E. Spörl

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.

Behandlung von Keratokonus durch Kollagenvernetzung

ZusammenfassungHintergrund. Ziel dieser Studie war der Nachweis, ob sich die Hornhaut bei Keratokonuspatienten mittels photochemischer Riboflavin/UVA-Kollagenvernetzung verfestigen lässt, um so die progressive Hornhautektasie zu stoppen. Patienten und Methode. Wir behandelten 16 Augen von 15 Patienten mit gesicherter Progression und überwiegend moderatem Keratokonus.Nach Epithelabrasio wurde Riboflavinlösung auf die Hornhaut appliziert und mit UVA aus 1 cm Abstand für 30 min bestrahlt.Nachkontrollen, die Visus,Hornhauttopographie und Messung der Endothelzelldichte einschlossen, erfolgten im 1. Jahr alle 3 Monate, danach alle 6 Monate, wobei der Nachbeobachtungszeitraum zwischen 1 und 3 Jahren lag. Ergebnisse. Eine Progression der Keratektasie konnte bei allen Patienten gestoppt werden. Bestkorrigierter Visus und der maximale Keratometerwert besserten sich gering in ca. 50% der Fälle.Bei allen Patienten blieben die Hornhauttransparenz und die Endothelzelldichte stabil. Schlussfolgerungen. Unsere Ergebnisse zeigen, dass möglicherweise die Quervernetzung des Kollagens eine geeignete konservative Behandlungsmöglichkeit ist,um das Fortschreiten des Keratokonus aufzuhalten. Weitere Studien sind geplant, um Spätkomplikationen auszuschließen und den Langzeiteffekt dieser Methode zu sichern.AbstractBackground. We were able to show a significant increase in corneal stiffness of rabbit and porcine eyes after combined riboflavin/UVA-induced collagen cross-linking. In this study,we tried to treat keratoconus patients with this method to stop the progression of corneal ectasia. Patients and methods. We treated 16 eyes of 15 patients with progressive keratoconus and mostly moderate keratectasia (48–56 dpt).After removal of the epithelium (7 mm Ø), riboflavin solution was applied on the cornea, which was irradiated with UVA (370 nm,3 mW/cm2) at a distance of 1 cm for 30 min.Post-operative follow-up controls were conducted every 3 months in the first year and then every 6 months, always including visual acuity testing, corneal topography and measurements of endothelial cell density.The follow-up time was between 1 and 3 years. Results. Progression of keratectasia was stopped in all patients.Best corrected visual acuity and the maximal keratometry values improved slightly in about 50% of the cases. In all patients corneal transparency, the degree of keratectasia registered by corneal topography and the density of endothelial cells remained unchanged within the follow-up time.No negative side-effects were observed. Conclusions. Our results show that collagen cross linking might be a useful conservative treatment modality to stop the progression of keratoconus.By this means the need for keratoplasty might be significantly reduced.Given the simplicity of the technique and minimal costs of the treatment it might also be well suited for developing countries.Further studies are envisaged to exclude long-term side effects and to evaluate the long term durability of the mechanical stiffness effect.

