Glutathione Protects the Developing Heart from Defects and Global DNA Hypomethylation Induced by Prenatal Alcohol Exposure.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is caused by prenatal alcohol exposure (PAE), the intake of ethanol (C2 H5 OH) during pregnancy. Features of FASD cover a range of structural and functional defects including congenital heart defects (CHDs). Folic acid and choline, contributors of methyl groups to one-carbon metabolism (OCM), prevent CHDs in humans. Using our avian model of FASD, we have previously reported that betaine, another methyl donor downstream of choline, prevents CHDs. The CHD preventions are substantial but incomplete. Ethanol causes oxidative stress as well as depleting methyl groups for OCM to support DNA methylation and other epigenetic alterations. To identify more compounds that can safely and effectively prevent CHDs and other effects of PAE, we tested glutathione (GSH), a compound that regulates OCM and is known as a "master antioxidant." METHODS/RESULTS: Quail embryos injected with a single dose of ethanol at gastrulation exhibited congenital defects including CHDs similar to those identified in FASD individuals. GSH injected simultaneously with ethanol not only prevented CHDs, but also improved survival and prevented other PAE-induced defects. Assays of hearts at 8 days (HH stage 34) of quail development, when the heart normally has developed 4-chambers, showed that this single dose of PAE reduced global DNA methylation. GSH supplementation concurrent with PAE normalized global DNA methylation levels. The same assays performed on quail hearts at 3 days (HH stage 19-20) of development, showed no difference in global DNA methylation between controls, ethanol-treated, GSH alone, and GSH plus ethanol-treated cohorts. CONCLUSIONS: GSH supplementation shows promise to inhibit effects of PAE by improving survival, reducing the incidence of morphological defects including CHDs, and preventing global hypomethylation of DNA in heart tissues.

BAC-EDTA transepithelial riboflavin-UVA crosslinking has greater biomechanical stiffening effect than standard epithelium-off in rabbit corneas.

Studies suggest that standard corneal collagen crosslinking (CXL) is a safe and effective treatment to stiffen the cornea for keratoconus and other ectatic corneal disorders. The purpose of the present study was to compare the biomechanical effects of transepithelial benzalkonium chloride-EDTA (BAC-EDTA) riboflavin-UVA crosslinking to standard epithelium-off riboflavin-UVA crosslinking in a rabbit model. Corneal stiffness was quantified using optical coherence elastography at two months after treatment. The mean lateral-to-axial displacement ratio for the BAC-EDTA transepithelial CXL group was lower (greater stiffness) [0.062 ± 0.042, mean ± SD] than epithelium-off CXL (mean ± SD: 0.065 ± 0.045) or untreated control eyes (0.069 ± 0.044). Using ANOVA with Tukey correction, a statistically significant difference was found between the BAC-EDTA transepithelial CXL group and standard epithelium-off CXL (p = 0.0019) or the untreated control (p < 0.0001) groups. A graph of the probability density functions for biomechanical stiffness also showed a greater shift in stiffening in the BAC-EDTA transepithelial CXL group than the standard epithelium-off CXL or untreated control group. These results demonstrated that the biomechanical stiffening effect produced by BAC-EDTA transepithelial CXL was greater than that produced by standard epithelium-off CXL in a rabbit model.

Biological and biomechanical responses to traditional epithelium-off and transepithelial riboflavin-UVA CXL techniques in rabbits.

PURPOSE: To compare the biological effects of riboflavin-ultraviolet A (UVA) corneal cross-linking (CXL) performed with a traditional epithelium-off method to several transepithelial methods in a rabbit model. Preliminary experiments on biomechanical rigidity were also performed. METHODS: Four treatment groups were included: (1) standard epithelium-off, (2) tetracaine transepithelial, (3) benzal-konium chloride-ethylenediaminetetraacetic acid (BKC-EDTA) transepithelial, and (4) femtosecond laser-assisted transepithelial riboflavin-UVA CXL. Six eyes from each treatment group and the untreated control group were analyzed at 24 hours and 2 months after treatment in wound healing studies. The TUNEL assay was performed to detect the extent of stromal cell death. Optical density was measured with a Scheimpflug analyzer. The corneal stiffening effect was quantitated in three eyes from each group using optical coherence elastography performed 2 months after treatments. RESULTS: Twenty-four hours after CXL, stromal cell death extended full corneal thickness with both standard epithelium-off CXL and femtosecond laser-assisted CXL, but only approximately one-third stromal depth after BKC-EDTA transepithelial CXL. Negligible stromal cell death was detected with tetracaine transepithelial CXL. Cell death results were statistically different between the BKC-EDTA transepithelial CXL and standard epithelium-off CXL groups (P < .0001). Significant corneal opacity differences were noted. Standard epithelium-off CXL had the greatest density and tetracaine transepithelial CXL had the least density compared to the control group after treatment. As measured with optical coherence elastography, a trend toward greater mean stiffening was observed with BKC-EDTA transepithelial CXL than with epithelium-off CXL, femtosecond laser-assisted CXL, or tetracaine transepithelial CXL, but the result did not reach statistical significance. All of the CXL treatment groups exhibited significantly smaller variance of stiffness compared to the control group. CONCLUSION: In the rabbit model, BKC-EDTA transepithelial CXL produced less stromal cell death and less risk of endothelial cell damage than standard epithelium-off CXL or femtosecond laser-assisted CXL. Additional study is needed to determine whether biomechanical stiffness is significantly different between the epithelium-off CXL and transepithelial CXL groups.

