David Tabibian

Corneal biomechanical properties at different corneal cross-linking (CXL) irradiances.

PURPOSE New corneal cross-linking (CXL) devices are capable of using higher UV-A light irradiances than used in original CXL protocols. The Bunsen-Roscoe law states that a photochemical reaction should stay constant if the delivered total energy is kept constant; however, little clinical data are available to support this hypothesis. METHODS We investigated the biomechanical properties of four groups (n = 50 each) of porcine corneas. Three groups were exposed to riboflavin 0.1 % and UV-A irradiation of equal total energy (3 mW/cm(2) for 30 minutes, 9 mW/cm(2) for 10 minutes, and 18 mW/cm(2) for 5 minutes). Controls were exposed to riboflavin 0.1% without irradiation. Youngs modulus of 5-mm wide corneal strips was used as an indicator of corneal stiffness. RESULTS We observed a decreased stiffening effect with increasing UV-A intensity. Youngs modulus at 10% strain showed significant differences between 3 mW/cm(2) and 9 mW/cm(2) (P = 0.002), 3 mW/cm(2) and 18 mW/cm(2) (P = 0.0002), 3 mW/cm(2) and the control group (P < 0.0001), and 9 mW/cm(2) and the control group (P = 0.015). There was no difference between 18 mW/cm(2) and the control group (P = 0.064) and between 9 mW/cm(2) and 18 mW/cm(2) (P = 0.503). CONCLUSIONS The biomechanical effect of CXL decreased significantly when using high irradiance/short irradiation time settings. Intrastromal oxygen diffusion capacity and increased oxygen consumption associated with higher irradiances may be a limiting factor leading to reduced treatment efficiency. Our results regarding the efficiency of high-irradiance collagen cross-linking (CXL) raise concerns about the clinical efficiency of the new high-irradiance CXL devices already used in clinical practice without proper validation.

Accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking as a first-line and sole treatment in early fungal keratitis.

PURPOSE To report the use of accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL) as a first-line treatment in a patient with an atypical fungal keratitis. METHODS Case report and literature review. RESULTS A patient who presented with a painful peripheral corneal infiltrate underwent PACK-CXL with a local limited abrasion and accelerated ultraviolet-A irradiation at 365 μm and 9 mW/cm² for 10 minutes. Cultures grew Aureobasidium pullulans. The corneal epithelium closed completely within 3 days and the infiltrate was completely eradicated without administration of antibiotics. CONCLUSIONS Accelerated PACK-CXL was successfully used as a first-line and sole treatment in a case of early fungal keratitis caused by Aureobasidium pullulans. Further characterization of the antifungal effect of PACK-CXL is needed in prospective studies.

Antibacterial efficacy of accelerated photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL).

PURPOSE To investigate whether optimized photoactivated chromophore for keratitis-corneal collagen cross-linking (PACK-CXL) treatment settings allow accelerating treatment while maintaining antibacterial efficacy. METHODS Staphylococcus aureus and Pseudomonas aeruginosa strains were irradiated with ultraviolet-A light of equal fluence but different intensity settings (18 mW/cm² for 5 minutes and 36 mW/cm² for 2.5 minutes). The killing rate was determined by comparing the number of colony-forming units between cross-linked specimens and non-irradiated controls. The potential additional effect of 0.001% benzalkonium chloride was also investigated. RESULTS The killing rates for Staphylococcus aureus were 92.5% ± 5.5% (5 minutes at 18 mW/cm²) and 94.4% ± 2.9% (2.5 minutes at 36 mW/cm²). For Pseudomonas aeruginosa, the killing rates were 93.2% ± 8.3% (5 minutes at 18 mW/cm²) and 92.9% ± 5.0% (2.5 minutes at 36 mW/cm²). The presence of benzalkonium chloride in the riboflavin solution did not increase the killing rate significantly. CONCLUSIONS The antibacterial efficacy of PACK-CXL follows the Bunsen-Roscoe law of reciprocity and can be maintained even when the irradiation intensity is considerably increased. These optimized settings may allow a shortened treatment time in the future for PACK-CXL and thus help facilitate the transition from the operating room to the slit lamp for treatment.

