L.E. Pillunat

Corneal Endothelial Cytotoxicity of Riboflavin/UVA Treatment in vitro

Recently, we have developed collagen crosslinking induced by combined riboflavin/UVA treatment, thus increasing the biomechanical rigidity of the cornea to treat progressive keratoconus. The present safety study was performed to evaluate possible cytotoxic effects of combined riboflavin/UVA treatment on the corneal endothelium in vitro. Endothelial cell cultures from porcine corneas were treated with 500 µM riboflavin solution, exposed to various endothelial UVA irradiances (370 nm) ranging from 0.1 to 1.6 mW/cm2 for 30 min and evaluated 24 h later using trypan blue staining and Yopro fluorescence staining. The effect of either treatment alone (UVA irradiation ranging from 0.2 to 6 mW/cm2) was also tested. An abrupt cytotoxic threshold irradiance level was found at 0.35 mW/cm2 after combined treatment with riboflavin plus UVA irradiation and at 4 mW/cm2 with UVA irradiation alone. Riboflavin alone was not toxic. A cytotoxic effect of the combined riboflavin/UVA treatment on corneal endothelial cells is to be expected with a corneal thickness of less than 400 µm. Therefore, pachymetry should be routinely performed before riboflavin/UVA treatment to exclude patients at risk.

Efficacy and safety of tafluprost 0.0015% versus latanoprost 0.005% eye drops in open-angle glaucoma and ocular hypertension: 24-month results of a randomized, double-masked phase III study

Purpose:  The objective of the study was to compare the long‐term efficacy and safety of tafluprost 0.0015% with latanoprost 0.005% eye drops in patients with open‐angle glaucoma or ocular hypertension.

[Biophysical principles of collagen cross-linking].

BACKGROUND The reduced mechanical stability of the cornea in keratoconus or in keratectasia after Lasik may be increased by photooxidative cross-linking of corneal collagen. The biophysical principles are compiled for the safe and effective application of this new treatment method. METHODS The setting of the therapy parameters should be elucidated from the absorption behaviour of the cornea. The safety of the method for the endothelium cells and the lens will be discussed. The induced cross-links are shown to be the result of changes in the physico-chemical properties of the cornea. RESULTS To reach a high absorption of the irradiation energy in the cornea, riboflavin of a concentration of 0.1% and UV light of a wavelength of 370 nm, corresponding to the relative maximum of absorption of riboflavin, were used. An irradiance of 3 mW/cm(2) and an irradiation time of 30 min lead to an increase of the mechanical stiffness. The endothelium cells will be protected due to the high absorption within the cornea, that means the damaging threshold of the endothelium cells will not be reached in a 400 microm thick stroma. As evidence for cross-links we can consider the increase of the biomechanical stiffness, the increased resistance against enzymatic degradation, a higher shrinkage temperature, a lower swelling rate and an increased diameter of collagen fibres. CONCLUSIONS The therapy parameters were tested experimentally and have been proven clinically in the corneal collagen cross-linking. These parameters should be respected to reach a safe cross-linking effect without damage of the adjacent tissues.

Biomechanical condition of the cornea as a new indicator for pathological and structural changes

AIM Several methods permit the measurement of geometric parameters of the cornea, but until now biomechanical conditions of the cornea have been ignored (e.g. in refractive corneal surgery). Besides the geometric condition, biomechanical properties of the cornea have been shown to influence applanation measurement of intra-ocular pressure (IOP) and epidemiological studies have identified corneal thickness as an independent risk factor for the development and progression of glaucoma. The aim of this investigation was to characterize the biomechanical properties of the cornea using the ocular response analyzer (ORA). METHODS The ocular response analyzer (ORA) is a new method available for non-contact measurement of the biomechanical properties of the cornea. We evaluated the reproducibility of measurements, the difference between static and dynamic factors and the impact of independent factors (e.g. IOP, age, CCT, swelling of the cornea) on 2,500 measurements of corneal hysteresis (CH) and corneal resistance factor (CRF). RESULTS In a large sample size we observed changes in CH and CRF after refractive surgery procedures (LASIK, UV-A cross-linking, keratoplasty) and in other corneal disorders (keratoconus, corneal dystrophies). CONCLUSIONS CRF and CH changes may reflect structural changes of the cornea. Thus, the ORA provides valuable information for a better understanding and characterization of the biomechanical condition of the cornea, especially with regard to diseases such as keratoconus and glaucoma.

