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.

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.

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.

In vivo use of a nanoknife for axon microsurgery.

OBJECTIVE: Microfabricated devices with nanoscale features have been proposed as new microinstrumentation for cellular and subcellular surgical procedures, but their effectiveness in vivo has yet to be demonstrated. In this study, we examined the in vivo use of 10 to 100 microm-long nanoknives with cutting edges of 20 nm in radius of curvature during peripheral nerve surgery. METHODS: Peripheral nerves from anesthetized mice were isolated on a rudimentary microplatform with stimulation microelectrodes, and the nanoknives were positioned by a standard micromanipulator. The surgical field was viewed through a research microscope system with brightfield and fluorescence capabilities. RESULTS: Using this assembly, the nanoknife effectively made small, 50 to 100 microm-long incisions in nerve tissue in vivo. This microfabricated device was also robust enough to make repeated incisions to progressively pare down the nerve as documented visually and by the accompanying incremental diminution of evoked motor responses recorded from target muscle. Furthermore, this nanoknife also enabled the surgeon to perform procedures at an unprecedented small scale such as the cutting and isolation of a small segment from a single constituent axon in a peripheral nerve in vivo. Lastly, the nanoknife material (silicon nitride) did not elicit any acute neurotoxicity as evidenced by the robust growth of axons and neurons on this material in vitro. CONCLUSION: Together, these demonstrations support the concept that microdevices deployed in a neurosurgical environment in vivo can enable novel procedures at an unprecedented small scale. These devices are potentially the vanguard of a new family of microscale instrumentation that can extend surgical procedures down to the cellular scale and beyond.

Microtechnology in medicine: the emergence of surgical microdevices.

With the emergence of technologies to fabricate and mass-produce microscale tools and micromachines, microsurgery stands to potentially benefit through the development of a fundamentally new class of instruments. These new instruments may provide the surgeon with access to the smallest reaches of the body and perform operations that are currently not possible with manually operated tools. These new devices can be variably constructed and configured based on a wide range of design possibilities and can be built to serve many different fundamental surgical functions requiring the manipulation and handling of small tissues and structures, including grasping, cutting, and monitoring. With these functionalities also comes a high degree of integration, allowing tools and space to be used efficiently. Adapted from the techniques of the microelectronics industry, the fabrication methods and materials produce structures that are mechanically strong and easy to reproduce on a large scale. Well-developed design and physical modeling tools mean that the process of instrument development and validation can be streamlined. Along with these new instruments comes the need to provide automated interfaces to effectively translate human operator intentions into the appropriate actuation and motion of these devices. These interfaces must include the capability to scale down human motions to the range of microns. Most likely, the operation of these new microsurgical devices will resemble the control schemes developed for robotic surgery. The control schemes will provide accurate motions while minimizing the chances of damaging tools or unnecessarily injuring tissues. Naturally, these new tools and surgical schemes will require a transition from the conventional paradigm. However, with new surgical capabilities that may allow direct intervention into the inner workings of a cell, MEMS and nanotechnology-based tools may become a crucial part of the arsenal for the next generation of surgeons. Invariably, future developments of this new class of instruments will depend in large part on needs identified by the surgeon and an understanding of the enabling properties of microtechnology and nanotechnology. Thus, recognition of the vast potentials of this new technology among clinicians will greatly help to accelerate the development and integration of new microdevices and novel procedures that address disease and injury with unprecedented precision.

EphB3: an endogenous mediator of adult axonal plasticity and regrowth after CNS injury.

Endogenous mechanisms underlying the remodeling of neuronal circuitry after mammalian CNS injury or disease remain primarily unknown. Here, we investigated axonal plasticity after optic nerve injury and found that macrophages recruited into the injury site and adult retinal ganglion cell (RGC) axons, which undergo injury-induced sprouting and terminal remodeling, were linked by their respective expression of a ligand and receptor pair active in axon guidance. Recruited macrophages specifically upregulated mRNA encoding the guidance molecule EphB3 and expressed EphB proteins capable of binding Ephrin B molecules in vivo and in vitro. Injured adult RGC axons in turn expressed EphrinB3, a known receptor for EphB3, and RGC axons bound recombinant EphB3 protein injected into the optic nerve. In vitro, EphB3 supported adult RGC axon outgrowth, and axons turned toward a source of this guidance molecule. In vivo, both reduction of EphB3 function in adult heterozygous animals and loss of function in homozygous animals greatly decreased RGC axon re-extension or sprouting after optic nerve injury. Comparisons of axon re-extension in EphB3 null and wild-type littermates showed that this loss of axonal plasticity was not attributable to a difference in intrinsic axon growth potential. Rather, the results indicated an essential role for local optic nerve-derived EphB3 in regulating adult RGC axon plasticity after optic nerve injury. Of note, the loss of EphB3 did not affect the ability of injured RGC axons to elaborate complex terminal branching, suggesting that additional EphB3-independent mechanisms governed adult axon branching triggered by CNS damage.

