A novel method for generating corneal haze in anterior stroma of the mouse eye with the excimer laser

Abstract

Refractive surgery is a popular method used to reduce or eliminate dependence on glasses and contact lenses. Corneal haze is one of the common complications observed after photorefractive keratectmomy (PRK). The objective of this study was to develop an in vivo mouse model that consistently produces moderate to severe corneal haze in the anterior stroma of the mouse cornea after excimer laser treatment to study myofibroblast biology and corneal wound healing in a genetically defined model. Regular-or irregular-phototherapeutic keratectomy (PTK) was performed on black C57BL/6 mice with the Summit Apex excimer laser (Alcon, Ft. Worth, TX). Different numbers of laser pulses (45; ablation depth ~10 μm) were fired on the central cornea, after scraping the epithelium prior to excimer laser ablation. Irregularity was generated by positioning a fine mesh screen in the path of laser after firing 50% of the pulses. Eyes were collected 1, 2, 3 or 4 weeks after the procedure. Haze formation was gauged with slit lamp biomicroscopy. Immunocytochemistry was used to determine number of myofibroblasts in the mouse cornea using antibodies specific for the myofibroblast marker alpha-smooth muscle actin (SMA). The numbers of SMA-positive cells/400X microscopic were determined by counting within the stroma. Statistical analysis was performed using analysis of variance (AVOVA) with the Bonferonni-Dunn adjustment for repeated measures. Regular-PTK with epithelial scrape (Group 3) and irregular-PTK with epithelial scrape (Group 4) in the mouse eyes were performed to produce corneal haze. Eyes collected 4 weeks after regular-or irregular-PTK after epithelial scrape showed 22 ± 6.6 (Group 3) or 34 ± 7.9 (Group 4) SMA-positive cells in the anterior cornea. The difference in the SMA-positive cells detected among the groups was statistically significant (p < 0.01). Less than 4 SMA-positive cells were detected in the tissue sections of the mouse eyes collected after 1, 2 or 3 weeks of regular (Group 3) or irregular PTK (Group 4) or controls (Group 1 and 2). The optimized PTK excimer laser conditions developed in this study produces haze selectively in anterior stroma of the mouse cornea immediately beneath the epithelial basement membrane. Irregular PTK performed after epithelial scrape by applying 45 laser pulses was found to be the most effective method to generate myofibroblasts. This PTK technique for inducing haze in mouse cornea in vivo provides a useful model for studying wound healing and myofibroblast biology in transgenic mice.

Quantitative Assessment of Ultrastructure and Light Scatter in Mouse Corneal Debridement Wounds

“…An earlier study found stromal haze to be produced in mouse corneas when BM was disrupted, leaving an irregular surface. 26 The current data confirm these results, showing that the haze accompanies a long-lasting change in stromal ECM organization. A significant increase in corneal light scatter was observed only when BM was disrupted (Figs.…”

“…25 Epithelial debridement leaving the BM intact reportedly leaves the cornea transparent, whereas disruption of the epithelial BM, particularly one that generates a rough surface on the stroma, heals with anterior stromal haze. 26 The status of the ECM after healing of these two wound types has not been evaluated. The present authors compared the two wound types using a manual procedure for epithelial removal that left the BM intact, as well as a deeper debridement procedure that removed epithelium, BM, and a small portion of anterior stroma.…”

Ectopic Epithelial Implants following Surface Ablation of the Cornea

“…The situation can be summarized as ''the physics works,'' but the biological generation of complications and deviations from the correction are still in need of improvement. While much is known about the cellular and molecular biology of corneal wound healing, [1][2][3][4][5][6] improvements beyond this basal knowledge are constantly sought in order to explain the observed variance in optical and refractive outcomes following SA-based surgeries.…”

“…The sections were rehydrated and washed 3 times in PBS. The slides were then incubated in 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining solution (2 mM MgCl 2 ; Sigma-Aldrich); 5 mM K 4 (CN) 6 -3H 2 O (Sigma-Aldrich); 5 mM K 3 (CN) 6 (Sigma-Aldrich); 1 mg/mL X-Gal (Promega, Madison, WI), in PBS pH 7.4 at 378C in a humidified chamber overnight. The slides were then washed 3 times with PBS, counterstained with nuclear fast red (Vector Labs, Burlingame, CA).…”

Corneal angiogenesis based on different protocols of alkaline cauterization in murine models

“…The procedures for the induction of corneal angiogenesis were performed on animals under dissociative anesthesia 18 . Antisepsis was performed, and proxymetacaine eye drops (Alcon, Sao Paulo, Brazil) were instilled to promote desensitization of the ocular surface.…”