Wesley C. Chang et al- In Vivo Use of a Nanoknife for Axon Microsurgery

8/3/2019 Wesley C. Chang et al- In Vivo Use of a Nanoknife for Axon Microsurgery

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IN VIVO USE OF A NANOKNIFE FORAXON MICROSURGERY

OBJECTIVE: Microfabricated devices with nanoscale features have been proposed asnew microinstrumentation for cellular and subcellular surgical procedures, but theireffectiveness in vivo has yet to be demonstrated. In this study, we examined the in vivouse of 10 to 100 m-long nanoknives with cutting edges of 20 nm in radius of curva-ture during peripheral nerve surgery.

METHODS: Peripheral nerves from anesthetized mice were isolated on a rudimentarymicroplatform with stimulation microelectrodes, and the nanoknives were positionedby a standard micromanipulator. The surgical field was viewed through a researchmicroscope system with brightfield and fluorescence capabilities.

RESULTS: Using this assembly, the nanoknife effectively made small, 50 to 100 m-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 visu-ally and by the accompanying incremental diminution of evoked motor responsesrecorded from target muscle. Furthermore, this nanoknife also enabled the surgeon toperform procedures at an unprecedented small scale such as the cutting and isolationof 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 evi-denced by the robust growth of axons and neurons on this material in vitro.

CONCLUSION:Together, these demonstrations support the concept that microdevicesdeployed in a neurosurgical environment in vivo can enable novel procedures at anunprecedented small scale. These devices are potentially the vanguard of a new fam-ily of microscale instrumentation that can extend surgical procedures down to the cel-lular 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

NEUROSURGERY VOLUME 61 | NUMBER 4 | OCTOBER2007 | 683

NEW INSTRUMENTATION

Wesley C. Chang, Ph.D.

Departments of Ophthalmologyand Physiology,Neuroscience Program

and Bioengineering Program,University of California, San Francisco,San Francisco, California

Elizabeth A. Hawkes, M.S.

Departments of Ophthalmologyand Physiology,Neuroscience Program

and Bioengineering Program,University of California, San Francisco,

San Francisco, California

Michel Kliot, M.D.

School of Medicine andDepartment 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 Ophthalmologyand Physiology,Neuroscience Programand Bioengineering Program,

University of California, San Francisco,San Francisco, California

Reprint requests:

Wesley C. Chang, Ph.D.,Departments of Ophthalmologyand Physiology,

Neuroscience Programand Bioengineering Program,University of California, San Francisco,

10 Koret Way, K-110,San Francisco, CA 94143.Email: [email protected]

Received, December 4, 2006.

Accepted, May 17, 2007.

Although axons play a critical role inrelaying information within the nerv-ous system, there is currently no thera-

peutic intervention that can directly repairthese key neural processes when severed ordamaged by trauma. Substantial researchefforts are underway to identify effective ther-apeutic strategies. Such approaches include

the suppression of growth-inhibitory mole-cules associated with myelin and scar tissues(8, 18, 19), the harnessing of axon guidancemolecules that are up-regulated after trauma(8, 12), and even the application of novel bio-engineered tissue scaffolds to guide new axonoutgrowth (7). All of these efforts share thegoal of stimulating the regeneration of injured

adult axons. Recently, we proposed an alterna-tive paradigm in which transected nerves aredirectly repaired by surgically reconnectingthe constituent axons to acutely reconstitutenerve function after damage. To demonstratethe feasibility of this concept, our researcheffort has presented early demonstrations ofseveral simple microfabricated devices that

cut, electrostatically translocate, and splicetogether individual axons in vitro (20).An important aspect of this microdevice-

based method of axon repair is the effective-ness of such small-scale instrumentation insurgical environments in vivo. Current uses ofmicrotechnology in surgical instrumentationinclude microscale motors, force-generating


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actuators, and catheter-mounted structures for microsurgicaltissue cutting, ophthalmic procedures, vascular surgeries, andtargeted drug delivery (1517). However, to operate on individ-ual cells or on subcellular elements such as axons, new surgi-cal microdevices are required with characteristic dimensions atthe micron scale. An important challenge will be to develop themeans to effectively deploy and manipulate these tiny devices

in a surgical setting. For instance, surgical microdevices willhave to be mechanically strong and robust for use in vivo, andit will be necessary to precisely position and manipulate thesedevices at scales much smaller than those possible with manu-ally operated tools. Such devices and capabilities will allowthe surgeon to perform novel microscale procedures that arecurrently not possible with existing surgical instrumentation.

In this study, we have investigated these issues by testing anaxon nanoknife (6, 20) that can be manipulated as a microde-vice in a surgical setting. This nanoknife consists of a thin layerof silicon nitride with a nanometer sharp cutting edge. In thecurrent demonstration, nanoknives were used to performperipheral nerve surgery in an anesthetized mouse. By usingthe nanoknife mounted on a micromanipulator in conjunction

with a simple, custom-assembled microplatform to hold andisolate an individual nerve, it was possible to precisely manip-ulate this surgical microdevice in the operating field and tomake targeted, effective cuts in the sciatic nerve while simulta-neously 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

enabled the first targeted cut-ting of a single axon in ananesthetized animal.

