Journal of Neuroscience Methods 336 (2020) 108634

Contents lists available at ScienceDirect

Journal of Neuroscience Methods
journal homepage: www.elsevier.com/locate/jneumethAcute in vivo testing of a polymer cuff electrode with integrated microfluidic
channels for stimulation, recording, and drug delivery on rat sciatic nerve

T

Sahar Elyahoodayana, Christopher Larsona, Angelica M. Coboa, Ellis Menga,b, Dong Songa,c,*a

Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA
Department of Electrical and Computer Engineering, University of Southern California, Los Angeles, CA, 90089, USAc
Neuroscience Graduate Program, University of Southern California, Los Angeles, CA, 90089, USAb

ARTICLE INFOABSTRACT

Keywords:
Electrophysiology
Peripheral nerve
Cuff electrode
Drug delivery

Background: Extraneural cuffs are among the least invasive peripheral nerve interfaces as they remain outside
the nerve. However, compared with more invasive interfaces, these electrodes may suffer from lower selectivity
and sensitivity since the targeted nerve fibers are more distanced from the electrodes.
New Method: A lyse-and-attract cuff electrode (LACE) was enabled by microfabrication and developed to improve selectivity and sensitivity while maintaining a cuff format. Its engineering design was described in previous work. LACE is a hybrid cuff that integrates both microelectrodes and microfluidic channels. The ultimate
goal is to increase fascicular selectivity and sensitivity by focal delivery via the microchannels of (1) lysing agent
to remove connective tissue separating electrodes from nerve fibers, and (2) neurotrophic factors to promoteaxonal sprouting of the exposed nerve fibers into microfluidic channels where electrodes are embedded. Here,
we focus on demonstrating in vivo function of microfluidics and microelectrodes in an acute preparation in which
we evaluate the ability to focally remove connective tissue and record and stimulate with microchannel-embedded microelectrodes neural activity in rat sciatic nerves.
Comparison with existing methods: While extraneural interfaces prioritize nerve health and intraneural interfaces
prioritize functionality, LACE represents a new extraneural approach which could potentially excel at both aims.
Results: Surgical implantation demonstrate preservation of LACE function following careful and minimal
handling. In vivo electrical evaluation demonstrates the ability of microelectrodes placed within microfluidic
channels to successfully stimulate and record compound action potentials from rat sciatic nerve. Furthermore,collagen-rich epineurium was focally removed following infusion of collagenase via microchannels and confirmed via microscopy.
Conclusion: The feasibility of using a cuff having integrated microelectrodes and microfluidics to stimulate,record, and deliver drug to focally lyse away the epineurium layer was demonstrated in acute experiments on rat
sciatic nerve.

1. Introduction
Peripheral nerve interfaces aim to provide therapies for various
diseases or restoration of lost functions through their interactions withthe peripheral nervous system. For example, electrical stimulation of
the peripheral nerve i.e. sciatic, ulnar, and occipital nerve have beenused to treat chronic pain (Sun and Morrell, 2014), (Picaza et al., 1977).
Therapeutic modulation of organ functions through vagus nerve stimulation (VNS) has demonstrated success in controlling blood pressure(Plachta et al., 2014), reducing body fat (Banni et al., 2012), suppressing epileptic seizures (Zabara, 1985), and treating arthritis (Giagka

and Serdijn, 2018), (Koopman et al., 2016). Furthermore, bidirectional
recording and stimulation from peripheral nerves has become an important approach for achieving closed-loop control of neural prostheses
such as robotic hands (Schultz and Kuiken, 2011)–(Tan et al., 2015) ora limb after spinal cord injury (Russell et al., 2019), (del Valle andNavarro, 2013). In all applications of peripheral nerve interfaces, selective recording and stimulation of nerve fibers within a nerve bundle
is highly desired so that targeted neural effects can be achieved while
undesired off-target effects are avoided. For example, motor control
requires recording and translating neural commands from efferent fibers within the nerve, while stimulating afferent fibers to send sensory

