Darryl R. Overby

Reduction of the Available Area for Aqueous Humor Outflow and Increase in Meshwork Herniations into Collector Channels Following Acute IOP Elevation in Bovine Eyes

PURPOSE To understand how hydrodynamic and morphologic changes in the aqueous humor outflow pathway contribute to decreased aqueous humor outflow facility after acute elevation of intraocular pressure (IOP) in bovine eyes. METHODS Enucleated bovine eyes were perfused at 1 of 4 different pressures (7, 15, 30, 45 mm Hg) while outflow facility was continuously recorded. Dulbecco PBS + 5.5 mM glucose containing fluorescent microspheres (0.5 mum, 0.002% vol/vol) was perfused to outline aqueous outflow patterns, followed by perfusion-fixation. Confocal images were taken along the inner wall (IW) of the aqueous plexus (AP) in radial and frontal sections. Percentage effective filtration length (PEFL; IW length exhibiting tracer labeling/total length of IW) was measured. Herniations of IW into collector channel (CC) ostia were examined and graded for each eye by light microscopy. RESULTS Increasing IOP from 7 to 45 mm Hg coincided with a twofold decrease in outflow facility (P < 0.0001), a 33% to 57% decrease in PEFL with tracer confined more to the vicinity of CC ostia, progressive collapse of the AP, and increasing percentage of CC ostia exhibiting herniations (from 15.6% +/- 6.5% at 7 mm Hg to 95% +/- 2.3% at 30 mm Hg [P < 10(-4)], reaching 100% at 45 mm Hg). CONCLUSIONS Decreasing outflow facility during acute IOP elevation coincides with a reduction in available area for aqueous humor outflow and the confinement of outflow to the vicinity of CC ostia. These hydrodynamic changes are likely driven by morphologic changes associated with AP collapse and herniation of IW of AP into CC ostia.

Electromagnetic needles with submicron pole tip radii for nanomanipulation of biomolecules and living cells

We describe the design and fabrication of a temperature-controlled electromagnetic microneedle (EMN) to generate custom magnetic field gradients for biomedical and biophysical applications. An electropolishing technique was developed to sharpen the EMN pole tip to any desired radius between 100 nm and 20 μm. The EMN can be used to apply strong static or dynamic forces (>50nN) to micrometer- or nanometer-sized magnetic beads without producing significant heating or needle movement. Large tip radii (20 μm) allow magnetic force application to multiple magnetic beads over a large area, while small radii (0.1–6 μm) can be used to selectively pull or capture single magnetic beads from within a large population of similar particles. The customizable EMN is thus well suited for micro- and nanomanipulation of magnetic particles linked to biomolecules or living cells.

Tools to study cell mechanics and mechanotransduction.

Analysis of how cells sense and respond to mechanical stress has been limited by the availability of techniques that can apply controlled mechanical forces to living cells while simultaneously measuring changes in cell and molecular distortion, as well as alterations of intracellular biochemistry. We have confronted this challenge by developing new engineering methods to measure and manipulate the mechanical properties of cells and their internal cytoskeletal and nuclear frameworks, and by combining them with molecular cell biological techniques that rely on microscopic analysis and real-time optical readouts of biochemical signaling. In this chapter, we describe techniques like microcontact printing, magnetic twisting cytometry, and magnetic pulling cytometry that can be systematically used to study the molecular basis of cellular mechanotransduction.

Morphometric Analysis of Aqueous Humor Outflow Structures with Spectral-Domain Optical Coherence Tomography

PURPOSE To describe morphometric details of the human aqueous humor (AH) outflow microvasculature visualized with 360-degree virtual castings during active AH outflow in cadaver eyes and to compare these structures with corrosion casting studies. METHODS The conventional AH outflow pathways of donor eyes (n = 7) and eyes in vivo (n = 3) were imaged with spectral-domain optical coherence tomography (SD-OCT) and wide-bandwidth superluminescent diode array during active AH outflow. Digital image contrast was adjusted to isolate AH microvasculature, and images were viewed in a 3D viewer. Additional eyes (n = 3) were perfused with mock AH containing fluorescent tracer microspheres to compare microvasculature patterns. RESULTS Observations revealed components of the conventional outflow pathway from Schlemms canal (SC) to the superficial intrascleral venous plexus (ISVP). The superficial ISVP in both our study and corrosion casts were composed of interconnected venules (10-50 μm) forming a hexagonal meshwork. Larger radial arcades (50-100 μm) drained the region nearest SC and converged with larger tortuous vessels (>100 μm). A 360-degree virtual casting closely approximated corrosion casting studies. Tracer studies corroborated our findings. Tracer decorated several larger vessels (50-100 μm) extending posteriorly from the limbus in both raw and contrast-enhanced fluorescence images. Smaller tracer-labeled vessels (30-40 μm) were seen branching between larger vessels and exhibited a similar hexagonal network pattern. CONCLUSIONS SD-OCT is capable of detailed morphometric analysis of the conventional outflow pathway in vivo or ex vivo with details comparable to corrosion casting techniques.