MATERIALS ANDMETHODS

Animals

The surgical demonstrationwas performed on adult femaleC57BL/6 mice or transgenic GFPmice (Charles River Laboratories,Inc., Wilmington, MA) anes-thetized with a mixture of keta-mine and xylazine (14). Thesedated animal was placed in aprone position with its hind legsfully extended. To expose the sci-atic nerve, an incision was madethrough the skin of the posteriorthigh and underlying hamstring

muscles. The sciatic nerve splitsinto several major branches (Fig.1A), the tibial and peroneal nerves,as well as a smaller branch. Cut-ting by the nanoknife was per-formed on the central largest

branch (Fig. 1A, arrow), whichinnervates the calf muscles and which we have identified as the tibial

branch of the sciatic nerve. To access the axons within the nerve, theensheathing epineurium and perineurium were gently removedthrough standard microdissection with handheld surgical forceps whileviewing under a dissection microscope.

Surgical Microplatforms

The isolation and stabilization of the nerve was performed using aspecial fixture mechanically isolated from the animal (Fig. 1B). Thisfixture was held and positioned over the animal by a micromanipula-tor and presented two separate flat platforms (Fig. 1C) for holding thenerve, one for stimulation (Fig. 1C, small arrow) and the other for nerveor axon cutting (Fig. 1C, arrowhead). These platforms consisted of twoelongated, rectangular chips of Pyrex glass (2 mm wide 0.5 mm thick

8 mm long; Corning, Corning, NY) that were arranged in paralleland held level at a position just a few millimeters above the hind limbof the animal. During the surgery, the segment of the sciatic nerve,with axons exposed, was lifted from the leg and draped over these twoplatforms (Fig. 1, CD). Along one edge of the proximal platform, a 500m-wide line of gold film was deposited to provide a conductor fornerve stimulation. This gold trace was electrically connected to a volt-age pulse generator and delivered electrical signals to the nerve. Theopposing pole was grounded to an Ag/AgCl2 electrode placed undera medial flap of skin on the leg.

The second, more distal platform, dedicated specifically for nerveand axon cutting, was separated from the stimulating platform by agap of 2 mm. This separation provided an isolated surface for themicrocutting of the nerve and also prevented unintended feed-throughof the electrical stimulation signals generated at the first platform so thesevered distal segment of nerve on the second platform could notreceive unintended stimulation.

684 | VOLUME 61 | NUMBER 4 | OCTOBER2007 www.neurosurgery-online.com

CHANG ET AL.

FIGURE 1. A custom surgical suite was assembled to enable cutting with nanoknives while allowing visual moni-toring through an overhead microscope. This suite also included the capability for stimulation of the target nerve andrecording of the resulting EMG. A, a wide view of the unmounted nerve (arrow) showing its anatomic position inthe posterior calf along with the target soleus muscle (asterisk). EMG was recorded from the soleus muscle at a posi-tion indicated by the asterisk. B, the surgical field showing the tibial branch of the sciatic nerve (arrow) mountedon the glass microplatforms. The green board supporting the microplatforms and facil itating the electrical connection

for stimulation is 2 1.5 cm. C, close-up of the surgical microplatforms (2-mm wide each) showing the embeddednerve stimulation electrode (arrow) and the nerve/axon cutting platform (arrowhead). The nerve is shown drapedon the surgical microplatforms, which are mechanically isolated from the animal. D, schematic diagram showing thesurgical field and supporting equipment, including the stimulation and recording instrumentation for nerve stimu-lation combined with electromyographic recording from the soleus muscle. The overhead microscopes objective lensis positioned just above the nerve straddling the microplatforms.

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During surgery, the nerve was kept moist by regular irrigation withphysiologically buffered media. However, it was necessary to keep the

buffered media within the boundaries of each platform because themedia itself can serve as a conduit for unintended feed-through ofelectrical signals.

Electrical Stimulation

The stimulation signal was a single monophasic, rectangular pulsewith 200-s duration and of varying amplitudes ranging from 100 mVto 2 V delivered at 5 Hz.

Observation

All surgical procedures were monitored in real time using anupright, boom-mounted microscope (Nikon Erect Image TrinocularTilting Head and Nosepiece with objective lenses mounted on SMS 20Diagnostics boom stand and with brightfield and fluorescence capabil-ities; Nikon Instruments, Garden City, NY), and surgical manipula-tions were performed directly under long working distance objectivelenses. Brightfield illumination was provided from above at obliqueangles to the microscopes optical axis, whereas fluorescence imagingwas enabled by an on-board mercury lamp along with appropriate fil-ters. With these provisions, it was possible to distinguish individual

axons in the mounted nerve. The operating field could be vieweddirectly by an observer through the microscope eyepieces or the imagecould be diverted to a mounted digital camera and then displayed andrecorded on a computer.