PACK‑CXL: Corneal Cross‑linking for Treatment of Infectious Keratitis

This article discusses corneal cross-linking (CXL) and how it transitioned from a modality for treating corneal ectatic disorders to an inventive means of treating infectious keratitis. Initially, CXL was successfully developed to halt the progression of ectatic diseases such as keratoconus, using the standard Dresden protocol. Later, indications were extended to treat iatrogenic ectasia developing after laser-assisted in situ keratomileusis (LASIK) and photo-refractive keratectomy (PRK). At the time, it had been postulated that the combination of ultraviolet light with riboflavin could not only biomechanically strengthen the cornea but also was capable of destroying living cells and organisms including keratocytes and pathogens. Thus a new and innovative concept of treatment for infectious keratitis emerged through the use of CXL technology. Initially only advanced infectious melting ulcers resisting standard microbicidal therapy were treated with CXL in addition to standard therapy. In subsequent studies CXL was also used to treat bacterial keratitis as first line therapy without the use of concomitant antibiotic therapy. With the increasing interest in CXL technology to treat infectious keratitis and to clearly separate its use from the treatment of ectatic disorders, a new term was adopted at the 9th CXL congress in Dublin for this specific indication: PACK-CXL (photoactivated chromophore for infectious keratitis). PACK-CXL has the potential to eventually become an interesting alternative to standard antibiotic therapy in treating infectious corneal disorders, and may help reduce the global burden of microbial resistance to antibiotics and other therapeutic agents.

PACK-CXL: Corneal cross-linking in infectious keratitis

BackgroundCorneal cross-linking (CXL) using ultraviolet light-A (UV-A) and riboflavin is a technique developed in the 1990’s to treat corneal ectatic disorders such as keratoconus. It soon became the new gold standard in multiple countries around the world to halt the progression of this disorder, with good long-term outcomes in keratometry reading and visual acuity. The original Dresden treatment protocol was also later on used to stabilize iatrogenic corneal ectasia appearing after laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK). CXL efficiently strengthened the cornea but was also shown to kill most of the keratocytes within the corneal stroma, later on repopulated by those cells.ReviewUltraviolet-light has long been known for its microbicidal effect, and thus CXL postulated to be able to sterilize the cornea from infectious pathogens. This cytotoxic effect led to the first clinical trials using CXL to treat advanced infectious melting corneal keratitis. Patients treated with this technique showed, in the majority of cases, a stabilization of the melting process and were able to avoid emergent à chaud keratoplasty. Following those primary favorable results, CXL was used to treat beginning bacterial keratitis as a first-line treatment without any adjunctive antibiotics with positive results for most patients. In order to distinguish the use of CXL for infectious keratitis treatment from its use for corneal ectatic disorders, a new term was proposed at the 9th CXL congress in Dublin to rename its use in infections as photoactivated chromophore for infectious keratitis -corneal collagen cross-linking (PACK-CXL).ConclusionPACK-CXL is now more frequently used to treat infections from various infectious origins. The original Dresden protocol is still used for this purpose. Careful modifications of this protocol could improve the efficiency of this technique in specific clinical situations regarding certain types of pathogens.

Increased Biomechanical Efficacy of Corneal Cross-linking in Thin Corneas Due to Higher Oxygen Availability.