Corresponding author at: University of Southern California, 403 Hedco Neuroscience Building, Los Angeles, CA, 90089, USA.
E-mail addresses: elyahood@usc.edu (S. Elyahoodayan), larsonce@usc.edu (C. Larson), acobo001@gmail.com (A.M. Cobo), ellis.meng@usc.edu (E. Meng),
dsong@usc.edu (D. Song).
⁎

https://doi.org/10.1016/j.jneumeth.2020.108634
Received 3 December 2019; Received in revised form 11 February 2020; Accepted 11 February 2020
Available online 14 February 20200165-0270/ Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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S. Elyahoodayan, et al.

Fig. 1. Peripheral nerve interfaces can be used for a) closed-loop control of prostheses or limbs after spinal cord injury, and b) modulation of various organs.

attract axonal sprouting towards the electrodes within the channels.
Reflective of this design concept, the device has been termed the lyseand-attract cuff electrode (LACE) (Cobo et al., 2019).
The engineering design, fabrication, packaging, electrical characterization and fluidic performance of LACE on a benchtop set-up were
demonstrated in a previous study (Cobo et al., 2019). In this paper, theimplantation procedure and a systematic evaluation of the LACE’s
capability to acutely record, stimulate, and deliver lysing agent to rat
sciatic nerves in vivo is described. LACE was implanted in an acutepreparation. Then evoked neural response to electrical stimulation withvarying magnitudes were recorded, validated, and characterized. Next,
nerve stimulation with electrochemically reversable stimulation pulses
to evoke muscle activation was performed. Finally, collagen fibers of
the epineurium layer were focally removed by delivery of collagenase
via the microfluidic channels. The acute effects of focal delivery were
evaluated using histology and label-free two-photon imaging and theseresults correlated to confirm focal lysing of the epineurium on thenerve’s surface. The findings indicate the electrodes’ recording andstimulation capabilities from within the microfluidic channels. They
further demonstrate the capability of LACE to perform chemical manipulation of the electrode-tissue interface.

feedback to the user. Furthermore, organ modulation via nerve stimulation, as in bioelectronic medicine, requires selective targeting of thespecific fibers within a nerve trunk that innervate the organ of interest(Fig. 1).
Extraneural cuff electrodes provide a minimally invasive method tointerface with the peripheral nerve as they wrap gently around thenerve and stimulate or record from multiple nerve fibers with non-penetrating surface electrodes. They enclose the circumference of thenerve with an insulative material to restrict ionic currents. Metal
electrodes on the inner wall of the cuff enable stimulation and recording of the encircled nerve fibers (Grill and Mortimer, 1996), (Marksand Loeb, 1976). More recent cuff electrodes take advantage of microfabrication techniques and are made from thin-film polymers andmetals. They consist of insulated thin-film metal traces, exposed electrode sites and integrated interconnects that allow external connection(Caravaca et al., 2017), (Rodríguez et al., 2000). Ideally, the cuff diameter can be adjustable to fit nerves of varying sizes, thereby allowing
surgical flexibility and better integration of the cuff with the nerve(Loeb and Peck, 1996), (Elyahoodayan et al., 2018).
Cuff electrodes induce minimal immune response when properly
installed (Navarro et al., 2005). However, electrodes on the surface ofthe cuff are separated from nerve fibers by the epineurium and perineurium and therefore suffer from lack of selectivity and sensitivity(Loeb and Peck, 1996), (Kang et al., 2015), (Hoffer and Kallesøe, 2001).
A variety of strategies have been investigated to improve selectivity andsensitivity (Larson and Meng, 2019), including extraneural cuffs which
reshape the nerve (Leventhal and Durand, 2003), (Schiefer et al., 2010),(Leventhal et al., 2006), (Caparso et al., 2009) and intrafascicularelectrodes which penetrate the nerve trunk (Lawrence et al., 2002),(Badia et al., 2011). Such approaches have varying degrees of success in
improving device functionality while maintaining long-term performance and nerve health. The latter penetrating approach is highly invasive and breaches the integrity of the nerve. To our knowledge, nonsurgical methods to focally remove epineurium and perineurium havenot been explored in conjunction with cuff electrodes.
To provide a means to evaluate such an approach towards achieving
fascicular selectivity and sensitivity, we developed a novel hybrid,
multi-functional cuff electrode (Cobo et al., 2019). The design consistsof a microfabricated thin-film polymer cuff with electrodes for recording and stimulation, and microfluidic channels to deliver drugs
focally. The microfluidic channels terminate in a well-defined outlet
and allow focal drug delivery of a chemical lysing agent to non-surgically remove connective tissue and thereby minimize the distance between the electrodes and nerve fibers without introducing mechanicaltrauma to the nerve. The resulting “window” provides greater access tothe underlying nerve fibers for electrodes on the cuff. This could possibly be enhanced further by delivery of neurotrophic factors in order to

