Endothelial Cell Loss Following Cataract Surgery Using Continuous Curvilinear Capsulorhexis or Precision Pulse Capsulotomy.

Purpose: To compare endothelial cell density (ECD), percentage of hexagonal cells (%Hex) and coefficient of variation (CV) in cell size following lens cataract surgery with phacoemulsification performed using Continuous Curvilinear Capsulorhexis (CCC) or Precision Pulse Capsulotomy (PPC). Patients and Methods: Sixty-seven subjects were randomly assigned to undergo lens cataract removal with the capsulotomy step performed using either CCC or PPC. Specular microscopy images were obtained pre-operatively, 1 month and 3 months after surgery. ECD, %Hex and CV were analyzed in a masked fashion by an independent reading center. Results: The mean percentage ECD loss at 1 month was 11.5% in the CCC group and 12.3% in the PPC group (P = 0.818; t-test). At 3 months, the mean percentage ECD loss was 11.7% in the CCC group and 12.4% in the PPC group (P = 0.815; t-test). The mean %Hex at 1 month was 54.3% in the CCC group and 54.7% in the PPC group (P = 0.695; t-test). At 3 months, the mean %Hex was 56.2% in the CCC group and 54.7% in the PPC group (P = 0.278; t-test). The CV at 1 month was 34.4% in the CCC group and 34.3% in the PPC group (P = 0.927; t-test). At 3 months, the CV was 32.7% in the CCC group and 33.4% in the PPC group (P = 0.864; t-test). Conclusion: No differences in ECD loss, %Hex and CV were observed between patients who received CCC or PPC. PPC use during cataract surgery does not result in any increased endothelial cell loss beyond that normally associated with this surgery.

Automated precision pulse capsulotomy vs manual capsulorhexis in white cataracts: reduction in procedural time and resource utilization.

PURPOSE: To compare the utility of precision pulse capsulotomy (PPC) with manual capsulorhexis for capsulotomy in white cataracts. SETTING: Hospital-based academic practice. DESIGN: Retrospective analysis of surgical case records and surgical videos from a single surgeon. METHODS: Cases involving intumescent and nonintumescent white cataracts were identified. Capsulotomy outcomes, surgical outcomes, procedural time, and resource utilization, as well as patient demographic and health data, were analyzed and subjected to statistical testing. RESULTS: 15 cases of white cataract (10 intumescent and 5 nonintumescent) performed using continuous curvilinear capsulorhexis (CCC) were compared with 20 cases (9 intumescent and 11 nonintumescent) performed using PPC. The cases covered a period of 14 months before and 30 months after surgeon adoption of PPC. There were no significant differences between the 2 groups in patient age, sex, ethnicity, ocular history, medical history, and medications. PPC resulted in complete capsulotomies without tags or tears and intracapsular intraocular lens implantation with 360-degree capsular overlap in all 20 cases. There was 1 CCC case resulting in the Argentinian flag sign. Compared with CCC, PPC white cataract cases also demonstrated significant advantages in capsulotomy time, reduced use of trypan blue and ophthalmic viscosurgical device, and less overall procedural time. CONCLUSIONS: PPC is a safe and highly effective method to create consistent capsulotomies in both intumescent and nonintumescent white cataracts. The use of PPC provides benefits of significant reductions in capsulotomy time, overall procedural time, and resource utilization, resulting in a streamlined treatment of these complex cataract surgery cases.

Use of P1-P4 Purkinje reflections as a surrogate sign for intraoperative patient fixation.

Circumferential, even anterior capsular overlap maximizes intraocular lens stability and posterior capsular opacification mitigation and provides best long-term outcomes for the cataract patient. P1 and P4 Purkinje reflections at patient fixation may provide a reliable marker for capsulotomy centration. However, patient fixation may be hindered during surgery because of anesthesia or light sensitivity. In this study, we demonstrate that the relationship between the P1 and P4 Purkinje reflections previewed prior to surgery when the patient is fixating may be recreated intraoperatively if fixation becomes difficult. The final position of P1 and P4 relative to one another at fixation is invariant in a given patient, but there are variations among patients. Knowledge of the P1 and P4 relationship can be used as a surrogate sign of patient fixation to assist in capsulotomy centration during cataract surgery.

