Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera

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.

Scleral remodelling in myopia and its manipulation: a review of recent advances in scleral strengthening and myopia control

“…Interestingly, however, while glyceraldehyde increases scleral stress and Young's modulus, as expected from cross-linking, it showed no significant effect in preventing myopia development (64). Other agents that have been investigated for enhancing scleral collagen cross-linking that may yet prove beneficial in preventing myopia development include methylglyoxal (65) and a range of formaldehyde releasers including sodium hydroxymethylglycinate (67,68).…”

Experimental myopia increases and scleral crosslinking using genipin inhibits cyclic softening in the tree shrew sclera

“…have shown that a much lower concentration of genipin is required to achieve a comparable scleral stiffening effect using glyceraldehyde (7 fold) or methylglyoxal (30 fold), which are alternative low‐cytotoxic collagen crosslinking agents. Recently, SXL was shown to reduce collagen fibril crimping, supporting the notion that SXL can counteract biomechanical and micro‐structural changes in the sclera during myopia . Only one study has investigated the biomechanics of the sclera in animal eyes with induced myopia and after SXL .…”

Topical Corneal Cross-Linking Solution Delivered Via Corneal Reservoir in Dutch-Belted Rabbits

“…This report is the first of using SMG in live animal corneas; the previous literature regarding this compound has been carried out in ex vivo systems. 7 , 8 SMG is a compound from a class of compounds known as formaldehyde-releasing agents (FARs). FARs are in widespread use as chemical preservatives in cosmetics and other personal care products, such as shampoos and conditioners.…”