Won Ryang Wee

Oxygen free radical damage in the cornea after excimer laser therapy

AIMS/BACKGROUND To evaluate the extent of oxygen radical damage in the cornea after excimer laser ablation. METHODS The 193 nm argon fluoride excimer laser was programmed for an average fluence of 150 mJ/cm2, with a firing rate of 5 Hz and an ablation zone diameter of 6 mm. Phototherapeutic keratectomy was performed to remove 30 μm of epithelium and 50 μm of stroma from the corneas of New Zealand white rabbits. Oxidative tissue damage after laser was determined by measuring oxidised lipids (conjugated dienes and ketodienes) in corneal lipid extracts, and by fast blue B staining to localise the lipid peroxide in the tissue. RESULTS Conjugated diene levels were 3.73 (SD 0.56) nmol per hemicornea in ablated corneas and 1.99 (0.33) nmol per hemicornea in normal corneas (p = 0.0044). Ketodiene levels were 2.72 (0.38) nmol per hemicornea in treated corneas and 0.91 (0.12) nmol per hemicornea in normal corneas (p < 0.001). Fast blue B staining disclosed that the tissue damage occurred primarily on the surface of the ablated cornea. CONCLUSION The presence of lipid peroxidation in the superficial corneal stroma in excimer laser treated corneas was demonstrated. This lipid peroxidation could be from oxygen free radicals generated by the infiltrating polymorphonuclear cells at the site of tissue damage.

Comparison of in vitro antiproliferative effects of steroids and nonsteroidal antiinflammatory drugs on human keratocytes.

Corticosteroids are currently administered after photorefractive keratectomy (PRK) to reduce corneal haze and myopic regression. However, the beneficial effects of corticosteroids are controversial, and they are associated with many side effects. In this study we compared the in vitro antiproliferative effects of nonsteroidal antiinflammatory drugs (NSAIDs), diclofenac and flurbiprofen, with those of dexamethasone on human keratocytes. Human keratocytes were incubated with various concentrations of diclofenac, flurbiprofen, and dexamethasone. The control samples were incubated under the same conditions except for the absence of drugs. Proliferation of the keratocytes was measured by [3H]thymidine uptake into DNA on days 1, 2, and 4. Diclofenac was the most potent agent, inducing dose-dependent inhibition at concentrations ≥101 mM on days 1 and 2, and ≥10-1 mM on day 4. Flurbiprofen followed closely, inhibiting keratocytes at and above 1 mM on day 1, 10-1 mM on day 2, and 10-4 mM on day 4. Dexamethasone was the least effective, exhibiting inhibition at and above 25 mM on day 1, 5 mM on day 2, and 1 mM on day 4 (Wilcoxon rank sum test, p ≤0.05). The ID5Oδ reflect the same trend (day 4: diclofenac=0.03 mM, flurbiprofen=0.2 mM, dexamethasone=3.2 mM). In addition, diclofenac and dexamethasone showed time-dependent antiproliferative effects. These results indicate that NSAIDs are more potent than corticosteroids in inhibiting proliferation of human keratocytes in vitro, and suggest a potential use of NSAIDs in modulating corneal wound healing after PRK

Effects of White-To-White Diameter and Anterior Chamber Depth on Implantable Collamer Lens Vault and Visual Outcome

