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NEW INSTRUMENTATION

IN VIVO USE OF A NANOKNIFE FOR AXON MICROSURGERY


Wesley C. Chang, Ph.D.
Departments of Ophthalmology and Physiology, Neuroscience Program and Bioengineering Program, University of California, San Francisco, San Francisco, California

Elizabeth A. Hawkes, M.S.


Departments of Ophthalmology and Physiology, Neuroscience Program and Bioengineering Program, University of California, San Francisco, San Francisco, California

Michel Kliot, M.D.


School of Medicine and Department of Neurological Surgery, University of Washington, Seattle, Washington, and Puget Sound Veterans Administration Health Care Center, Seattle, Washington

David W. Sretavan, M.D., Ph.D.


Departments of Ophthalmology and Physiology, Neuroscience Program and Bioengineering Program, University of California, San Francisco, San Francisco, California Reprint requests: Wesley C. Chang, Ph.D., Departments of Ophthalmology and Physiology, Neuroscience Program and Bioengineering Program, University of California, San Francisco, 10 Koret Way, K-110, San Francisco, CA 94143. Email: changw@vision.ucsf.edu Received, December 4, 2006. Accepted, May 17, 2007.

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 m-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 brighteld and uorescence capabilities. RESULTS: Using this assembly, the nanoknife effectively made small, 50 to 100 mlong 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.
KEY WORDS: Axon, Microelectromechanical systems, Microfabrication, Microsurgery, Nanoknife, Nanoneurosurgery, Nanotechnology, Subcellular surgery
Neurosurgery 61:683692, 2007
DOI: 10.1227/01.NEU.0000280070.51586.9F

www.neurosurgery-online.com

lthough axons play a critical role in relaying information within the nervous system, there is currently no therapeutic intervention that can directly repair these key neural processes when severed or damaged by trauma. Substantial research efforts are underway to identify effective therapeutic strategies. Such approaches include the suppression of growth-inhibitory molecules associated with myelin and scar tissues (8, 18, 19), the harnessing of axon guidance molecules that are up-regulated after trauma (8, 12), and even the application of novel bioengineered tissue scaffolds to guide new axon outgrowth (7). All of these efforts share the goal of stimulating the regeneration of injured

adult axons. Recently, we proposed an alternative paradigm in which transected nerves are directly repaired by surgically reconnecting the constituent axons to acutely reconstitute nerve function after damage. To demonstrate the feasibility of this concept, our research effort has presented early demonstrations of several simple microfabricated devices that cut, electrostatically translocate, and splice together individual axons in vitro (20). An important aspect of this microdevicebased method of axon repair is the effectiveness of such small-scale instrumentation in surgical environments in vivo. Current uses of microtechnology in surgical instrumentation include microscale motors, force-generating

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enabled the rst targeted cutting of a single axon in an anesthetized animal.

MATERIALS AND METHODS


The surgical demonstration was performed on adult female C57BL/6 mice or transgenic GFP mice (Charles River Laboratories, Inc., Wilmington, MA) anesthetized with a mixture of ketamine and xylazine (14). The FIGURE 1. A custom surgical suite was assembled to enable cutting with nanoknives while allowing visual monisedated animal was placed in a toring through an overhead microscope. This suite also included the capability for stimulation of the target nerve and prone position with its hind legs recording of the resulting EMG. A, a wide view of the unmounted nerve (arrow) showing its anatomic position in fully extended. To expose the scithe posterior calf along with the target soleus muscle (asterisk). EMG was recorded from the soleus muscle at a posiatic nerve, an incision was made tion indicated by the asterisk. B, the surgical eld showing the tibial branch of the sciatic nerve (arrow) mounted through the skin of the posterior on the glass microplatforms. The green board supporting the microplatforms and facilitating the electrical connection thigh and underlying hamstring for stimulation is 2 1.5 cm. C, close-up of the surgical microplatforms (2-mm wide each) showing the embedded muscles. The sciatic nerve splits nerve stimulation electrode (arrow) and the nerve/axon cutting platform (arrowhead). The nerve is shown draped into several major branches (Fig. on the surgical microplatforms, which are mechanically isolated from the animal. D, schematic diagram showing the 1A), the tibial and peroneal nerves, surgical eld and supporting equipment, including the stimulation and recording instrumentation for nerve stimuas well as a smaller branch. Cutlation combined with electromyographic recording from the soleus muscle. The overhead microscopes objective lens ting by the nanoknife was peris positioned just above the nerve straddling the microplatforms. formed on the central largest branch (Fig. 1A, arrow), which innervates the calf muscles and which we have identied as the tibial actuators, and catheter-mounted structures for microsurgical branch of the sciatic nerve. To access the axons within the nerve, the tissue cutting, ophthalmic procedures, vascular surgeries, and ensheathing epineurium and perineurium were gently removed targeted drug delivery (1517). However, to operate on individthrough standard microdissection with handheld surgical forceps while ual cells or on subcellular elements such as axons, new surgiviewing under a dissection microscope.

