Ultraviolet-visible light spectral transmittance of rabbit corneas after riboflavin/ultraviolet-A (365 nm) corneal collagen cross-linking

Abstract

Purpose: To determine the effect of riboflavin/ultraviolet-A (365 nm) corneal collagen cross-linking on the transmission of the ultraviolet-visible (UV-VIS) light spectrum through the cornea.

Methods: Twelve New Zealand white male rabbits were used in this research. Cross-linking was performed unilaterally on the right eyes of the animals while only the epithelium was removed on the left eyes as the control. Seven weeks after cross-linking, the animals were euthanized, and the enucleated eyes were processed for transmission spectroscopy. To confirm that the cross-linking procedures was done successfully on the right corneas, the tensile force-extension relationship was measured using six corneas from three of the rabbits after the transmission spectrum was determined.

Results: Seven weeks after cross-linking, ten of the 12 rabbits had clear corneas in the cross-linked and control eyes. The two rabbits with neovascularization and granular opacities in the right corneas were not included in subsequent measurements. In the cross-linked corneas, transmittance was 87.57% at 650 nm, and decreased continuously as the wavelength shortened. From 315 nm, the transmittance rapidly decreased and was 35.52% at 300 nm. In the control corneas, transmittance was 95.95% at 650 nm and decreased continuously as the wavelength shortened. Below 315 nm, the transmittance rapidly decreased, to 40.29% at 300 nm. The transmittance of the cross-linking corneas was 10%-20% lower than that of the control corneas. The difference was 8.38% at 650 nm and increased as the wavelength shortened, reaching a maximum of 20.59% at 320 nm, and decreased rapidly to 4.77% at 300 nm. The tensile force-extension relationship showed that a greater force was necessary to extend the cross-linking corneas over 500 µm than that of the control corneas.

Conclusions: The transmittance of the cross-linked corneas was 10%-20% lower than that of the control corneas. The difference increased as the wavelength decrease, reaching a maximum at 320 nm and then decreasing rapidly. Ultraviolet collagen cross-linking exhibited a protective effect against ultraviolet penetration.

Figure 1

A spectrophotometer used for measuring the ultraviolet-visible light transmittance spectrum. A: The spectrophotometer was set up so that the cornea and microscope cover glass could be placed perpendicularly to the measuring beam. B: The cover glass was placed on the cuvette holder. C: The corneal sample was placed on the cover glass with the epithelium directed downward.

Figure 2

Measurement of corneal stiffness. A: A stress-strain curve was obtained using the Biomaterial Universal testing machine. B: The 8.0-mm diameter corneas were clamped 2.0 mm on each side.

Figure 3

Corneal edema after cross-linking. A right cornea (OD) 2 days after cross-linking showing severe edema and opacity compared to the control cornea (OS). At approximately 1 week after cross-linking, corneal edema was not observed with the naked eye.

Figure 4

Photographs of corneas before enucleation, 7 weeks after cross-linking. Ten of 12 rabbits exhibited clear corneas in the cross-linked (A) and control eyes (B).

Figure 5

Complications after cross-linking. A: In one rabbit (#3), corneal neovascularization was found at the center of the right cornea. B: In another rabbit (#5), granular-type opacities were observed at the center of the right cornea. The left corneas of both rabbits were clear.

Figure 6

The average transmission spectra of ten right (cross-linking, solid line) and left (control, dashed line) eyes from ten rabbits. The transmittance of the cross-linked corneas was 4.77%–21.28% lower than that of the control corneas. The error bars mean the standard deviation of the transmission percentage between animals in each group. In general, the individual variation among the cross-linked corneas was larger than that among the control corneas.

Figure 7

The difference between the transmittance of the control corneas and that of the cross-linked corneas from ten rabbits. The difference was 8.383% at 650 nm and increased as the wavelength decreased, reaching a maximum of 20.591% at 320 nm, and decreasing rapidly to 4.770% at 300 nm. Additionally, a local hump was found between 425 and 455 nm, and a focal maximum at 350 nm.

Figure 8

The difference between the transmittance of the control corneas and that of the cross-linked corneas of three rabbits (#6, #9, and #11) and the other seven rabbits. The solid line represents those whose corneal thickness differences were less than 7 µm (#6, #9, and #11), and the dashed line represents the remaining rabbits. The transmittance difference of the three rabbits was 3%–4% less than the others, but the overall pattern was similar between the groups.

Figure 9

The average force required to extend the cornea over 500 µm in the cross-linking group and the control group. In the control groups (dashed line), extension of the cornea required a gradual and uniform increase in the force. By comparison, greater force was necessary to extend the cross-linked corneas (solid line) over 500 µm. The error bars represent the standard deviation of force between animals in each group.

Figure 10

The hematoxylin and eosin–stained images of the cross-linked and control corneas. There were few keratocytes in the anterior stroma of the cross-linked corneas (A) compared to the control corneas (B). Neither the epithelia nor Descemet’s membrane showed any significant differences (hematoxylin and eosin, 40X).

Figure 11

A representative result from terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining in the cross-linked corneas. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive apoptotic cells were found at the posterior stroma of the cross-linked corneas.