Cutting With the Nanoknife

For microcutting of the isolated nerves, a nanoknife previouslydeveloped for the cutting of individual axons in vitro was used (6). Thepyramid-shaped nanoknife was fabricated by conformal molding ofchemical vapor-deposited silicon nitride over silicon etched preciselyalong specific crystal planes, resulting in an ultrasharp cutting edge atthe apex (20 nm edge radius of curvature as determined by scanningelectron microscopy), small enough to target individual axons. Thisdevice has been used in vitro to cut both axons from cultured neuronsand from harvested adult nerves (6). For the surgical procedures in the

present study, the knife was mounted on a supporting rod and thenpositioned by a commercially available, precision micromanipulator(MP-285; Sutter Instruments, Novato, CA), which allowed the nano-knife to be precisely aimed at one or a few axons at a time. Surgicaldemonstrations were also performed in transgenic mice expressinggreen fluorescent protein (GFP). Retention of the cytoplasmically dis-solved GFP after cutting was used to determine whether or not the sev-ered ends of the axons resealed after cutting. This analysis of resealingafter single axon cutting has been previously reported (6).

Evoked EMG Response

To record the evoked EMG signals triggered by nerve stimulation, asharp tungsten needle was inserted into the calf soleus muscle. Theopposite pole was grounded at the same Ag/AgCl2 probe used withthe stimulating circuit. Signals generated at the tungsten probe werefed to an AC amplifier (DAM-80; World Precision Instruments Inc.,Sarasota, FL) and amplified 1000 times. The amplified signals were inturn read and recorded by an oscilloscope (TEK 3012B; Tektronix,Beaverton, OR).

For EMG recordings, the amplitude of tibial nerve stimulation wasfirst varied to find the approximate threshold, below which no actionpotential and, therefore, no EMG signal could be detected. Nerve stim-ulation was then elicited using a stimulus fourfold stronger than the

identified threshold. To capture and display accurate EMG signals, wesubtracted the background stimulation artifacts, which were measured

by recording from the muscle in an actual surgical configuration butwith the sciatic nerve completely severed between the platform and themuscle while still draped over the platform. (EMGs with this back-ground subtraction are displayed in all figures.) Although this proce-dure eliminated signal artifacts substantially, some remnant of signal

feed-through remained owing to slight variations in the artifactsbetween recordings.

Evaluation of Biocompatibility

To investigate any potential acute toxicity of silicon nitride, bothmouse hippocampal neurons and retinal explants were cultured onflat substrates coated with the same chemical vapor-deposited siliconnitride used to construct the nanoknives. Briefly, hippocampal cellswere harvested from the brains of E16 mouse embryos and plated onsubstrates that were preabsorbed with poly-L-lysine using standardprotocols (5). Mouse retinal explants from E14 embryos were plated onlaminin-coated substrates, again using published protocols (3, 21). Bothcultures were maintained in 37C and 5% CO2/95% air in their respec-tive nutrient media along with appropriate supplements.

RESULTS

Test Microdevice

The test microdevice is a microscale cutting instrument withnanoscale features that was originally designed for the precisesevering of axons under well-controlled research conditions invitro (6). This pyramid-shaped microdevice consists of amechanically strong, 1 m-thick shell of silicon nitride (Fig. 2A)and was mounted onto a thin metal rod attached to a glassmicropipette holder (Fig. 2B). The pipette holder was, in turn,mounted to a robotic X-Y-Z micromanipulator to move themicrodevice into position and to deliver the cutting stroke.Because the nanoknife was used for targeted cutting of singleaxons in a peripheral nerve, the precise movement provided bya micromanipulator was necessary. In the current assembly forthis surgical demonstration, it was possible to station onemicromanipulator system exclusively to hold the microplat-form supporting the nerve and then station another microma-nipulator on the opposite side of the surgical field to presentand manipulate the nanoknife for nerve cutting. Knife posi-tioning with the micromanipulator and nerve (or axon) cut-ting using the microplatform as a cutting board was easy toperform and could be observed through the microscope.

Deployment in the Surgical Field

The placement of the nerve on the microplatform allowedthe nerve to be lifted away from the leg musculature and be

mechanically stabilized. When placed on the platform andlifted, the nerve was put under some tension and was heldstationary and physically isolated from the animal. Conse-quently, small movements of the animal resulting from heart-

beat, respiration, and involuntary muscle movements werenot transmitted to the nerve. The microplatform also allowedthe nerve to be maintained in an aqueous environment whileeliminating contact with other tissues, thus ensuring that the


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electrical nerve stimulation did not feed through to the mus-cles through other pathways in the animal. Although mechan-ical manipulations were required to place the intact nerve onthe platform and to prepare the exposed axons for cutting,nerve function itself was not disrupted because nerve stimu-lation using the built-in electrode on the platform readily trig-gered observable twitching of the calf muscle and an easilydetectable EMG signal.

