James V. Jester

Three-dimensional distribution of transverse collagen fibers in the anterior human corneal stroma.

PURPOSEnRecent investigations of human corneal structure and biomechanics have shown that stromal collagen fibers (lamellae) are organized into a complex, highly intertwined three-dimensional meshwork of transverse oriented fibers that increases stromal stiffness and controls corneal shape. The purpose of this study was to characterize the three-dimensional distribution of transverse collagen fibers along the major meridians of the cornea using an automated method to rapidly quantify the collagen fibers angular orientation.nnnMETHODSnThree eyes from three donors were perfusion-fixed under pressure, excised, and cut into four quadrants. Quadrants were physically sectioned using a vibratome and scanned using nonlinear optical high-resolution macroscopy. Planes were analyzed numerically using software to identify collagen fiber angles relative to the corneal surface, stromal depth, and radial position within the anterior 250 μm of the stroma.nnnRESULTSnThe range of fiber angles and the fiber percentage having an angular displacement greater than ±3.5° relative to the corneal surface (transverse fibers) was highest in the anterior stroma and decreased with depth. Numerical analysis showed no significant differences in fiber angles and transverse fibers between quadrants, meridians, and radial position.nnnCONCLUSIONSnThese results match our previous observation of a depth-dependent gradient in stromal collagen interconnectivity in the central cornea, and show that this gradient extends from the central cornea to the limbus. The lack of a preferred distribution of angled fibers with regard to corneal quadrant or radial position likely serves to evenly distribute loads and to avoid the formation of areas of stress concentration.

From nano to macro: Studying the hierarchical structure of the corneal extracellular matrix

In this review, we discuss current methods for studying ocular extracellular matrix (ECM) assembly from the nano to the macro levels of hierarchical organization. Since collagen is the major structural protein in the eye, providing mechanical strength and controlling ocular shape, the methods presented focus on understanding the molecular assembly of collagen at the nanometre level using X-ray scattering through to the millimetre to centimetre level using non-linear optical (NLO) imaging of second harmonic generated (SHG) signals. Three-dimensional analysis of ECM structure is also discussed, including electron tomography, serial block face scanning electron microscopy (SBF-SEM) and digital image reconstruction. Techniques to detect non-collagenous structural components of the ECM are also presented, and these include immunoelectron microscopy and staining with cationic dyes. Together, these various approaches are providing new insights into the structural blueprint of the ocular ECM, and in particular that of the cornea, which impacts upon our current understanding of the control of corneal shape, pathogenic mechanisms underlying ectatic disorders of the cornea and the potential for corneal tissue engineering.

A Comparative Study of Vertebrate Corneal Structure: The Evolution of a Refractive Lens

PURPOSEnAlthough corneal curvature plays an important role in determining the refractive power of the vertebrate eye, the mechanisms controlling corneal shape remain largely unknown. To address this question, we performed a comparative study of vertebrate corneal structure to identify potential evolutionarily based changes that correlate with the development of a corneal refractive lens.nnnMETHODSnNonlinear optical (NLO) imaging of second-harmonic-generated (SHG) signals was used to image collagen and three-dimensionally reconstruct the lamellar organization in corneas from different vertebrate clades.nnnRESULTSnSecond-harmonic-generated images taken normal to the corneal surface showed that corneal collagen in all nonmammalian vertebrates was organized into sheets (fish and amphibians) or ribbons (reptiles and birds) extending from limbus to limbus that were oriented nearly orthogonal (ranging from 77.7°-88.2°) to their neighbors. The slight angular offset (2°-13°) created a rotational pattern that continued throughout the full thickness in fish and amphibians and to the very posterior layers in reptiles and birds. Interactions between lamellae were limited to sutural fibers in cartilaginous fish, and occasional lamellar branching in fish and amphibians. There was a marked increase in lamellar branching in higher vertebrates, such that birds ≫ reptiles > amphibians > fish. By contrast, mammalian corneas showed a nearly random collagen fiber organization with no orthogonal, chiral pattern.nnnCONCLUSIONSnOur data indicate that nonmammalian vertebrate corneas share a common orthogonal collagen structural organization that shows increased lamellar branching in higher vertebrate species. Importantly, mammalian corneas showed a different structural organization, suggesting a divergent evolutionary background.

Measurement of an Elasticity Map in the Human Cornea.

Purpose The biomechanical properties of the cornea have an important role in determining the shape of the cornea and visual acuity. Since the cornea is a nonhomogeneous tissue, it is thought that the elastic properties vary throughout the cornea. We aim to measure a map of corneal elasticity across the cornea. Methods An acoustic radiation force elasticity microscope (ARFEM) was used to create a map of corneal elasticity in the human cornea. This ARFEM uses a low frequency, high intensity acoustic force to displace a femtosecond laser-generated microbubble, while using a high frequency, low intensity ultrasound to monitor the position of the microbubble within the cornea. From the displacement of the bubble and the magnitude of the acoustic radiation force, the local value of corneal elasticity is calculated in the direction of the displacement. Measurements were conducted at 6 locations, ranging from the central to peripheral cornea at anterior and posterior depths. Results The mean anterior elastic moduli were 4.2 ± 1.2, 3.4 ± 0.7, and 1.9 ± 0.7 kPa in the central, mid, and peripheral regions, respectively, while the posterior elastic moduli were 2.3 ± 0.7, 1.6 ± 0.3, and 2.9 ± 1.2 kPa in the same radial locations. Conclusions We found that there is a unique distribution of elasticity axially and radially throughout the cornea.