Animals

cal microdevices are required with characteristic dimensions at the micron scale. An important challenge will be to develop the means to effectively deploy and manipulate these tiny devices in a surgical setting. For instance, surgical microdevices will have to be mechanically strong and robust for use in vivo, and it will be necessary to precisely position and manipulate these devices at scales much smaller than those possible with manually operated tools. Such devices and capabilities will allow the surgeon to perform novel microscale procedures that are currently not possible with existing surgical instrumentation. In this study, we have investigated these issues by testing an axon nanoknife (6, 20) that can be manipulated as a microdevice in a surgical setting. This nanoknife consists of a thin layer of silicon nitride with a nanometer sharp cutting edge. In the current demonstration, nanoknives were used to perform peripheral nerve surgery in an anesthetized mouse. By using the nanoknife mounted on a micromanipulator in conjunction with a simple, custom-assembled microplatform to hold and isolate an individual nerve, it was possible to precisely manipulate this surgical microdevice in the operating eld and to make targeted, effective cuts in the sciatic nerve while simultaneously monitoring the entire process visually. Furthermore, progressive cuts to the nerve resulted in an accompanying, incremental reduction in the evoked electromyography (EMG) signal. Finally, this custom-assembled surgical suite also

Surgical Microplatforms
The isolation and stabilization of the nerve was performed using a special xture mechanically isolated from the animal (Fig. 1B). This xture was held and positioned over the animal by a micromanipulator and presented two separate at platforms (Fig. 1C) for holding the nerve, one for stimulation (Fig. 1C, small arrow) and the other for nerve or axon cutting (Fig. 1C, arrowhead). These platforms consisted of two elongated, rectangular chips of Pyrex glass (2 mm wide 0.5 mm thick 8 mm long; Corning, Corning, NY) that were arranged in parallel and held level at a position just a few millimeters above the hind limb of the animal. During the surgery, the segment of the sciatic nerve, with axons exposed, was lifted from the leg and draped over these two platforms (Fig. 1, CD). Along one edge of the proximal platform, a 500 m-wide line of gold lm was deposited to provide a conductor for nerve stimulation. This gold trace was electrically connected to a voltage pulse generator and delivered electrical signals to the nerve. The opposing pole was grounded to an Ag/AgCl2 electrode placed under a medial ap of skin on the leg. The second, more distal platform, dedicated specically for nerve and axon cutting, was separated from the stimulating platform by a gap of 2 mm. This separation provided an isolated surface for the microcutting of the nerve and also prevented unintended feed-through of the electrical stimulation signals generated at the rst platform so the severed distal segment of nerve on the second platform could not receive unintended stimulation.

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During surgery, the nerve was kept moist by regular irrigation with physiologically buffered media. However, it was necessary to keep the buffered media within the boundaries of each platform because the media itself can serve as a conduit for unintended feed-through of electrical signals.

Electrical Stimulation
The stimulation signal was a single monophasic, rectangular pulse with 200-s duration and of varying amplitudes ranging from 100 mV to 2 V delivered at 5 Hz.

identied threshold. To capture and display accurate EMG signals, we subtracted the background stimulation artifacts, which were measured by recording from the muscle in an actual surgical conguration but with the sciatic nerve completely severed between the platform and the muscle while still draped over the platform. (EMGs with this background subtraction are displayed in all gures.) Although this procedure eliminated signal artifacts substantially, some remnant of signal feed-through remained owing to slight variations in the artifacts between recordings.

Evaluation of Biocompatibility
To investigate any potential acute toxicity of silicon nitride, both mouse hippocampal neurons and retinal explants were cultured on at substrates coated with the same chemical vapor-deposited silicon nitride used to construct the nanoknives. Briey, hippocampal cells were harvested from the brains of E16 mouse embryos and plated on substrates that were preabsorbed with poly-L-lysine using standard protocols (5). Mouse retinal explants from E14 embryos were plated on laminin-coated substrates, again using published protocols (3, 21). Both cultures were maintained in 37 C and 5% CO2/95% air in their respective nutrient media along with appropriate supplements.

Observation
All surgical procedures were monitored in real time using an upright, boom-mounted microscope (Nikon Erect Image Trinocular Tilting Head and Nosepiece with objective lenses mounted on SMS 20 Diagnostics boom stand and with brighteld and uorescence capabilities; Nikon Instruments, Garden City, NY), and surgical manipulations were performed directly under long working distance objective lenses. Brighteld illumination was provided from above at oblique angles to the microscopes optical axis, whereas uorescence imaging was enabled by an on-board mercury lamp along with appropriate lters. With these provisions, it was possible to distinguish individual axons in the mounted nerve. The operating field could be viewed directly by an observer through the microscope eyepieces or the image could be diverted to a mounted digital camera and then displayed and recorded on a computer.

RESULTS
Test Microdevice
The test microdevice is a microscale cutting instrument with nanoscale features that was originally designed for the precise severing of axons under well-controlled research conditions in vitro (6). This pyramid-shaped microdevice consists of a mechanically strong, 1 m-thick shell of silicon nitride (Fig. 2A) and was mounted onto a thin metal rod attached to a glass micropipette holder (Fig. 2B). The pipette holder was, in turn, mounted to a robotic X-Y-Z micromanipulator to move the microdevice into position and to deliver the cutting stroke. Because the nanoknife was used for targeted cutting of single axons in a peripheral nerve, the precise movement provided by a micromanipulator was necessary. In the current assembly for this surgical demonstration, it was possible to station one micromanipulator system exclusively to hold the microplatform supporting the nerve and then station another micromanipulator on the opposite side of the surgical eld to present and manipulate the nanoknife for nerve cutting. Knife positioning with the micromanipulator and nerve (or axon) cutting using the microplatform as a cutting board was easy to perform and could be observed through the microscope.

