TUNEL-positive cells were observed 3 h after surgery and the numbers decreased thereafter. Epithelial proliferation began at 12 h and was greater at 48 h, while stromal cell proliferation began at 24 h and was greater at 72 h. The hen cornea is anatomically similar to the human cornea, and the manner in which it heals is a good model for studying different surgical techniques and pharmacologic assays.

Introduction

Refractive surgery has increased exponentially in popularity during the past decade, and millions of people worldwide have chosen this alternative to reduce their dependence of glasses and contact lenses. To increase the safety, security, and predictability of refractive procedures, new diagnostic tools, surgical instruments, and techniques have been developed; however, refractive surgery has limitations and complications that should be addressed. The study of corneal wound healing related to refractive surgery procedures and their pharmacologic modulation is an attractive line of research aimed at reducing complications and increasing the quality of vision. This is especially important because cellular and molecular biology has provided powerful weapons for gaining an understanding of the corneal wound-healing process.

Because there are obvious limitations in humans, animal models allow research to be conducted with sufficient samples and under controlled parameters. Wilson et al. (2001) described a cascade of events in rabbits that comprises the corneal wound-healing response. Those authors reported apoptosis of the keratocytes during the first hours after surgery; 24 h after surgery apoptosis is followed by two simultaneous cellular processes: the removal of damaged cells and extracellular matrix. Dead cells are removed by macrophages and damaged extracellular matrix by metalloproteinases (Ye and Azar, 1998, Ye et al., 2000) and the plasmin system (Berman et al., 1980). Both processes are regulated by several cytokines synthesized and released by the epithelium, i.e., transforming growth factor (TGF)-β and interleukin 1 and 6 (Girard et al., 1991, Malecaze et al., 1997). The synthesis of new tissue requires proliferation, migration, and differentiation of epithelial cells, which are stimulated by growth factors secreted by keratocytes, epithelial growth factor (EGF), keratinocyte growth factor, and hepatocyte growth factor (Klenkler and Sheardown, 2004, Wilson et al., 1994). The synthesis of new tissue also requires proliferation of the remaining keratocytes (Zieske, 2000) that are regulated by EGF, TGF-β, platelet-derived growth factor, and fibroblast growth factor, secreted by epithelial and inflammatory cells (Baldwin and Marshall, 2002) and the synthesis of a new extracellular matrix secreted by fibroblasts.

When the basement membrane is damaged, the remaining keratocytes undergo mitosis and differentiation and become myofibroblasts due to release of TGF-β by the epithelium (Stramer et al., 2003). Myofibroblasts are cells with migration and wound-contraction capabilities that are characterized by expression of alpha smooth-muscle actin (SMA). Finally, the elimination of some cells involved in wound healing and remodeling of disordered collagen results in return to the normal state.

Photorefractive keratectomy (PRK) is a safe and effective procedure to correct refractive errors for cases with epithelial basement membrane dystrophy and high myopia. It is also a good model by which to study wound healing.

Several animal models are available to study corneal wound healing, most of them in rabbits (Mohan et al., 2003), rats (Power et al., 1995), and monkeys (Del Pero et al., 1990, Malley et al., 1990). Rabbits are not a good model because the cornea lacks Bowman's layer and the corneas are highly reactive to pharmacologic and surgical treatments. Rats have a small and pointed cornea and monkeys are difficult to handle, expensive, and have complicated housing requirements. Avian corneas are similar to human corneas and have a Bowman's layer. Hens, which are inexpensive, easy to handle and anesthetize, and do not require complicated housing, have been used in refractive surgery by our research group (Merayo-Lloves et al., 2001, Torres et al., 2005) and others (Fowler et al., 2004).

The goals of this study were to describe the wound-healing process after PRK in the avian animal model and relate the clinical course and the objective measurement of transmittance of light to the events that occur during wound healing.

Section snippets

Animals

Forty-four Iber Braun adult hens, Gallus gallus domesticus (weight, 2 kg) were used. Animals were cared for following the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

The hens were anaesthetized with an intramuscular injection of ketamine hydrochloride (37.5 mg/kg; Ketolar, Parke-Davis S.A., Barcelona, Spain) and xylazine hydrochloride (5 mg/kg; Rompun, Bayer AG, Leverkusen, Germany) followed by topical application of 0.5% tetracaine chlorhydrate and 1 

Clinical course and complications

There were no infections or corneal melting during follow-up. Visualization of epithelial defects was enhanced by fluorescein staining and recorded photographically using a surgical microscope. Immediately after 7.5-mm deepithelization and corneal laser ablation, a rough surface with concentric ring patterns was observed. The new epithelium covered half of the ulcer at 24 h; on the second day, no epithelial defect was present, but a rough surface was observed.

On day 4 postoperatively, incipient

Discussion

This study presents a comprehensive picture of the corneal wound-healing process after refractive surgery as a result of the linking and correlating of clinical, biophysical, and biologic parameters in a hen model that mimics human disease. Previous studies in human have focused on clinical follow-up by confocal microscopy; however, this technique cannot detect cell death and does not quantify proliferation or determine cell type.

Regarding animal models, different studies have examined

Acknowledgements

The authors thank H. Martinez, MD, for his clinical follow-up; Angel Garcia Barcia for veterinary assistance; Marta González Parra for technical assistance; and Luis Santiago Bucero for technical assistance with electron microscopy.

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    Supported in part by grant FIS-PI:01/0270 and Red Temática de Investigación Cooperativa en Oftalmología CO3/13, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Madrid, Spain. Alcon-Cusi SA (Barcelona, Spain) donated the experimental excimer laser.

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