2. Experimental methods
2.1. Design and fabrication
LACE (Fig. 2) is a flexible thin-film polymer device featuring metalelectrodes for recording and stimulation, microfluidic channels for drugdelivery, and an adjustable locking mechanism for precise fitting

Fig. 2. Fabricated Parylene C cuff electrode with integrated microfluidic
channels. A red photoresist layer is shown in the channels for visibility.
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Fig. 3. Magnified view of the microfluidic
outlets and the electrode arrangement (left)
and illustrated cross section of one channel andits electrodes (right). Electrodes E1, E3, E5,
and E7 are partially occluded by the microfluidic channels with the outlets directly above
the electrodes. Electrodes E2, E4, E6, and E8
are fully occluded by the channel (not used inexperiments described in this paper). The surface electrode (ES) is fully exposed.

around the nerve. It is fabricated from Parylene C using surface micromachining techniques on a temporary silicon wafer carrier. Four
microfluidic channels are spaced 350 μm apart on the circumference ofthe nerve, each with a 20 μm H × 250 μm W cross-section and 200 μmdiameter outlets. The electrode layout is shown in Fig. 3. Eight metalelectrodes (300 μm × 1500 μm) made from 200 nm-thick platinum arearranged as a bipolar pair inside each microfluidic channel. One electrode of each pair is directly beneath the microfluidic outlet while thesecond is approximately 4 mm away from the outlet. A ninth electrodeof the same dimensions is located outside the channels (the surface
electrode). The locking mechanism consists of a tab which is led bysuture through a slit which catches on a set of ratchet-like teeth. The
series of locking teeth allow for adjustable sizing to fit nerves of
1.1–1.5 mm diameter in 0.1 mm increments.

2.3. Recording set-up
The connection scheme for the electrophysiological recording setup
is illustrated in Fig. 5a. Evoked compound action potentials (CAP), asummation of action potentials generated by multiple fibers, were recorded differentially between the surface electrode and E7. A large
platinum wire was placed on the exposed muscle of the animal near theincision as ground. The recording amplifier (A–M systems, model 1700)
was set to 80 dB gain with a 10 Hz – 10 kHz band pass filter. All signals
were digitized and recorded with a recording system (Digidata 1322A,
Molecular Devices) and data were saved with pClamps9 software(Molecular Devices) using a 100 kHz sampling frequency.
Bipolar needle electrodes were inserted into the nerve 20 mm fromthe recording site and were connected to an external stimulator(Multichannel Systems STG4000). Biphasic, cathodic-first pulses with afixed duration of 200 μs (Cogan, 2008), and amplitudes of 100–700 μA
in steps of 50 μA were delivered.
After normal CAPs were recorded, a negative control experiment
was performed to verify the neural nature of the recorded signals.
Lidocaine was applied to the sciatic nerve distal from the stimulationsite. Lidocaine is a local anesthetic that prevents the generation andconduction of action potentials in a nerve by binding to and blocking
fast inactivation voltage-gated Na + channels (Vedantham and Cannon,
1999). Therefore, application of lidocaine would abolish neural activity
to demonstrate that the recorded data was indeed neural activity, notnoise or artifact from stimulation.
The nerve was then stimulated while the leg muscle was observed
for twitching. Twenty minutes after application of lidocaine, no muscle
twitch in response to nerve stimulation was observed. Nerve recordings
were then performed with the same stimulation paradigm using LACE
(Fig. 5b). Subsequently, while nerve conduction was still blocked bylidocaine, the leg muscle was stimulated directly using needle electrodes, producing a twitch. While directly stimulating the muscle, recording from the nerve was performed to verify whether EMG activity
contaminated the previous CAP recordings (Fig. 5c).