Specific Hydraulic Conductivity of Corneal Stroma as Seen by Quick-Freeze/Deep-Etch

Previous studies of the hydraulic conductivity of connective tissues have failed to show a correspondence between ultrastructure and specific hydraulic conductivity. We used the technique of quick-freeze/deep-etch to examine the ultrastructure of the corneal stroma and then utilized morphometric studies to compute the specific hydraulic conductivity of the corneal stroma. Our studies demonstrated ultrastructural elements of the extracellular matrix of the corneal stroma that are not seen using conventional electron microscopic techniques. Furthermore, we found that these structures may be responsible for generating the high flow resistance characteristic of connective tissues. From analysis of micrographs corrected for depth-of-field effects, we used Carmen-Kozeny theory to bound a morphometrically determined specific hydraulic conductivity of the corneal stroma between 0.46 x 10(-14) and 10.3 x 10(-14) cm2. These bounds encompass experimentally measured values in the literature of 0.5 x 10(-14) to 2 x 10(-14) cm2. The largest source of uncertainty was due to the depth-of-field estimates that ranged from 15 to 51 nm; a better estimate would substantially reduce the uncertainty of these morphometrically determined values.

Studies on depth‐of‐field effects in microscopy supported by numerical simulations

Micrographs are two‐dimensional (2D) representations of three‐dimensional (3D) objects. When the depth‐of‐field of a micrograph is comparable with or larger than the characteristic dimension of objects within the micrograph, measured 2D parameters (e.g. particle number density, surface area of particles, fraction of open space) require stereological correction to determine the correct 3D values. Here, we develop a stereological theory using a differential approach to relate the 3D volume fraction and specific surface area to the 2D projected area and perimeter fractions, accounting for the influence of depth‐of‐field. The stereological theory is appropriate for random isotropic arrangements of non‐interpenetrating particles and is valid for convex geometries (e.g. spheres, spheroids, cylinders). These geometrical assumptions allow the stereological formulae to be expressed as a set of algebraic equations incorporating a single parameter to describe particle shape that is tightly bounded between 1.5π and 2π. The stereological theory may also be applied to arrangements of interpenetrating convex particles, and for this case, the resulting stereological formulae become identical to the formulae previously presented by Miles. To test the accuracy of the stereological theory, random computational arrangements of non‐interpenetrating and interpenetrating spheres or cylinders are analysed, and the projected area and perimeter fractions are numerically determined as a function of depth‐of‐field. The computational results show very good agreement with the theoretical predictions over a broad range of depth‐of‐field, volume fraction and particle geometry for both non‐interpenetrating and interpenetrating particles, demonstrating the overall accuracy of the stereological theory. Applications of the stereological theory towards analysis of biological tissues and extracellular matrix are discussed.

Hydrodynamics of aqueous humor outflow

We present a hydrodynamic model of the aqueous outflow pathway that can, for the first time, account for the generation of outflow resistance based upon anatomical measurements. The hydraulic conductivity of the extracellular matrix in the juxtacanalicular connective tissue (JCT) is determined by combining advanced morphometric techniques with electron microscopy images taken using quick-freeze/deep-etch to preserve ultrastructural detail. A hydrodynamic interaction between pores of the endothelium of Schlemms canal, breaks in the basement membrane, and this morphometrically-determined hydraulic conductivity lead to predictions that are consistent with experimentally-measured outflow resistances.

Development of a Two-Color Fluorescent Tracer Technique to Study Aqueous Outflow Patterns and Outflow Resistance in Human Eyes

Glaucoma is a leading cause of blindness, and elevated intraocular pressure (IOP) characteristic of glaucoma is caused by increased aqueous humor outflow resistance. Studies have localized the bulk of outflow resistance to particular regions along the outflow pathway — namely, the inner wall endothelium of Schlemm’s canal and its underlying juxtacanalicular tissue (JCT) [1] — but the hydrodynamic details of how aqueous humor flows through these tissues and how these tissues generate outflow resistance are not well understood.Copyright

A stereologic technique to quantify the specific hydraulic conductivity of extracellular matrix using electron microscopy

Publisher Summary This chapter describes a new stereo-logic technique to quantify the specific hydraulic conductivity K, of extracellular matrix (ECM) directly from electron micrographs. This approach utilizes Darcys law and Carmen-Kozeny theory to express K in terms of ECM porosity (s: ratio of void volume to total volume) and specific surface area (a: ratio of wetted surface area of total volume). A stereo-logic technique is developed to define s and K, in terms of the measurable pore area and perimeter on a micrograph of known depth-of-field. Monte Carlo simulations of random polymer arrangements are performed to demonstrate the accuracy of the stereo-logic theory. The chapter focuses upon the development of a quantitative stereo logic technique to determine the specific hydraulic conductivity of ECM directly from electron micrographs. It validates the stereo logic theory by showing that the theoretical predictions are consistent with results from Monte Carlo simulations of random polymer arrangements, which are representative of the ultra-structure generally observed within ECM. These findings support the application of the stereologic theory as an analytical tool to quantify the specific hydraulic conductivity of ECM directly from electron micrographs. Moreover, a recent study has shown that this stereologic technique is able to successfully predict K for bovine corneal stroma.