Kollagenvernetzung mit Riboflavin und UVA-Licht bei Keratokonus – Dresdner Ergebnisse

PURPOSE The aim of this long-term retrospective study was to prove a long-term halting effect of riboflavin and UVA-induced collagen cross-linking in progressive keratoconus. METHODS Since 1998, within an ethics-committee-approved study (EK 310 499), patients with progressive keratoconus and a minimal corneal thickness of 400 microm have received cross-linking treatment. An increase of the maximum K-value by > or =1 D within the previous year, a patients statement of deteriorating visual acuity, or the need for a new contact lens fitting more than once in 2 years was considered progression. The maximum follow-up time was 7.5 years. At the first examination and all follow-up examinations, refraction, best corrected visual acuity, corneal topography, and ultrasound pachymetry were recorded. RESULTS The analysis included 153 eyes of 111 patients, with a minimal follow-up of 12 months. Keratectasia significantly decreased in the 1st year by 2.29 D, in the 2nd year by 3.27 D, and in the 3rd year by 4.34 D. Visual acuity improved significantly in at least one line or remained stable (i.e., no line loss) in the 1st year in 48.9% and 23.8%, respectively; in the 2nd year in 50.7% and 29.6%, respectively; and in the 3rd year in 60.6% and 36.4%, respectively. We saw no severe side effects. Three patients showed continuous progression of keratoconus and received cross-linking treatment again. Despite the small number of patients with a follow-up longer than 3 years, therefore limiting the statistical assertions, our results indicate long-term stabilization or improvement after collagen cross-linking. CONCLUSION With regard to the size of our cohort and the follow-up time, no comparable data have been published in the literature. The results of this study indicate that collagen cross-linking appears to be an effective therapeutic option for progressing keratoconus. Besides the clinical benefit, there are enormous economic and psychosocial benefits. Cross-linking is an outpatient, minimally invasive, cost-effective treatment involving minimal effort for the persons concerned.

[Collagen cross-linking with riboflavin and UVA light in keratoconus. Results from Dresden].

PURPOSE The aim of this long-term retrospective study was to prove a long-term halting effect of riboflavin and UVA-induced collagen cross-linking in progressive keratoconus. METHODS Since 1998, within an ethics-committee-approved study (EK 310 499), patients with progressive keratoconus and a minimal corneal thickness of 400 microm have received cross-linking treatment. An increase of the maximum K-value by > or =1 D within the previous year, a patients statement of deteriorating visual acuity, or the need for a new contact lens fitting more than once in 2 years was considered progression. The maximum follow-up time was 7.5 years. At the first examination and all follow-up examinations, refraction, best corrected visual acuity, corneal topography, and ultrasound pachymetry were recorded. RESULTS The analysis included 153 eyes of 111 patients, with a minimal follow-up of 12 months. Keratectasia significantly decreased in the 1st year by 2.29 D, in the 2nd year by 3.27 D, and in the 3rd year by 4.34 D. Visual acuity improved significantly in at least one line or remained stable (i.e., no line loss) in the 1st year in 48.9% and 23.8%, respectively; in the 2nd year in 50.7% and 29.6%, respectively; and in the 3rd year in 60.6% and 36.4%, respectively. We saw no severe side effects. Three patients showed continuous progression of keratoconus and received cross-linking treatment again. Despite the small number of patients with a follow-up longer than 3 years, therefore limiting the statistical assertions, our results indicate long-term stabilization or improvement after collagen cross-linking. CONCLUSION With regard to the size of our cohort and the follow-up time, no comparable data have been published in the literature. The results of this study indicate that collagen cross-linking appears to be an effective therapeutic option for progressing keratoconus. Besides the clinical benefit, there are enormous economic and psychosocial benefits. Cross-linking is an outpatient, minimally invasive, cost-effective treatment involving minimal effort for the persons concerned.

Enzymatischer Nachweis der Tiefenabhängigkeit der Vernetzungswirkung von Riboflavin/UVA an der Hornhaut

PURPOSE It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically. METHOD Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior. RESULTS The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h. CONCLUSIONS The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.

Enhanced pressure in the central retinal vein decreases the perfusion pressure in the prelaminar region of the optic nerve head.