Precision pulse capsulotomy: performance metrics and utility in routine and complex cases.

PURPOSE: To evaluate precision pulse capsulotomy (PPC) performance. SETTING: University and private practice in the United States and South Korea. DESIGN: Multicenter retrospective analysis. METHODS: The surgical videos of 337 cataract surgeries with PPC capsulotomy performed by 4 surgeons at 4 centers were used to assess capsulotomy outcomes including completion rate, diameter, roundness (ovality), and quality of capsular overlap. RESULTS: PPC use resulted in 99.4% free-floating capsulotomies from 337 cases. Video image analysis in a subset (n = 52) yielded a mean capsulotomy diameter of 5.0 mm ± 0.16 mm SD (95% CI, 4.96-5.04 mm). Capsulotomies were round to slightly oval at the end of the case with a mean ovality of 3.0% ± 2.86% (95% CI, 2.22%-3.78%; 360 degrees capsular overlap was obtained in 98% of cases. The offset of the capsulotomy center with the intraocular lens (IOL) optic center was 197 µm ± 122 µm (SD) (95% CI, 148-246 µm). PPC was used successfully in traumatic cataracts with compromised anterior and posterior capsule, phacodonesis, intumescent cataract with constricted pupil, and zonular dialysis and in penetrating keratoplasty with open-sky extracapsular cataract extraction. CONCLUSIONS: Surgeons obtained good PPC capsulotomy outcomes in routine and challenging cases. Little variation was observed in achieving free-floating capsulotomies with approximately 5.0 mm diameter and complete capsular overlap. Variation was observed in the amount of offset between the capsulotomy center and the center of the IOL optic. PPC was useful in cases with multiple comorbidities that challenge capsulotomy performance.

Fabry-Pérot optical sensor and portable detector for monitoring high-resolution ocular hemodynamics.

Our understanding of ocular hemodynamics and its role in ophthalmic disease progression remains unclear due to the shortcomings of precise and on-demand biomedical sensing technologies. Here, we report high-resolution in vivo assessment of ocular hemodynamics using a Fabry-Pérot cavity-based micro-optical sensor and a portable optical detector. The designed optical system is capable of measuring both static intraocular pressure and dynamic ocular pulsation profiles in parallel. Through a dynamic intensity variation analysis method which improves sensing resolution by 3-4 folds, our system is able to extract systolic/diastolic phases from a single ocular pulsation profile. Using a portable detector, we performed in vivo studies on rabbits and verified that ophthalmic parameters obtained from our optical system closely match with traditional techniques such as tonometry, electrocardiography, and photo-plethysmography.

Multifunctional biophotonic nanostructures inspired by the longtail glasswing butterfly for medical devices.

Numerous living organisms possess biophotonic nanostructures that provide colouration and other diverse functions for survival. While such structures have been actively studied and replicated in the laboratory, it remains unclear whether they can be used for biomedical applications. Here, we show a transparent photonic nanostructure inspired by the longtail glasswing butterfly (Chorinea faunus) and demonstrate its use in intraocular pressure (IOP) sensors in vivo. We exploit the phase separation between two immiscible polymers (poly(methyl methacrylate) and polystyrene) to form nanostructured features on top of a Si3N4 substrate. The membrane thus formed shows good angle-independent white-light transmission, strong hydrophilicity and anti-biofouling properties, which prevent adhesion of proteins, bacteria and eukaryotic cells. We then developed a microscale implantable IOP sensor using our photonic membrane as an optomechanical sensing element. Finally, we performed in vivo testing on New Zealand white rabbits, which showed that our device reduces the mean IOP measurement variation compared with conventional rebound tonometry without signs of inflammation.

Biocompatible Multifunctional Black-Silicon for Implantable Intraocular Sensor.

