Cheryl R. Hann

Ultrastructural Localization of Collagen IV, Fibronectin, and Laminin in the Trabecular Meshwork of Normal and Glaucomatous Eyes

Purpose: To determine whether differences in the ultrastructural characteristics or composition of the basement membranes of the trabecular lamellae and Schlemm’s canal exist in normal eyes and eyes with primary open-angle glaucoma (POAG). Basement membranes play key roles in the attachment of the overlying trabecular cells and Schlemm’s canal cells. Methods: Electron microscopy used in conjunction with immunogold labeling was used to examine the ultrastructure of the basement membranes in the trabecular meshwork and to determine the presence of collagen IV, laminin, and fibronectin in 6 normal eyes and 6 eyes with POAG. To determine which cells in the meshwork synthesized these molecules in situ hybridization was studied in an additional 8 normal eyes. Results: No distinctive ultrastructural changes were found in the basement membranes of glaucomatous eyes, whether early or advanced disease, when compared with normal eyes. Label for all three proteins was present in the basement membranes of the trabecular lamellae, Schlemm’s canal, and in scattered patches within the juxtacanalicular tissue. Laminin and fibronectin were most abundant in the periphery of the sheath material surrounding the elastic tendons in the juxtacanalicular tissue. In contrast to previously published light microscopic studies, no increase in fibronectin was found in glaucoma. Regions of the basement membrane of the canal underlying giant vacuoles were similar to regions without giant vacuoles in both appearance and labeling. In situ hybridization revealed that mRNA for all three proteins was present in most trabecular cells throughout the meshwork; no regional differences in cellular labeling within were observed. Conclusion: The ultrastructural characteristics and immunogold labeling of basement membranes were similar in normal and glaucomatous eyes; no additional structures were labeled in POAG eyes that were not also labeled in normal eyes. Label of the patches of amorphous fibrogranular material within the juxtacanalicular tissue suggests it is basement membrane in origin, while the sheath material which is known to accumulate in POAG was not heavily labeled and does not appear to be basement membrane in origin.

Second-generation trabecular meshwork bypass stent (istent inject) increases outflow facility in cultured human anterior segments

PURPOSE To determine whether a second-generation trabecular meshwork (TM) bypass stent (iStent inject) influences outflow facility in cultured human anterior segments. DESIGN Prospective laboratory investigation using normal human donor eyes. METHODS Human anterior segments (n = 7) were placed in perfusion organ culture. One or 2 iStent inject stents were inserted into the TM within the nasal and/or superior quadrants using a specially designed injector. Anterior segments were returned to culture and perfused for an additional 24 hours. Morphology of the TM and Schlemm canal (SC) was assessed by scanning electron microscopy (SEM) and 3-dimensional micro-computed tomography (3D micro-CT). RESULTS Insertion of 1 iStent inject into the nasal or superior quadrant of the TM increased outflow facility from 0.16 ± 0.05 μL/min/mm Hg to 0.38 ± 0.23 μL/min/mm Hg (P < .03, n = 7), with concurrent pressure reduction from 16.7 ± 5.4 mm Hg to 8.6 ± 4.4 mm Hg. Addition of a second iStent inject further increased outflow facility to 0.78 ± 0.66 μL/min/mm Hg (n = 2). SEM showed the iStent inject flange compressed against the uveal region of the TM, the thorax securely inserted within the TM, and the head located in the lumen of SC. Dilation of SC was noted around the iStent inject head and SC cell disruption was observed at the iStent inject insertion site. 3D micro-CT confirmed iStent inject placement. CONCLUSION iStent inject, a second-generation bypass stent, increased outflow facility in human anterior segment culture. The iStent inject is a promising new device to lower intraocular pressure via TM bypass.

HUMAN CORNEAL ENDOTHELIAL CELL TRANSPLANTATION IN A HUMAN EX VIVO MODEL

PURPOSE To determine the effects of incorporating superparamagnetic microspheres (SPMs) into cultured human corneal endothelial cells (HCECs) and to describe preliminary experiments of HCEC transplantation, facilitated by SPMs and an external magnetic field, in a human anterior segment ex vivo model. METHODS HCECs were cultured as monolayers and incorporated with magnetite oxide SPMs (900, 300, and 100 nm) at different concentrations. Cell viability, migration toward a magnetic field, and light transmittance were measured after incorporation of the SPMs. HCEC transplantation into the eyes of human recipients was investigated by subjecting anterior segments in organ culture to an external magnetic field. Light and electron microscopy were used to assess HCEC attachment to corneal stroma. RESULTS SPMs were incorporated into the cytoplasm of HCECs after overnight incubation. None of the SPMs affected the short-term viability of cultured HCECs (P > 0.14, n = 6) or their light transmittance (P > 0.06, n = 5), although there was a trend toward decreased transmittance with the higher concentration of 900-nm SPMs. Cell migration toward a magnetic field was higher for HCECs with incorporated SPMs than for HCECs without SPMs (P < or = 0.01, n = 6), with dose-response relationships evident for the 300- and 100-nm SPMs. SPMs facilitated the attachment of HCECs to the corneal stroma in the human anterior segment model with minimal change in intracameral (intraocular) pressure. CONCLUSIONS SPMs facilitate migration of HCECs toward a magnetic source and attachment of cells to the corneal stroma without affecting cell viability or light transmittance. The human anterior segment model can be used to study HCEC transplantation.