2.2. Surgical technique
Surgery was performed on male Wistar rats (>9 weeks old,
300−400 g). All animal experiments were approved by theInstitutional Animal Care and Use Committee (IACUC) and theDepartment of Animal Resources of the University of Sothern California(DAR, USC). Animals were anesthetized with 0.2 mL/100 g of ketamineand xylazine. Anesthesia was maintained throughout the surgical procedure by inhalation of a mixture of oxygen and isoflurane.
A midsagittal incision was made at the right thigh. Junction between muscles was identified and pulled apart by blunt dissection. The
sciatic nerve was exposed; blood was rinsed off using 1× phosphatebuffered saline (PBS). LACE was then implanted around the right sciatic
nerve with a 4.0 suture needle. The suture was knotted at one end andthe needle was threaded through a hole in the electrode tab (Fig. 4a).
After LACE was guided under and then around the nerve, the sutureneedle was passed through the first slit and locked in place by pullingon the suture at one end and the FFC cable connected to the electrode at
the opposite end (Fig. 4b). This technique allows for easy implantation
with minimal to no damage to the electrode or tissue.

Fig. 4. a) LACE threaded underneath sciatic nerve. b) LACE locked around the sciatic nerve.
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Fig. 5. Acute in vivo recording setup is shown on the right of eachpanel, and the corresponding recordings from LACE are shown on
the left. a) LACE was used to record neural activities from thenerve. E7 was used as the working electrode and SE was used as
the reference electrode. Stimulation pulses were applied to thenerve 20 mm from the recording site using needle electrodes. The
stimulation pulses were charge-balanced anodic-first biphasic
pulses with a fixed duration of 200 μs, and pulse amplitudes
ranging from 100 μA to 700 μA in steps of 50 μA. The recording
amplifier was set to a gain of 80 dB, with 10 Hz-10 kHz band passfilter. b) Lidocaine was applied to the nerve distal from the stimulation electrodes and (a) was repeated. c) Lidocaine was again
applied to the nerve, the leg muscle was stimulated at 700 μA, andactivity was recorded from the nerve using LACE. d) CAPs elicitedwith stimulations of varying amplitude were recorded using LACE.
e) and f) are verification of recorded nerve response. e)
Application of lidocaine to the nerve abolished neural responses
while stimulation artifacts were unchanged. f) EMG artifacts areabsent during direct muscle stimulation.

2.4. Stimulation set-up

tissue anatomy (Vijayaraghavan et al., 2014). Collagen is a noncentrosymmetric molecule emitting exactly half of the wavelength ofthe excitation wavelength (Zoumi et al., 2002), (Campagnola et al.,2002) and provides sufficient signal for SHG imaging. Since the peripheral nerve is rich in collagen, SHG is a suitable technique to image
collagen fibrils at high resolution (Sinclair et al., 2012).
The tissue was whole mounted in 1% clear agarose gel. SHG imaging was carried out on a commercial scanning Zeiss LSM510NLO twophoton microscope. The excitation wavelength was set to 900 nm and
SHG signal was collected using a 420−460 nm bandpass filter. All
images were acquired with a 25x/1.05 numerical aperture water immersion lens. The nerve was tile-scanned with a field of view of
600 × 600 μm. The axial and lateral resolution was set to 10 μm.
After 2 P-SHG imaging, agarose was removed and the tissue wasmounted in optimal cutting temperature (OCT) compound and frozen at−80 °C. It was then sectioned at 30 μm on a cryostat and treated by H&
E staining to observe the quality and location of lysing activity. Stained
samples were imaged using a DM2500 microscope and a DFC450 digital
camera (Leica Microsystem, Cambridge, UK). The lysed regions were
measured by Leica Application Suite Version 4.