Depth-resolved Corneal Biomechanical Changes Measured Via Optical Coherence Elastography Following Corneal Crosslinking.

Purpose: To evaluate depth-resolved changes of corneal biomechanical properties in eyes with corneal ectasia after corneal crosslinking (CXL) using optical coherence elastography. Methods: In a prospective pilot series of eyes with corneal ectasia, a custom high-speed swept source optical coherence tomography system was used to image the cornea before and 3 months after CXL during a low-speed applanating deformation while monitoring applanation force. Cross-correlation was applied to track frame-by-frame two-dimensional optical coherence tomography speckle displacements, and the slope of force versus local axial displacement behavior during the deformation was used to produce a two-dimensional array of axial stiffness (k). These values were averaged for anterior (ka) and posterior (kp) stromal regions and expressed as a ratio (ka/kp) to assess depth-dependent differences in stiffness. CXL was performed according to the Dresden protocol with a system approved by the U.S. Food and Drug Administration. Results: Four eyes from four patients with keratoconus (n = 3) or post-LASIK ectasia (n = 1) underwent optical coherence elastography before and 3 months after CXL. The mean ka/kp was 1.03 ± 0.07 before CXL compared with 1.34 ± 0.17 after the CXL procedure. All four eyes demonstrated at least a 20% increase in the ka/kp. Conclusions: Preferential stiffening of the anterior stroma with the standard CXL protocol was demonstrated with optical coherence elastography in live human subjects. Translational Relevance: Although ex vivo studies have demonstrated anterior stiffening effects after CXL using various destructive and nondestructive methods, this report presents the first evidence of such changes in serial live human measurements.

Depth-Dependent Corneal Biomechanical Properties in Normal and Keratoconic Subjects by Optical Coherence Elastography.

Purpose: Compare depth-resolved biomechanical properties in normal and keratoconic corneas in live human subjects using optical coherence elastography (OCE). Methods: In a prospective series of normal and keratoconus (KC) eyes, a corneal perturbation was applied by a custom swept-source OCE system using a transparent flat lens coupled to force transducers. Cross-correlation was applied to track frame-by-frame OCT speckle displacement. Regional displacements for the anterior and posterior stroma were plotted in force versus displacement (k) graphs. A spatial biomechanical property ratio (ka/kp ) was defined by dividing the maximum total displacement by the maximum force for the anterior (ka ) and posterior cornea (kp) and was compared between normal and KC groups with the Mann-Whitney U test. Area under the receiver operating characteristics curve (AUROC) for differentiating normal and KC eyes was calculated for ka/kp , kmax, and thinnest point of corneal thickness (TPCT). Results: Thirty-six eyes were analyzed (21 eyes of 12 normal subjects and 15 KC eyes of 12 subjects). The ka/kp for the normal group was 1.135 ± 0.07 (mean ± standard deviation) and 1.02 ± 0.08 for the KC group (P < 0.001), indicating a relative deficit in anterior stromal stiffness in KC eyes. AUROC was 0.91 for ka /kp , 0.95 for kmax, and 1 for TPCT. Conclusions: Significant differences in depth-dependent corneal biomechanical properties were observed between normal and KC subjects. Translational Relevance: OCE was applied for the first time to human KC subjects and revealed alterations in the normal anterior-to-posterior stromal stiffness gradient, a novel and clinically accessible disease biomarker.

Noninvasive Assessment of Corneal Crosslinking With Phase-Decorrelation Optical Coherence Tomography.