Observation During the Surgical Procedure

Because conventional surgical or tissue dissection micro-scopes lack the necessary magnification to observe individualaxons, an upright, boom-mounted research microscopeequipped with 10 eyepieces was adapted for direct overheadobservation of the surgical field with both brightfield and flu-orescence imaging. This research microscope was equippedwith 4 to 20 long working distance objective lenses that pro-

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

Cutting With the Nanoknife

The exposed tibial branch of the mouse sciatic nerve asviewed through the microscope during a cutting sequence isshown in Figure 3. In Figure 3A, the black profile is a portion ofthe nanoknife brought in from above into the surgical field andprepositioned to the side of the nerve. At this position, thenanoknife is raised above both the microplatform and thenerve, resulting in the knife profile being slightly out of focusrelative to the nerve.

To test whether or not this microdevice can be used tomake small cuts in a living nerve, the nanoknife was broughtover the desired position and lowered onto the nerve (Fig.3B). The silicon nitride material is sufficiently optically trans-lucent to allow visualization of the nerve through the nano-knife itself. This property of the microdevice was useful in

allowing the operator to roughly judge nanoknife contactwith the nerve and dimpling of nerve tissue during the cut-ting. The amount of vertical displacement of the nanoknifeduring cutting could also be determined more accuratelyfrom the micromanipulator controller.

The resulting incision from one nerve cutting experiment isshown in Figure 3C (arrow). The incision measured approxi-mately 100 m in length, corresponding to the length of the

686 | VOLUME 61 | NUMBER 4 | OCTOBER 2007 www.neurosurgery-online.com

CHANG ET AL.

FIGURE 2. A diagram of the axon nanoknife showing device configura-

tion and the ultrasharp cutting edge (apex of the elongated pyramidalstructure, green). B, wider view of the mounted knife (boxed), held by amicropipette holder, which in turn is held in the micromanipulator.

A

B

FIGURE 3. Visualization and use of the axon nanoknife for microscalenerve cutting is demonstrated. A, the intact nerve as viewed through the

microscope with a total magnification 100. The nanoknife with a100-m long cutting edge is held above the nerve off to one side at thebeginning of the procedure (upper right). B, the nanoknife is brought overthe desired position and lowered to make the cut. C, a cut approximately100 m long (arrow) is visible in the nerve. D, the cutting sequence wasrepeated using the same nanoknife to make a second cut in the nerve(arrow). AD, scale bar, 200 m.

A B

C D


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nanoknife cutting edge of 100 m. The cutting sequence wasrepeated, and a second incision of approximately the same sizewas made (Fig. 3D, arrow) next to the original. We estimate thatin each cut, perhaps 50 to 100 axons are severed, but this quan-tification remains to be verified objectively.

To assess the physiological state of the cut ends of axons sev-ered by the nanoknife, this cutting procedure was also repeatedin a GFP transgenic animal in which all cells contained freelydissolved cytoplasmic GFP (Fig. 4A). Under fluorescence imag-ing, it was possible to identify the site of the cut (Fig. 4B,arrow). At this cut site, the severed axons remained brightlyfluorescent, indicating the retention of cytoplasmic GFP. Thisability of severed axons to reseal its cut ends has been previ-

ously demonstrated in vitro (6) and is presumably the result ofthe 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 inde-pendent visual confirmation that the stimulation worked andwas above threshold. Concurrently, the recording probe

inserted into the target muscle recorded a signal consisting ofan artifact coinciding with the stimulating pulse followed by anEMG waveform beginning approximately 1 ms later. When the

background (control) waveform was subtracted from this rawrecording, the artifact was largely eliminated, and the trueshape and duration of the EMG wave could be discerned.Control waveforms have been subtracted from all EMG record-ings shown in the figures. Typically, the waveform lasted justover 2 ms (Fig. 5). By varying the magnitude of the stimulationpulse, it was determined that, for this system, the threshold forevoking a response in the muscle was between 300 and 400mV delivered at the platform. Below this threshold, therecorded muscle signal was nearly identical to the backgroundcontrol and no muscle twitching was observed.

Repeated Use of the Nanoknife for Targeted Cutting

The precision and durability of the nanoknives was demon-strated in experiments in which a single nanoknife was usedrepeatedly to progressively pare down a nerve. For this exer-cise, a shorter knife with a cutting edge of 10 m was used.From our observations, we estimate that perhaps only a fewdozen axons are severed with each cutting stroke. In additionto visual observations of the cutting sequence and the tissueincisions, the successive removal of functional axons withinthe nerve was verified simultaneously with EMG recordingfrom the innervated calf muscle (Fig. 5). The intact nerve sup-ported by the microplatform at the start of the procedure isshown in Figure 5A. Figure 5B shows the EMG recording fol-lowing the delivery of a subthreshold stimulus, whereas Figure5C shows the EMG waveform recorded after delivery of asupramaximal stimulus. Figure 5F shows the muscle responseresulting from stimulation of an intact nerve and after a largenumber of axons had been cut by the nanoknife (Fig. 5, D andF). We estimate that approximately one-half to two-thirds of

the nerve had been eliminated at this point. As a result of thesmall amount of tension on the nerve when it is placed ontothe microplatform, the ends of severed axons retract from thefield of view while the remaining uncut axons remain in thesurgical field. The progressive diminution in the EMG wave-form at various stages of this cutting procedure is shown inFigure 5, GI. Note that the EMG recordings in Figure 5, E, F, Hand Iwere obtained using a fourfold suprathreshold stimulus.Figure 3H shows the EMG corresponding to the situationshown in Figure 5G, in which only a few axons are left on themicroplatform. The severing of these remaining axons withthe nanoknife resulted in the elimination of detectable EMGsignal (Fig. 5I).

