Marcelo V. Netto

Wound healing in the cornea: a review of refractive surgery complications and new prospects for therapy.

Purpose: The corneal wound healing response is of particular relevance for refractive surgical procedures since it is a major determinant of efficacy and safety. The purpose of this review is to provide an overview of the healing response in refractive surgery procedures. Methods: Literature review. Results: LASIK and PRK are the most common refractive procedures; however, alternative techniques, including LASEK, PRK with mitomycin C, and Epi-LASIK, have been developed in an attempt to overcome common complications. Clinical outcomes and a number of common complications are directly related to the healing process and the unpredictable nature of the associated corneal cellular response. These complications include overcorrection, undercorrection, regression, corneal stroma opacification, and many other side effects that have their roots in the biologic response to surgery. The corneal epithelium, stroma, nerves, inflammatory cells, and lacrimal glands are the main tissues and organs involved in the wound healing response to corneal surgical procedures. Complex cellular interactions mediated by cytokines and growth factors occur among the cells of the cornea, resulting in a highly variable biologic response. Among the best characterized processes are keratocyte apoptosis, keratocyte necrosis, keratocyte proliferation, migration of inflammatory cells, and myofibroblast generation. These cellular interactions are involved in extracellular matrix reorganization, stromal remodeling, wound contraction, and several other responses to surgical injury. Conclusions: A better understanding of the complete cascade of events involved in the corneal wound healing process and anomalies that lead to complications is critical to improve the efficacy and safety of refractive surgical procedures. Recent advances in understanding the biologic and molecular processes that contribute to the healing response bring hope that safe and effective pharmacologic modulators of the corneal wound healing response may soon be developed.

Effect of Prophylactic and Therapeutic Mitomycin C on Corneal Apoptosis, Cellular Proliferation, Haze, and Long-term Keratocyte Density in Rabbits

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 224 New 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 alpha-SMA. RESULTS TUNEL-positive apoptotic cells marginally increased in all mitomycin C groups whereas 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.

Femtosecond Laser and Microkeratome Corneal Flaps: Comparison of Stromal Wound Healing and Inflammation

PURPOSE To examine early postoperative wound healing in rabbit corneas that had LASIK flaps formed with three different models (15 KHz, 30 KhZ, and 60 KHz) of a femtosecond laser compared with flaps formed with a microkeratome. METHODS Thirty-nine rabbit eyes were randomized to receive either no surgery or corneal flaps formed with one of the lasers or the microkeratome. Sixteen eyes also had lamellar cuts with no side cuts with the 30 KHz laser. Animals were sacrificed and corneas processed as frozen sections or fixed for transmission electron microscopy. Frozen sections were evaluated with the TUNEL assay to detect apoptosis, immunocytochemistry for Ki67 to detect cell mitosis, and immunocytochemistry for CD11b to detect mononuclear cells. RESULTS Rabbit corneas that had flaps formed with the 15 KHz laser had significantly more stromal cell death, greater stromal cell proliferation, and greater monocyte influx in the central and peripheral comea at 24 hours after surgery than corneas that had flaps formed with the 30 KHz or 60 KHz laser or the microkeratome. Results of the 60 KHz laser and microkeratome were not significantly different for any of the parameters at 24 hours, except for mitotic stromal cells at the flap margin. Transmission electron microscopy revealed that the primary mode of stromal cell death at 24 hours after laser ablation was necrosis. CONCLUSIONS Stromal cell necrosis associated with femtosecond laser flap formation likely contributes to greater inflammation after LASIK performed with the femtosecond laser, especially with higher energy levels that result in greater keratocyte cell death.

Gene therapy in the cornea

Technological advances in the field of gene therapy has prompted more than three hundred phase I and phase II gene-based clinical trials for the treatment of cancer, AIDS, macular degeneration, cardiovascular, and other monogenic diseases. Besides treating diseases, gene transfer technology has been utilized for the development of preventive and therapeutic vaccines for malaria, tuberculosis, hepatitis A, B and C viruses, AIDS, and influenza. The potential therapeutic applications of gene transfer technology are enormous. The cornea is an excellent candidate for gene therapy because of its accessibility and immune-privileged nature. In the last two decades, various viral vectors, such as adeno, adeno-associated, retro, lenti, and herpes simplex, as well as non-viral methods, were examined for introducing DNA into corneal cells in vitro, in vivo and ex vivo. Most of these studies used fluorescent or non-fluorescent marker genes to track the level and duration of transgene expression in corneal cells. However, limited studies were directed to evaluate prospects of gene-based interventions for corneal diseases or disorders such as allograft rejection, laser-induced post-operative haze, herpes simplex keratitis, and wound healing in animal models. We will review the successes and obstacles impeding gene therapy approaches used for delivering genes into the cornea.

Wavefront analysis comparison of LASIK outcomes with the femtosecond laser and mechanical microkeratomes.