Fig. 7a illustrates the stimulation set-up. The rat sciatic nerve iscomposed of tibial and peroneal divisions which innervate the thigh,leg and foot muscle. For stimulation testing, the stimulus was applied to
E5 as the working electrode and surface electrode as the return. Motor
responses were monitored from compound muscle potentials of theextensor digitorum longus muscle recorded by bipolar needle electrodes. The bipolar recording electrodes were positioned to record
differentially along the longitudinal direction of the muscle fibers.
Again, a negative control was performed by applying lidocaine to thenerve and recording responses from the same muscle (Fig. 7b).
2.5. Drug delivery
Before implantation, the microfluidic channels were primed withcollagenase as described previously (Cobo et al., 2019). Once LACE was
implanted, collagenase was infused at 0.1 μl/min for 10 min. This flowrate was previously determined by benchtop experiments with dye on a
nerve phantom, which resulted in localized delivery with no diffusion(Cobo et al., 2019). The reaction time of collagenase to lyse the epineurium is approximately one hour (Rydevik et al., 1985). Thus, onehour after delivery, the nerve was excised, and the rat was euthanizedwith compressed carbon dioxide gas. The tissue was fixed by immersion
in 10 % formalin solution for an hour in 4 °C to preserve morphology. It
was then transferred to 30 % sucrose solution to dehydrate it overnight.
Two-photon (2 P) second harmonic generation (SHG) microscopy wasused to examine the nerve structure. SHG does not require labeling withmolecular probes and is thus a non-destructive technique to image

3. Results
3.1. Fabrication results
The electrodes were fabricated, packaged, and characterized as
described in (Cobo et al., 2019). Benchtop tests verified function of the
microfluidic channels and locking mechanism. Cyclic voltammetry (CV)was performed in 0.05 M H2SO4 to clean the electrodes of any residues
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Rectified and Integrated CAP

that may remain after fabrication. CV and electrochemical impedance
spectroscopy (EIS) were then performed in phosphate buffered saline(PBS) to assess stimulation characteristics. From integration of the CVwith a scan rate of 250 mV/s, cathodal charge storage capacity of theplatinum electrodes was 1.1 ± 0.1 mC/cm2 (mean ± SE, n = 4) and
impedance at 1 kHz was 1.8 ± 0.11kΩ (n = 6). Devices were disinfected by soaking for 15 min in isopropanol.
3.2. Acute recording test