Untersuchungen zur Verfestigung der Hornhaut am Kaninchen

Hintergrund: Eine mechanische Stabilisierung der Hornhaut beim Keratokonus könnte das Fortschreiten dieser Erkrankung verzögern. Die an der Hornhaut von enukleierten Schweineaugen optimierten Vernetzungsmethoden zur Erhöhung der Festigkeit wurden im Langzeitversuch am Kaninchenmodell auf Beständigkeit und biologische Verträglichkeit getestet. Methode: Von 28 Kaninchen wurde das rechte Auge behandelt, das linke Auge diente jeweils als Kontrolle. Nach der mechanischen Entfernung des Epithels wurden 19 Augen mit Riboflavin-Dextranlösung getropft und anschließend mit UV-Strahlung (365 nm, Bestrahlungsstärke 2 mW/cm2) für 45 min exponiert. Auf 9 Augen wirkte ein Gemisch aus Methocel und 0,075% Glutaraldehyd für 20 min ein. Die Reaktion der Hornhaut wurde mit der Spaltlampenphotographie dokumentiert. Nach 1 Monat wurden 20 Tiere und nach 3 Monaten 8 Tiere getötet, die Augen enukleiert und von Streifen (Breite 5 mm, Länge 8 mm) der Hornhäute der Spannungs-Dehnungszusammenhang gemessen. Ergebnisse: Vier Wochen nach der Vernetzung konnte eine signifikant höhere Festigkeit in den mit Riboflavin+UV behandelten Hornhäuten nachgewiesen werden. Die vernetzten und unvernetzten Hornhäute unterschieden sich bei einer Dehnung von 6% in der Spannung um den Faktor 1,61±0,75 (p=0,041). Nach 12 Wochen lag diese Festigkeitszunahme noch bei 1,3±0,48 (p=0,07). Es kam zu keiner sichtbaren Entzündungsreaktion oder Beeinträchtigung der Transparenz. Die mit Glutaraldehyd behandelten Hornhäute wiesen zwar auch eine Zunahme der Festigkeit auf (Faktor 1,3±0,66), aber der Effekt war statistisch nicht signifikant (p=0,319), sodass ein Langzeittest dieser Methode nicht gerechtfertigt war. Schlussfolgerungen: Die Kombination von Riboflavin+UV-Strahlung ist geeignet zur Erzeugung von mindestens temporären Vernetzungen in der Hornhaut bei Kaninchen ohne klinische Zeichen für Nebenwirkungen wie Trübung oder Entzündung.Background: The mechanical stabilization of the cornea in keratoconus may delay progression of this disease. The cross-linking techniques optimized in corneas of enucleated porcine eyes were investigated under in vivo conditions in rabbits to estimate the biocompatibility and duration of the stiffening effect. Methods: Twenty-eight rabbits were treated monocularly, the fellow eye serving as control. The epithelium was mechanically removed and 19 eyes were treated with riboflavin plus ultraviolet irradiation (365 nm, 2 mW/cm2) for 45 min and 9 eyes with 0.075% glutaraldehyde for 20 min. After treatment, the eyelids were sutured for 3 days. The healing process was controlled by slit-lamp examination and photographically documented. After 1 month, 20 animals and after 3 months 8 animals were sacrificed, the eyes enucleated, and the stress-strain relation of the corneas measured and compared to the fellow eye. Results: The epithelium was closed after 4–5 days. The transparency of the corneas remained clear during follow-up, and there were no signs of inflammatory reaction. Stress for a strain of 6% was higher in the treated corneas by a factor of 1.3±0.66 (P=0.319) in the glutaraldehyde group and by a factor of 1.6±0.75 (P=0.0408) in the riboflavin group at 1 month, and by 1.3±0.48 (P=0.07) at 3 months after treatment. Conclusions: The cross-linking technique using riboflavin plus UV irradiation is suitable for at least temporarily stiffening the cornea in vivo and seems to be a promising method for conservative treatment of keratectasia.

Artificial stiffening of the cornea by induction of intrastromal cross-links

Purpose: To increase the stability of the cornea by artificial cross-linking (radiation or chemical agents) and to investigate a future therapy for keratoconus. Materials and methods: The epithelium of enucleated porcine eyes was removed. Ten eyes in each of eight test groups were treated with UV light (λ = 254 nm), 0.5 % riboflavin and UV light (365 nm), blue light (436 nm) and sunlight, and the chemical agents glutaraldehyde (1 % and 0.1 %, 10 min) and Karnovskys solution (0.1 %, 10 min). Strips of 5 mm in width and 9 mm in length were cut from each cornea and the stress-strain behaviour of the strips was measured. For comparison, eight groups of ten untreated corneas each were measured by the same method. Results: Compared to untreated corneas riboflavin and UV irradiation as well as glutaraldehyde and Karnovskys solution treatment resulted in significantly increased stiffness of the cornea (p < 0.05). Conclusions: The biomechanical behaviour of the cornea can be altered by low-concentration glutaraldehyde, Karnovskys solution, and by riboflavin and UV irradiation, which offers potential conservative treatment of keratoconus. To optimize this effect further investigation is necessary regarding the dose-effect relation and the in-vivo conditions.Hintergrund: Die biomechanische Festigkeit der Hornhaut soll durch künstliche Quervernetzung der Kollagenfibrillen (Strahlenvernetzung, chemische Vernetzung) erhöht werden, um eine evtl. konservative Therapie des Keratokonus zu prüfen. Methode: Von enukleierten Schweineaugen wurde das Epithel entfernt. Je 10 Augen in 8 Testgruppen wurden mit UV-Strahlung (λ = 254 nm), einer 0,5 %igen Riboflavinlösung und UV-Strahlung (365 nm), blauem Licht (436 nm) und Sonnenlicht sowie mit chemischen Vernetzern – Glutaraldehyd (1 % und 0,1 %, 10 min) und Karnovsky- Lösung (0,1 %, 10 min) – behandelt. Als Standard dienten jeweils Kontrollgruppen mit 10 unbehandelten Hornhäuten. Aus jeder Hornhaut wurde ein zentraler Streifen von 5 mm Breite und 9 mm Länge geschnitten und die Spannungs-Dehnungs-Kurven gemessen. Ergebnisse: Bei UV-Bestrahlung von mit Riboflavin vorbehandelten Hornhäuten nimmt die Dehnbarkeit signifikant ab (p < 0,05). Auch die mit Glutaraldehyd oder Karnovsky-Lösung behandelten Hornhäute zeigen eine signifikante Zunahme der Festigkeit (p < 0,05). Schlußfolgerung: UV-Strahlen und Riboflavin sowie niedrigkonzentriertes Glutaraldehyd oder Karnovsky-Lösung führen zu einer Verfestigung der Hornhaut wahrscheinlich aufgrund von Vernetzungen der Kollagene oder der Proteoglykane. Weitere Untersuchungen sind jedoch bezüglich der Optimierung der Dosis-Wirkungs-Beziehung und der In-vivo-Bedingungen erforderlich.