Although we have shown that the EMG signal diminishes

monotonically with the paring down of the nerve, the exactrelationship between the fraction of the nerve eliminated andthe diminution of the EMG is undetermined and indeed varies

between different trials. This uncertainty results from the waythat the EMG signal is constructed, a summation of signalsfrom the individual innervated muscle cells. The strongest con-tributors to the composite EMG signal are from the cells clos-est to the recording probe, whereas more distant muscle cells



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

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contribute less. Thus, cutting axons that map to muscle near theprobe results in a larger diminution of the signal that wouldoccur when the exact same cut is made in axons mapping tomuscle cells further away.

With repeated use, the cutting performance of the nanoknifewas not noticeably diminished. Breakage of the nanoknife itselfduring cutting was only observed in instances in which opera-tor error led to the unintended impact of the nanoknife with themicroplatform or some other nearby surface. The most frequentlimitation to the lifetimes of the nanoknives was not in fact

breakage of the silicon nitride microdevice, but the breakage atthe joint where the microdevice is attached to its mounting rod.

Surgical Cutting of Single Axons

A major goal of this study was to determine whether or notnanoknife use could enable novel cellular or subcellular-scalesurgical procedures that are not currently feasible using exist-ing instrumentation. We tested the precision and cutting per-formance of the nanoknife on individual axons in an in vivosurgical environment. A sequence of images illustrating the useof a nanoknife on a single axon is shown in Figure 6. In thisexample, the nanoknife was operated in the same manner as

described previously for mak-ing incisions in whole nerves.Figure 6, B and C, shows thenanoknife moved into posi-tion for axon cutting, whereasFigure 6D shows the resultingcut (arrow) in the targetedaxon. Asecond cut was subse-quently made in the sameaxon resulting in the isolationof a short 30-m segment (Fig.6E, arrow). This axon segmentwas moved away from theparent axon to aid in visuali-zation (Fig. 6F, arrow).

Biocompatibility ofSilicon Nitride forNeurons

During the course of ourexperimentation, we did notencounter instances of appar-ent acute deleterious effects ofsilicon nitride on nerves orsurrounding tissues. To exam-ine this issue of biocompatibil-ity in more detail, we placedactual nanoknives in tissueculture dishes and seededmouse retinal explant tissue orhippocampal neurons directlyonto silicon nitride knives todetermine whether this mate-rial was suitable as a neuronal

growth substrate. The results showed that after 2 days in culturedirectly on silicon nitride, hippocampal neurons that wereseeded as isolated neurons exhibited significant process out-growth (Fig. 7A). Likewise, a fairly dense network of axongrowth could be observed from the explanted retinal tissue (Fig.7B). The appearance of the retinal axons and hippocampal neu-rons were very similar to what has been reported when they aregrown on tissue culture glass (3, 5, 21).

DISCUSSION

In this study, we subjected silicon nitride nanoknives to test-ing under the operative conditions of peripheral nerve surgeryin a living anesthetized mouse. The main objectives of this

work were to examine whether or not this new type ofmicroscale neurosurgical instrumentation could be used effec-tively in vivo and to verify the ability of these microdevices todeliver surgical precision at unprecedented cellular and subcel-lular length scales. The results demonstrated that by using arudimentary microplatform and off-the-shelf components suchas micromanipulators and research microscopes, nanoknivescan be deployed and used effectively for microscale peripheral


CHANG ET AL.

FIGURE 5. Nerve images and EMG recordings showing the progressive loss of evoked muscle response signal dur-ing nerve section using an axon nanoknife. A, image showing the intact tibial branch of the sciatic nerve supportedon the microplatform. B, application of a subthreshold stimulus (200 mV) resulted in no detectable EMG signal. C,the resulting EMG signal after application of a suprathreshold stimulus (500 mV) is shown. D, view of the nerve aftermost of its axons have been pared away using the nanoknife is demonstrated. E, the EMG waveform recorded in thesoleus muscle after stimulation of the intact nerve is shown with a stimulus fourfold above threshold. F, the EMGwaveform was recorded from the remaining axons shown in D. This waveform is reduced in amplitude compared withthat obtained from the intact nerve. G, continued axon cutting using the same nanoknife left just a few axons remain-ing on the microplatform. H, the EMG waveform was recorded from stimulation of the axons remaining in G. I, noapparent EMG signal is observed after all of the axons have been cut using the nanoknife.


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nerve surgery. In addition to precise 50 to 100 m incisions ina peripheral nerve, and the incremental paring down of a sin-gle nerves muscle response, novel subcellular-scale neurosur-gical procedures enabled by this novel surgical instrumentationincluded the precise targeted cutting and isolation of a seg-ment from a single axon in vivo. All procedures could beobserved and monitored in real-time, allowing user feedback,image capture, and EMG recording.