Cutting With the Nanoknife


For microcutting of the isolated nerves, a nanoknife previously developed for the cutting of individual axons in vitro was used (6). The pyramid-shaped nanoknife was fabricated by conformal molding of chemical vapor-deposited silicon nitride over silicon etched precisely along specic crystal planes, resulting in an ultrasharp cutting edge at the apex (20 nm edge radius of curvature as determined by scanning electron microscopy), small enough to target individual axons. This device has been used in vitro to cut both axons from cultured neurons and from harvested adult nerves (6). For the surgical procedures in the present study, the knife was mounted on a supporting rod and then positioned by a commercially available, precision micromanipulator (MP-285; Sutter Instruments, Novato, CA), which allowed the nanoknife to be precisely aimed at one or a few axons at a time. Surgical demonstrations were also performed in transgenic mice expressing green uorescent protein (GFP). Retention of the cytoplasmically dissolved GFP after cutting was used to determine whether or not the severed ends of the axons resealed after cutting. This analysis of resealing after single axon cutting has been previously reported (6).

Evoked EMG Response


To record the evoked EMG signals triggered by nerve stimulation, a sharp tungsten needle was inserted into the calf soleus muscle. The opposite pole was grounded at the same Ag/AgCl2 probe used with the stimulating circuit. Signals generated at the tungsten probe were fed to an AC amplier (DAM-80; World Precision Instruments Inc., Sarasota, FL) and amplied 1000 times. The amplied signals were in turn read and recorded by an oscilloscope (TEK 3012B; Tektronix, Beaverton, OR). For EMG recordings, the amplitude of tibial nerve stimulation was rst varied to nd the approximate threshold, below which no action potential and, therefore, no EMG signal could be detected. Nerve stimulation was then elicited using a stimulus fourfold stronger than the

Deployment in the Surgical Field


The placement of the nerve on the microplatform allowed the nerve to be lifted away from the leg musculature and be mechanically stabilized. When placed on the platform and lifted, the nerve was put under some tension and was held stationary and physically isolated from the animal. Consequently, small movements of the animal resulting from heartbeat, respiration, and involuntary muscle movements were not transmitted to the nerve. The microplatform also allowed the nerve to be maintained in an aqueous environment while eliminating contact with other tissues, thus ensuring that the

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FIGURE 3. Visualization and use of the axon nanoknife for microscale nerve cutting is demonstrated. A, the intact nerve as viewed through the microscope with a total magnification 100. The nanoknife with a 100-m long cutting edge is held above the nerve off to one side at the beginning of the procedure (upper right). B, the nanoknife is brought over the desired position and lowered to make the cut. C, a cut approximately 100 m long (arrow) is visible in the nerve. D, the cutting sequence was repeated using the same nanoknife to make a second cut in the nerve (arrow). AD, scale bar, 200 m.

vided a working distance between the front of the objective and the tissue of 7.4 to 17 mm. Operation of the nanoknife in the surgical setting as described required a minimum working distance of approximately 5 mm.

Cutting With the Nanoknife


FIGURE 2. A diagram of the axon nanoknife showing device conguration and the ultrasharp cutting edge (apex of the elongated pyramidal structure, green). B, wider view of the mounted knife (boxed), held by a micropipette holder, which in turn is held in the micromanipulator.

electrical nerve stimulation did not feed through to the muscles through other pathways in the animal. Although mechanical manipulations were required to place the intact nerve on the platform and to prepare the exposed axons for cutting, nerve function itself was not disrupted because nerve stimulation using the built-in electrode on the platform readily triggered observable twitching of the calf muscle and an easily detectable EMG signal.

Observation During the Surgical Procedure


Because conventional surgical or tissue dissection microscopes lack the necessary magnication to observe individual axons, an upright, boom-mounted research microscope equipped with 10 eyepieces was adapted for direct overhead observation of the surgical eld with both brighteld and uorescence imaging. This research microscope was equipped with 4 to 20 long working distance objective lenses that pro-

The exposed tibial branch of the mouse sciatic nerve as viewed through the microscope during a cutting sequence is shown in Figure 3. In Figure 3A, the black prole is a portion of the nanoknife brought in from above into the surgical eld and prepositioned to the side of the nerve. At this position, the nanoknife is raised above both the microplatform and the nerve, resulting in the knife prole being slightly out of focus relative to the nerve. To test whether or not this microdevice can be used to make small cuts in a living nerve, the nanoknife was brought over the desired position and lowered onto the nerve (Fig. 3B). The silicon nitride material is sufficiently optically translucent to allow visualization of the nerve through the nanoknife itself. This property of the microdevice was useful in allowing the operator to roughly judge nanoknife contact with the nerve and dimpling of nerve tissue during the cutting. The amount of vertical displacement of the nanoknife during cutting could also be determined more accurately from the micromanipulator controller. The resulting incision from one nerve cutting experiment is shown in Figure 3C (arrow). The incision measured approximately 100 m in length, corresponding to the length of the

