Detecting Mechanical Anisotropy of the Cornea Using Brillouin Microscopy.

Purpose: The purpose of this study was to detect the mechanical anisotropy of the cornea using Brillouin microscopy along different perturbation directions. Methods: Brillouin frequency shift of both whole globes (n = 10) and cornea punches (n = 10) were measured at different angles to the incident laser, thereby probing corneal longitudinal modulus of elasticity along different directions. Frequency shift of virgin (n = 26) versus cross-linked corneas (n = 15) over a large range of hydration conditions were compared in order to differentiate the contributions to Brillouin shift due to hydration from those due to stromal tissue. Results: We detected mechanical anisotropy of corneas, with an average frequency shift increase of 53 MHz and 96 MHz when the instrument probed from 0° to 15° and 30° along the direction of the stromal fibers. Brillouin microscopy did not lose sensitivity to mechanical anisotropy up to 96% water content. We experimentally measured and theoretically modeled how mechanical changes independent of hydration affect frequency shift as a result of corneal cross-linking by isolating an approximately 100 MHz increase in frequency shift following a cross-linking procedure purely due to changes of stromal tissue mechanics. Conclusions: Brillouin microscopy is sensitive to mechanical anisotropy of the stroma even in highly hydrated corneas. The agreement between model and experimental data suggested a quantitative relationship between Brillouin frequency shift, hydration state of the cornea, and stromal tissue stiffness. Translational Relevance: The protocol and model validated throughout this study offer a path for comprehensive measurements of corneal mechanics within the clinic; allowing for improved evaluation of the long-term mechanical efficacy of cross-linking procedures.

Multifunctional synthetic Bowman's membrane-stromal biomimetic for corneal reconstruction.

As the outermost layer of the eye, the cornea is vulnerable to physical and chemical trauma, which can result in loss of transparency and lead to corneal blindness. Given the global corneal donor shortage, there is an unmet need for biocompatible corneal substitutes that have high transparency, mechanical integrity and regenerative potentials. Herein we engineered a dual-layered collagen vitrigel containing biomimetic synthetic Bowman's membrane (sBM) and stromal layer (sSL). The sBM supported rapid epithelial cell migration, maturation and multilayer formation, and the sSL containing tissue-derived extracellular matrix (ECM) microparticles presented a biomimetic lamellar ultrastructure mimicking the native corneal stroma. The incorporation of tissue-derived microparticles in sSL layer significantly enhanced the mechanical properties and suturability of the implant without compromising the transparency after vitrification. In vivo performance of the vitrigel in a rabbit anterior lamellar keratoplasty model showed full re-epithelialization within 14 days and integration of the vitrigel with the host tissue stroma by day 30. The migrated epithelial cells formed functional multilayer with limbal stem cell marker p63 K14 expressed in the lower layer, epithelial marker K3 and K12 expressed through the layers and tight junction protein ZO-1 expressed by the multilayers. Corneal fibroblasts migrated into the implants to facilitate host/implant integration and corneal stromal regeneration. In summary, these results suggest that the multi-functional layers of this novel collagen vitrigel exhibited significantly improved biological performance as corneal substitute by harnessing a fast re-epithelialization and stromal regeneration potential.

Biomechanical Impact of Localized Corneal Cross-linking Beyond the Irradiated Treatment Area.

PURPOSE: To investigate the stiffening effect of localized corneal cross-linking (L-CXL) within and beyond the irradiated region in three dimensions. METHODS: Ten porcine eyes were debrided of epithelium and incrementally soaked with 0.1% riboflavin solution. Using a customized, sharp-edged mask, half of the cornea was blocked while the other half was exposed to blue light (447 nm). The three-dimensional biomechanical properties of each cornea were then measured via Brillouin microscopy. An imaging system was used to quantify the optimal transition zone between cross-linked and non-cross-linked sections of the cornea when considering light propagation and scattering. RESULTS: A broad transition zone of 610 µm in width was observed between the fully cross-linked and non-cross-linked sections, indicating the stiffening response extended beyond the irradiated region. Light propagation and the scattering induced by the riboflavin-soaked cornea accounted for a maximum of 25 and 159 ± 3.2 µm, respectively. CONCLUSIONS: The stiffening effect of L-CXL extends beyond that of the irradiated area. When considering L-CXL protocols clinically, it will be important to account for increased stiffening in surrounding regions. [J Refract Surg. 2019;35(4):253-260.].