1
0.5
0

-0.5

Evoked CAP responses were recorded from rat sciatic nerves using
LACE (n = 3). Fig. 5d shows a representative example of overlaid CAP
responses elicited by stimuli with varying amplitudes. The CAP responses increase in magnitude with the stimulus magnitude, indicatingthat stronger stimuli recruit more nerve fibers which constructively add
to produce a larger CAP response at the recording site. Since the recording is bipolar, there are two phases to the CAP activity: a positive
followed by a negative phase. This arises from the connection scheme,
having the recording electrode proximal to the stimulation electrodeconnected to the non-inverting input of the differential amplifier andthe distal electrode connected to the inverting input. The temporaldispersion of the CAP waveform is due to variability in conduction
velocity across nerve fibers (Liang et al., 1991), (Maynard et al., 1997).
Larger myelinated axons have faster conduction velocity due to smaller
axial resistance. Also, large diameter axons have lower threshold to
extracellular current due to smaller axial resistance, allowing more
efficient passage of longitudinal extracellular current (Lloyd et al.,1946), (Huxley and Stämpeli, 1949).
Fig. 5e verifies the neural nature of the recorded signals. After application of lidocaine, elimination of responses following stimulation
confirmed that the recorded signals shown in Fig. 8a were indeed
neural. Furthermore, the leg muscle was stimulated with a needle
electrode to evoke EMG activity. Fig. 5f demonstrates that, after application of lidocaine to the nerve proximal from the neuromuscularjunction, only stimulation artifacts
were recorded by LACE, while no response was observed. This result
further verifies that the signals shown in Fig. 8a were not EMG or
contaminated by EMG.
Each neural response to its respective stimulus was rectified andintegrated. The mean over three trials for each response was computed
and the obtained values were normalized by the maxima and plottedversus the stimulus magnitude (Fig. 6a). Here, the threshold stimulation
current for eliciting CAP was 100 μA. The plot demonstrates a steep
slope for stimuli below 200 μA and a very shallow slope for stimuli
greater than 200 μA suggesting gradual saturation of number of recruited fibers above 200 μA.
Fig. 6b illustrates the latency-to-onset of CAP versus the stimulation
amplitude. Since large diameter fibers are recruited first and have fasterconduction velocity than small diameter fibers, this plot is relatively
flat. At a distance of 20 mm between stimulation and recording electrodes, the latencies provide an approximation of conduction velocitiesof 16−21 m/s. Conduction velocities are consistent with values previously reported in rat sciatic nerve experiments (Yu et al., 2014), (Xueet al., 2015).

a

1.5

0

100

200

300

400

500

600

700

Latency to Onset (ms)

Stimulus Intensity (µA)

b

1.4
1.2
1
0.8

0

100

200

300

400

500

600

700

Stimulus Intensity (µA)
Fig. 6. Recruitment curve of sciatic nerve fibers. Error bars are standard error
mean values from three rats. a) Rectified and integrated values of each CAP
activity following stimulation. The integrated CAPs are normalized by the
maxima and plotted versus the stimulus intensity. b) Latency to onset versus thestimulus intensity.

As before, Fig. 7d is a verification of recorded neural activities. Afterapplication of lidocaine, the absence of any activity following stimulation confirms that the recorded signals are biological.
3.4. Drug delivery
A rendering of LACE installed on a nerve is illustrated in Fig. 8 a.
Histological results demonstrated effects of collagenase treatment on
the morphology of sciatic nerves. Tissue closest to the microfluidic
channel openings demonstrated obvious disruption of epineurium(Fig. 8b) whereas tissue further away from the microfluidic channel
openings was unaffected (Fig. 8C). The lysed region was ∼300 μm ofthe nerve’s perimeter, which is comparable to the diameter of the microfluidic channel outlet (200 μm). This result suggests that the chosen
flow rate and duration produced a focal distribution of collagenase
effects on the surface of the nerve with ∼50 μm diffusion beyond theedge of the outlet.
Tile-scanned SHG images were acquired using a 900 nm excitation
wavelength. The acquired scans were then z-stacked in Imaris image
analysis software to construct a 3D rendering (Fig. 9a). The green regions indicate emission from collagen fibers, while diminished signal isrepresentative of collagen absence. A dark circular region with a diameter of 290 nm, enlarged in Fig. 9b, showed a clear, focal removal ofcollagen. Again, this shape and dimension is comparable to the outlet of
the LACE microfluidic channel (Fig. 9c) and agrees with the lysed region found in transverse histological sections.

3.3. Acute stimulation test
The stimulation tests after acute implantation verified that LACE
can deliver enough charge to the nerve to cause muscle contractions.
Fig. 7 c shows an example of recordings from extensor digitorum longus
muscle with increasing stimulation intensity. The recording is frommultiple motor unit action potentials, adding up temporally and spatially to generate complex waveforms. The maximum stimulus applied
across the electrode is 0.04 μC. This is well below the charge storage
capacitance of LACE at ∼4.5 μC, ensuring delivery of electrochemicallyreversable stimulation pulses.

