Dr. Pooja Khamar, K17849, Dr. Rohit Shetty
Abstract:
Purpose:
Study molecular factors and effect of prophylactic application of epigenetic modulation on scarring in photorefractive keratectomy (PRK).
Methods & Materials:
Corneal epithelium samples collected intra- op were studied for gene expression profile from 6 corneal haze eyes. Fibrosis was studied in vitro using differential doses of TGFβ on cultured corneal epithelial cells. In-vivo PRK model was used to study drugs to treat post PRK corneal scarring.
Results:
WNT pathway and PREX1(oxidative stress marker) levels were altered in patient epithelium with haze and could modulate wound healing in vitro. Pro-fibrotic expression was altered by histone deacetylase (HDAC) inhibitor SAHA. PRK model eyes show significant reduction in postoperative haze without keratocyte cell death when SAHA was applied.
Conclusion:
Novel genes are involved in pathogenesis of haze post PRK. Modulation of haze predisposing pathways may be mediated by HDAC modulators to prevent haze post PRK in future.
Introduction:
Myopia is the most common cause of refractive errors among the young adults and also accounts for major cause of visual morbidity across the south-east Asia. (1) Intolerable cosmetic issues with spectacle correction and ocular surface side effects with contact lenses, Laser assisted corneal treatment for myopia gain major attraction among the clinician and as well as patients. Corneal surface ablation surgeries came into view after novel innovation of excimer laser. Till date many innovations came into place in this direction starting from PRK to Laser- in-situ keratomileusis (LASIK) to SMILE and many to come in future. PRK uses 193 nm ultraviolet laser light to ablate the corneal tissue and thus corrects the refractive errors (2) It treats at the level of anterior corneal stroma after debriding the epithelium and thus it remodels the stromal architecture. Corneal haze and deposition of extracellular matrix or fibrosis in sub-epithelial space are the common cause of reduced visual quality after PRK. This is associated with post-operative discomfort, night vision problems like halo and glare.(3) Though PRK has very good outcomes, with advent innovation of LASIK use of it has been limited in selected cases like in thin corneas, post radial keratotomy, post penetrating keratoplasty cases , in persons involving in sports and combat situations (to decrease the chances of flap related complications) and in post LASIK cases to treat residual refractive errors.(4,5,6) There is sufficient literature support stating that cytokine Transforming Growth factor- beta (TGF-b) has been associated with transformation of quiescent keratocytes into corneal fibroblasts and myofibroblasts.(7)With introduction of vorinostat, a deacetylase (HDAC) inhibitor in ophthalmic practice, targeted intervention at molecular level would be the novel therapeutic approach to address the specific problem of corneal haze after PRK.(8)
Materials & Methods:
The study received ethical clearance from institutional review board and strictly adhered to the guidelines laid down by Declaration of Helsinki. All patient samples were collected after obtaining informed, written consent as per institutional and ethics board guidelines. The study cohort was selected from patients who reported to our clinic.
Study cohort:
The study comprised 121 patients who underwent PRK at our institution. Inclusion criteria were as follows: Patients above 18 years of age, with stable refraction for the past 12 months or more, with 7.00 D of spherical equivalent refractive error or less as determined by manifest subjective refraction with a visual acuity correctable to at least 6/6 in both eyes and eyes that underwent PRK for -2.00 D to -7.00 D MRSE with 6.0-mm optical zones. Patients with clinical signs of progressive or unstable myopia, keratoconus or abnormal corneal topography or history of previous ocular surgery, with significant corneal or anterior segment pathologies, history of ocular infection or episode of allergy for at least 6 months prior to surgery, history of keloid, connective tissue disorder or hypertrophic scars, Pregnant women, nursing mothers or women on contraceptive pills or hormone replacement therapy during the time of surgery, on chronic systemic corticosteroids or immunosuppressive therapy that may affect corneal would healing or remodelling were excluded from the study.
Patients had been instructed to discontinue hard or RGP lenses for at least 2 weeks and soft contact lenses for at least 1 week before preoperative examination. Mitomycin-C (MMC) (write concentration) was used for patients with ablation depth higher than 75 micron.
Surgical procedure and postoperative follow up:
All patients who met the above mentioned criteria underwent standard PRK procedure by single surgeon after ensuring strict asepsis. The eyelids were retracted with a lid speculum and the eyelashes were everted using sterile surgical eye drape. Topical anesthetic eye drop 0.5% proparacaine hydrochloride (Paracaine, Sunways, (India) Pvt. Ltd) was instilled once before and after applying the eye drape. The epithelium over central 9 mm of cornea was debrided using a atraumatic, mechanical scraper. The epithelium removed was collected in a sterile microfuge tube in balanced salt solution (BSS) and stored at -80 degree Celsius. Thereafter the refractive correction was performed using 193 nm excimer laser with Wavelight EX500 (Alcon Laboratories, Fort Worth, TX). A bandage soft contact lens (BCL; Ciba Vision, Duluth, GA) was applied after clearing the stromal debris with balanced salt solution, which was usually removed 3 to 5 days after surgery. Topical Moxifloxacin hydrochloride 0.5% eye drops (Vigamox, Alcon, Bayer Pharma AG) and topical fluorometholone 0.1% (FML, Allergan, Inc ) was prescribed. FML was tapered gradually over 12 weeks. Postoperative follow-up occurred at 5 days, 2 weeks, 1 month, 3 months, 6 months, 1 year, and 10 to 12 years. Stromal haze was clinically assessed subjectively based on the haze grading system reported by Fantes et al [60]: grade 0, completely clear cornea; grade 0.5, trace haze, seen with careful oblique illumination with slit- lamp bio-microscopy; grade 1, more prominent haze, not interfering with visibility of fine iris details; grade 2, mild obscuration of iris details; grade 3, moderate obscuration of the iris and lens; and grade 4, completely opaque stroma in the area of ablation.