Multifunctional black-silicon (b-Si) integrated on the surface of an implantable intraocular pressure sensor significantly improves sensor performance and reliability in six-month in vivo studies. The antireflective properties of b-Si triples the signal-to-noise ratio and increases the optical readout distance to a clinically viable 12 cm. Tissue growth and inflammation response on the sensor is suppressed demonstrating desirable anti-biofouling properties.

Real-Time In Vivo Intraocular Pressure Monitoring using an Optomechanical Implant and an Artificial Neural Network.

Optimized glaucoma therapy requires frequent monitoring and timely lowering of elevated intraocular pressure (IOP). A recently developed microscale IOP-monitoring implant, when illuminated with broadband light, reflects a pressure-dependent optical spectrum that is captured and converted to measure IOP. However, its accuracy is limited by background noise and the difficulty of modeling non-linear shifts of the spectra with respect to pressure changes. Using an end-to-end calibration system to train an artificial neural network (ANN) for signal demodulation we improved the speed and accuracy of pressure measurements obtained with an optically probed IOP-monitoring implant and make it suitable for real-time in vivo IOP monitoring. The ANN converts captured optical spectra into corresponding IOP levels. We achieved an IOP-measurement accuracy of ±0.1 mmHg at a measurement rate of 100 Hz, which represents a ten-fold improvement from previously reported values. This technique allowed real-time tracking of artificially induced sub-1 s transient IOP elevations and minor fluctuations induced by the respiratory motion of the rabbits during in vivo monitoring. All in vivo sensor readings paralleled those obtained concurrently using a commercial tonometer and showed consistency within ±2 mmHg. Real-time processing is highly useful for IOP monitoring in clinical settings and home environments and improves the overall practicality of the optical IOP-monitoring approach.

A microscale optical implant for continuous in vivo monitoring of intraocular pressure.

Intraocular pressure (IOP) is a key clinical parameter in glaucoma management. However, despite the potential utility of daily measurements of IOP in the context of disease management, the necessary tools are currently lacking, and IOP is typically measured only a few times a year. Here we report on a microscale implantable sensor that could provide convenient, accurate, on-demand IOP monitoring in the home environment. When excited by broadband near-infrared (NIR) light from a tungsten bulb, the sensor's optical cavity reflects a pressure-dependent resonance signature that can be converted to IOP. NIR light is minimally absorbed by tissue and is not perceived visually. The sensor's nanodot-enhanced cavity allows for a 3-5 cm readout distance with an average accuracy of 0.29 mm Hg over the range of 0-40 mm Hg. Sensors were mounted onto intraocular lenses or silicone haptics and secured inside the anterior chamber in New Zealand white rabbits. Implanted sensors provided continuous in vivo tracking of short-term transient IOP elevations and provided continuous measurements of IOP for up to 4.5 months.

Regulation of axon regeneration by the RNA repair and splicing pathway.

Mechanisms governing a neuron's regenerative ability are important but not well understood. We identify Rtca (RNA 3'-terminal phosphate cyclase) as an inhibitor of axon regeneration. Removal of Rtca cell-autonomously enhanced axon regrowth in the Drosophila CNS, whereas its overexpression reduced axon regeneration in the periphery. Rtca along with the RNA ligase Rtcb and its catalyst Archease operate in the RNA repair and splicing pathway important for stress-induced mRNA splicing, including that of Xbp1, a cellular stress sensor. Drosophila Rtca and Archease had opposing effects on Xbp1 splicing, and deficiency of Archease or Xbp1 impeded axon regeneration in Drosophila. Moreover, overexpressing mammalian Rtca in cultured rodent neurons reduced axonal complexity in vitro, whereas reducing its function promoted retinal ganglion cell axon regeneration after optic nerve crush in mice. Our study thus links axon regeneration to cellular stress and RNA metabolism, revealing new potential therapeutic targets for treating nervous system trauma.

Ectopic vesicular glutamate release at the optic nerve head and axon loss in mouse experimental glaucoma.