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inserted into the target muscle recorded a signal consisting of an artifact coinciding with the stimulating pulse followed by an EMG waveform beginning approximately 1 ms later. When the background (control) waveform was subtracted from this raw recording, the artifact was largely eliminated, and the true shape and duration of the EMG wave could be discerned. Control waveforms have been subtracted from all EMG recordings shown in the gures. Typically, the waveform lasted just over 2 ms (Fig. 5). By varying the magnitude of the stimulation pulse, it was determined that, for this system, the threshold for evoking a response in the muscle was between 300 and 400 mV delivered at the platform. Below this threshold, the recorded muscle signal was nearly identical to the background control and no muscle twitching was observed.

Repeated Use of the Nanoknife for Targeted Cutting


The precision and durability of the nanoknives was demonstrated in experiments in which a single nanoknife was used repeatedly to progressively pare down a nerve. For this exercise, a shorter knife with a cutting edge of 10 m was used. From our observations, we estimate that perhaps only a few dozen axons are severed with each cutting stroke. In addition to visual observations of the cutting sequence and the tissue incisions, the successive removal of functional axons within the nerve was verified simultaneously with EMG recording from the innervated calf muscle (Fig. 5). The intact nerve supported by the microplatform at the start of the procedure is shown in Figure 5A. Figure 5B shows the EMG recording following the delivery of a subthreshold stimulus, whereas Figure 5C shows the EMG waveform recorded after delivery of a supramaximal stimulus. Figure 5F shows the muscle response resulting from stimulation of an intact nerve and after a large number of axons had been cut by the nanoknife (Fig. 5, D and F). We estimate that approximately one-half to two-thirds of the nerve had been eliminated at this point. As a result of the small amount of tension on the nerve when it is placed onto the microplatform, the ends of severed axons retract from the eld of view while the remaining uncut axons remain in the surgical eld. The progressive diminution in the EMG waveform at various stages of this cutting procedure is shown in Figure 5, GI. Note that the EMG recordings in Figure 5, E, F, H and I were obtained using a fourfold suprathreshold stimulus. Figure 3H shows the EMG corresponding to the situation shown in Figure 5G, in which only a few axons are left on the microplatform. The severing of these remaining axons with the nanoknife resulted in the elimination of detectable EMG signal (Fig. 5I). Although we have shown that the EMG signal diminishes monotonically with the paring down of the nerve, the exact relationship between the fraction of the nerve eliminated and the diminution of the EMG is undetermined and indeed varies between different trials. This uncertainty results from the way that the EMG signal is constructed, a summation of signals from the individual innervated muscle cells. The strongest contributors to the composite EMG signal are from the cells closest to the recording probe, whereas more distant muscle cells

FIGURE 4. The nerve of a GFP transgenic mouse is demonstrated before being cut (A) and 3 minutes after being cut (B). The cut is indicated by the arrow. The retention of the cytoplasmic GFP indicated that the cut ends readily resealed. Scale bar, 200 m.

nanoknife cutting edge of 100 m. The cutting sequence was repeated, and a second incision of approximately the same size was made (Fig. 3D, arrow) next to the original. We estimate that in each cut, perhaps 50 to 100 axons are severed, but this quantication remains to be veried objectively. To assess the physiological state of the cut ends of axons severed by the nanoknife, this cutting procedure was also repeated in a GFP transgenic animal in which all cells contained freely dissolved cytoplasmic GFP (Fig. 4A). Under uorescence imaging, it was possible to identify the site of the cut (Fig. 4B, arrow). At this cut site, the severed axons remained brightly uorescent, indicating the retention of cytoplasmic GFP. This ability of severed axons to reseal its cut ends has been previously demonstrated in vitro (6) and is presumably the result of the preservation of membrane self-repair mechanisms (2).

EMG Recording
When the nerve was stimulated at low frequencies of 5 Hz, the innervated calf muscles twitched visibly, providing an independent visual conrmation that the stimulation worked and was above threshold. Concurrently, the recording probe

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described previously for making incisions in whole nerves. Figure 6, B and C, shows the nanoknife moved into position for axon cutting, whereas Figure 6D shows the resulting cut (arrow) in the targeted axon. A second cut was subsequently made in the same axon resulting in the isolation of a short 30-m segment (Fig. 6E, arrow). This axon segment was moved away from the parent axon to aid in visualization (Fig. 6F, arrow).