Microdevice Robustness

For use in microsurgery, microdevices must be mechanicallystrong to permit repeated use. The robust performance of thenanoknife is attributable in part to the material properties of sil-icon nitride (ultimate strength, 28 GPa), which is actuallystronger 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.

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

Microinstrumentation Use in Vivo

A fundamental question concerning the surgical use ofmicrodevices that have characteristic sizes of only several tensor hundreds of microns is whether or not such small instru-mentation can be deployed satisfactorily and used efficiently

in an operative field. For the current study, we have chosen toaddress this question using the relatively accessible tibial branch of the mouse sciatic nerve in a demonstration ofmicroscale nerve and single axon cutting. The key to deploy-ment of the nanoknife in this instance was the use of a rudi-mentary microplatform that provided support for the nerveand allowed the nerve to be mechanically isolated. Themicroplatform also served as an axon microcutting surface,

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FIGURE 6. Precise targeted cutting of a single axon is performed usingthe nanoknife. A, view of an axon (center bottom) isolated from the tib-ial 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 sideat 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 locationindicated by the arrow. The nanoknife has been removed from the surgi-

cal field. E, the cutting sequence was repeated using the same nanoknife tomake a second cut in the axon (arrow). F, the small segment in betweenthe two incisions made in D and E was moved aside and is indicated bythe arrow. AF, scale bar, 200 m.

A B

C D

E F

FIGURE 7. Fluorescent images showing hippocampal neurons (A) andneurite outgrowth (B) from embryonic retinal explants cultured on siliconnitride substrates. The neurons used in these experiments express a solu-ble cytoplasmic GFP protein and are fluorescent. AB, scale bar, 100 m.

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allowing the nerve to remain moist while isolated from othertissues. The spatial stability of the resulting operative fieldduplicated the well-controlled in vitro environment in whichwe have previously used similar cutting microdevices suc-cessfully (6, 20). Lastly, the microplatform also contained anembedded electrode for nerve stimulation. Although thismicroplatform strategy was designed for microdevice use inperipheral nerve surgery, a similar strategy can also be triedfor the more challenging use of surgical microdevices in cen-tral nervous system regions such as the spinal cord.

Improvements to Off-the-shelf Components

Except for the nanoknife and the microplatform, all otherinstrumentation used in this study is commercially available.Although they serve the needs of this initial study, morerefined equipment could be developed specifically to enablethe efficient use of surgical microdevices. One area of need isthe development of miniaturized micromanipulators to econ-omize on space utilization around the operative field. Anotherarea of critical need is the development of surgical micro-scopes with sufficient magnification to visualize axons andthat provide sufficient working distances between the opticalelements and the tissue, similar to the boom-mounted micro-scope used in this study but more specifically designed formicrosurgery. The design of future surgical microscopes forcellular-scale neurosurgery should also include on-boardlighting and likely also incorporate fluorescence imaging,which provides more contrast and far better signal-to-noiseratios over brightfield imaging. Another useful feature would

be a mechanism to coordinate the spatial positioning andmovement of the surgical field (animal), the operatingmicrodevices, and the field of view of the surgical microscope.Finally, ease of use by the operator and other ergonomicissues must be considered and incorporated into more refined

systems compatible with clinical use.Issues of Biocompatibility

An obvious concern of any prospective medical device is thesuitability of the constituent materials for the target tissue.Evaluations of biocompatibility include identifying any acutetoxins that have an immediate, detrimental effect on the hosttissue as well as longer-term responses of the host tissue result-ing from the prolonged presence of the materials. For siliconnitride, an essential material in many microdevices, biocompat-ibility has been demonstrated both in vitro with tissue slicesand in vivo (9, 10, 22). In our own tests, we demonstrated thatindividual neurons grow and extend neurites directly on siliconnitride-coated substrates with outgrowths and neuronal sur-

vival comparable to those on standard culture glass, indicatingthe absence of any acute toxicity. Furthermore, extensive stud-ies of silicon nitride with implantable microdevices such asminiature electrode and neurorecording arrays (10) haveshown that this material can be implanted in an animal and leftfor many weeks without eliciting adverse responses from hosttissues. One study (9) explicitly evaluated silicon nitride, alongwith other materials, against a battery of standard biocompat-

ibility tests and considered the material as a nonirritant.Taken together, these findings establish silicon nitride as suit-able for use with neural tissues, especially in cases requiringonly brief contact with tissues.