Although clinical and experimental observations indicate that the optic nerve head (ONH) is a major site of axon degeneration in glaucoma, the mechanisms by which local retinal ganglion cell (RGC) axons are injured and damage spreads among axons remain poorly defined. Using a laser-induced ocular hypertension (LIOH) mouse model of glaucoma, we found that within 48 h of intraocular pressure elevation, RGC axon segments within the ONH exhibited ectopic accumulation and colocalization of multiple components of the glutamatergic presynaptic machinery including the vesicular glutamate transporter VGLUT2, several synaptic vesicle marker proteins, glutamate, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex and active zone cytomatrix components, as well as ultrastructurally identified, synaptophysin-containing vesicles. Ectopic vesicle exocytosis and glutamate release were detected in acute preparations of the LIOH ONH. Immunolocalization and analysis using the ionotropic receptor channel-permeant cation agmatine indicated that ONH axon segments and glia expressed glutamate receptors, and these receptors were more active after LIOH compared with controls. Pharmacological antagonism of glutamate receptors and neuronal activity resulted in increased RGC axon sparing in vivo. Furthermore, in vivo RGC-specific genetic disruption of the vesicular glutamate transporter VGLUT2 or the obligatory NMDA receptor subunit NR1 promoted axon survival in experimental glaucoma. As the inhibition of ectopic glutamate vesicular release or glutamate receptivity can independently modify the severity of RGC axon loss, synaptic release mechanisms may provide useful therapeutic entry points into glaucomatous axon degeneration.

Rabbit models for continuous curvilinear capsulorhexis instruction.

PURPOSE: To develop a rabbit model for continuous curvilinear capsulorhexis (CCC) instruction. SETTING: University of California San Francisco, San Francisco, California, USA. DESIGN: Experimental study. METHODS: Isolated rabbit lenses were immersed in 2% to 8% paraformaldehyde (PFA) fixative from 15 minutes to 6 hours. Rabbit eyes were treated by substituting aqueous with 2% to 4% PFA for 30 minutes to 6 hours, followed by washes with a balanced salt solution. Treated lenses and eyes were held in purpose-designed holders using vacuum. A panel of 6 cataract surgeons with 5 to 15 years of experience performed CCC on treated lenses and eyes and responded to a questionnaire regarding the utility of these models for resident teaching using a 5-item Likert scale. RESULTS: The expert panel found that rabbit lenses treated with increasing amounts of fixative simulated CCC on human lens capsules from the third to the seventh decade of life. The panel also found fixative-treated rabbit eyes to simulate some of the experience of CCC within the human anterior chamber but noted a shallower anterior chamber depth, variation in pupil size, and corneal clouding under some treatment conditions. CONCLUSIONS: Experienced cataract surgeons who performed CCC on these rabbit models strongly agreed that isolated rabbit lenses treated with fixative provide a realistic simulation of CCC in human patients and that both models were useful tools for capsulorhexis instruction. Results indicate that rabbit lenses treated with 8% PFA for 15 minutes is a model with good fidelity for CCC training. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Involvement of EphB/Ephrin-B signaling in axonal survival in mouse experimental glaucoma.

PURPOSE: To examine the functional significance of EphB/ephrin-B upregulation in mouse experimental glaucoma. METHODS: In a loss-of-function approach, mouse mutants lacking EphB2 (EphB2(-/-)) or EphB3 (EphB3(-/-)) protein, and mutants expressing EphB2 truncated in the C-terminus (EphB2(lacZ/lacZ)) were subjected to laser-induced ocular hypertension (LIOH), an experimental mouse model of glaucoma. The number of optic nerve axons was counted in paraphenylenediamine (PPD)-stained sections and compared between EphB mutants and wild type littermates. In a gain-of-function approach, retina/optic nerve explants obtained from LIOH-treated animals were exposed to EphB2-Fc recombinant proteins or Fc control proteins. Tissue sections through the optic nerve head (ONH) were labeled with neuron-specific anti-tubulin ß-III antibody to determine axonal integrity. RESULTS: Both EphB2 and EphB3 null mutant mice exhibited more severe axonal degeneration than wild type littermates after treatment with LIOH. Mutant mice in which the C-terminal portion of EphB2 is truncated had an intermediate phenotype. Application of EphB2-Fc recombinant protein to LIOH-treated optic nerve explants resulted in greater sparing of tubulin ß-III-containing retinal ganglion cell (RGC) axons. CONCLUSIONS: These results provide genetic evidence in mice that both EphB/ephrin-B forward and reverse signaling feed into an endogenous pathway to moderate the effects of glaucomatous insult on RGC axons. LIOH-induced axon loss is maintained in retina/optic nerve explants after removal from an ocular hypertensive environment. Exogenous application of EphB2 protein enhances RGC axon survival in explants, suggesting that modulation of Eph/ephrin signaling may be of therapeutic interest.