Biocompatibility of Silicon Nitride for Neurons


During the course of our experimentation, we did not encounter instances of apparent acute deleterious effects of silicon nitride on nerves or FIGURE 5. Nerve images and EMG recordings showing the progressive loss of evoked muscle response signal dursurrounding tissues. To examing nerve section using an axon nanoknife. A, image showing the intact tibial branch of the sciatic nerve supported on the microplatform. B, application of a subthreshold stimulus (200 mV) resulted in no detectable EMG signal. C, ine this issue of biocompatibilthe resulting EMG signal after application of a suprathreshold stimulus (500 mV) is shown. D, view of the nerve after ity in more detail, we placed most of its axons have been pared away using the nanoknife is demonstrated. E, the EMG waveform recorded in the actual nanoknives in tissue soleus muscle after stimulation of the intact nerve is shown with a stimulus fourfold above threshold. F, the EMG culture dishes and seeded waveform was recorded from the remaining axons shown in D. This waveform is reduced in amplitude compared with mouse retinal explant tissue or that obtained from the intact nerve. G, continued axon cutting using the same nanoknife left just a few axons remainhippocampal neurons directly ing on the microplatform. H, the EMG waveform was recorded from stimulation of the axons remaining in G. I, no onto silicon nitride knives to apparent EMG signal is observed after all of the axons have been cut using the nanoknife. determine whether this material was suitable as a neuronal growth substrate. The results showed that after 2 days in culture contribute less. Thus, cutting axons that map to muscle near the directly on silicon nitride, hippocampal neurons that were probe results in a larger diminution of the signal that would seeded as isolated neurons exhibited signicant process outoccur when the exact same cut is made in axons mapping to growth (Fig. 7A). Likewise, a fairly dense network of axon muscle cells further away. growth could be observed from the explanted retinal tissue (Fig. With repeated use, the cutting performance of the nanoknife 7B). The appearance of the retinal axons and hippocampal neuwas not noticeably diminished. Breakage of the nanoknife itself rons were very similar to what has been reported when they are during cutting was only observed in instances in which operagrown on tissue culture glass (3, 5, 21). tor error led to the unintended impact of the nanoknife with the microplatform or some other nearby surface. The most frequent limitation to the lifetimes of the nanoknives was not in fact DISCUSSION breakage of the silicon nitride microdevice, but the breakage at the joint where the microdevice is attached to its mounting rod. In this study, we subjected silicon nitride nanoknives to testing under the operative conditions of peripheral nerve surgery Surgical Cutting of Single Axons in a living anesthetized mouse. The main objectives of this A major goal of this study was to determine whether or not work were to examine whether or not this new type of nanoknife use could enable novel cellular or subcellular-scale microscale neurosurgical instrumentation could be used effecsurgical procedures that are not currently feasible using existtively in vivo and to verify the ability of these microdevices to ing instrumentation. We tested the precision and cutting perdeliver surgical precision at unprecedented cellular and subcelformance of the nanoknife on individual axons in an in vivo lular length scales. The results demonstrated that by using a surgical environment. A sequence of images illustrating the use rudimentary microplatform and off-the-shelf components such of a nanoknife on a single axon is shown in Figure 6. In this as micromanipulators and research microscopes, nanoknives example, the nanoknife was operated in the same manner as can be deployed and used effectively for microscale peripheral

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FIGURE 6. Precise targeted cutting of a single axon is performed using the nanoknife. A, view of an axon (center bottom) isolated from the tibial branch of the sciatic nerve and resting on the microplatform is shown. B, the nanoknife (upper right) is elevated above the axon off to one side at the beginning of the procedure. C, the nanoknife is lowered to make a precise cut in the axon of interest. D, the axon is severed at the location indicated by the arrow. The nanoknife has been removed from the surgical eld. E, the cutting sequence was repeated using the same nanoknife to make a second cut in the axon (arrow). F, the small segment in between the two incisions made in D and E was moved aside and is indicated by the arrow. AF, scale bar, 200 m.

FIGURE 7. Fluorescent images showing hippocampal neurons (A) and neurite outgrowth (B) from embryonic retinal explants cultured on silicon nitride substrates. The neurons used in these experiments express a soluble cytoplasmic GFP protein and are uorescent. AB, scale bar, 100 m.

nerve surgery. In addition to precise 50 to 100 m incisions in a peripheral nerve, and the incremental paring down of a single nerves muscle response, novel subcellular-scale neurosurgical procedures enabled by this novel surgical instrumentation included the precise targeted cutting and isolation of a segment from a single axon in vivo. All procedures could be observed and monitored in real-time, allowing user feedback, image capture, and EMG recording.

A single device has been used previously to cut more than 200 axons in vitro (6).

Microinstrumentation Use in Vivo


A fundamental question concerning the surgical use of microdevices that have characteristic sizes of only several tens or hundreds of microns is whether or not such small instrumentation can be deployed satisfactorily and used efficiently in an operative field. For the current study, we have chosen to address this question using the relatively accessible tibial branch of the mouse sciatic nerve in a demonstration of microscale nerve and single axon cutting. The key to deployment of the nanoknife in this instance was the use of a rudimentary microplatform that provided support for the nerve and allowed the nerve to be mechanically isolated. The microplatform also served as an axon microcutting surface,

Microdevice Robustness
For use in microsurgery, microdevices must be mechanically strong to permit repeated use. The robust performance of the nanoknife is attributable in part to the material properties of silicon nitride (ultimate strength, 28 GPa), which is actually stronger than bulk steel (ultimate strength, 0.5 GPa). Moreover, silicon nitride is not subject to plastic deformation (4), which would tend to dull cutting edges after repeated use.

