Anne Huhtala

Eight-year follow-up of photorefractive keratectomy for myopia.

PURPOSE: We evaluated 8-year results of excimer laser photorefractive keratectomy (PRK) for myopia in terms of stability and late complications. METHODS: Ninety-two myopic eyes of 55 patients were treated with a single-step method using an Aesculap-Meditec MEL 60 excimer laser with a 5.0-mm ablation zone. Treated eyes were divided into three groups according to preoperative refraction: low myopes (≤-6.00 D), medium myopes (-6.10 to -10.00 D), and high myopes (>-10.00 D). RESULTS: Change in myopic regression stabilized in all myopia groups within 12 months, although a small myopic shift occurred up to 8 years after PRK. Mean change in refraction between 2 and 8 years was -0.42 ± 0.48 D for low myopes, -0.37 ± 0.34 D for medium myopes, and -0.41 ± 0.50 D for high myopes. The percentage of eyes within ±1.00 D of emmetropia 8 years after PRK was 78.3% in the low myopia group, 68.8% in the medium myopia group, and 57.1% in the high myopia group. One eye lost 2 lines of best spectacle-corrected visual acuity due to irregular astigmatism. In 13.0% of eyes, a residual trace corneal haze was observed, which had no effect on visual acuity. Apart from the loss of 2 lines of BSCVA in one eye, there were no other late complications during the study period. CONCLUSIONS: The mean change in refraction between 2 and 8 years was less than -0.50 D, regardless of preoperative refraction, and may be attributed to natural age-related refractive change. The appearance of residual corneal haze after 8 years correlated with the amount of myopic correction. PRK was a safe and stable surgical procedure in this group of patients.

Comparison of an Immortalized Human Corneal Epithelial Cell Line and Rabbit Corneal Epithelial Cell Culture in Cytotoxicity Testing

The cytotoxicity of benzalkonium chloride (BAC) and disodium edetate (EDTA) was evaluated in vitro in rabbit corneal epithelial primary cells and in the immortalized human corneal epithelial cell line SV40. Cell injury was assessed by lactate dehydrogenase (LDH) leakage and by reduction of the tetrazolium salt WST-1 to formazan by mitochondrial metabolic activity. Cell cultures were exposed to test compounds both in serum-free and in serum-containing medium. Although WST-1 and LDH tests measured different physiological endpoints, they yielded comparable results. However, the LDH test seemed less reliable due to great variation. The use of serum was found to result in lower toxicity of the compounds in both tests. The rabbit primary cell culture and the human corneal cell line were quite similar in their responses to BAC and EDTA. The human cell line is a promising in vitro alternative in oculotoxicity testing.

The Cytotoxic Effects of Preserved and Preservative-Free Prostaglandin Analogs on Human Corneal and Conjunctival Epithelium In Vitro and the Distribution of Benzalkonium Chloride Homologs in Ocular Surface Tissues In Vivo

Purpose: To investigate the cytotoxicity of benzalkonium chloride (BAC)-containing ophthalmic solutions of prostaglandin analogs (latanoprost, travoprost, bimatoprost, and preservative-free (PF) tafluprost), BAC mixture (BACmix) and BAC homologs with different alkyl chain lengths using human corneal epithelial (HCE) and conjunctival epithelial (IOBA-NHC) cell cultures. The distribution of BAC homologs in rabbit ocular surface tissues in vivo was examined. Methods: The cells were exposed for one hour to prostaglandin analogs, BACmix and three homologs. Cytotoxicity was assessed with the WST-1 and lactate dehydrogenase (LDH) assays for cellular viability and cell membrane integrity. BAC 0.02% solution was instilled on the rabbit eye daily for 14 days and the concentrations of BAC homologs in external ocular tissues were determined. Results: The order of decreasing cytotoxicity in the WST-1 test was latanoprost ≥ travoprost > bimatoprost ≥ PF tafluprost. IOBA-NHC cells were more sensitive than HCE cells. In HCE, only latanoprost diluted to 10% increased LDH leakage. In IOBA-NHC, LDH leakage was statistically significant with 3–10% travoprost and 10% latanoprost. The order of decreasing cytotoxicity of preservatives was C14 > C12 > BACmix > C16 in HCE and C12 > C14 > BACmix > C16 in IOBA-NHC. Following treatment with BAC 0.02% solution, the amounts of BAC-C12, -C14 and -C16 in rabbit cornea and conjunctiva, respectively were: 0.37 ± 0.08 and 2.64 ± 0.27 ng/mg; 0.42 ± 0.07 and 4.77 ± 0.43 ng/mg; 0.04 ± 0.01 and 0.54 ± 0.05 ng/mg. Conclusions: The cytotoxic effects of latanoprost, travoprost, and bimatoprost were dependent on the BAC concentration in their formulations. BACmix was cytotoxic at the concentrations above those corresponding to 0.001% BAC in ophthalmic medications. PF tafluprost was the least toxic of the drugs tested. Within studied BAC homologs, those with longer alkyl chain and higher lipophility penetrated effectively into rabbit external ocular tissues.