Axon repair: surgical application at a subcellular scale.

Injury to the nervous system is a common occurrence after trauma. Severe cases of injury exact a tremendous personal cost and place a significant healthcare burden on society. Unlike some tissues in the body that exhibit self healing, nerve cells that are injured, particularly those in the brain and spinal cord, are incapable of regenerating circuits by themselves to restore neurological function. In recent years, researchers have begun to explore whether micro/nanoscale tools and materials can be used to address this major challenge in neuromedicine. Efforts in this area have proceeded along two lines. One is the development of new nanoscale tissue scaffold materials to act as conduits and stimulate axon regeneration. The other is the use of novel cellular-scale surgical micro/nanodevices designed to perform surgical microsplicing and the functional repair of severed axons. We discuss results generated by these two approaches and hurdles confronting both strategies.

Axonal/glial upregulation of EphB/ephrin-B signaling in mouse experimental ocular hypertension.

PURPOSE: To use a laser-induced ocular hypertension (LIOH) mouse model to examine the optic nerve head (ONH) expression of EphB/ephrin-B, previously shown to be upregulated in glaucomatous DBA/2J mice. To relate ephrin-B reverse signaling with states of axonal response to disease. METHODS: LIOH was induced unilaterally in CD-1 mice by laser photocoagulation of limbal and episcleral veins. Intraocular pressure (IOP) was measured with a tonometer. EphB/ephrin-B mRNA expression was assessed by in situ hybridization on eyecup cryosections and real-time PCR. Cell specific markers were used to identify the cellular origin of EphB/ephrin-B expression. Activation of ephrin-B signaling was investigated with a phosphospecific antibody on cryosections and retinal whole-mounts. RESULTS: Upregulation of EphB/ephrin-B expression occurred early within a day of IOP elevation. A transient increase of phosphorylation-dependent ephrin-B (pEB) reverse signaling was observed in ONH axons, microglia, and some astrocytes. Morphologically unaffected retinal ganglion cell (RGC) axons differed from axons with reactive aberrant trajectories by exhibiting increased pEB activation, whereas pEB levels in morphologically affected axons were comparable to those of controls. CONCLUSIONS: An Eph-ephrin signaling network is activated at the ONH after LIOH in CD-1 mice, either before or coincident with the initial morphologic signs of RGC axon damage reported previously. Of note, ephrin-B reverse signaling was transiently upregulated in RGC axons at the ONH early in their response to IOP elevation but was downregulated in axons that had been damaged by glaucomatous injury and exhibited aberrant trajectories. Ephrin-B reverse signaling may mark RGC axons for damage or confer a protective advantage against injury.

Laser-induced ocular hypertension in albino CD-1 mice.

PURPOSE: To establish a laser-induced model of ocular hypertension (LIOH) in albino CD-1 mice and to characterize the sequence of pathologic events triggered by intraocular pressure (IOP) elevation. METHODS: LIOH was induced unilaterally in CD-1 mice by laser photocoagulation of limbal and episcleral veins 270 degrees to 300 degrees circumferentially, sparing the nasal aspect and the long ciliary arteries. IOP was measured with a rebound tonometer. Hematoxylin and eosin-stained plastic sections were used for morphometric analysis of retinal layers, and retinal whole-mounts were immunostained with anti-Brn-3b to quantify retinal ganglion cell (RGC) gene expression ion and density. Axonal and myelin morphologies were characterized using appropriate antibodies, and axon counts were obtained from paraphenylenediamine-stained optic nerve sections. RESULTS: LIOH resulted in IOP doubling within 4 hours after laser treatment, which returned to normal by 7 days. Axon degenerative changes, reactive plasticity, and aberrant regrowth were detected at the optic nerve head (ONH) as early as 4 days after treatment. By 7 days, axon number was significantly reduced in the myelinated optic nerve, with concurrent signs of myelin degradation. At 14 days, Brn-3b(+) RGC density was reduced, with neuronal loss confined to the RGC layer and no apparent effects on other retinal layers. CONCLUSIONS: Laser photocoagulation of limbal and episcleral veins induces transient ocular hypertension in albino CD-1 mice. The ensuing retinal and optic nerve pathologic events recapitulated key features of glaucoma and placed ONH RGC axon responses as an early manifestation of damage. LIOH in albino mice may be useful as a mouse model to examine mechanisms of RGC and axon glaucomatous injury.