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allowing the nerve to remain moist while isolated from other tissues. The spatial stability of the resulting operative field duplicated the well-controlled in vitro environment in which we have previously used similar cutting microdevices successfully (6, 20). Lastly, the microplatform also contained an embedded electrode for nerve stimulation. Although this microplatform strategy was designed for microdevice use in peripheral nerve surgery, a similar strategy can also be tried for the more challenging use of surgical microdevices in central nervous system regions such as the spinal cord.

ibility tests and considered the material as a nonirritant. Taken together, these ndings establish silicon nitride as suitable for use with neural tissues, especially in cases requiring only brief contact with tissues.

Cellular and Subcellular-scale Surgery


In this study, we performed what we believe is the rst subcellular-scale surgery on a single axon in a living anesthetized animal. Although the simple cutting of single axons in itself has limited clinical applications, the development of microdevices for precise axon cutting is the rst part of a long-term research program that examines the feasibility of conducting the surgical repair of individual damaged or severed axons as a means of achieving functional recovery after nervous system injury independent of axonal regeneration (20). We have previously proposed that axon cutting would be followed by the alignment of severed axon ends and subsequent axon splicing by electrofusion to reconstitute functional axons. Although preliminary proof of principle has been obtained for these latter steps, the execution of an entire axon repair sequence in vivo will require much future research and development. However, it is worthwhile to consider that new microscale surgical instrumentation can extend surgical therapy beyond the current organ and tissue levels into the realm of cells or even subcellular components. This cell level surgical capability may invariably have other uses in clinical neurosurgery and in basic research where the isolation or manipulation of specic parts of a neuron may have substantial scientic interest.

Improvements to Off-the-shelf Components


Except for the nanoknife and the microplatform, all other instrumentation used in this study is commercially available. Although they serve the needs of this initial study, more refined equipment could be developed specifically to enable the efficient use of surgical microdevices. One area of need is the development of miniaturized micromanipulators to economize on space utilization around the operative field. Another area of critical need is the development of surgical microscopes with sufficient magnification to visualize axons and that provide sufficient working distances between the optical elements and the tissue, similar to the boom-mounted microscope used in this study but more specifically designed for microsurgery. The design of future surgical microscopes for cellular-scale neurosurgery should also include on-board lighting and likely also incorporate fluorescence imaging, which provides more contrast and far better signal-to-noise ratios over brightfield imaging. Another useful feature would be a mechanism to coordinate the spatial positioning and movement of the surgical field (animal), the operating microdevices, and the field of view of the surgical microscope. Finally, ease of use by the operator and other ergonomic issues must be considered and incorporated into more refined systems compatible with clinical use.

Micro- and Nanotechnology in Neurosurgery


It is increasingly recognized that not only will micro- and nanotechnology greatly impact the current practice of medicine in both diagnosis and therapy, these emerging disciplines can also potentially enable new neurosurgical procedures at unprecedented small scales (1, 1113, 17). Currently, optimal treatment is not possible in many cases as a result of a mismatch between tissue size and surgical instrumentation. New microfabricated surgical instruments with nanoscale features that can overcome these limitations will allow a new level of access at the cell and tissue level that is not possible with current technology. Microdevices fabricated using methods adapted from the semiconductor industry are already used in common everyday products such as automobile airbag sensors, inkjet printer heads, and micromirror arrays in digital light processing projectors. As a result of their inherent small mass and robust mechanical properties, such micromachines can be operated at extremely high speeds and have proven to be very durable and reliable. However, the use of microdevices as cellular or subcellular-scale operative instruments brings about a different set of challenges. Although the manufacture of miniature-scaled neurosurgical devices is based on welldeveloped and reasonably mature fabrication technology, the actual performance of such microinstrumentation in vivo and their potential use in cellular and subcellular-scale neurosurgical procedures have yet to be optimized and should represent an important new direction in microsurgery.

Issues of Biocompatibility
An obvious concern of any prospective medical device is the suitability of the constituent materials for the target tissue. Evaluations of biocompatibility include identifying any acute toxins that have an immediate, detrimental effect on the host tissue as well as longer-term responses of the host tissue resulting from the prolonged presence of the materials. For silicon nitride, an essential material in many microdevices, biocompatibility has been demonstrated both in vitro with tissue slices and in vivo (9, 10, 22). In our own tests, we demonstrated that individual neurons grow and extend neurites directly on silicon nitride-coated substrates with outgrowths and neuronal survival comparable to those on standard culture glass, indicating the absence of any acute toxicity. Furthermore, extensive studies of silicon nitride with implantable microdevices such as miniature electrode and neurorecording arrays (10) have shown that this material can be implanted in an animal and left for many weeks without eliciting adverse responses from host tissues. One study (9) explicitly evaluated silicon nitride, along with other materials, against a battery of standard biocompat-

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Disclosure
David W. Sretavan, M.D., Ph.D., is a founder with a nancial interest in a biotechnology company developing microdevices for applications in microsurgery.

Acknowledgments
This research was supported by the Sandler Family Supporting Foundation and the That Man May See Foundation. David W. Sretavan, M.D., Ph.D., is a recipient of Research to Prevent Blindness Lew R. Wasserman Merit Award. Additional support to the Sretavan Laboratory from the National Eye Institute and the Research to Prevent Blindness Foundation is gratefully acknowledged. All devices were fabricated at the University of California Berkeley Microfabrication Laboratory.