Preferential fluid flow in the human trabecular meshwork near collector channels

PURPOSE To determine whether preferential pathways exist within the human trabecular meshwork, pigmented and nonpigmented regions adjacent to and between collector channels were examined, and the configuration of the juxtacanalicular tissue (JCT) was analyzed. METHODS Healthy whole human eyes were perfused at 10 or 25 mm Hg with 0.5 mum fluorescent beads. Tissue wedges of pigmented and nonpigmented meshwork (with and without collector channels) were dissected from each eye and examined by confocal microscopy. Bead concentration adjacent to and between collector channels was quantified. The configuration of the JCT adjacent to collector channels from whole eyes perfused at 20 mm Hg was analyzed by light microscopy. RESULTS Eyes perfused at 25 mm Hg had more beads adjacent to collector channels in pigmented than in nonpigmented regions (4.9%+/-3.5% vs. 1.1%+/-0.9%; P=0.02). In pigmented regions without collector channels, bead concentration was decreased by fivefold (4.9%+/-3.5% vs. 0.96%+/-0.88%; P=0.04). Perfusion at 25 mm Hg increased beads by threefold under pigmented collector channels compared with the same regions in eyes perfused at 10 mm Hg. Expansion of the JCT occurred more often under collector channels at 25 mm Hg than at 10 mm Hg (44% vs. 17%; P=0.01). The JCT region under collector channels was expanded compared with JCT regions between adjacent collector channels (1053+/-424 microm(2) vs. 571+/-216 microm(2); P<0.001). CONCLUSIONS Increased levels of beads in pigmented trabecular meshwork adjacent to collector channels suggest preferential flow pathways are present in human trabecular meshwork. At elevated pressure, the JCT region under collector channels is expanded, possibly because of increased fluid flow.

The Elastin Fiber System between and Adjacent to Collector Channels in the Human Juxtacanalicular Tissue

PURPOSE To determine the composition and investigate the elastin fiber system in the juxtacanalicular tissue adjacent to and between collector channel orifices in normal human eyes. METHODS Normal human eyes (71.0 ± 8.6 years; mean ± SD; n = 4) were perfusion fixed at low (10 mm Hg) and high pressure (20 mm Hg) with 3% paraformaldehyde/0.1 M phosphate buffer. Frontal serial sections were cut from paraffin blocks, and regions with and without collector channels were selected. Sections were stained using Weigerts resorcin-fuchsin stain with oxidation. Immunohistochemistry was performed using antibodies against elastin, fibrillin-1, and microfibrillar-associated protein-1/2. RESULTS Elastin, elaunin, and oxytalan fibers were identified within the juxtacanalicular tissue of the inner and outer walls in low- and high-pressure eyes. These fibers were found at collector channel orifices, between collector channels, and within collector channel walls. Fibrillin-1 was located at the base and lateral edges of Schlemms canal endothelial cells. Microfibrillar-associated protein-1/2 was found with elastin-like fibers at the base of Schlemms canal endothelium cells, in the juxtacanalicular tissue, and in the uveal region. CONCLUSIONS Elastin, elaunin, oxytalan, and elastin-associated proteins fibrillin-1 and microfibrillar-associated protein-1/2 were identified within the juxtacanalicular tissue of the inner and outer walls and within collector channel walls of human eyes perfused at low and high pressure. No differences in labeling patterns for elastin, elaunin, and oxytalan were found in the juxtacanalicular tissue adjacent to or between collector channel orifices. The elastin fiber system appears to have a significant role in the support and distensibility of the juxtacanalicular region under collector channels.