PURPOSE To evaluate differences related to ocular aberrations after customized LASIK for myopia using three different microkeratomes. METHODS Charts of 410 patients who underwent customized LASIK with the Alcon LADARVision4000 excimer laser were reviewed. Patients were stratified according to the device used to create the flap: Moria M2 microkeratome, Bausch & Lomb Hansatome microkeratome, or IntraLase laser. The difference between the wavefront pre- and postoperative value received a positive or a negative sign if the change occurred toward or away from zero, respectively, and it was compared to preoperative minus postoperative manifest refraction spherical equivalent (MRSE). RESULTS Patients showed increase in the aberration level after LASIK with the three devices used in this study. IntraLase spherical aberration change tended to be better than mechanical microkeratomes for higher MRSE values (IntraLase compared to Hansatome, P < or = .023 for MRSE values > or = 4.00 diopters [D]; IntraLase compared to Moria, P < or = .015 for MRSE values > or = 2.00 D). For total aberrations, the improvement values for IntraLase tended to be higher than those for Moria (IntraLase compared to Moria, P < or = .021 for MRSE values > or = 3.00 D). For total higher order aberrations, IntraLase values tended to be better than Moria and Hansatome microkeratomes (IntraLase compared to Hansatome, P < or = .047 for MRSE values between 3.00 and 8.00 D; IntraLase compared with Moria, P < or = .002 for MRSE values > or = 2.00 D). Change in coma root-mean-square was similar for the three groups. CONCLUSIONS The findings suggest the femtosecond laser provides a better platform for LASIK than the commonly used microkeratomes analyzed in this study.

Surface wave elastometry of the cornea in porcine and human donor eyes.

PURPOSE To introduce a nondestructive technique for characterization of corneal stiffness, determine measurement precision, and investigate comparative stiffness values along central, radial, and circumferential vectors in porcine corneas. The effects of epithelial debridement, relaxing incisions, and crosslink-mediated stiffening on surface wave velocity are also studied. METHODS A handheld prototype system was used to measure ultrasound surface wave propagation time between two fixed-distance transducers along a ten-position map. Repeatability was assessed with replicate measurements in 6 porcine corneas. In 12 porcine globes with controlled intraocular pressure (IOP), serial measurements were performed before and after epithelial removal, then after 250- and 750-microm-deep relaxing incisions. In human globes with constant intravitreal pressure, central wave velocity and transcorneal IOP measurements were compared before and after collagen cross-linking. RESULTS Measurement repeatability across all regions was between 2.2% and 8.1%. Epithelial removal resulted in increases in measured stiffness in 67% of eyes, but statistical power was insufficient to detect a systematic change. Wave velocity across a central incision decreased significantly after 250-microm keratotomy (P < .001), but did not undergo a significant further decrease with deeper keratotomy. Meridional stiffness changes consistent with coupling effects were detected after keratotomy. Surface wave velocity and transcorneal IOP measurements increased markedly after collagen cross-linking despite maintenance of a constant IOP. CONCLUSIONS Handheld corneal elastometry provides a repeatable measure of regional stiffness changes after relaxing incisions and collagen cross-linking in in vitro experiments. Surface wave elastometry allows focal assessment of corneal biomechanical properties that are relevant in refractive surgery, ectatic disease, and glaucoma.

Collagen crosslinking with riboflavin and ultraviolet-A in eyes with pseudophakic bullous keratopathy

PURPOSE: To evaluate the safety and efficacy of corneal collagen crosslinking (CXL) in patients with painful pseudophakic bullous keratopathy (PBK). SETTING: University of São Paulo, São Paulo and Sadalla Amin Ghanem Eye Hospital, Joinville, Santa Catarina, Brazil. METHODS: This prospective study included consecutive eyes with PBK that had CXL. After a 9.0 mm epithelial removal, riboflavin 0.1% with dextran 20% was applied for 30 minutes followed by ultraviolet‐A irradiation (370 nm, 3 mW/cm2). Therapeutic contact lenses were placed for 1 week. Corneal transparency, central corneal thickness (CCT), and ocular pain were assessed preoperatively and 1 and 6 months postoperatively. Statistical analysis was by paired t tests. RESULTS: Fourteen patients (14 eyes) with a mean age 71.14 years ± 11.70 (SD) (range 53 to 89 years) were enrolled. Corneal transparency was better in all eyes 1 month after surgery. At 6 months, corneal transparency was similar to preoperative levels (P = .218). The mean CCT was 747 μm preoperatively and 623 μm at 1 month; the decrease was statistically significant (P<.001). At 6 months, the mean CCT increased to 710 μm, still significantly thinner than preoperatively (P = .006). Pain scores at 6 months were not significantly different than preoperatively (P = .066). CONCLUSIONS: Corneal CXL significantly improved corneal transparency, corneal thickness, and ocular pain 1 month postoperatively. However, it did not seem to have a long‐lasting effect in decreasing pain and maintaining corneal transparency in patients with PBK. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Pupil size in refractive surgery candidates.