REFERENCES
1. Apuzzo ML, Liu CY: 2001: Things to come. Neurosurgery 49:765778, 2001. 2. Bement WM, Yu HY, Burkel BM, Vaughan EM, Clark AG: Rehabilitation and the single cell. Curr Opin Cell Biol 19:95100, 2007. 3. Birgbauer E, Oster SF, Severin CG, Sretavan DW: Retinal axon growth cones respond to EphB extracellular domains as inhibitory axon guidance cues. Development 128:30413048, 2001. 4. Bostock R, Collier J, Jansen R-JE, Jones R, Moore D, Townsend J: Silicon nitride microclips for the kinematic location of optic bres in silicon V-shaped grooves. J Micromech Microeng 8:343360, 1998. 5. Brewer GJ, Torricelli JR, Evege EK, Price PJ: Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35:567576, 1993. 6. Chang WC, Keller CG, Sretavan DW: Isolation of neuronal substructures and precise neural microdissection using a nanocutting device. J Neurosci Methods 152:8390, 2006. 7. Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So KF, Schneider GE: Nano neuro knitting: Peptide nanober scaffold for brain repair and axon regeneration with functional return of vision. Proc Natl Acad Sci U S A 103:50545059, 2006. 8. Goldshmit Y, Galea MP, Wise G, Bartlett PF, Turnley AM: Axonal regeneration and lack of astrocytic gliosis in EphA4-deficient mice. J Neurosci 24:10,06410,073, 2004. 9. Kotzar G, Freas M, Abel P, Fleischman A, Roy S, Zorman C, Moran JM, Melzak J: Evaluation of MEMS materials of construction for implantable medical devices. Biomaterials 23:27372750, 2002. 10. Kristensen BW, Noraberg J, Thibaud P, Koudelka-Hep M, Zimmer J: Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures. Brain Res 896:117, 2001. 11. Leary SP, Liu CY, Apuzzo ML: Toward the emergence of nanoneurosurgery: Part IInanomedicine: Diagnostics and imaging at the nanoscale level. Neurosurgery 58:805823, 2006. 12. Leary SP, Liu CY, Apuzzo ML: Toward the emergence of nanoneurosurgery: Part IIInanomedicine: Targeted nanotherapy, nanosurgery, and progress toward the realization of nanoneurosurgery. Neurosurgery 58:10091026, 2006. 13. Leary SP, Liu CY, Yu C, Apuzzo ML: Toward the emergence of nanoneurosurgery: Part Iprogress in nanoscience, nanotechnology, and the comprehension of events in the mesoscale realm. Neurosurgery 57:606634, 2005. 14. Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y: Green mice as a source of ubiquitous green cells. FEBS Lett 407:313319, 1997. 15. Polla DL, Erdman AG, Robbins WP, Markus DT, Diaz-Diaz J, Rizq R, Nam Y, Brickner HT, Wang A, Krulevitch P: Microdevices in medicine. Annu Rev Biomed Eng 2:551576, 2000. 16. Rebello KJ: Applications of MEMS in surgery. Proceedings of the IEEE 92:4355, 2004. 17. Roy S, Ferrara LA, Fleischman AJ, Benzel EC: Microelectromechanical systems and neurosurgery: A new era in a new millennium. Neurosurgery 49:779798, 2001. 18. Schwab ME: Repairing the injured spinal cord. Science 295:10291031, 2002. 19. Silver J, Miller JH: Regeneration beyond the glial scar. Nat Rev Neurosci 5:146156, 2004. 20. Sretavan DW, Chang W, Hawkes E, Keller C, Kliot M: Microscale surgery on single axons. Neurosurgery 57:635646, 2005. 21. Suh LH, Oster SF, Soehrman SS, Grenningloh G, Sretavan DW: L1/Laminin modulation of growth cone response to EphB triggers growth pauses and regulates the microtubule destabilizing protein SCG10. J Neurosci 24:19761986, 2004. 22. Voskerician G, Shive MS, Shawgo RS, von Recum H, Anderson JM, Cima MJ, Langer R: Biocompatibility and biofouling of MEMS drug delivery devices. Biomaterials 24:19591967, 2003.

COMMENTS

hang et al. have eloquently presented their work in a most exciting area of medicine, the micro- and nanoworld as it pertains to manipulation and alteration of neural structures. They have demonstrated that axon surgery is indeed possible. This seems farfetched but so have many advances in neurosurgery during their early formative years. The major obstacles to the advancement of this field will be related to the development of surgical platforms and the performance of multiple microtasks in rapid succession or simultaneously. The latter is required to accomplish clinically relevant surgical procedures in multiple axons in a timely manner. The work of Chang et al., however, suggests that such further refinements are not all that far off. The authors are to be commended for their vision, their forward-thinking efforts, and their creativity. Edward C. Benzel Cleveland, Ohio