Anatomic Changes in Schlemm's Canal and Collector Channels in Normal and Primary Open-Angle Glaucoma Eyes Using Low and High Perfusion Pressures

PURPOSE To examine the anatomy of Schlemms canal (SC) and collector channels (CCs) in normal human and primary open-angle glaucoma (POAG) eyes under low and high perfusion pressure. METHODS In normal (n = 3) and POAG (n = 3) eye pairs, one eye was perfused at 10 mm Hg while the fellow eye was perfused at 20 mm Hg for 2 hours. Eyes were perfusion fixed at like pressures, dissected into quadrants, embedded in Epon Araldite, and scanned by three-dimensional micro-computed tomography (3D micro-CT). Schlemms canal volume, CC orifice area, diameter, and number were measured using ANALYZE software. RESULTS Normal eyes showed a larger SC volume (3.3-fold) and CC orifice area (9962.8 vs. 8825.2 μm(2)) and a similar CC diameter (34.3 ± 17.8 vs. 32.7 ± 13.0 μm) at 10 mm Hg compared to 20 mm Hg. In POAG eyes, SC volume (2.0-fold), CC orifice area (8049.2 μm(2)-6468.4 μm(2)), and CC diameter (36.2 ± 19.1 vs. 29.0 ± 13.8 μm) were increased in 10 mm Hg compared to 20 mm Hg perfusion pressures. Partial and total CC occlusions were present in normal and POAG eyes, with a 3.7-fold increase in total occlusions in POAG eyes compared to normal eyes at 20 mm Hg. Visualization of CCs increased by 24% in normal and by 21% in POAG eyes at 20 mm Hg compared to 10 mm Hg. Schlemms canal volume, CC area, and CC diameter were decreased in POAG eyes compared to normal eyes at like pressures. CONCLUSIONS Compensatory mechanisms for transient and short periods of increased pressure appear to be diminished in POAG eyes. Variable response to pressure change in SC and CCs may be a contributing factor to outflow facility change in POAG eyes.

Aqueous Humor Rapidly Stimulates Myocilin Secretion from Human Trabecular Meshwork Cells

Myocilin, a protein associated with the development of glaucoma, is expressed in most eye tissues with highest expression observed in trabecular meshwork cells. In culture, primary human trabecular meshwork cells incubated in 10% fetal bovine serum have reduced myocilin expression compared to in vivo, but incubation in human aqueous humor, their normal in vivo nutrient source, restores myocilin expression to near in vivo levels. To investigate the mechanism by which human aqueous humor stimulates myocilin accumulation in conditioned media from normal human trabecular meshwork cells, three independent trabecular meshwork cell lines were cultured in Dulbeccos Modified Eagles Medium (DMEM) containing various supplements: fetal bovine serum (10%), human serum (0.2%), porcine aqueous humor (50%), bovine serum albumin (0.1%), dexamethasone (10(-7)M), human aqueous humor (50%) or heat-inactivated human aqueous humor (50%). Conditioned media from cultured primary trabecular meshwork cells following incubation in human aqueous humor showed significant accumulation of myocilin in a time- (15 min) and dose-dependent manner (half maximal effective concentration ∼ 30%) while intracellular myocilin levels decreased. Minimal myocilin accumulation was observed in conditioned media isolated from trabecular meshwork cells cultured in DMEM containing fetal bovine or human serum, bovine serum albumin, porcine aqueous humor, dexamethasone or DMEM alone. Heat inactivation of human aqueous humor nearly eliminated human aqueous humor-stimulated myocilin secretion. Inhibitors of new protein synthesis, gene transcription, the endoplasmic reticulum/Golgi system and endocytic/exocytic secretory pathways failed to inhibit human aqueous humor-stimulated myocilin secretion. Using immunolabeling and transmission electron microscopy, myocilin was found associated with 70-90 nm vesicle-like structures within the cytoplasm of human aqueous humor treated trabecular meshwork cells. These studies suggest that myocilin secretion from trabecular meshwork cells occurs in a Golgi-independent manner following human aqueous humor treatment. Heat-labile factors in human aqueous humor are responsible for the time- and dose-dependent release of myocilin from vesicle-like structures within the cytoplasm of trabecular meshwork cells.