Interlamellar cohesion after corneal crosslinking using riboflavin and ultraviolet A light

Aims Collagen crosslinking treatment of progressive keratoconus using the photosensitiser riboflavin and ultraviolet A light of 370 nm wavelength has been shown to increase significantly the tensile strength of corneal collagen by about 300%. In keratoconus, interlamellar and interfibrillar slippage have been proposed as pathogenetic mechanisms. Therefore, the aim of this study was to assess the impact of collagen crosslinking on the interlamellar cohesive force. Methods 72 post mortem porcine eyes were divided into six different treatment groups: the untreated control group, the standard crosslinking group, the hypo-osmolar crosslinking group, the stromal swelling group, the formaldehyde group and the α-amylase group. An anterior 9×4 mm strip of 400 μm thickness was prepared using a lamellar rotating microkeratome. For interlamellar cohesive force measurements a splitting plane was created at 50% depth. Force–distance profiles were recorded using a microcomputer-controlled biomaterial testing machine. Results The mean interlamellar cohesive force was 0.24 N/mm in the untreated control group, 0.26 N/mm in the standard crosslinking group, 0.25 N/mm in the hypo-osmolar crosslinking group, 0.23 N/mm in hydrated corneas, 0.27 N/mm in the formaldehyde group without statistically significant difference. Only the values of the α-amylase group were statistically significantly lowered by 31.5% to 0.16 N/mm. Conclusions Surprisingly, corneal crosslinking does not increase the interlamellar cohesive force. In the α-amylase group the cohesive force was mainly decreased because of the digestion of proteoglycans. Crosslinking seems to stabilise only inter- and intrafibrillar, but not interlamellar cohesion.

[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.

Biomechanical changes in the anterior lens capsule after trypan blue staining

Purpose: To examine the effect of trypan blue staining on the biomechanical behavior of the porcine anterior lens capsule. Setting: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. Methods: Fifty‐five anterior lens capsules from porcine cadaver eyes were used. Two parallel 8.0 mm × 4.0 mm large capsule strips were prepared from each capsule. After trypan blue staining for various time intervals combined with exposure to white light (6000 lux) or with no light exposure, biomechanical stress‐strain measurements were performed using an automated material tester. Untreated specimens and specimens treated with glutaraldehyde 0.1% were used as controls. The absorption spectrum of trypan blue 0.1% solution and the emission spectrum of the light source were measured. Results: After treatment with light and trypan blue, at 25% strain, there was a statistically significant increase in stress of up to 70.1% and in elastic stiffness of 47% and a decrease in the ultimate mechanical strain of up to 13%. There were no biomechanical changes in capsules with trypan blue staining in the absence of light or after a short illumination time of 30 seconds, indicating a light‐dependent process. After 30 minutes of glutaraldehyde 0.1% treatment, there was an increase in stress of 321.6% at 25% strain and a decrease in the ultimate strain of 47.6%. The emission spectrum of the light source included the absorption peak for trypan blue at 580 nm. Conclusions: Trypan blue staining of the lens capsule combined with light irradiation for at least 1 minute led to an increase in elastic stiffness at 25% strain and a reduction in the ultimate extensibility. This effect is probably due to the photosensitizing action of trypan blue, leading to light‐induced collagen crosslinking of the capsule collagen similar to age‐related crosslinking. Nucleus expression might be impeded by the increased capsule stiffness. Continuous curvilinear capsulorhexis is facilitated.

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.