Assessment Of Efficacy Of A Novel Crosslinking Protocol With Intracameral Oxygen (Bubble-Cxl) In Increasing The Corneal Stiffness Using Atomic Force Microscopy

The environmental oxygen level plays a critical role in corneal crosslinking (CXL), a treatment method to increase corneal biomechanical stability. In this study, we introduce a new CXL method (Bubble-CXL), in which intracameral oxygen serves as an additional oxygen source during eye treatment. The efficiency of this new method was compared with the efficiency of the standard CXL method.

 Three fresh porcine eye pairs were included in this study. One eye of each pair was treated with standard CXL, whereas in the partner eye, intracameral oxygen was injected prior to CXL and removed at the end of the procedure. The Young’s modulus of each cornea was measured using atomic force microscopy.

The epithelium was removed for eyes treated with Bubble-CXL, and intracameral oxygen was injected until 80% of the anterior chamber was filled. Next, 0.1% riboflavin solution was instilled every 5 min for 30 min. The cornea was irradiated with ultraviolet A light (with an irradiance of 3 mW∙cm⁻² and a wavelength of 370 nm) for 30 min, while 0.1% riboflavin solution was continuously instilled every 5 min. After CXL treatment, the intracameral oxygen was removed. A whole central corneal punch of 6 mm diameter and an average height of 0.87 ± 0.07 mm was extracted after crosslinking and fixed on a glass slide with epoxy glue. Stiffness measurements were performed on the day after CXL treatment.

Young’s moduli were compared between corneas of the same pair of eyes. For corneas treated with conventional CXL, similar Young’s moduli were found (91.65 ± 22.07 kPa, 100.58 ± 54.78 kPa, 103.31 ± 42.67 kPa (AV ± SD)), whereas corneas treated with Bubble-CXL collectively showed significantly higher Young’s moduli (130.46 ± 59.42 kPa, 322.51 ± 83.61 kPa, 129.06 ± 36.71 kPa (AV ± SD)), when compared to the partner eye treated with conventional CXL (Figure 2C, boxplot diagrams, Table 1). Young’s moduli distributions were constructed from experimental probability density functions (pdfs; equivalents of continuous histograms) (Figure 2C), revealing a shift towards higher Young’s moduli for corneas treated with Bubble-CXL for all pairs of eyes.

 All analyzed corneas treated with intracameral oxygen showed significantly higher Young’s modulus and thus an increased stiffness compared to the cornea of the partner eye treated with the standard protocol. Using intracameral oxygen in CXL therapy may increase crosslinking efficiency and improve biomechanical corneal properties.