Wesley C. Chang

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.

Microtechnology and nanotechnology in nerve repair

Abstract 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 nerves 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.

IN VIVO USE OF A NANOKNIFE FOR AXON MICROSURGERY

OBJECTIVEMicrofabricated 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 μm-long nanoknives with cutting edges of 20 nm in radius of curvature during peripheral nerve surgery. METHODSPeripheral 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. RESULTSUsing this assembly, the nanoknife effectively made small, 50 to 100 μm-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. CONCLUSIONTogether, 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.

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.

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.

A tribute to dr. David Kline: A new approach to an old peripheral nerve problem-splicing instead of regenerating disrupted axons

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Applications of a novel cell micropatterning process for long-term neuron and cell culture

We show several applications for a unique method for creating user-friendly, micropatterned substrates for neuron and cell culture. With neurons, we demonstrated that our micropatterns can separate cell bodies and axons while organizing them in orderly arrays for easy experimentation. These micropatterned neuron cultures also persisted for weeks, enabling maturation and development of presynaptic structures and potentially allowing the explicit design of neuronal circuits. Beyond neurons, we also demonstrated the culture of fibroblasts on the patterned substrates and showed that these cell types likewise remained viable and highly compliant to a variety micropatterns.

Microdevice components for a cellular microsurgery suite

We describe microfabricated tools that will enable cellular microsurgery for direct repair of injured nerves leading to restoration of function. Our proposed neural repair strategy uses a suite of novel microfabricated tools to cut, manipulate, align and then reconnect individual axons (nerve cell processes) with micron-scale precision. Each of these functions has been individually demonstrated using prototype devices. Additionally, we have developed assembly techniques to integrate the required tools onto a single, 3D multifunctional MEMS platform, designed to facilitate the semi-autonomous execution of all of the required surgical functions in proper sequences.

Precision MEMS Nano-Cutting Device for Cellular Microsurgery

An important tool for biological research and microsurgery is a microdevice for the cutting and isolation of subcellular neuronal components such as axons and dendrites for analysis or microsurgery. We have fabricated an easy-to-use, inexpensive and robust MEMS device with a nanoscale cutting tool that performs highly reproducible cutting of axons and dendrites. The device consists of a knife with an 20 nm-sharp edge ranging from 10-200 microns in length and is formed from molding conformally deposited silicon nitride over a potassium hydroxide-etched trench in -oriented single crystal silicon. Knife surfaces are coated with a thin layer of liquid perfluorinated polyether to prevent adhesion of debris from cut targets. The knife is assembled onto a microfabricated suspension and frame consisting of serpentine flexures of single crystal silicon. These supporting structures help to properly orient the knife and control cutting force. We have used this assembled nano-cutting device to make reliable cuts of individual living dendrites and unmyelinated and myelinated axons from both adult and embryonic animal tissue. The cutting device was able to target and cut specific cell processes within a complex field and without disturbing surrounding structures. The cuts were sharp and repeatable, and microdevice’s performance was undiminished with repeated use.