Stromal haze, myofibroblasts, and surface irregularity after PRK

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Abstract

The aim of this study was to investigate the relationship between the level of stromal surface irregularity after photorefractive keratectomy (PRK) and myofibroblast generation along with the development of corneal haze.

Variable levels of stromal surface irregularity were generated in rabbit corneas by positioning a fine mesh screen in the path of excimer laser during ablation for a variable percentage of the terminal pulses of the treatment for myopia that does not otherwise generate significant opacity. Ninety-six rabbits were divided into eight groups:

Groups/TreatmentIIIIIIIVVVIVIIVIII
PRK ablation−4.5D−4.5D−4.5D−4.5D−4.5D−9.0D−9.0DUnwounded
Induced irregularity10%30%50%50%+SM+SM

SM, smoothing PTK

Slit lamp analysis and haze grading were performed in all groups. Rabbits were sacrificed at 4 hr or 4 weeks after surgery and histochemical analysis was performed on corneas for apoptosis (TUNEL assay), myofibroblast marker alpha-smooth muscle actin (SMA), and integrin α4 to delineate the epithelial basement membrane.

Slit-lamp grading revealed severe haze formation in corneas in groups IV and VI, with significantly less haze in groups II, III, and VII and insignificant haze compared with the unwounded control in groups I and V. Analysis of SMA staining at 4 weeks after surgery, the approximate peak of haze formation in rabbits, revealed low myofibroblast formation in group I (1.2±0.2 cells/400× field) and group V (1.8±0.4), with significantly more in groups II (3.5±1.8), III (6.8±1.6), VII (7.9±3.8), IV (12.4±4.2) and VI (14.6±5.1). The screened groups were significantly different from each other (p<0.05), with myofibroblast generation increasing with higher surface irregularity in the −4.5 diopter PRK groups. The −9.0 diopter PRK group VI had significantly more myofibroblast generation than the −9.0 diopter PRK with PTK-smoothing group VII (p<0.01). Areas of basement membrane disruption were demonstrated by staining corneas for integrin α4 and were prominent in corneas with grade I or higher haze. SMA-positive myofibroblasts tended to be present sub-adjacent to basement membrane defects. Late apoptosis was detected at 1 month after surgery within clusters of myofibroblasts in the sub-epithelial stroma.

In conclusion, these results demonstrated a relationship between the level of corneal haze formation after PRK and the level of stromal surface irregularity. PTK-smoothing with methylcellulose was an effective method to reduce stromal surface irregularity and decreased both haze and associated myofibroblast density. We hypothesize that stromal surface irregularity after PRK for high myopia results in defective basement membrane regeneration and increased epithelium-derived TGFβ signalling to the stroma that increases myofibroblast generation. Late apoptosis appears to have a role in the disappearance of myofibroblasts and haze over time.

Introduction

The corneal wound healing process following corneal refractive procedures involves a very complex and sometimes unpredictable biological response. After photorefractive keratectomy (PRK), the organization of the extracellular matrix is altered in the anterior stroma, and along with changes in cellular density and phenotype, can be associated with the production of disorganized extracellular matrix components. The final result is a decrease in tissue transparency-referred to as corneal haze or opacity. In most patients the level of stromal opacity that develops following PRK is not clinically significant, but in some, especially with higher levels of correction, the opacity can be severe. The generation of corneal myofibroblast cells has recently been identified as the primary biological event responsible for the formation of corneal haze (Jester et al., 1999a, Jester and Ho-Chang, 2003, Maltseva et al., 2001). Myofibroblasts are highly contractile cells with reduced transparency attributable to decreased intracellular crystallin production (Jester et al., 1999b).

Several factors have been suggested to contribute to haze formation after PRK, including the length of time required for epithelial defect healing, the depth of the ablation, irregularity of the postoperative stromal surface, removal of the epithelial basement membrane, or ablation of Bowman's layer (Tang and Liao, 1997, Vinciguerra et al., 1998aa,b; Moller-Pedersen et al., 1998, Nakamura et al., 2001, Serrao et al., 2003, Stramer et al., 2003, Kuo et al., 2004). There, however, has been no conclusive evidence supporting any particular hypothesis.