Aqueous Humor Outflow: Dynamics and Disease

This review summarizes the goals and outcomes of the eighth annual ARVO/Pfizer Ophthalmic Research Institute conference that was held on May 4th and 5th, 2012, at the Embassy Suites Fort Lauderdale, Fort Lauderdale, Florida. This conference series has been funded by the ARVO Foundation for Eye Research with the help of a generous grant from Pfizer Ophthalmics. Funding from the ARVO/Pfizer Ophthalmics Research Institute has allowed a series of “think tanks” for the leading experts on various subspecialties of ophthalmology research. These meetings have helped to define various challenges and potential solutions to issues relevant to the field of ophthalmology. In 2012, the emphasis of the conference was on the physiological role of the conventional outflow pathway in aqueous humor dynamics in normal and glaucomatous eyes. In particular, clinical observations associated with aqueous humor outflow in health and disease, animal models in the understanding of aqueous outflow, role of mechanosensing in aqueous humor fluid flow, and new paradigms in cell and extracellular matrix crosstalk within the conventional outflow pathway were discussed. The primary goal of the conference was to bring together a diverse group of experts who are pioneers in conventional outflow biology and related nonocular areas of research with the hope of utilizing the groups knowledge and experience to evaluate the current understanding of aqueous outflow regulation and problems thereof. The invited group consisted of 29 investigators directly involved in conventional outflow research. Also present were three invited outside experts in cellular mechanics and cytoskeleton structures (Jeffrey Fredberg, PhD, Harvard School of Public Health, Boston, MA, USA), molecular mechanisms of cell adhesion and mechanosensing (Benjamin Geiger, PhD, Weizmann Institute of Science, Rehovot, Israel), and murine genetics (Richard Libby, PhD, University of Rochester Medical Center, Rochester, NY, USA). In addition, the conference was attended by 27 observers who participated in discussion of key topics at the end of each presentation. Together, the group was tasked with reviewing the current scientific dogma regarding aqueous outflow diseases and identifying the most pressing research questions and needs in the current funding environment. Above all, the group was asked to think “outside the box” to develop future research directions. The conference generated a list of highly relevant but currently unanswered questions with the hope that solutions to these issues would improve understanding of the role of the conventional outflow pathway in normal and glaucoma eyes. The meeting was organized into four sessions: (a) clinical insights into conventional outflow dysfunction, (b) the use of mice as model systems for conventional outflow, (c) mechanosensing within the conventional outflow pathway, and (d) the role of the extracellular matrix and signaling in conventional outflow dynamics. Speakers presented their thoughts on the preselected topics and provided key research goals that have been addressed or need to be addressed in the near future. The sessions were followed by discussion aimed at summarizing and interpreting the current knowledge as well as identifying unique research questions that were as yet unanswered. Anatomy and Physiology of the Conventional Aqueous Humor Outflow Pathway The conventional outflow pathway is mainly a pressure-driven system. Under homeostatic conditions, this pathway regulates the drainage of aqueous humor from the anterior chamber of the eye, thereby maintaining a constant intraocular pressure (IOP).1 Relevant tissues of the anterior segment that are anatomically involved in IOP control include the ciliary muscles, trabecular meshwork (TM), Schlemms canal (SC), collector channels, and aqueous veins. The ciliary muscle is composed of smooth muscle fibers that have a true elastic net of tendons that anchor into the choroid posteriorly and the scleral spur, TM, and inner wall of SC anteriorly.2,3 It is widely accepted that contraction of the ciliary muscle causes expansion of the TM and opening of SC, which subsequently increases the conductivity of aqueous humor (AH) through the TM. All these, along with reports of nerve innervation in the TM, indicate that the TM is a self-regulating tissue with both afferent and efferent nervous components responsible for controlling its functions.2,4

ATP-sensitive potassium (KATP) channel activation decreases intraocular pressure in the anterior chamber of the eye.

PURPOSE. ATP-sensitive potassium channel (K(ATP)) openers target key cellular events, many of which have been implicated in glaucoma. The authors sought to determine whether K(ATP) channel openers influence outflow facility in human anterior segment culture and intraocular pressure (IOP) in vivo. METHODS. Anterior segments from human eyes were placed in perfusion organ culture and treated with the K(ATP) channel openers diazoxide, nicorandil, and P1075 or the K(ATP) channel closer glyburide (glibenclamide). The presence, functionality, and specificity of K(ATP) channels were determined by RT-PCR, immunohistochemistry, and inside-out patch clamp in human trabecular meshwork (TM) tissue or primary cultures of normal human trabecular meshwork (NTM) cells. The effect of diazoxide on IOP in anesthetized Brown Norway rats was measured with a rebound tonometer. RESULTS. K(ATP) channel openers increased outflow facility in human anterior segments (0.14 ± 0.02 to 0.26 ± 0.09 μL/min/mm Hg; P < 0.001) compared with fellow control eyes (0.22 ± 0.11 to 0.21 ± 0.11 μL/min/mm Hg; P > 0.5). The effect was reversible, with outflow facility returning to baseline after drug removal. The addition of glyburide inhibited diazoxide from increasing outflow facility. Electrophysiology confirmed the presence and specificity of functional K(ATP) channels. K(ATP) channel subunits K(ir)6.1, K(ir)6.2, SUR2A, and SUR2B were expressed in TM and NTM cells. In vivo, diazoxide significantly lowered IOP in Brown Norway rats. CONCLUSIONS. Functional K(ATP) channels are present in the trabecular meshwork. When activated by K(ATP) channel openers, these channels increase outflow facility through the trabecular outflow pathway in human anterior segment organ culture and decrease IOP in Brown Norway rat eyes.

Antigen Retrieval of Basement Membrane Proteins from Archival Eye Tissues

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no “new” components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.