Optical effects of anti-TGFβ treatment after photorefractive keratectomy in a cat model.

Increasing awareness of the potential risk of iatrogenic keratectasia after laser in situ keratomileusis (LASIK) has led to a renewed interest in photorefractive keratectomy (PRK) and prompted the development of advanced surface ablation techniques like laser subepithelial keratomileusis (LASEK)1 and Epi-LASIK2. One major disadvantage of surface ablations over LASIK is the more pronounced wound healing response, whose functional consequences include a longer visual rehabilitation period, regression and haze. Since these side effects can significantly limit the treatment of higher myopia (reviewed in3-5), several attempts have been made to decrease their occurrence through preservation of the epithelial layer (e.g. LASEK and Epi-LASIK) and pharmacological modulation of wound healing, as proposed in the early days of PRK6. Topical steroids have been used widely7, but their effects on haze and refractive regression remain controversial8-11. Mitomycin C, a cytostatic agent originally introduced for chemotherapy of malignant tumors, has also been shown to attenuate wound healing after PRK, particularly in cases with higher susceptibility for regression and haze, i.e. those involving the treatment of higher myopia12-15. However, safety concerns and side effects have afflicted the use of steroids (elevation of intraocular pressure, cataract induction and delayed epithelial healing) and Mitomycin C (cytotoxic, possibly mutagenic, limited data on long-term keratocyte integrity), prompting the search for alternatives. One means of modulating corneal wound-healing is via the inhibition of transforming growth factor β (TGFβ)16, 17. TGFβ is a multifunctional cytokine released by the lacrimal gland, the corneal epithelium and conjunctival cells18. TGFβ promotes keratocyte proliferation19, 20, migration21, differentiation into myofibroblasts that express α-smooth muscle actin (αSMA - reviewed in22) and the deposition of extracellular matrix proteins19. TGFβ has been shown to play a crucial role in the development of haze after PRK, so that application of anti-TGFβ antibodies to the eye reduces both corneal reflectivity (haze) and fibrosis after PRK in rabbits16, 17. However, stromal re-growth still occurred, suggesting that at least in the rabbit, stromal regeneration may be controlled by TGFβ-independent mechanisms17. Other than haze, the optical consequences of blocking TGFβ after PRK16, 17 have never yet been examined. The cat model used in the present study is unique in allowing simultaneous investigation of biological and optical aspects of corneal wound healing after PRK23-25. However, before testing the in situ effects of anti-TGFβ treatment in the cat, we first measured the response of feline corneal keratocytes to TGFβ stimulation in vitro, to verify that they behaved similarly to keratocytes from rabbits, pigs and humans. In vivo experiment were then carried out to test the hypothesis that blocking TGFβ activity in the cat eye after PRK: (1) decreases transformation of corneal keratocytes into contractile myofibroblasts, (2) decreases haze (corneal reflectivity) by reducing the incidence of reflective myofibroblasts in the ablation optical zone, (3) decreases refractive regression by slowing keratocyte proliferation and the generation of new extracellular matrix in the stroma, and (4) decreases the induction of higher order aberrations (HOAs) by decreasing the fine-grained, contractile influence of myofibroblasts on the corneal surface.