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: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.

Transforming growth factor-beta (TGFβ) is a pleiotropic multifunctional cytokine that regulates several essential cellular processes in many parts of the body including the cornea. Three isoforms of TGFβ are known in mammals and the human cornea expresses all of them. TGFβ1 has been shown to play a central role in scar formation in adult corneas whereas TGFβ2 and TGFβ3 have been implicated to play a critical role in corneal development and scarless wound healing during embryogenesis. The biological effects of TGFβ in the cornea have been shown to follow SMAD dependent as well as SMAD-independent signaling pathways depending upon cellular responses and microenvironment. Corneal TGFβ expression is necessary for maintaining corneal integrity and corneal wound healing. On the other hand, TGFβ is perhaps the most important cytokine in the pathogenesis of fibrotic disease in the cornea. Although the transformation of keratocytes to myofibroblasts induced by TGFβ is largely believed to cause corneal fibrosis or scarring, the precise molecular mechanism(s) involved in this process is still unknown. Currently no drugs are available to treat corneal scarring effectively without causing significant side effects. Many approaches to treat TGFβ-mediated corneal scarring are under investigation. These include blocking of TGFβ, TGFβ receptor, TGFβ function and/or TGFβ maturation. Other strategies such as modulating keratocyte proliferation, apoptosis, transcription and DNA condensation are also being investigated. The potential of gene therapy to neutralize the pathologic effects of TGFβ has also been demonstrated recently.

PURPOSE-To determine the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) and to examine the effects of dosage and duration of exposure. METHODS-In 224New Zealand rabbits, −9.0 diopter PRK with mitomycin C or balanced salt solution was performed. Haze level was graded at the slit-lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks, or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67 and α-SMA. RESULTS-TUNEL-positive apoptotic cells marginally increased in all mitomycin C groupswhereas Ki67-positive mitotic cells decreased significantly following mitomycin C application. A greater decrease in myofibroblasts was noted with prophylactic mitomycin C treatment than therapeutic mitomycin C treatment. There was, however, an anterior stromal acellular zone (approximately 20% of the total stroma) in eyes treated with mitomycin C, which persisted to the maximum follow-up of 6 months.CONCLUSIONS-Mitomycin C treatment induces apoptosis of keratocytes and myofibroblasts, but the predominate effect in inhibiting or treating haze appears to be at the level of blocked replication of keratocytes or other progenitor cells of myofibroblasts. Treatment with 0.002% mitomycin C for 12 seconds to 1 minute appears to be just as effective as higher concentrations for longer duration in the rabbit model. However, a persistent decrease in keratocyte density in the anterior stroma could be a warning sign for future complications and treatment should be reserved for patients with significant risk of developing haze after PRK.Photorefractive keratectomy (PRK) has proven to be a safe and effective procedure to correct low to moderate levels of myopia, hyperopia, or astigmatism. It continues to represent a good alternative to LASIK for many patients, and in some situations, PRK remains the procedure of choice. 1 However, PRK continues to have downsides, including increased postoperative pain, a stronger healing response, and most significantly, the possibility of subepithelial corneal opacity or "haze" formation following corrections for high myopia. 2,3 The native conformation of the extracellular matrix is altered after PRK. Along with changes in cellular density and phenotype, there is variable production of disorganized extracellular

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PURPOSE Trichostatin A (TSA), a histone deacetylase inhibitor, has been shown to suppress TGF-β-induced fibrogenesis in many nonocular tissues. The authors evaluated TSA cytotoxicity and its antifibrogenic activity on TGF-β– driven fibrosis in the cornea with the use of in vitro and in vivo models. METHODS Human corneal fibroblasts (HSFs) were used for in vitro studies, and New Zealand White rabbits were used for in vivo studies. Haze in the rabbit cornea was produced with photorefractive keratectomy (PRK) using excimer laser. Trypan blue exclusion and MTT assays evaluated TSA cytotoxicity to the cornea. Density of haze in the rabbit eye was graded with slit lamp biomicroscopy. Real-time PCR, immunoblotting, or immunocytochemistry was used to measure α-smooth muscle actin (SMA), fibronectin, and collagen type IV mRNA or protein levels. TUNEL assay was used to detect cell death. RESULTS TSA concentrations of 250 nM or less were noncytotoxic and did not alter normal HSF morphology or proliferation. TGF-β1 treatment of HSF significantly increased mRNA and protein levels of SMA (9-fold), fibronectin (2.5-fold), and collagen type IV (2-fold). TSA treatment showed 60% to 75% decreases in TGF-β1-induced SMA and fibronectin mRNA levels and 1.5- to 3.0-fold decreases in protein levels but had no effect on collagen type IV mRNA or protein levels in vitro. Two-minute topical treatment of TSA on rabbit corneas subjected to -9 D PRK significantly decreased corneal haze in vivo. CONCLUSIONS TSA inhibits TGF-β1-induced accumulation of extracellular matrix and myofibroblast formation in the human cornea in vitro and markedly decreases haze in rabbit cornea in vivo. (Invest Ophthalmol Vis Sci. 2009;50:2695–2701) DOI: 10.1167/iovs.08-2919