Purpose: There is strong evidence that abnormalities in corneal biomechanical play a causal role in corneal ectasias, such as keratoconus. Additionally, corneal crosslinking (CXL) treatment, which halts progression of keratoconus, directly appeals to corneal biomechanics. However, existing methods of corneal biomechanical assessment have various drawbacks: dependence on IOP, long acquisition times, or limited resolution. Here, we present a method that may avoid these limitations by using optical coherence tomography (OCT) to detect the endogenous random motion within the cornea, which can be associated with stromal crosslinking. Methods: Phase-decorrelation OCT (PhD-OCT), based in the theory of dynamic light scattering, is a method to spatially resolve endogenous random motion by calculating the decorrelation rate, Γ, of the temporally evolving complex-valued OCT signal. PhD-OCT images of ex vivo porcine globes were recorded during CXL and control protocols. In addition, human patients were imaged with PhD-OCT using a clinical OCT system. Results: In both the porcine cornea and the human cornea, crosslinking results in a reduction of Γ (P < 0.0001), indicating more crosslinks. This effect was repeatable in ex vivo porcine corneas (change in average Γ = -41.55 ± 9.64%, n = 5) and not seen after sham treatments (change in average Γ = 2.83 ± 12.56%, n = 5). No dependence of PhD-OCT on IOP was found, and correctable effects were caused by variations in signal-to-noise ratio, hydration, and motion. Conclusions: PhD-OCT may be a useful and readily translatable tool for investigating biomechanical properties of the cornea and for enhancing the diagnosis and treatment of patients.

Live human assessment of depth-dependent corneal displacements with swept-source optical coherence elastography.

PURPOSE: To assess depth-dependent corneal displacements in live normal subjects using optical coherence elastography (OCE). METHODS: A corneal elastography method based on swept-source optical coherence tomography (OCT) was implemented in a clinical prototype. Low amplitude corneal deformation was produced during OCT imaging with a linear actuator-driven lens coupled to force transducers. A cross-correlation algorithm was applied to track frame-by-frame speckle displacement across horizontal meridian scans. Intra-measurement force and displacement data series were plotted against each other to produce local axial stiffness approximations, k, defined by the slope of a linear fit to the force/displacement data (ignoring non-axial contributions from corneal bending). Elastographic maps displaying local k values across the cornea were generated, and the ratio of mean axial stiffness approximations for adjacent anterior and posterior stromal regions, ka/kp, was calculated. Intraclass correlation coefficients (ICC) were used to estimate repeatability. RESULTS: Seventeen eyes (ten subjects) were included in this prospective first-in-humans translational study. The ICC was 0.84. Graphs of force vs. displacement demonstrated that, for simultaneously acquired measurements involving the same applied force, anterior stromal displacements were lower (suggesting stiffer behavior) than posterior stromal displacements. Mean ka was 0.016±0.004 g/mm and mean kp was 0.014±0.004 g/mm, giving a mean ka/kp ratio of 1.123±0.062. CONCLUSION: OCE is a clinically feasible, non-invasive corneal biomechanical characterization method capable of resolving depth-dependent differences in corneal deformation behavior. The anterior stroma demonstrated responses consistent with stiffer properties in compression than the posterior stroma, but to a degree that varied across normal eyes. The clinical capability to measure these differences has implications for assessing the biomechanical impact of corneal refractive surgeries and for ectasia risk screening applications.

Corneal Deformation Response and Ocular Geometry: A Noninvasive Diagnostic Strategy in Marfan Syndrome.

PURPOSE: To evaluate corneal air-puff deformation responses and ocular geometry as predictors of Marfan syndrome. DESIGN: Prospective observational clinical study. METHODS: Sixteen investigator-derived, 4 standard Ocular Response Analyzer (ORA), and geometric variables from corneal tomography and optical biometry using Oculus Pentacam and IOL Master were assessed for discriminative value in Marfan syndrome, measuring right eyes of 24 control and 13 Marfan syndrome subjects. Area under the receiver operating characteristic (AUROC) curve was assessed in univariate and multivariate analyses. RESULTS: Six investigator-derived ORA variables successfully discriminated Marfan syndrome. The best lone disease predictor was Concavity Min (Marfan syndrome 47.5 ± 20, control 69 ± 14, P = .003; AUROC = 0.80). Corneal hysteresis (CH) and corneal resistance factor (CRF) were decreased (Marfan syndrome CH 9.45 ± 1.62, control CH 11.24 ± 1.21, P = .01; Marfan syndrome CRF 9.77 ± 1.65, control CRF 11.03 ± 1.72, P = .01) and corneas were flatter in Marfan syndrome (Marfan syndrome Kmean 41.25 ± 2.09 diopter, control Kmean 42.70 ± 1.81 diopter, P = .046). No significant differences were observed in central corneal thickness, axial eye length, or intraocular pressure. A multivariate regression model incorporating corneal curvature and hysteresis loop area (HLA) provided the best predictive value for Marfan syndrome (AUROC = 0.85). CONCLUSIONS: This study describes novel biodynamic features of corneal deformation responses in Marfan syndrome, including increased deformation, decreased bending resistance, and decreased energy dissipation capacity. A predictive model incorporating HLA and corneal curvature shows greatest potential for noninvasive clinical diagnosis of Marfan syndrome.

