Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Abstract

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon's space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Scleral structure and biomechanics

“…Second harmonic generation (SHG) imaging exploits the coherent scattering event whereby two incident photons are converted into a single photon of half the original wavelength. SHG signal emission is an intrinsic property of biological materials containing large repetitive, noncentrosymmetric units that include collagen, and indeed SHG has been used extensively to probe the organisation of scleral collagen fibril bundles (Brown et al, 2007;Cone-Kimball et al, 2013;Jones et al, 2015;Keyes et al, 2011;Pijanka et al, 2012;Teng et al, 2006;Zyablitskaya et al, 2018) (see also Fig 1D). The ability of SHG to image large tissue volumes has enabled full 3D reconstructions of the ex vivo human ONH to be built (Winkler et al, 2010), while its application in monitoring real-time pressure-induced LC and PPS deformations (Midgett et al, 2018;Sigal et al, 2014a) is enhancing our understanding of the role of IOP in glaucoma biomechanics.…”

Changes in Collagen Structure and Permeability of Rat and Human Sclera After Crosslinking

“…More recently, crosslinking of scleral tissue has been proposed as a mechanism to treat myopia, although the data are limited. 10 , 18 It is important to understand how the sclera reacts to crosslinking. Here, we observed that the effects of crosslinking on permeability persist at a significant distance away from the treatment area.…”

Comparison of morphological changes of corneal collagen fibers treated with collagen crosslinking agents using second harmonic generation images

“…Despite advances in clinical and biochemical studies of the effects of corneal CXL, the direct nondestructive observation of morphologic features of corneas after CXL treatment without extrinsic labeling is necessary. To tackle this problem, in recent years, second harmonic generation (SHG) microscopy has become a valid alternative [15][16][17][18].…”