Isolation of neuronal substructures and precise neural microdissection using a nanocutting device.

We describe a set of microfabricated nanocutting devices with a cutting edge of less than 20 nm radius of curvature that enables high precision microdissection and subcellular isolation of neuronal structures. With these devices, it is possible to isolate functional substructures from neurons in culture such as segments of axons and dendrites, dendritic spines and Nodes of Ranvier. By fine-tuning the mechanical compliance of these devices, they can also act as alternatives to costly laser capture microdissection workstations for harvesting specific neuronal populations from tissue sections for analysis. The small size of the device (1 mm2x100 microm) allows convenient insertion into researcher specific experimental set-ups. Its ease of use and possibility for batch fabrication makes this a highly effective and versatile tool for tissue microdissection and the microanalysis of neuronal function.

Microscale surgery on single axons.

OBJECTIVE: The lack of meaningful axon regeneration after central nervous system damage and poor functional recovery after serious peripheral nervous system nerve injuries have been long-standing problems of substantial interest to both neurosurgeons and neurobiologists. As an alternative to strategies that seek to promote the regeneration of adult axons, our research group has taken advantage of advances in microtechnology to develop a paradigm of direct axon repair involving the substitution of damaged axon regions with healthy segments from donor axons. METHODS: This repair methodology uses a novel combination of microtechnology, electrokinetic axon manipulation, and the well-established biological principle of cell fusion. These three fields of research have been integrated in a multidisciplinary approach to develop a solution for a significant clinical problem that currently has no specific treatment. RESULTS: The findings reported here provide some initial proof of principle for the core technologies we intend to use for axon repair. Functional recovery from nerve damage of course is clinically challenging, and many obstacles would need to be overcome before such axon repair procedures can be contemplated for therapeutic use. We identify some of the clinical issues that must be addressed for microtechnology-assisted axon repair to transition from the realm of research into actual surgical settings. CONCLUSION: It is hoped that each advance in axon repair technology will spur additional research to provide us with a comprehensive understanding on how best to pursue neurosurgical intervention at the microscale.

Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans.

The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.

An oligodendrocyte lineage-specific semaphorin, Sema5A, inhibits axon growth by retinal ganglion cells.

In the mammalian CNS, glial cells repel axons during development and inhibit axon regeneration after injury. It is unknown whether the same repulsive axon guidance molecules expressed by glia and their precursors during development also play a role in inhibiting regeneration in the injured CNS. Here we investigate whether optic nerve glial cells express semaphorin family members and, if so, whether these semaphorins inhibit axon growth by retinal ganglion cells (RGCs). We show that each optic nerve glial cell type, astrocytes, oligodendrocytes, and their precursor cells, expressed a distinct complement of semaphorins. One of these, sema5A, was expressed only by purified oligodendrocytes and their precursors, but not by astrocytes, and was present in both normal and axotomized optic nerve but not in peripheral nerves. Sema5A induced collapse of RGC growth cones and inhibited RGC axon growth when presented as a substrate in vitro. To determine whether sema5A might contribute to inhibition of axon growth after injury, we studied the ability of RGCs to extend axons when cultured on postnatal day (P) 4, P8, and adult optic nerve explants and found that axon growth was strongly inhibited. Blocking sema5A using a neutralizing antibody significantly increased RGC axon growth on these optic nerve explants. These data support the hypothesis that sema5A expression by oligodendrocyte lineage cells contributes to the glial cues that inhibit CNS regeneration.

L1/Laminin modulation of growth cone response to EphB triggers growth pauses and regulates the microtubule destabilizing protein SCG10.

During development, EphB proteins serve as axon guidance molecules for retinal ganglion cell axon pathfinding toward the optic nerve head and in midbrain targets. To better understand the mechanisms by which EphB proteins influence retinal growth cone behavior, we investigated how axon responses to EphB were modulated by laminin and L1, two guidance molecules that retinal axons encounter during in vivo pathfinding. Unlike EphB stimulation in the presence of laminin, which triggers typical growth cone collapse, growth cones co-stimulated by L1 did not respond to EphB. Moreover, EphB exposure in the presence of both laminin and L1 resulted in a novel growth cone inhibition manifested as a pause in axon elongation with maintenance of normal growth cone morphology and filopodial activity. Pauses were not associated with loss of growth cone actin but were accompanied by a redistribution of the microtubule cytoskeleton with increased numbers of microtubules extending into filopodia and to the peripheral edge of the growth cone. This phenomenon was accompanied by reduced levels of the growth cone microtubule destabilizing protein SCG10. Antibody blockade of SCG10 function in growth cones resulted in both changes in microtubule distribution and pause responses mirroring those elicited by EphB in the presence of laminin and L1. These results demonstrate that retinal growth cone responsiveness to EphB is regulated by co-impinging signals from other axon guidance molecules. Furthermore, the results are consistent with EphB-mediated axon guidance mechanisms that involve the SCG10-mediated regulation of the growth cone microtubule cytoskeleton.

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development.

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.