PURPOSE The pressure in the central retinal vein (CRVP) has been shown to be higher in glaucoma patients than in controls. Until now, these measurements have been performed in arbitrary units or in units of ophthalmodynamometric force. In our study, a contact lens dynamometer, calibrated in mm Hg, was used to calculate the retinal perfusion pressure. METHODS A total of 27 patients with primary open angle glaucoma (POAG) and 27 healthy control subjects were included in the study. The IOP measurement included Goldmann applanation tonometry, whereas the pressure enhancement measurement consisted of contact lens dynamometry. results: the pressures are given in mm hg, and are expressed as the mean ± SD for the control subjects versus the POAG patients: IOP 14.4 ± 2.7 vs. 15.4 ± 2.9, systolic blood pressure 141 ± 20.1 vs. 153 ± 16.5 (P = 0.013), central retinal vein threshold pressure (CRVTP) 11.9 ± 3.8 vs. 16.8 ± 5.0, CRVP 15.0 ± 2.7 vs. 17.9 ± 4.2, and retinal perfusion pressure (PPret) standard 84 ± 12.2 vs. 94 ± 9.1 and new 83 ± 12.2 vs. 91 ± 9.6. The differences in PPret between using the new versus the standard method are 0.55 ± 1.33 vs. -2.5 ± 3.89 (P = 0.041 and P = 0.002, respectively). The PPret was at least 5.0 mm Hg lower in 5 of the 27 POAG patients when the new calculation method was used. CONCLUSIONS The perfusion pressure in the retina and prelaminar region of the optic nerve head (ONH) may be lower than expected because the CRVP may be higher. The pressure measurement in the central retinal vein may be a step toward a better understanding of ONH pathophysiology.

Okuläre Hämodynamik und Gesichtsfeld beim Glaukom unter Dorzolamid-Therapie

ZusammenfassungZielsetzung. Untersucht wird der Einfluss einer lokalen Dorzolamid-Applikation auf das Gesichtsfeld und die okuläre Hämodynamik beim Glaukom. Methodik. In einer retrospektiven, nicht maskierten klinischen Studie wurden 28 Patienten (28 Augen) mit primär chronischem Offenwinkelglaukom (PCOG), die 3-mal täglich 1 Dorzolamid-Augentropfen erhielten, über den Verlauf von 9 Monaten hinsichtlich folgender Parameter untersucht: Augeninnendruck, pulsatiler okulärer Blutfluss (POBF) und Humphrey 30-2 Gesichtsfeld. Der POBF wurde mit dem Pneumotonometer nach Langham (OBF-System, U.K.) ermittelt. Die statistische Analyse erfolgte mit dem Wilroxon-Test, die Signifikanzniveaus wurden nach Bonferoni-Holm adjustiert. Ergebnisse. Der Augeninnendruck sank unter der Dorzolamid-Therapie signifikant von 18 mmHg auf 15,5 mmHg nach 9 Monaten, und die Gesichtsfelddefekte verringerten sich signifikant um 18% gegenüber dem Ausgangswert. Es zeigte sich eine signifikante Änderung des POBF (Ausgangswert: 543 μl/min; 9 Monate: 675 μl/min). Schlussfolgerungen. Die Resultate zeigen die erwartete Augeninnendrucksenkung. Visuelle Funktion und POBF verbesserten sich signifikant. Eine Erklärung dafür könnte das Vorliegen eines analogen vasodilatatorischen Effektes sein, der bei systemischer Carboanhydrasehemmerapplikation beobachtet wurde.AbstractObjective. It has been shown that oral carbonic anhydrase inhibitors improve visual function in glaucoma. Furthermore topical dorzolamide might improve ocular hemodynamics, as was demonstrated previously. This study was undertaken to evaluate whether topical dorzolamide affects visual function and ocular hemodynamics in glaucoma. Methods. In a retrospective, open clinical trial, dorzolamide eye drops were administered to 28 patients with confirmed primary open angle glaucoma (POAG) in both eyes, 3 times daily for a mean follow up of 9 months. One eye was randomly chosen for evaluation. IOP, blood pressure, heart rate, pulsatile ocular blood flow (POBF) and Humphrey 30–2 visual fields were measured at baseline and after the start of the therapy. POBF was determined by pneumotonography. For statistical analysis the Wilcoxon-matched-paired test and the Bonferoni-Holm adjustment were used. Results. In dorzolamide-treated patients the IOP dropped from 18 mmHg to 15.5 mmHg after 9 months therapy (p<0.01) and the visual field improved significantly by 18% (p<0.05). A statistically significant change was found for POBF from 543 μl/min to 675 μl/min (p<0.05). Conclusions. The results showed the expected drop in intraocular pressure. Visual function and pulsatile ocular blood flow improved significantly which might be explained by an analogous, vasodilatory effect as was observed in orally applied carbonic anhydrase inhibitors.

[Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas].

PURPOSE It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically. METHOD Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior. RESULTS The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h. CONCLUSIONS The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.