PURPOSE To assess pupil size measurements obtained under scotopic and mesopic conditions with the Procyon pupillometer and under photopic conditions with the Humphrey videokeratographer. METHODS The pupil sizes of 96 candidates for refractive surgery (192 eyes) were measured with the Procyon pupillometer PS2000 SA and the Humphrey Atlas 992 corneal topographer. Anisocoria and pupillary unrest were analyzed according to gender (two groups: 51 females and 45 males), age (five groups: 20 to 30 yr, 31 to 40 yr, 41 to 50 yr, 51 to 60 yr, older than 60 yr) and level of refraction (five groups: >-6.00 D SE, -6.00 to -3.00 D SE, -3.00 to 0 D SE, 0 to +2.50 D SE, +2.50 to +5.00 D SE). RESULTS The median value of pupil diameter measured with the Procyon pupillometer at the scotopic (0.04 lux), mesopic-low (0.4 lux), and mesopic-high (4 lux) levels of illumination were 6.54+/-0.88 mm; 5.62+/-0.95 mm, and 4.09+/-0.76 mm, respectively. The median pupil size with the Humphrey topographer was 3.65+/-0.62 mm. Pupillary unrest was highest at the mesopic-high level of illumination, with a median value of 0.31+/-0.34 mm. Median pupil size measured with both instruments at all light levels dropped significantly after the fifth decade of life (P<.05, ANOVA). CONCLUSIONS The Procyon pupillometer and Humphrey videokeratographer revealed an inverse correlation between the pupil size and the age, but no relationship with gender or level of refraction. The Procyon pupillometer provides an objective method for measuring pupil size at controlled light levels with a permanent printed record.

Topographic, corneal wavefront, and refractive outcomes 2 years after collagen crosslinking for progressive keratoconus.

Purpose: The aim was to report the corneal higher-order aberrations (HOA), the topographic metrics, and the visual and refractive outcomes 2 years after performing collagen crosslinking (CXL) for progressive keratoconus. The correlation among corneal HOAs, topographic metrics, and visual acuity changes was also investigated. Methods: This is a prospective case series involving 42 eyes from 32 patients with progressive keratoconus treated with CXL. The main outcomes measured at baseline and 6, 12, and 24 months after treatment were uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), refractive changes, topographic data, and corneal aberrations. Results: Two years after CXL treatment, the UDVA (P < 0.001), CDVA (P < 0.001), and spherical equivalent (P = 0.048) improved significantly. The corneal topographic data revealed significant decreases in apical keratometry (P < 0.001), differential keratometry (P = 0.031), and central keratometry (P = 0.003) compared with the baseline measurements. Aberration analyses revealed a significant reduction in coma (P = 0.016), trefoil (P = 0.018), secondary astigmatism (P < 0.001), quatrefoil (P = 0.031), secondary coma (P < 0.001), and secondary trefoil (P = 0.001). Corneal HOA (except quatrefoil) demonstrated a significant correlation with postoperative CDVA; the highest correlations were for coma (rho = 0.703, P < 0.001), secondary astigmatism (rho = 0.519, P = 0.001), and total HOA (rho = 0.487, P = 0.001). However, the corneal HOA changes were not statistically associated with improved visual acuity. After treatment, the reduction in apical keratometry was the only variable that correlated with the improvement in the CDVA (rho = 0.319, P = 0.042). Conclusions: After 2 years, CXL was found to be effective in improving the UDVA, CDVA, topographic metrics, and most corneal HOAs in eyes with progressive keratoconus. A significant reduction was observed in apical keratometry, and this reduction directly correlated with an improvement in visual acuity.

Wavefront analysis in normal refractive surgery candidates

PURPOSE To quantify the higher order aberrations of refractive surgery candidates and compare the wavefront-determined refractions with manifest refractions refined with a +/- 0.25 Jackson cross cylinder. METHODS Results of 226 consecutive patients (418 eyes) were analyzed with the WaveScan WavePrint system (VISX, Santa Clara, Calif). Only patients with normal eyes without previous surgery were included. RESULTS The mean spherical equivalent refraction determined with wavefront analysis was -3.40 +/- 3.14 diopters (D) (range: -10.72 to +5.41 D). The largest amount of higher order aberrations was detected with : a 6-mm pupil diameter (coma 0.14 +/- 0.08 microm; trefoil 0.10 +/- 0.07 microm; spherical aberrations 0.09 +/- 0.07 microm). The mean root-mean-square of higher order aberrations and total aberrations were 0.23 +/- 0.11 microm and 4.00 +/- 2.45 microm, respectively. No statistically significant correlation was noted between higher order aberrations and gender (P = 0.7) or between higher order aberration and refractive level (P > .59). The mean differences in spherical equivalent refraction, sphere, and cylinder between WaveScan measurements and manifest refraction were 0.36 +/- 0.41 D, 0.40 +/- 0.44 D, and 0.28 +/- 0.32 D, respectively. CONCLUSIONS This study provides reference values for higher order aberrations in normal refractive surgery candidates. Wavefront analysis also proved to be a valuable tool for objectively measuring preoperative refractive error.