PURPOSE To compare the currently available ultraviolet-A (UV-A) corneal cross-linking (CXL) treatment protocols for thin corneas with respect to oxygen, UV fluence, and osmotic pressure. METHODS Freshly enucleated murine (n = 16) and porcine (n = 16) eyes were used. The dependency on oxygen and the amount of UV absorption were evaluated using different CXL protocols, including standard CXL, contact lens-assisted CXL (caCXL), and CXL after corneal swelling. The CXL protocol was adapted from the treatment parameters of the human cornea to fit the thickness of murine and porcine corneas. Immediately after CXL, the corneas were subjected to biomechanical testing, including preconditioning, stress relaxation at 0.6 MPa, and stress-strain extensiometry. A two-element Prony series was fitted to the relaxation curves for viscoelastic characterization. RESULTS Standard CXL was most efficient; prior corneal swelling reduced the long-term modulus by 6% and caCXL by 15% to 20%. Oxygen reduction decreased the long-term modulus G∞ by 14% to 15% and the instantaneous modulus G0 by 2% to 5%, and increased the short-term modulus G2 by 22% to 31%. Reducing the amount of absorbed UV energy decreased the long-term modulus G∞ by 5% to 34%, the instantaneous modulus G0 by 7% to 29%, and the short-term modulus G2 by 17% to 20%. The amount of absorbed UV light was more important in porcine than in murine corneas. CONCLUSIONS The higher oxygen availability in thin corneas potentially increases the overall efficacy of riboflavin UV-A CXL compared to corneas of standard thickness. Clinical protocols for thin corneas should be revised to implement these findings.

The Effect of Standard and High-Fluence Corneal Cross-Linking (CXL) on Cornea and Limbus

PURPOSE When treating peripheral ectatic disease-like pellucid marginal degeneration (PMD), corneal cross-linking with UV-A and riboflavin (CXL) must be applied eccentrically to the periphery of the lower cornea, partly irradiating the corneal limbus. Here, we investigated the effect of standard and double-standard fluence corneal cross-linking with riboflavin and UV-A (CXL) on cornea and corneal limbus in the rabbit eye in vivo. METHODS Epithelium-off CXL was performed in male New Zealand White rabbits with two irradiation diameters (7 mm central cornea, 13 mm cornea and limbus), using standard fluence (5.4 J/cm(2)) and double-standard fluence (10.8 J/cm(2)) settings. Controls were subjected to epithelial removal and riboflavin instillation, but were not irradiated with UV-A. Following CXL, animals were examined daily until complete closure of the epithelium, and at 7, 14, 21, and 28 days. Animals were killed and a corneoscleral button was excised and processed for light microscopy and immunohistochemistry. RESULTS For both irradiation diameters and fluences tested, no signs of endothelial damage or limbal vessel thrombosis were observed, and time to re-epithelialization was similar to untreated controls. Histological and immunohistochemical analysis revealed no differences in the p63 putative stem cell marker expression pattern. CONCLUSIONS Even when using fluence twice as high as the one used in current clinical CXL settings, circumferential UV-A irradiation of the corneal limbus does not alter the regenerative capacity of the limbal epithelial cells, and the expression pattern of the putative stem cell marker p63 remains unchanged. This suggests that eccentric CXL may be performed safely in PMD.

Establishing Corneal Cross-Linking With Riboflavin and UV-A in the Mouse Cornea In Vivo: Biomechanical Analysis

PURPOSE To establish corneal cross-linking (CXL) with riboflavin and UV-A in in the mouse cornea in vivo and to develop tools to measure the biomechanical changes observed. METHODS A total of 55 male C57BL/6 wild-type mice (aged 5 weeks) were divided into 14 groups. Standard CXL parameters were adapted to the anatomy of the mouse cornea, and riboflavin concentration (0.1%-0.5%) and fluence series (0.09-5.4 J/cm²) were performed on the assumption of the endothelial damage thresholds. Untreated and riboflavin only corneas were used as controls. Animals were killed at 30 minutes and at 1 month after CXL. Corneas were harvested. Two-dimensional (2D) biomechanical testing was performed using a customized corneal holder in a commercially available stress-strain extensometer/indenter. Both elastic and viscoelastic analyses were performed. Statistical inference was performed using t-tests and specific mathematical models fitted to the experimental stress-strain and stress-relaxation data. Adjusted P values by the method of Benjamini and Hochberg are reported. RESULTS For all CXL treatment groups, stress-relaxation showed significant differences (P < 0.0001) after 120 seconds of constant strain application, with cross-linked corneas maintaining a higher stress (441 ± 40 kPa) when compared with controls (337 ± 39 kPa). Stress-strain analysis confirmed these findings but was less sensitive to CXL-induced changes: at 0.5% of strain, cross-linked corneas remained at higher stress (778 ± 111 kPa) when compared with controls (659 ± 121 kPa). CONCLUSIONS Cross-linking was induced in the mouse cornea in vivo, and its biomechanical effect successfully measured. This could create opportunities to study molecular pathways of CXL in transgenic mice.

