Abstract
PurposeSecond harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea. Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects.MethodsSingle sub-Tenon's (sT) injections (400 μL) using SMG (40–400 mM) were made at the equatorial 12 o'clock position of the right eye of cadaveric rabbit heads (n = 16 pairs). After 3.5 hours, confocal microscopy (CM) was performed using 860 nm two-photon excitation and 400 to 450 nm emission. Pixel density and fiber bundle “waviness” analyses were performed on the images. Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. Comparison experiments with riboflavin photochemical crosslinking were done.ResultsTherapeutic tissue crosslinking localization studies indicated that crosslinking changes occurred at the site of injection and in adjacent sectors. Second harmonic generation signals revealed large fibrous collagenous bundled structures that displayed various degrees of waviness. Histogram analysis showed a nearly 6-fold signal increase in 400 mM SMG over 40 mM. This corresponded to a ΔTm = 13°C for 400 mM versus ΔTm = 4°C for 40 mM. Waviness analysis indicated increased fiber straightening as a result of SMG CXL.ConclusionsSecond harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG. These changes provide insights into the macromolecular changes that are induced by therapeutic crosslinking technology and may provide a method to evaluate connective tissue protein changes induced by scleral crosslinking therapies.
Highlights
Second harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea
Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects
Second harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG
Summary
Single sub-Tenon’s (sT) injections (400 lL) using SMG (40–400 mM) were made at the equatorial 12 o’clock position of the right eye of cadaveric rabbit heads (n 1⁄4 16 pairs). Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. All chemical solutions and buffers were prepared fresh using Millipore water (double distilled, de-ionized water, q 1⁄4 18.2 MXcm at 25 8C) on the day of crosslinking. The incubation time was 3.5 hours at room temperature (188–208C), at which time the tissue was dissected from the globes, washed in PBS three times and left to soak in fresh solution while immediately transporting for microscopy. We wanted to avoid cold storage conditions to allow the reactions to proceed under simulated physiologic conditions. We wanted to provide time to allow the compound to fully permeate the tissues as well as allow for the crosslinking reactions to go to completion at a temperature approaching physiologic. No microscopy findings (as viewed by microscopy) were noted that would suggest significant tissue degradation
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