Cellular and Subcellular-scale Surgery

In this study, we performed what we believe is the first sub-cellular-scale surgery on a single axon in a living anesthetizedanimal. Although the simple cutting of single axons in itself haslimited clinical applications, the development of microdevicesfor precise axon cutting is the first part of a long-term researchprogram that examines the feasibility of conducting the surgi-cal repair of individual damaged or severed axons as a meansof achieving functional recovery after nervous system injuryindependent of axonal regeneration (20). We have previouslyproposed that axon cutting would be followed by the align-ment of severed axon ends and subsequent axon splicing byelectrofusion to reconstitute functional axons. Although pre-liminary proof of principle has been obtained for these lattersteps, the execution of an entire axon repair sequence in vivowill require much future research and development. However,it is worthwhile to consider that new microscale surgical instru-mentation can extend surgical therapy beyond the currentorgan and tissue levels into the realm of cells or even subcellu-lar components. This cell level surgical capability may invari-ably have other uses in clinical neurosurgery and in basicresearch where the isolation or manipulation of specific parts ofa neuron may have substantial scientific interest.

Micro- and Nanotechnology in Neurosurgery

It is increasingly recognized that not only will micro- andnanotechnology greatly impact the current practice of medi-cine in both diagnosis and therapy, these emerging disciplinescan also potentially enable new neurosurgical procedures at

unprecedented small scales (1, 1113, 17). Currently, optimaltreatment is not possible in many cases as a result of a mis-match between tissue size and surgical instrumentation. Newmicrofabricated surgical instruments with nanoscale featuresthat can overcome these limitations will allow a new level ofaccess at the cell and tissue level that is not possible with cur-rent technology. Microdevices fabricated using methodsadapted from the semiconductor industry are already used incommon everyday products such as automobile airbag sen-sors, inkjet printer heads, and micromirror arrays in digitallight processing projectors. As a result of their inherent smallmass and robust mechanical properties, such micromachinescan 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 bringsabout a different set of challenges. Although the manufactureof miniature-scaled neurosurgical devices is based on well-developed and reasonably mature fabrication technology, theactual performance of such microinstrumentation in vivo andtheir potential use in cellular and subcellular-scale neurosurgi-cal procedures have yet to be optimized and should representan important new direction in microsurgery.

690 | VOLUME 61 | NUMBER 4 | OCTOBER2007 www.neurosurgery-online.com

CHANG ET AL.


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Disclosure

David W. Sretavan, M.D., Ph.D., is a founder with a financial inter-est in a biotechnology company developing microdevices for applica-tions in microsurgery.

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the single cell. Curr Opin Cell Biol 19:95100, 2007.

3. Birgbauer E, Oster SF, Severin CG, Sretavan DW: Retinal axon growth conesrespond to EphB extracellular domains as inhibitory axon guidance cues.

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4. Bostock R, Collier J, Jansen R-JE, Jones R, Moore D, Townsend J: Siliconnitride microclips for the kinematic location of optic fibres in silicon V-shapedgrooves.J Micromech Microeng 8:343360, 1998.

5. Brewer GJ, Torricelli JR, Evege EK, Price PJ: Optimized survival of hippocam-pal neurons in B27-supplemented Neurobasal, a new serum-free mediumcombination.J Neurosci Res 35:567576, 1993.

6. Chang WC, Keller CG, Sretavan DW: Isolation of neuronal substructures andprecise 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 nanofiber scaffold for brain repair and axon

regeneration with functional return of vision. Proc Natl Acad Sci U S A103:50545059, 2006.

8. Goldshmit Y, Galea MP, Wise G, Bartlett PF, Turnley AM: Axonal regenerationand lack of astrocytic gliosis in EphA4-deficient mice. J Neurosci24:10,06410,073, 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 progresstoward the realization of nanoneurosurgery. Neurosurgery 58:10091026,

2006.13. Leary SP, Liu CY, Yu C, Apuzzo ML: Toward the emergence of nanoneuro-

surgery: Part Iprogress in nanoscience, nanotechnology, and the comprehen-

sion 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 IEEE92:4355, 2004.

17. Roy S, Ferrara LA, Fleischman AJ, Benzel EC: Microelectromechanical sys-

tems and neurosurgery: A new era in a new millennium. Neurosurgery49:779798, 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 Neurosci5:146156, 2004.

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

21. Suh LH, Oster SF, Soehrman SS, Grenningloh G, Sretavan DW: L1/Lamininmodulation of growth cone response to EphB triggers growth pauses and reg-

ulates the microtubule destabilizing protein SCG10.J Neurosci 24:19761986,2004.

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Langer R: Biocompatibility and biofouling of MEMS drug delivery devices.Biomaterials 24:19591967, 2003.

AcknowledgmentsThis research was supported by the Sandler Family Supporting Foundation

and the That Man May See Foundation. David W. Sretavan, M.D., Ph.D., is arecipient 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 Micro-fabrication Laboratory.

COMMENTS

Chang et al. have eloquently presented their work in a most excitingarea of medicine, the micro- and nanoworld as it pertains tomanipulation and alteration of neural structures. They have demon-strated that axon surgery is indeed possible. This seems farfetched butso have many advances in neurosurgery during their early formativeyears. The major obstacles to the advancement of this field will berelated to the development of surgical platforms and the performanceof multiple microtasks in rapid succession or simultaneously. The lat-ter is required to accomplish clinically relevant surgical procedures inmultiple axons in a timely manner. The work of Chang et al., however,suggests that such further refinements are not all that far off. Theauthors are to be commended for their vision, their forward-thinkingefforts, and their creativity.

