Person:

Kochevar, Irene

Purpose To investigate the effectiveness of antimicrobial blue light (aBL) as an alternative or adjunctive therapeutic for infectious keratitis. Methods: We developed an ex vivo rabbit model and an in vivo mouse model of infectious keratitis. A bioluminescent strain of Pseudomonas aeruginosa was used as the causative pathogen, allowing noninvasive monitoring of the extent of infection in real time via bioluminescence imaging. Quantitation of bacterial luminescence was correlated to colony-forming units (CFU). Using the ex vivo and in vivo models, the effectiveness of aBL (415 nm) for the treatment of keratitis was evaluated as a function of radiant exposure when aBL was delivered at 6 or 24 hours after bacterial inoculation. The aBL exposures calculated to reach the retina were compared to the American National Standards Institute standards to estimate aBL retinal safety. Results: Pseudomonas aeruginosa keratitis fully developed in both the ex vivo and in vivo models at 24 hours post inoculation. Bacterial luminescence in the infected corneas correlated linearly to CFU (R2 = 0.921). Bacterial burden in the infected corneas was rapidly and significantly reduced (>2-log10) both ex vivo and in vivo after a single exposure of aBL. Recurrence of infection was observed in the aBL-treated mice at 24 hours after aBL exposure. The aBL toxicity to the retina is largely dependent on the aBL transmission of the cornea. Conclusions: Antimicrobial blue light is a potential alternative or adjunctive therapeutic for infectious keratitis. Further studies of corneal and retinal safety using large animal models, in which the ocular anatomies are similar to that of humans, are warranted.

Purpose To examine whether Nrf2-regulated antioxidant defense and p53 are activated in human corneal endothelial cells (CEnCs) by environmental levels of ultraviolet A (UV-A), a known stimulator of oxidative stress. Methods: Immortalized human CEnCs (HCEnCi) were exposed to UV-A fluences of 2.5, 5, 10, or 25 J/cm2, then allowed to recover for 3 to 24 hours. Control HCEnCi did not receive UV-A. Reactive oxygen species (ROS) were measured using H2DCFDA. Cell cytotoxicity was evaluated by lactate dehydrogenase (LDH) release. Levels of Nrf2, HO-1, NQO-1, p53, and caspase3 were detected by immunnoblotting or real-time PCR. Activated caspase3 was measured by immunoblotting and a fluorescence assay. Results: Exposure of HCEnCi to 5, 10, and 25 J/cm2 UV-A increased ROS levels compared with controls. Nrf2, HO-1, and NQO-1 mRNA increased 1.7- to 3.2-fold at 3 and 6 hours after irradiation with 2.5 and 5 J/cm2 UV-A. At 6 hours post irradiation, UV-A (5 J/cm2) enhanced nuclear Nrf2 translocation. At 24 hours post treatment, UV-A (5, 10, and 25 J/cm2) produced a 1.8- to 2.8-fold increase in phospho-p53 and a 2.6- to 6.0-fold increase in activated caspase3 compared with controls, resulting in 20% to 42% cell death. Conclusions: Lower fluences of UV-A induce Nrf2-regulated antioxidant defense and higher fluences activate p53 and caspase3, indicating that even near-environmental levels of UV-A may affect normal CEnCs. This data suggest that UV-A may especially damage cells deficient in antioxidant defense, and thus may be involved in the etiology of Fuchs' endothelial corneal dystrophy (FECD).

Purpose Scleral cross-linking (SXL) with a photosensitizer and light is a potential strategy to mechanically reinforce the sclera and prevent progressive axial elongation responsible for severe myopia. Current approaches for light delivery to the sclera are cumbersome, do not provide uniform illumination, and only treat a limited area of sclera. To overcome these challenges, we developed flexible optical waveguides optimized for efficient, homogeneous light delivery. Methods: Waveguides were fabricated from polydimethylsiloxane elastomer. Blue light (445 nm) is coupled into the waveguide with an input fiber. Light delivery efficiency from the waveguide to scleral tissue was measured and fit to a theoretical model. SXL was performed on fresh porcine eyes stained with 0.5% riboflavin, using irradiances of 0, 25, and 50 mW/cm2 around the entire equator of the eye. Stiffness of scleral strips was characterized with tensiometry. Results: Light delivery with a waveguide of tapered thickness (1.4–0.5 mm) enhanced the uniformity of light delivery, compared to a flat waveguide, achieving a coefficient of variation of less than 10%. At 8% strain, sclera cross-linked with the waveguides at 50 mW/cm2 for 30 minutes had a Young's modulus of 10.7 ± 1.0 MPa, compared to 5.9 ± 0.5 MPa for no irradiation, with no difference in stiffness between proximally and distally treated halves. The stiffness of waveguide-irradiated samples did not differ from direct irradiation at the same irradiance. Conclusions: We developed flexible waveguides for periscleral cross-linking. We demonstrated efficient and uniform stiffening of a 5-mm-wide equatorial band of scleral tissue.