One of the most intriguing aspects of haze formation is that it rarely occurs in eyes that have lower levels of myopia corrected with PRK, even though the procedure is otherwise identical. Thus, eyes that have PRK for less than six diopters of myopia rarely develop significant haze. As the level of correction increases beyond six diopters, however, the incidence of clinically significant haze increases to as high as 2–4% (Lipshitz et al., 1997, Hersh et al., 1997, Shah et al., 1998, Siganos et al., 1999). Studies of PRK in rabbits confirmed that there was also a relationship between the level of correction and haze and myofibroblast formation in this model (Mohan et al., 2003). Any hypothesis proposed to account for haze formation should incorporate this clinical observation.

It has long been noted that abnormalities associated with surface irregularity abnormalities, such as topographic central islands and peninsulas, increase with increasing levels of correction after PRK. Vinciguerra et al., 1998a, Vinciguerra et al., 1998b reported a clinical correlation between the irregularity of the ablated surface after PRK and the incidence of corneal haze in a group of eighty eyes and noted a decrease in the incidence of haze when PRK included a stromal PTK-smoothing procedure. Taylor et al. (1994) performed scanning electron microscopy (SEM) in rabbit corneas that had different levels of excimer laser ablation and showed that there was an increase in irregularity of the ablated surface as the depth of the ablation increased. More recently, Horgan et al. (1999) demonstrated with SEM that phototherapeutic keratectomy (PTK) could be used to reduce stromal irregularity after PRK in porcine corneas. A lower incidence of corneal haze was also noted after PRK followed by PTK-smoothing by Serrao et al. (2003) in a ten-patient contralateral-eye study.

The present study tested the hypothesis that corneal haze and myofibroblast generation are related to the level of stromal surface irregularity after PRK using a rabbit model in which reproducible levels of surface irregularity were generated. The effect of PTK-smoothing on myofibroblast generation was also examined. Finally, the effect of surface irregularity after PRK on basement membrane regeneration was also explored.

Section snippets

Animals and surgery

The Animal Control Committee at the Cleveland Clinic Foundation approved all of the animal studies included in this work. All animals were treated in accordance with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Anaesthesia was achieved by intramuscular injection of ketamine hydrochloride (30 mg kg−1) and xylazine hydrochloride (5 mg kg−1). In addition, topical proparacaine hydrochloride 1% (Alcon, Ft. Worth, TX, USA) was applied to each eye just before

Corneal haze evaluated by biomicroscopy

Corneal haze was noted beginning at 2 weeks and appeared to peak around 4 weeks after surgery in all groups. The quantitative assessment of haze was significantly greater in the PRK for −9.0 diopters of myopia group VI than in the PRK for −4.5 diopters of myopia group I. When stromal surface irregularity was induced in the −4.5 diopter PRK groups, a marked increase in haze was observed at 4 weeks (Table 2, Table 3; Fig. 2), with the increase in corneal haze formation being directly related to

Discussion

The results of this study demonstrate that there is a relationship between the level of corneal haze formation after PRK, and associated anterior stromal myofibroblasts, and the level of stromal surface irregularity remaining after surface ablation. In corneas with surface irregularity, there also appear to be defects in the basement membrane after healing of the epithelium and these defects appear to correspond to areas where myofibroblasts are generated in the anterior stroma. In addition,

Acknowledgements

Supported in part by US Public Health Service grants EY 10056 and EY 15638 from the National Eye Institute, National Institutes of Health, Bethesda, MD.

References (26)

  • J.V. Jester et al.

    The cellular basis of corneal transparency: evidence for ‘corneal crystallins’

    J. Cell Sci.

    (1999)
  • I.C. Kuo et al.

    Late-onset corneal haze and myopic regression after photorefractive keratectomy (PRK)

    Cornea

    (2004)
  • T.U. Linna et al.

    Effect of myopic LASIK on corneal sensitivity and morphology of subbasal nerves

    Invest. Ophthalmol. Vis. Sci.

    (2000)
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