Edward C. BenzelCleveland, Ohio

In this report, the authors describe the use of a microfabricatednanoknife with a cutting edge measuring 20 nanometers for use invivo. Previous work from the same group has reported the use of thesame nanoknife as applied in vitro for single axon surgery. This rep-resents an extension of the work to the in vivo environment forperipheral nerve surgery in a mouse model. The authors were able toconstruct a surgical setup such that the operative substrate wasimmobilized from the normal respiration and movements of themouse. The nanoknife was used to make progressive cuts in the sci-atic nerve, with electrophysiological recordings demonstrating the

progress dimunition of motor responses from the target muscle. Inaddition, single axon surgery was demonstrated in vivo. Finally, bio-compatibility of the device was also demonstrated by cell cultureexperiments. This is a highly important report in that it represents thefirst demonstration of subcellular surgery in vivo. Although it is notquite surgery on the nanoscale, this demonstration represents a majorreduction in the scale of surgery in a living animal. I look forward tofuture work from the authors with demonstration of the sequentialsteps of single axon repair.

Charles Y. LiuLos Angeles, California

This project uses a nanoblade to cut nervous tissue and axons. Theirdevice appears similar to single-cell extraction techniques inwhich a micropipette tip controlled by a robotic micromanipulator is

used to selectively aspirate axons, dendrites, or cell bodies from neu-rons in culture. As with previously published in vitro experiments (1),once an axon has been isolated, it can be precisely transected. Usinga mouse sciatic nerve, the authors had to first separate an axon fromthe nerve before cutting it on the platform. Therefore, in situ singleaxon transection has not been demonstrated thus far. Limitationsinclude a wide nanoblade and, perhaps, difficulty differentiating indi-vidual axons.




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The authors ultimate goal is to use this device to directly repairaxons. However, the current clinical applications of direct axon repairappear quite limited. For example, what is the time window for axonrepair before Wallerian degeneration? Additional pathophysiologicalstudies are needed to answer questions such as these. Regardingperipheral nerve repair, alignment of the basal lamina delineating theendoneurial tubes may be an alternative application that is more read-

ily applicable to current practice.Despite these concerns, the authors are commended for their for-ward-thinking concept. As their technology advances parallel to simul-taneous discoveries in neuronal protection, intraoperative imaging,and device production, potential nanotechnology applications in neu-rosurgery will surely appear over time.

Stephen M. RussellNew York, New York

1. Sretavan DW, Chang W, Hawkes E, Keller C, Kliot M: Microscale surgery on

single axons. Neurosurgery 57:635646, 2005.

W

ith the advent of the operating microscope and microsurgicaltechniques, Millesi (4) and Millesi et al. (5) improved clinical

results and popularized the use of nerve grafts for nerve repair. Todate, surgical techniques for nerve repair have been at the tissue orcable level. Even in a fascicular nerve repair, there are literally severalhundred to a few thousand axons in the proximal stump and a simi-lar number of endoneurial pathways in the distal stump (3). With themost meticulous repairs, the endoneurial tubes can never be reap-proximated exactly, and this results in mismatching of regeneratingaxons at the site of suture, or within the graft, leading to inappropri-ate (non-specific) and incomplete reinnervation and subsequent poorrecovery in function (1). Misdirection at the repair site is common,and precise reinnervation is only possible in clean and focal crushinjuries rather than ones in which the nerve is severed and thenrepaired with a suture (6).

Techniques to match regenerating axons with reciprocal and appro-priate targets in the distal nerve stump are currently only partially

possible with experimental techniques that maximize topographic

alignment (2). Further technological advances will require micro-surgery 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 per-form nerve and axon microsurgery. Essentially, the data demonstratethat precise and targeted severance of a portion of a mouse tibial nerveand individual axons from the same tibial nerve can be achieved using

the prototype device. It is notable that a significant engineering aspectis the development of the entire assembly for the micromanipulation,a microplatform in which the nerve is positioned, followed by theactual use of this cutting device. Each of these is a fundamental engi-neering issue that the investigators have solved admirably. Yet, thetranslation of this device from the type of proof of principle demon-strated herein to a practical tool for human nerve surgery remains chal-lenging.

A major shortcoming with even the most microscopic form of nerverepair is the biological constraint of cellular misalignment, which can-not be easily overcome by further progress in microsurgical techniques.The authors have provided the first glimpse of the technologies thatmay enable more precise surgery at the nanoscale level. I look forwardto further studies by these investigators as they endeavor to advancenerve repair into the cellular domain.

Rajiv MidhaCalgary, Canada

1. Brushart TM: The mechanical and humoral control of specificity in nerverepair, 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 periph-

eral 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 tech-

niques: A review. Neurol Med Chir (Tokyo) 40:187199, 2000.

4. Millesi H: Brachial plexus injuries. Nerve grafting. Clin Orthop Relat Res237:3642, 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.


CHANG ET AL.

Documents

Wesley C. Chang et al- In Vivo Use of a Nanoknife for Axon Microsurgery