Geldanamycin increases 4-hydroxynonenal (HNE)-induced cell death in human retinal pigment epithelial cells

Development of age-related macular degeneration (AMD) is associated with functional abnormalities and cell death in retinal pigment epithelial (RPE) cells attributable to oxidative stress. To minimize the adverse effects of oxidative stress, cells activate their defence systems, e.g., via increased expression of heat shock protein (Hsp), activation of stress sensitive AP-1 and NF-kappaB transcription factors. In this study, we examined the accumulation of Hsp70 protein, activation of AP-1 and NF-kappaB transcription factors in human ARPE-19 cells subjected to a 4-hydroxynonenal (HNE)-induced oxidative stress. In addition, the influence of Hsp90 inhibitor geldanamycin (GA) was studied in HNE-treated cells. Mitochondrial metabolic activity and apoptosis were determined to evaluate cell death in the ARPE-19 cells. The ARPE-19 cells showed increased accumulation of Hsp70 protein before of the cytotoxic hallmarks appearing in response to HNE. In contrast, increased DNA-binding activities of AP-1 or NF-kappaB transcription factors were not seen under HNE insults. Interestingly, GA significantly increased cell death in the HNE-treated cells, which was involved in caspase-3 independent apoptosis. This study reveals that the Hsps have an important role in the cytoprotection of RPE cells subjected to HNE-derived oxidative stress.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

LASIK flap creation with the Ziemer femtosecond laser in 787 consecutive eyes.

PURPOSE To present the flap characteristics and short-term efficacy and safety of 787 consecutive LASIK procedures with the FEMTO LDV femtosecond laser (Ziemer Ophthalmic Systems) for the treatment of refractive errors. METHODS Seven hundred eighty-seven consecutive eyes of 405 previously non-operated patients were treated with the FEMTO LDV. Intended flap thickness was 110 microm and intended flap diameter varied from 8.5 to 9.5 mm. Refractive treatment was performed with the WaveLight ALLEGRETTO WAVE Concerto 500 Hz excimer laser. All eyes were wavefront-optimized. RESULTS The mean flap thickness, measured by ultrasound pachymetry, was 90.0+/-5.5 microm (range: 67 to 107 microm) in right eyes and 90.1+/-4.6 microm (range: 77 to 106 microm) in left eyes. Mean flap diameter was 9.1+/-0.2 mm (range: 8.4 to 9.9 mm) in right eyes and 9.1+/-0.2 mm (range: 8.0 to 10.0 mm) in left eyes. Increasing flap thickness was correlated with increasing corneal thickness in right eyes and flatter keratometric value K(1) in left eyes. The most common complication was minor bleeding during the procedure (12.7%). All other complications were rare (8.4%), and none prevented further laser ablation. CONCLUSIONS The Ziemer FEMTO LDV laser created thinner LASIK flaps than intended but with a low standard deviation and minimal intraoperative complications.

A collaborative evaluation of the cytotoxicity of two surfactants by using the human corneal epithelial cell line and the WST-1 test

This study was undertaken to investigate the use of the in vitro test WST-1, an assay of cell proliferation and viability, for a preliminary safety evaluation of topical ophthalmic preparations. The cytotoxicity of two surfactants, benzalkonium chloride (BAC) and polyoxyethylene-20-stearyl ether (Brij78, PSE) was independently investigated in four laboratories in the EU by using an immortalized human corneal epithelial (HCE) cell line. The HCE cells were exposed to BAC and PSE for 5 min, 15 min, and 1 hour, and the results of the HCE-WST-1 tests were collected and compared. After one-hour exposure, the EC(50) values in BAC-treated cells in the presence of serum ranged between 0.0650 +/- 0.0284 (mean +/- SD) mM, and those in the absence of serum 0.0296 +/- 0.0081 mM. The corresponding values for PSE were 0.0581 +/-.0300 mM and 0.0228 +/-.0063 mM. There were variations in the results between different laboratories, with coefficients of variation ranging from 31 to 121%, mean 58%. The use of one-hour exposure time is to be preferred, and the elimination of serum in the culture medium is recommended to avoid both underestimation of toxic effects and variability of the test results.