n this report, the authors describe the use of a microfabricated nanoknife with a cutting edge measuring 20 nanometers for use in vivo. Previous work from the same group has reported the use of the same nanoknife as applied in vitro for single axon surgery. This represents an extension of the work to the in vivo environment for peripheral nerve surgery in a mouse model. The authors were able to construct a surgical setup such that the operative substrate was immobilized from the normal respiration and movements of the mouse. The nanoknife was used to make progressive cuts in the sciatic nerve, with electrophysiological recordings demonstrating the progress dimunition of motor responses from the target muscle. In addition, single axon surgery was demonstrated in vivo. Finally, biocompatibility of the device was also demonstrated by cell culture experiments. This is a highly important report in that it represents the first demonstration of subcellular surgery in vivo. Although it is not quite surgery on the nanoscale, this demonstration represents a major reduction in the scale of surgery in a living animal. I look forward to future work from the authors with demonstration of the sequential steps of single axon repair. Charles Y. Liu Los Angeles, California

his project uses a nanoblade to cut nervous tissue and axons. Their device appears similar to single-cell extraction techniques in which a micropipette tip controlled by a robotic micromanipulator is used to selectively aspirate axons, dendrites, or cell bodies from neurons in culture. As with previously published in vitro experiments (1), once an axon has been isolated, it can be precisely transected. Using a mouse sciatic nerve, the authors had to first separate an axon from the nerve before cutting it on the platform. Therefore, in situ single axon transection has not been demonstrated thus far. Limitations include a wide nanoblade and, perhaps, difficulty differentiating individual axons.

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The authors ultimate goal is to use this device to directly repair axons. However, the current clinical applications of direct axon repair appear quite limited. For example, what is the time window for axon repair before Wallerian degeneration? Additional pathophysiological studies are needed to answer questions such as these. Regarding peripheral nerve repair, alignment of the basal lamina delineating the endoneurial tubes may be an alternative application that is more readily applicable to current practice. Despite these concerns, the authors are commended for their forward-thinking concept. As their technology advances parallel to simultaneous discoveries in neuronal protection, intraoperative imaging, and device production, potential nanotechnology applications in neurosurgery will surely appear over time. Stephen M. Russell New York, New York

1. Sretavan DW, Chang W, Hawkes E, Keller C, Kliot M: Microscale surgery on single axons. Neurosurgery 57:635646, 2005.

ith the advent of the operating microscope and microsurgical techniques, Millesi (4) and Millesi et al. (5) improved clinical results and popularized the use of nerve grafts for nerve repair. To date, surgical techniques for nerve repair have been at the tissue or cable level. Even in a fascicular nerve repair, there are literally several hundred to a few thousand axons in the proximal stump and a similar number of endoneurial pathways in the distal stump (3). With the most meticulous repairs, the endoneurial tubes can never be reapproximated exactly, and this results in mismatching of regenerating axons at the site of suture, or within the graft, leading to inappropriate (non-specific) and incomplete reinnervation and subsequent poor recovery in function (1). Misdirection at the repair site is common, and precise reinnervation is only possible in clean and focal crush injuries rather than ones in which the nerve is severed and then repaired with a suture (6). Techniques to match regenerating axons with reciprocal and appropriate targets in the distal nerve stump are currently only partially possible with experimental techniques that maximize topographic

alignment (2). Further technological advances will require microsurgery to be performed at a cellular level. It is in this context that the current report by Chang et al. is relevant. They illustrate the in vivo use of a microfabricated nanoknife to perform nerve and axon microsurgery. Essentially, the data demonstrate that precise and targeted severance of a portion of a mouse tibial nerve and individual axons from the same tibial nerve can be achieved using the prototype device. It is notable that a signicant engineering aspect is the development of the entire assembly for the micromanipulation, a microplatform in which the nerve is positioned, followed by the actual use of this cutting device. Each of these is a fundamental engineering issue that the investigators have solved admirably. Yet, the translation of this device from the type of proof of principle demonstrated herein to a practical tool for human nerve surgery remains challenging. A major shortcoming with even the most microscopic form of nerve repair is the biological constraint of cellular misalignment, which cannot be easily overcome by further progress in microsurgical techniques. The authors have provided the rst glimpse of the technologies that may enable more precise surgery at the nanoscale level. I look forward to further studies by these investigators as they endeavor to advance nerve repair into the cellular domain. Rajiv Midha Calgary, Canada

1. Brushart TM: The mechanical and humoral control of specificity in nerve repair, in Gelberman RH (ed): Operative Nerve Repair and Reconstruction. Philadelphia, J.B. Lippincott, 1991, pp 215230. 2. de Medinaceli L, Rawlings RR: Is it possible to predict the outcome of peripheral nerve injuries? A probability model based on prosoects for regenerating neurites. Biosystems 20:243258, 1987. 3. Matsuyama T, Mackay M, Midha R: Peripheral nerve repair and grafting techniques: A review. Neurol Med Chir (Tokyo) 40:187199, 2000. 4. Millesi H: Brachial plexus injuries. Nerve grafting. Clin Orthop Relat Res 237:3642, 1988. 5. Millesi H, Meissl G, Berger A: The interfascicular nerve-grafting of the median and ulnar nerves. J Bone Joint Surg Am 54:727750, 1972. 6. Nguyen QT, Sanes JR, Lichtman JW: Pre-existing pathways promote precise projection patterns. Nat Neurosci 5:861867, 2002.

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