This study examined the effects of BMP7 gene transfer on corneal wound healing and fibrosis inhibition in vivo using a rabbit model. Corneal haze in rabbits was produced with the excimer laser performing -9 diopters photorefractive keratectomy. BMP7 gene was introduced into rabbit keratocytes by polyethylimine-conjugated gold nanoparticles (PEI2-GNPs) transfection solution single 5-minute topical application on the eye. Corneal haze and ocular health in live animals was gauged with stereo- and slit-lamp biomicroscopy. The levels of fibrosis [α-smooth muscle actin (αSMA), F-actin and fibronectin], immune reaction (CD11b and F4/80), keratocyte apoptosis (TUNEL), calcification (alizarin red, vonKossa and osteocalcin), and delivered-BMP7 gene expression in corneal tissues were quantified with immunofluorescence, western blotting and/or real-time PCR. Human corneal fibroblasts (HCF) and in vitro experiments were used to characterize the molecular mechanism mediating BMP7’s anti-fibrosis effects. PEI2-GNPs showed substantial BMP7 gene delivery into rabbit keratocytes in vivo (2×104 gene copies/ug DNA). Localized BMP7 gene therapy showed a significant corneal haze decrease (1.68±0.31 compared to 3.2±0.43 in control corneas; p<0.05) in Fantes grading scale. Immunostaining and immunoblot analyses detected significantly reduced levels of αSMA (46±5% p<0.001) and fibronectin proteins (48±5% p<0.01). TUNEL, CD11b, and F4/80 assays revealed that BMP7 gene therapy is nonimmunogenic and nontoxic for the cornea. Furthermore, alizarin red, vonKossa and osteocalcin analyses revealed that localized PEI2-GNP-mediated BMP7 gene transfer in rabbit cornea does not cause calcification or osteoblast recruitment. Immunofluorescence of BMP7-transefected HCFs showed significantly increased pSmad-1/5/8 nuclear localization (>88%; p<0.0001), and immunoblotting of BMP7-transefected HCFs grown in the presence of TGFβ demonstrated significantly enhanced pSmad-1/5/8 (95%; p<0.001) and Smad6 (53%, p<0.001), and decreased αSMA (78%; p<0.001) protein levels. These results suggest that localized BMP7 gene delivery in rabbit cornea modulates wound healing and inhibits fibrosis in vivo by counter balancing TGFβ1-mediated profibrotic Smad signaling.

This study examined the gene transfer efficiency and toxicity of 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNP) in the human cornea in vitro and rabbit cornea in vivo. PEI2-GNP with nitrogen-to-phosphorus (N/P) ratios of up to 180 exhibited significant transgene delivery in the human cornea without altering the viability or phenotype of these cells. Similarly, PEI2-GNP applied to corneal tissues collected after 12 h, 72 h, or 7 days exhibited appreciable gold uptake throughout the rabbit stroma with gradual clearance of GNP over time. Transmission electron microscopy detected GNP in the keratocytes and the extracellular matrix of the rabbit corneas. Additionally, slitlamp biomicroscopy in live animals even 7 days after topical PEI2-GNP application to the cornea detected no inflammation, redness, or edema in rabbit eyes in vivo, with only moderate cell death and immune reactions. These results suggest that PEI2-GNP are safe for the cornea and can be potentially useful for corneal gene therapy in vivo.

Tissue-targeted AAV5-mediated decorin gene therapy is effective and safe for treating corneal fibrosis in vivo.