Additive Effect of Repeated Corneal Collagen Cross-linking in Keratoconus

PURPOSE To report the long-term clinical outcome in a patient diagnosed as having bilateral progressive keratoconus who received a single corneal collagen cross-linking (CXL) treatment in the right eye and repeated CXL in the left eye. METHODS Observational case report. Topographical changes were assessed by high-resolution Scheimpflug imaging. The right eye underwent a standard epithelium-off CXL procedure in February 2008, followed by the left eye 4 weeks later. In 2012, the left eye was treated with CXL for a second time. Irradiation was performed in all cases at a fluence of 5.4 J/cm². Energy settings were 30 minutes @ 3 mW/cm² for the CXL procedures performed in 2008, and 10 minutes @ 9 mW/cm² for the second CXL procedure of the left eye that was performed in 2012. RESULTS The right eye that underwent a single CXL procedure showed a flattening of keratometry values between 2008 and 2012, followed by stabilization. The left eye showed a similar flattening effect between 2008 and 2012, followed by another flattening effect after the second CXL procedure and accompanied by a distinct increase in corrected distance visual acuity. CONCLUSIONS Following repeated CXL, the corneal stroma and endothelium remained inconspicuous, and postoperative haze and visibility of the stromal demarcation line was similar to what is usually observed after a single CXL procedure. Whether the additive flattening effect of the anterior surface observed in this single case goes along with an additive increase in biomechanical stiffness remains to be seen.

Repeated Cross-linking After a Short Time Does Not Provide Any Additional Biomechanical Stiffness in the Mouse Cornea In Vivo

PURPOSE To study whether repeated collagen cross-linking (CXL) performed in vivo in mice shows an additive effect on mechanical corneal stiffness. METHODS In this experimental study, epithelium-off CXL was performed in a total of 18 eyes from male C57BL/6 mice, with 0.27%-riboflavin solution applied for 20 minutes, followed by ultraviolet-A (UVA) irradiation (365 nm, 9mW/cm2) for 2:50 minutes (fluence 1.53 J/cm2). CXL was performed as either a single (1×CXL) or a repeated (2×CXL) treatment. Un-irradiated corneas served as controls. In the 2×CXL group, the procedure was performed on day 1 and day 4 to ensure complete reepithelialization between sessions. Biomechanical analysis was performed on day 7. Corneas were harvested with a small scleral ring and mounted on a customized two-dimensional flap holder. The biomechanical measurement consisted of three parts: (1) pre-conditioned during three cycles from 0.04 to 0.4 N, (2) stress relaxation during 120 seconds following 0.4 N force application, and (3) stress-strain curve until break. RESULTS After the relaxation period of 120 seconds, highly significant differences (P < .001) were found between the controls and both 1×CXL corneas and 2×CXL corneas. No significant difference (P = .70) was detected between the 1×CXL and 2×CXL groups. The stress remaining after relaxation was 355 ± 25.2 kPa in the control group, 457 ± 34.1 kPa in the 1×CXL group, and 463 ± 22.2 kPa in the 2×CXL group. No significant differences in the stress-strain curves were found between the conditions. CONCLUSIONS Repeated CXL 3 days after the first procedure does not further increase corneal stiffness in mice in vivo. [J Refract Surg. 2017;33(1):56-60.].