PURPOSE To investigate the effects of anterior chamber depth (ACD) and white-to-white (WTW) diameter on vault in eyes with implantable collamer lenses (ICL V4, STAAR Surgical AG) and to evaluate the effect of vault on visual outcomes and other biometric changes. METHODS Twenty-eight eyes of 16 patients who underwent ICL implantation were retrospectively reviewed. Patients were divided into two groups (A1, A2) according to preoperative ACD (> or = 3.3 mm and < 3.3 mm, respectively), two groups (B1, B2) based on WTW diameter (> or = 11.55 mm and < 11.55 mm, respectively), and two groups (C1, C2) according to the difference of ICL diameter and sulcus length (> or = 0.25 mm and < 0.25 mm, respectively). Vault, endothelial cell density, and visual performance were compared between each set of groups. Correlations of vault with WTW diameter, ACD, and diametric difference of the ICL from the sulcus were analyzed. RESULTS The high ACD and WTW groups showed significantly higher vaults (0.59 +/- 0.32 mm and 0.57 +/- 0.36 mm, respectively) than the low ACD and WTW groups (0.26 +/- 0.17 mm and 0.25 +/- 0.14 mm, respectively; P = .01 and .01), whereas the differences of diameter between the sulcus and ICL were not related to significant vault differences. The WTW diameter wascorrelated more closely to the vault than the ACD or sulcus diameter (r = 0.70, P < .001), whereas the vault difference did not affect visual quality or endothelial cell density. CONCLUSIONS A high WTW diameter or ACD is likely to render a high vault, regardless of any difference in the ICL and sulcus diameters.

Contrast sensitivity after LASIK, LASEK, and wavefront-guided LASEK with the VISX S4 laser

PURPOSE To evaluate the changes in contrast sensitivity and glare after corneal refractive surgery for moderate myopia. METHODS The study comprised 267 eyes of 149 patients who underwent LASIK, laser epithelial keratomileusis (LASEK), or wavefront-guided LASEK with the VISX STAR S4. Contrast sensitivity was measured with VCTS 6500 before surgery and at 2 and 6 months postoperatively. Glare was evaluated using questionnaires and the ALC glare test at last follow-up. Contrast sensitivity at 2 and 6 months was compared with preoperative contrast sensitivity in each group. Glare sensitivity in each group was compared to normal controls. RESULTS Contrast sensitivity did not change after LASIK (P > .05). Contrast sensitivity of 3 and 6 cycles per degree (cpd) at 2 months increased after LASEK (P < .05) and contrast sensitivity of 6, 12, and 18 cpd at 2 months and 3, 6, 12, and 18 cpd at 6 months improved in wavefront-guided LASEK (P < .05). Glare test and questionnaires did not show any significant difference between the groups. CONCLUSIONS Wavefront-guided LASEK induced significant increases in contrast sensitivity of moderate myopes at high spatial frequencies under mesopic conditions by 6 months. Glare was not affected by the different refractive procedures.

Effect of artificial tears on cultured keratocytes in vitro

We investigated the effects of artificial tears on cultured rabbit and human keratocytes in vitro. The cells were exposed to seven nonpreserved commercially available artificial tear formulations and examined under phasecontrast microscopy for 60 min. After 5-min exposures to the solutions, rabbit keratocytes were fixed for transmission electron microscopy (TEM). In rabbit keratocytes, phase-contrast microscopy and TEM demonstrated that Aqua Site (CIBA Vision Ophthalmics, Atlanta, GA, U.S.A.), Hypo Tears PF (Johnson & Johnson, Claremont, CA, U.S.A.), and Tears Naturale Free (Alcon, Humacao, Puerto Rico, U.S.A.) immediately induced intracytoplasmic vacuoles and cell swelling, and subsequent cell degeneration. Rabbit cells exposed to the other artificial tears, which contained Ca2+ and did not contain EDTA, maintained their normal shape and appearance for 60 min. Phase-contrast microscopy of human keratocytes showed that Aqua Site and Hypo Tears PF induced mild and delayed cellular swelling, but the other artificial tears tested did not affect the cell shape for the entire 60-min observation period. Electrolyte balance and osmolarity of artificial tears appear to be critical for keratocyte survival. Maintenance of keratocyte integrity may be an important factor to consider when selecting an artificial tear preparation to be used when corneal epithelium is not intact.