Simulating the Mechanics of Lens Accommodation via a Manual Lens Stretcher.

The goal of this protocol is to mimic the biomechanics of physiological accommodation in a cost-efficient, practical manner. Accommodation is achieved through the contraction of the ciliary body and relaxation of zonule fibers, which results in the thickening of the lens necessary for near vision. Here, we present a novel, simple method in which accommodation is replicated by tensing the zonules connected to the lens capsule via a manual lens stretcher (MLS). This method monitors the radial stretching achieved by a lens when subjected to a consistent force and allows for a comparison of accommodating lenses, which can be stretched, to non-accommodating lenses, which cannot be stretched. Importantly, the stretcher couples to the zonules directly, and not to the sclera of the eye, thus only requiring the lens, zonules, and ciliary body rather than the entire globe sample. This difference can significantly decrease the cost of acquiring donor cadaver lenses by about 62% compared to acquiring an entire globe.

Mechanical outcome of accelerated corneal crosslinking evaluated by Brillouin microscopy.

PURPOSE: To quantify corneal mechanical changes induced by corneal crosslinking (CXL) procedures of different ultraviolet-A (UVA) intensity and exposure time using Brillouin microscopy. SETTINGS: University of Maryland, College Park, Maryland, USA. DESIGN: Experimental study. METHODS: Porcine cornea samples were debrided of epithelia and soaked with riboflavin 0.1% solution. Samples were exposed to a standard 5.4 J/cm2 of UVA radiation with varying intensity and exposure time as follows: 3 mW/cm2 for 30.0 minutes, 9 mW/cm2 for 10.0 minutes, 34 mW/cm2 for 2.65 minutes, and 50 mW/cm2 for 1.80 minutes. Using Brillouin microscopy, the Brillouin modulus for each sample was computed as a function of radiation intensity/exposure time. For validation, the Young's modulus was found with the stress-strain test and compared at each irradiation condition. RESULTS: The standard 3 mW/cm2 irradiance condition produced a significantly larger increase in corneal Brillouin modulus than the 9 mW/cm2 (P ≤ .05), 34 mW/cm2 (P ≤ .01), and 50 mW/cm2 (P ≤ .01) conditions. Depth analysis showed similar anterior sections of the standard and 9 mW/cm2 conditions but significantly less stiffening in the central and posterior of the 9 mW/cm2 condition. The stiffening of the standard protocol was significantly larger in all sections of the 34 mW/cm2 and 50 mW/cm2 conditions (P ≤ .01). The overall change in Brillouin-derived Brillouin modulus correlated with the increase in Young's modulus (R2 = 0.98). CONCLUSIONS: At a constant UVA light dose, accelerating the irradiation process decreased CXL stiffening. Brillouin analysis showed that accelerated protocols were especially ineffective in the deeper portions of the cornea.

Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™.

The repeats-in-toxin family of toxins includes proteins produced by Gram negative bacteria such as Escherichia coli (α-hemolysin), Bordetella pertussis (adenylate cyclase toxin), and Aggregatibacter actinomycetemcomitans (LtxA), which contribute to the pathogenesis of these organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In its association with target cells, LtxA binds to a receptor, lymphocyte function-associated antigen-1, as well as membrane lipids and cholesterol, before being internalized via a lysosomal-mediated pathway. The motivation for this project comes from our discovery that DRAQ5™, a membrane-permeable nuclear stain, prevents the internalization of LtxA in a Jurkat T cell line. We hypothesized that DRAQ5™, in crossing the plasma membrane, alters the properties of the membrane to inhibit LtxA internalization. To investigate how DRAQ5™ interacts with the lipid membrane to prevent LtxA internalization, we used studied DRAQ5™-mediated membrane changes in model membranes using a variety of techniques, including differential scanning calorimetry and fluorescence spectroscopy. Our results suggest that DRAQ5™ inhibits the activity of LtxA by decreasing the fluidity of the cellular lipid membrane, which decreases LtxA binding. These results present an interesting possible anti-virulence strategy; by altering bacterial toxin activity by modifying membrane fluidity, it may be possible to inhibit the pathogenicity of A. actinomycetemcomitans.