Intraoperative corneal epithelium samples were studied for gene expression profiling from six eyes that developed haze postoperatively and ten eyes of age matched controls. Patient sample profiling was performed using microarray followed by validation by QPCR. Total RNA was isolated using Qiagen’s RNeasy minikit Cat#74104 and RNA quality (QA/QC) was checked using bioanalyzer tape station and RNA integrity number was determined. Genes with at least 2 fold change and p-value <0.05 were considered for constructing the gene-gene network, Interactions were identified using STRING database and network was constructed using Cytoscape 3.2.1. Human corneal epithelial cells line were cultured using sterile conditions and various genes were studied from literature shown to be associated with haze development were used to construct the gene-function-pathway modulation network
In vitro studies performed on human corneal epithelial (HCE) cells with differential doses of TGFb for analysing structural and pro-fibrotic genes and regulators of signalling cascades. Rabbit PRK model was used to study haze post refractive surgery and topical instillation drugs were tested. To induce haze in a rabbit cornea, −9.00-diopter (D) PRK with a 6-mm ablation zone was performed on the central corneal stroma with an excimer laser. HDACi (25 μm) or balanced salt solution was topically applied on the stroma for 5 minutes immediately after PRK. Contralateral eyes served as controls.
Results
In spite of normal vision after surgery, patients may develop corneal haze over a period of time. Our microarray experiments revealed 1100 up and 1780 down regulated genes from the pooled set of control and pre-disposed patients. Differentially expressed genes with a cut off > ±1.5 fold change were considered significant. Few were highly significant like IL11 was 3.92 fold, ADAM11 was 3.52, PXDN was 2.39, PREX1 was 1.80 fold up regulated and SOX17 was -1.87, FOS was -2.70, JUN was -3.43, CXCL10 was -3.65 folds down regulated. This suggests that Gene expression (known and novel genes) profiles were significantly altered in haze predisposed patient epithelium. Structural genes (COL I, COL IV, MMP2 and 14 FN1) were elevated in haze patients and correlated with in-vitro model system. PREX1, an oxidative stress marker if elevated could modulate wound healing in vitro. Altered WNT pathway was observed in haze predisposed epithelium.
HDACi (histone deacetylase inhibitors) showed anti fibrotic effect on mRNA levels of fibronectin. Transforming growth factor beta 1-treated HCE cells showed 3-fold increase in fibronectin mRNA levels. Thus targeting this pathway in turn reduces fibronectin activity and thus haze could be altered by epigenetic regulators such as HDAC (histone deacetylase) inhibitors. Therefore, in vivo studies of HDACi in rabbit PRK model shows significant reduction in postoperative haze without concomitant keratocyte cell death.
Discussion
Maintenance of corneal avascularity and transparency is the key to good visual outcome after any intervention(9) After any injury either accidental or surgical a lot of wound healing pathways are activated. As literature suggests that break in epithelial continuity can lead to activation of a lot of pro-fibrotic genes which results in activation and release of many cytokines which in turn activate myofibroblasts and other tissue remodelling cells.(10)In corneal healing one of the most important cytokine which mediates such activities is TGF-b1. TGF-b1 induces fibrosis in cornea via Smad signalling. Histone acetylation is reported to regulate Smad mediated gene expression.(8)
Different ways to deal with epithelium debridement have been postulated to decrease postoperative corneal haze formation. The common methods include mechanical scrapping by scrapper, alcoholic debridement and Trans epithelial laser.(11) Last two have better outcomes as compare to first one in terms of long term corneal clarity. Pain and post-operative haze are the common side effects after epithelial debridement. After use of ethanol to remove epithelium, increase in inflammation in anterior stromal keratocyte has been observed and that may lead to haze formation.(11) Contrary to that, some of the literature suggest that it produces less inflammation and also less post-operative haze. Laser assisted epithelial removal decreases keratocyte apoptosis and thus maintaining normal stromal keratocytes in turn reduces corneal haze.
Use of Mitomycin-C during the procedure to reduce postoperative haze has been established.(12) It is an anti-metabolite which reduces the activity of myofibroblast and thus reducing the fibrotic activities. Studies showed that there is no endothelial toxicity but some amount of anterior stromal keratocyte loss has been found. Post operatively use of steroid has been advocated once the epithelium healed to suppress the cytokine activities.(13) Secondary glaucoma and secondary infection should be looked for when patient has been on such therapy. Bandage contact lenses are useful in early epithelial healing (14) but have no effect on post-operative scarring.
PRK in cases of high myopia and with larger zone of ablation have been associated with higher chances of post-operative corneal haze. (15) Nearly 25% of the post-operative corneal haze patients have visual disturbance and can be night mare for the surgeons. None of the above mentioned approaches has addressed this long term sequale in appropriate manner with minimal side-effects. We have found that expression of structural genes which are responsible for synthesis of collagen –I and IV are increased in in vivo samples associated with increased inflammatory marker PREX1. Literature supports the role of TGF-B1 in the activation of pro fibrotic activities. We have tested a novel therapeutic molecule Histone deacetylase inhibitor in rabbit PRK eyes which decreases the TGF-B expression. We found that it reduces post-operative corneal haze without affecting normal stromal keratocyte
Conclusion:
WNT pathway and deregulated PREX1 gene are involved in the pathogenesis of haze post PRK. Modulation of such pro fibrotic and haze predisposing pathways may be mediated by epigenetic factors. HDACi eye drops could be an effective modulator to prevent haze post PRK for low myopia in future.
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