Serial biomechanical comparison of edematous, normal, and collagen crosslinked human donor corneas using optical coherence elastography.

PURPOSE: To noninvasively evaluate the effects of corneal hydration and collagen crosslinking (CXL) on the mechanical behavior of the cornea. SETTING: Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, USA. DESIGN: Experimental study. METHODS: An optical coherence elastography (OCE) technique was used to measure the displacement behavior of 5 pairs of debrided human donor globes in 3 serial states as follows: edematous, normal thickness, and after riboflavin-ultraviolet-A-mediated CXL. During micromotor-controlled axial displacements with a curved goniolens at physiologic intraocular pressure (IOP), serial optical coherence tomography scans were obtained to allow high-resolution intrastromal speckle tracking and displacement measurements over the central 4.0 mm of the cornea. RESULTS: With no imposed increase in IOP, the mean lateral to imposed axial displacement ratios were 0.035 µm/µm ± 0.037 (SD) in edematous corneas, 0.021 ± 0.02 µm/µm in normal thickness corneas, and 0.014 ± 0.009 µm/µm in post-CXL corneas. The differences were statistically significant (P<.05, analysis of variance) and indicated a 40% increase in lateral stromal resistance with deturgescence and a further 33% mean increase in relative stiffness with CXL. CONCLUSIONS: Serial perturbations of the corneal hydration state and CXL had significant effects on corneal biomechanical behavior. With an axially applied stress from a nonapplanating contact lens, displacements along the direction of the collagen lamellae were 2 orders of magnitude lower than axial deformations. These experiments show the ability of OCE to quantify clinically relevant mechanical property differences under physiologic conditions. FINANCIAL DISCLOSURES: Proprietary or commercial disclosures are listed after the references.

Capturing structure and function in an embryonic heart with biophotonic tools.

Disturbed cardiac function at an early stage of development has been shown to correlate with cellular/molecular, structural as well as functional cardiac anomalies at later stages culminating in the congenital heart defects (CHDs) that present at birth. While our knowledge of cellular and molecular steps in cardiac development is growing rapidly, our understanding of the role of cardiovascular function in the embryo is still in an early phase. One reason for the scanty information in this area is that the tools to study early cardiac function are limited. Recently developed and adapted biophotonic tools may overcome some of the challenges of studying the tiny fragile beating heart. In this chapter, we describe and discuss our experience in developing and implementing biophotonic tools to study the role of function in heart development with emphasis on optical coherence tomography (OCT). OCT can be used for detailed structural and functional studies of the tubular and looping embryo heart under physiological conditions. The same heart can be rapidly and quantitatively phenotyped at early and again at later stages using OCT. When combined with other tools such as optical mapping (OM) and optical pacing (OP), OCT has the potential to reveal in spatial and temporal detail the biophysical changes that can impact mechanotransduction pathways. This information may provide better explanations for the etiology of the CHDs when interwoven with our understanding of morphogenesis and the molecular pathways that have been described to be involved. Future directions for advances in the creation and use of biophotonic tools are discussed.

Method for optical coherence elastography of the cornea.

The material properties of the cornea are important determinants of corneal shape and refractive power. Corneal ectatic diseases, such as keratoconus, are characterized by material property abnormalities, are associated with progressive thinning and distortion of the cornea, and represent a leading indication for corneal transplantation. We describe a corneal elastography technique based on optical coherence tomography (OCT) imaging, in which displacement of intracorneal optical features is tracked with a 2-D cross-correlation algorithm as a step toward nondestructive estimation of local and directional corneal material properties. Phantom experiments are performed to measure the effects of image noise and out-of-plane displacement on effectiveness of displacement tracking and demonstrated accuracy within the tolerance of a micromechanical translation stage. Tissue experiments demonstrate the ability to produce 2-D maps of heterogeneous intracorneal displacement with OCT. The ability of a nondestructive optical method to assess tissue under in situ mechanical conditions with physiologic-range stress levels provides a framework for in vivo quantification of 3-D corneal elastic and viscoelastic resistance, including analogs of shear deformation and Poisson's ratio that may be relevant in the early diagnosis of corneal ectatic disease.