Corneal Flap Measurements in Laser in situ Keratomileusis Using the Moria M2 Automated Microkeratome

PURPOSE To evaluate accuracy and predictability and factors that influence the dimensions of the laser in situ keratomileusis (LASIK) corneal flap created with the Moria M2 automated microkeratome (Moria SA, Antony, France). METHODS The flap thickness of 454 eyes of 243 consecutive patients was measured using subtraction ultrasonic pachymetry during LASIK with the Moria M2 microkeratome head 130 designed to create a 160-microm-thick flap. Flap dimensions were evaluated and measurements were correlated with preoperative parameters. A stepwise regression analysis was used to determine the factors that influenced actual flap thickness. RESULTS The preoperative spherical equivalent refraction of the 454 eyes ranged from -12.125 diopters (D) to +6.25 D. Patient age ranged from 18 to 57 years (mean age: 31.3 +/- 8.8 years). Mean preoperative keratometric power K1 was 44.31 +/- 1.59 D and K2 was 43.32 +/- 1.54 D. Mean preoperative central comeal thickness was 552.4 +/- 32.5 microm (range: 466 to 665 microm). With an attempted thickness of 160 microm, the Moria M2 flap thickness ranged from 77 to 209 microm (mean: 153.3 +/- 19.0 microm). Mean horizontal flap diameter was 9.2 +/- 0.2 mm and mean hinge length 4.6 +/- 0.3 mm. Increasing flap thickness was found to correlate with increasing preoperative comeal thickness, younger patient age, and flatter preoperative keratometric power K1. CONCLUSIONS Although the standard deviation of the flap thickness was relatively small, remarkable individual variation was noted. Therefore, the intraoperative calculation of the remaining stromal bed is recommended. Furthermore, the consideration of central corneal thickness, patient age, and preoperative keratometry are helpful parameters to avoid too deep ablation.

The Effects of 5-Fluorouracil on Ocular Tissues In Vitro and In Vivo after Controlled Release from a Multifunctional Implant

PURPOSE To evaluate the effects of 5-fluorouracil (5-FU) on ocular cells in vitro and the effects of degradable 5-FU-loaded poly(DL-lactide-co-glycolide; PDLGA) 50:50 implant in the rabbit eye in vivo. METHODS Cytotoxicity was assessed with a tetrazolium salt WST-1 cell proliferation/viability test and a lactate dehydrogenase (LDH) leakage test in rabbit corneal stromal fibroblasts (SIRCs), bovine corneal endothelial cells (BCECs), human conjunctival epithelial cells (IOBA-NHCs), human retinal pigment epithelial cells (ARPE-19), and human corneal epithelial cells (HCECs). The 5-FU-loaded PDLGA implants were surgically placed in rabbit eyes with a deep sclerectomy technique and the histopathology of the eyes was examined. RESULTS In vitro, 5-FU affected cell proliferation and survival in a time- and dose-dependent manner. In the WST-1 test, adverse effects in serum-free conditions started from 0.0005 mg/mL 5-FU in SIRCS and HCECs, whereas in other cell types, 0.005 mg/mL 5-FU hindered cell proliferation. In serum-free conditions 72-hour 5 mg/mL 5-FU treatment decreased cell viability to 40% in BCECs and to 10% to 15% in other cell types. 5-FU had no or very minor effects on LDH leakage. In vivo, the 5-FU implant showed no signs of toxicity in cornea and retina, whereas in the conjunctival stroma near the implantation site, some inflammatory cells and a marked subepithelial condensation of stromal connective tissue was observed during the postoperative period of 4 weeks. CONCLUSIONS 5-FU had a broad therapeutic range, and the 5-FU implant showed only minor tissue reactions in conjunctiva at the surgical site. 5-FU is a possible candidate for controlled drug release.

Bilateral comparison of corneal flap dimensions with the Moria M2 reusable head and single use head microkeratomes.

PURPOSE To compare the Moria (Antony, France) M2 automated microkeratome with the head 130 to a new disposable single use head to evaluate complications, accuracy, and safety of the procedure. METHODS Ninety-eight eyes of 49 consecutive patients were operated with the Moria M2 microkeratome. One eye was operated with the metallic head 130 and the other with a plastic single use head, both designed to create a 160-microm flap. Intraoperative flap dimensions were correlated to preoperative parameters and evaluated 1 month postoperatively. RESULTS With the head 130, mean thickness was 153.3 microm (standard deviation [SD] 13.3, range: 102 to 179 microm). When using a single use head, mean thickness was 148.0 microm (SD 9.8, range: 120 to 170 microm). Occasional iron particles were observed in one eye with both head types. No true epithelial ingrowth was detected in any of the eyes, but epithelial dots at the wound edge occurred in one eye, when using the head 130, but not in the eyes operated with a single use head. CONCLUSIONS On average, both head types created thinner flaps than attempted. Single use heads produced thinner flaps than the head 130. Accuracy in flap thickness in terms of standard deviation was significantly better in single use heads than in the head 130. Single use heads also had fewer microkeratome-related complications. In clinical practice, the single use head was easier to use because no assembly was required. Plastic single use heads also worked more smoothly than the metallic head 130.