Holmium:YAG Laser Thermokeratoplasty for Astigmatism in Rabbits

BACKGROUND Holmium:YAG laser thermokeratoplasty has generated considerable interest as a technique for correcting hyperopia. In this study, the effect of holmium:YAG laser on inducing astigmatism according to application patterns was evaluated. METHODS An experimental study based on the results of astigmatic holmium:YAG laser thermokeratoplasty using the Summit OmniMed laser system (Summit Technology Inc, Waltham, Mass) in 36 rabbit eyes is presented. We divided the rabbits into four groups: arcuate, reverse arcuate, linear, and control group according to application patterns. All rabbits were followed for 3 months and cycloplegic refractive measurements were carried out. RESULTS The average surgically induced astigmatism was 1.86 diopters (D) for the arcuate group, 2.93 D for the reverse arcuate group, and 1.31 D for the linear group. No significant complications related to the operation were noted. CONCLUSION The reverse arcuate pattern of holmium:YAG laser thermokeratoplasty is most effective in inducing astigmatism in rabbits.

Keratocyte-Populated Collagen Gel as an In Vitro Model of Excimer Laser Keratectomy

BACKGROUND To develop an in vitro model to study the effects of excimer laser keratectomy on corneal stromal cells, we evaluated two types of collagen gel populated with keratocytes. METHODS Keratocyte-populated collagen gels were prepared with type I collagen in 6-well plates or in culture plate inserts, the bottom of which consisted of a nitrocellulose membrane, contained within 6-well plates. The gels were ablated by the 193-nm excimer laser, set to ablate 50, 100, or 200 microns deep, and was observed under a phase-contrast microscope for 2 days. RESULTS Keratocytes cultured in collagen gel developed cytoplasmic processes and formed networks of interconnected cells. Cells within the ablated area in the 6-well plates began to lose their cytoplasmic processes and became round approximately 3 hours after excimer laser ablation. These cellular changes were more prominent in the gels ablated to a depth of 200 microns. Cells outside of the ablation zones in the 6-well plates and the culture plate inserts remained intact. CONCLUSIONS These results suggest the use of keratocyte-populated collagen gel as an in vitro model of cellular response to excimer laser keratectomy and also suggest that gel prepared in culture plate inserts is the preferred method.

Effects of gamma-interferon on keratocyte-induced collagen gel contraction and keratocyte proliferation

PURPOSE Gamma-interferon has been shown to be an effective immunoregulatory polypeptide that can modulate fibroblastic response. We investigated the effects of gamma-interferon on keratocyte proliferation and keratocyte-induced collagen gel contraction. METHODS Gamma-interferon in concentrations of 0.01, 1, 100, and 1000 U/ml of media was added to keratocytes embedded in polymerized type I collagen and the gel area was measured after 5 days with an image analysis system. The rate of keratocyte proliferation within and outside the collagen gel under the influence of gamma-interferon was also investigated. RESULTS Keratocyte-induced collagen gel contraction was significantly inhibited at all concentrations above 0.01 U/ml. The keratocyte proliferation was not affected by low and moderate concentrations and was significantly stimulated at concentration of 1000 U/ml. CONCLUSION Keratocyte-induced collagen gel contraction is inhibited by gamma-interferon and the mechanism of this effect is not inhibition of keratocyte proliferation by gamma-interferon.

Dichlorotriazinyl Aminofluorescein Inhibits Human Keratocyte Proliferation in Vitro

PURPOSE Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo experimentation. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. METHODS Human keratocytes in 96 well plates were exposed to different concentrations of DTAF (10e-7, 10e-6, 10e-5, 10e-4, 10e-3, 10e-2, and 10e-1 mg/ml of media). Exposure times of 1 and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after exposure to the drug using a coulter-counter and a hemocytometer. RESULTS The proliferation of keratocytes after 24 hours of exposure to the drug was inhibited in a dose dependent manner by DTAF, but 1 hour exposure of keratocytes to the drug did not inhibit keratocyte proliferation. CONCLUSION These results suggest that DTAF has inhibitory effects on human keratocyte proliferation after 24 hours of exposure, while exposure limited to 1 hour does not induce such a change.