Single cell and neural process experimentation using laterally applied electrical fields between pairs of closely apposed microelectrodes with vertical sidewalls.

As biomedical research has moved increasingly towards experimentation on single cells and subcellular structures, there has been a need for microscale devices that can perform manipulation and stimulation at a correspondingly small scale. We propose a microelectrode array (MEA) featuring thickened microelectrodes with vertical sidewalls (VSW) to focus electrical fields horizontally on targets positioned in between paired electrodes. These microelectrodes were fabricated using gold electroplating that was molded by photolithographically patterned SU-8 photoresist. Finite element modeling showed that paired VSW electrodes produce more uniform electrical fields compared to conventional planar microelectrodes. Using paired microelectrodes, 3 microm thick and spaced 10 microm apart, we were able to perform local electroporation of individual axonal processes, as demonstrated by entry of EGTA to locally chelate intra-axonal calcium, quenching the fluorescence of a pre-loaded calcium indicator dye. The same electrode configuration was used to electroporate individual cells, resulting in the targeted transfection of a transgene expressing a cytoplasmically soluble green fluorescent protein (GFP). In addition to electroporation, our electrode configuration was also capable of precisely targeted field stimulation on individual neurons, resulting in action potentials that could be tracked by optical means. With its ability to deliver well-characterized electrical fields and its versatility, our configuration of paired VSW electrodes may provide the basis for a new tool for high-throughput and high-content experimentation in broad areas of neuroscience and biomedical research.

Microtechnology and nanotechnology in nerve repair.

OBJECTIVE: This review will describe the novel contributions to the field of nerve repair from the emerging disciplines of microtechnology and nanotechnology. METHOD: This broad review will cover the advances described in the literature of the medical and biological fields and the engineering and physical sciences. The authors have also included their own work in this field. DISCUSSION: Microtechnology and nanotechnology are providing two fundamentally different pathways for pursuing nerve repair: (1) microstructured scaffolds to promote regeneration and (2) direct repair by reconnecting axons. In the first instance, many of the traditional techniques for microfabrication of microelectronics have been applied to the development of implantable tissue scaffolds with precisely formed architectures. Combined with nanotechnological capabilities to control their surface chemistries, these tissue constructs have been designed to create a microenvironment within nerve tissue to optimally promote the outgrowth of neurites. With some initial successes in animal models, these next generation tissue scaffolds may provide a marked improvement over traditional nerve grafts in the ability to overcome nerve degenerative processes and to coax nerve regeneration leading to restoration of at least some nerve function. A second, completely different repair strategy aims to directly repair nerves at the microscale by acutely reconnecting severed or damaged axons immediately after injury and potentially forestalling the usual downstream degenerative processes. This strategy will take advantage of the traditional capabilities of microfabrication to create microelectromechanical systems that will serve as ultramicrosurgical tools that can operate at the micron scale and reliably manipulate individual axons without incurring damage. To bring about some restoration of a nerve's function, axon repair will have to be performed repetitively on a large scale and soon after injury. Development work is currently underway to bring about the feasibility of this technique. CONCLUSION: With the emergence of microtechnology and nanotechnology, new methods for repairing nerves are being explored and developed. There have been two fundamental benefits from the technologies of the ultrasmall scale: (1) enhancement of regeneration using new tissue scaffold materials and architecture; (2) direct repair of nerves at the scale of single neurons and axons.