Characterization of Quiescent Epithelial Cells in Mouse Meibomian Glands and Hair Follicle/Sebaceous Glands by Immunofluorescence Tomography

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Confocal Microscopic Analysis of a Rabbit Eye Model of High-Incidence Recurrent Herpes Stromal Keratitis

Purpose: Using CJLAT, a chimeric herpes simplex virus (HSV-1) that produces a high incidence of herpes stromal keratitis (HSK) in latently infected rabbits, and in vivo confocal microscopy (CM), we characterized the cellular events that precede the development of HSK. Methods: Thirty days after infection, in vivo CM was performed daily for 10 days and then weekly for up to 80 days after infection. Results: We detected 3 types of subclinical corneal lesions before HSK was clinically apparent: (1) small epithelial erosions; (2) regenerating epithelium overlying small cell infiltrates within the basal epithelial cell layer; and (3) dendritic-like cells within the basal epithelial layer overlying stromal foci containing infiltrating cells. Sequential in vivo CM observations suggested that subclinical foci resolved over time but were larger and more abundant with CJLAT than with wild-type HSV-1 McKrae. Active HSK was observed only with CJLAT and was initially associated with a large epithelial lesion overlying stromal immune cell infiltrates. Conclusions: These results suggest that replication in the cornea of reactivated virus from the trigeminal ganglia produces epithelial lesions, which recruit immune cell infiltrates into the basal epithelial layer and anterior stroma. The virus is usually cleared rapidly eliminating viral antigens before the arrival of the immune cells, which disperse. However, if the virus is not cleared rapidly, or if an additional reactivation results in an additional round of virus at the same site before the immune cells disperse, then the immune cells are stimulated and may induce an immunopathological response leading to the development of HSK.

A microfabricated, optically accessible device to study the effects of mechanical cues on collagen fiber organization

As the primary structural protein of our bodies, fibrillar collagen and its organizational patterns determine the biomechanics and shape of tissues. While the molecular assembly of individual fibrils is well understood, the mechanisms determining the arrangement of fibers and thus the shape and form of tissues remain largely unknown. We have developed a cell culture model that successfully recapitulates early tissue development and the de novo deposition of collagen fibers to investigate the role of mechanical cues on collagen fiber alignment. The devices used a thin, collagen-coated deformable PDMS membrane inside a tissue culture well built on microscope-grade coverslips. Deformations and strains in the PDMS membrane were quantified by tracking fluorescent bead displacement and through the use of a COMSOL model. Cyclical strains were applied to serum-cultured rabbit corneal cells at 0.5xa0Hz for 24–48xa0h and showed a preferred alignment after 36xa0h of cyclical loading. Cells cultured with ascorbic acid under methylcellulose serum-free conditions deposited a collagenous matrix that was visible under live second harmonic generation microscopy at week 4. Our microfabricated tissue culture system allows for the controllable application of a wide range of stress profiles to cells, and for the observation and quantification of cells and de novo collagen formation in vitro. Future studies will involve the fabrication of models to study the formation and organization of collagen in ocular diseases.

Robust segmentation of corneal fibers from noisy images

Corneal collagen structure, which plays an important role in determining visual acuity, has drawn a lot of research attention to exploring its geometric properties. Advancement of nonlinear optical (NLO) imaging provides a potential way for capturing fiber-level structure of cornea, however, the artifacts introduced by the NLO imaging process make image segmentation on such images a bottleneck for further analysis. Especially, the existing methods fail to preserve the branching points which are important for mechanical analysis. In this paper, we propose a hybrid image segmentation method, which integrates seeded region growing and iterative voting. Results show that our algorithm outperforms state-of-the-art techniques in segmenting fibers from background while preserving branching points. Finally, we show that, based on the segmentation result, branching points and the width of fibers can be determined more accurately than the other methods, which is critical for mechanical analysis on corneal structure.

Indentation Testing of the Optic Nerve Head and Posterior Sclera

The mechanical behavior of the optic nerve head (ONH) and the surrounding sclera play important roles in the development of optic neuropathy. We assessed the indentation behavior of the ONH and the surrounding sclera in unfixed human autopsy eyes from individuals between 27 and 87 years of age. Our testing device utilized a round-tipped 250 µm diameter stainless steel probe to measure the force applied in discrete 50 µm steps from the surface of the tissues to a depth of 400 µm. Thirteen eyes from eight individuals were indented in various locations within and around the ONH and the posterior sclera. Tissue thickness, force and depth were recorded at each position tested. Data was analyzed by evaluating the recorded force as a function of the depth of indentation. The data showed that, for both the sclera and the ONH, the force rose exponentially with increased depth of indentation. Apparent stiffness values were estimated using 2 different equations that assess soft biologic tissues. Results showed that significantly higher levels of force were required with aging to indent the posterior sclera, with a corresponding increase in stiffness values. However, no significant increase in indentation force as a function of age was noted for the ONH. Interestingly, the data suggests that some individuals have relatively large differences in the stiffness between the ONH and the posterior sclera, while others show less difference regardless of age. This data supports the notion that some individuals have relatively softer ONH tissue, and as the sclera becomes stiffer with age, this difference is magnified. This difference may be important in understanding the biomechanical contribution to the individually different susceptibility to glaucoma.