Novel high-resolution micropatterning for neuron culture using polylysine adsorption on a cell repellant, plasma-polymerized background.

The ability to organize individual neurons and their processes in culture provides important benefits to both basic neuroscience research applications and the development of biomedical microdevices. While numerous methods have been used to produce such micropatterning of neurons and cells in general, there has yet been no method to simultaneously provide high-resolution patterns with high compliance of cells to desired patterns and good manufacturability. To develop such a process, this work used a plasma polymerized, nonfouling poly ethylene oxide (PEO)-like film to provide a cell repellant substrate on which cell adhesive micropatterns can be selectively laid down. While the use of plasma polymerized, organic films have been used for cell micropatterning, this process exploits the often-overlooked tendency for the surface of this PEO-like material to adsorb polylysine from aqueous solution while remaining nonfouling with respect to other species, such as bovine serum albumin (BSA) and immunoglobulin G (IgG). When the adsorption of polylysine was enhanced by brief plasma oxidation, which slightly alters the surface chemistry of the material, simple photolithographic liftoff could be used to micropattern stable, cell adhesive areas on an otherwise cell repellant background. We showed that the application of photolithography itself on the PEO-like material did not alter its chemical properties, nor did it result in the erosion of the micropatterned polylysine on its surface. Hippocampal neurons from embryonic mice flourished on these micropatterned substrates and exhibited viability comparable to neurons cultured on polylysine coated glass. Furthermore, the compliance of cell bodies and outgrowing neurites to the micropatterns was nearly perfect. In addition to providing cell adhesive regions, the micropatterned polylysine coating also served as a template mediating the immobilization of other bioactive species such as IgG and laminin. Using this "piggybacking" of laminin on polylysine, we were also able to culture and micropattern retinal ganglion cells (RGC).

Upregulation of EphB2 and ephrin-B2 at the optic nerve head of DBA/2J glaucomatous mice coincides with axon loss.

PURPOSE: To identify genes with upregulated expression at the optic nerve head (ONH) that coincides with retinal ganglion cell (RGC) axon loss in glaucomatous DBA/2J mice. To further demonstrate that the proteins encoded by these genes bind to RGC axons and influence fundamental axon physiology. METHODS: In situ hybridization and cell-type-specific immunolabeling were performed on ONH sections from DBA/2J mice (3 to 11 months old) and C57Bl/6NCrl mice (10 months old). EphB2-Fc and ephrin-B2-Fc chimeric proteins were applied to adult RGC axons in vitro and in vivo at the ONH to demonstrate protein binding on axons. EphB2-Fc or control Fc protein was applied in a bath or locally to axons preloaded with the calcium indicator Fluo-4-AM, and changes in intra-axonal calcium were determined. RESULTS: EphB2 and ephrin-B2 were specifically upregulated at the ONH of DBA/2J mice starting at 9 months of age, but not in age-matched C57Bl/6NCrl mice or in DBA/2J animals that did not have axon loss. EphA4 was also present at the ONH, but no difference in expression was detected between unaffected and affected animals. EphB2 was expressed by F4/80(+), MOMA2(+), ED1(-) macrophage-like cells, ephrin-B2 was expressed by Iba-1(+) microglia and GFAP(+) astrocytes, whereas EphA4 was expressed by GFAP(+) astrocytes. EphB2-Fc and ephrin-B2-Fc protein bound to RGC axons in culture and to ONH RGC axons in vivo. Adult RGC axons in vitro elevated intra-axonal calcium in response to EphB2-Fc but not to control Fc protein. CONCLUSIONS: The expression of EphB2 and ephrin-B2 is upregulated at the ONH of glaucomatous DBA/2J mice coinciding with RGC axon loss. The direct binding of EphB2 and ephrin-B2 on adult RGC axons at the ONH and the ability of EphB2 to elevate intra-axonal calcium indicate that these proteins may affect RGC axon physiology in the setting of glaucoma and thus affect the development or progression of the disease.