4. Discussion
In this study, we developed the surgical technique to implant LACE
in rat sciatic nerves with minimal handling of the device and the tissue.
The adjustable locking mechanism allowed for simple adjustment of the
LACE around nerves of various diameters. After implantation, device
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Fig. 7. Acute in vivo stimulation setup is shownon the right panel, and corresponding recorded
evoked EMG shown on the left panel. a) E5 wasused as the working stimulation electrode andSE was used as the reference. Anodic-first stimulation pulses set to a fixed pulse duration of
200 μs and amplitudes ranging from 40 μA to
200 μA were applied. EMG was recorded bipolarly using needle electrodes in the extensor
digitorum longus muscle in the rat’s right hindleg. The recording amplifier was set to a gain of
60 dB with 10 Hz-10 kHz band pass filter. b)
Lidocaine was applied proximal from the neuromuscular junction and (a) was repeated. c)
EMG activity recorded following nerve stimulation. d) Verification of the recorded muscle
response. Application of lidocaine to the nerve
abolished EMG response while the stimulus
artifact was unchanged.

integrity and function were maintained. This enabled demonstration of
LACE’s capability to record neural activity from rat sciatic nerves, andto deliver charge to stimulate the nerve and evoke muscle response. The

recorded signals were confirmed to have neural origin by comparing
evoked potentials before and after application of lidocaine to the nerve.
Leg muscle innervated by the implanted nerve was also stimulated toFig. 8. Demonstration of the effect of lysing agent on exterior connective tissue of the nerve. a) Rendering of the LACE installed on the nerve. b) Histology slides after
delivery of collagenase show that it effectively disrupted the epineurium near the microchannel outlet. c) Away from the outlets, the epineurium was intact.
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demonstrated in both sensory and motor fibers (Šámal et al., 2006),(Liu et al., 2014). In contrast to these microsurgical techniques, LACE
has the advantageous capability of enzymatically removing the connective tissue, thereby reducing the risk of surgical nerve injury(Rydevik et al., 1985). The long term response of the nerve to collagenase and the ability to induce axonal sprouting remain to be investigated.
The main objective of this study was to test LACE and its feasibility
to perform acute in vivo stimulation, recording and drug delivery on rat
sciatic nerve. This study is the first demonstration of the LACE’s potential to offer a new, drug delivery-based extraneural approach toperipheral nerve interfacing for developing hybrid bionic systems andbioelectronic medicines.
In future studies, we will design packaging for chronic LACE implantation including electrical and drug delivery components. We willthen perform functional testing of LACE in chronic implantation conditions including nerve health and stability of the interface after delivery of collagenase to elucidate possible level of axonal inflammationand fibrosis. We will also evaluate the electrodes for cross-talk and
impedance spectra to quantify leakage current, electrode performance,
and wiring integrity for chronic electrophysiological recording andelectrical stimulation experiments. We expect that these results will be
one step closer to offering a solution for selective nerve stimulation andrecording for biomedicine application.
Acknowledgements
This work was sponsored by the Defense Advanced Research
Projects Agency (DARPA) BTO under the auspices of Drs. Doug Weberand Eric Van Gieson through the DARPA Contracts Management Office
Cooperative Agreement HR0011-15-2-0006. C.L. was partially supported by the University of Southern California Provost Fellowship. The
authors would like to thank Dr. Victor Pikov (Medipace), Mr. Jason
Miranda (Galvani), and Dr. Srikanth Vasudevan (FDA) for their guidance, and Dr. Thai Truong, Peter Luu and the USC Translational
Imaging Center for their imaging assistance.

Fig. 9. a) 2 P-SHG images of whole mount nerve tissue with excitation wavelength of 900 nm. b) A circular dark region, magnified, indicates absence ofcollagen fibers. Comparison of the dimensions of the dark region (290 μm
diameter) with c) the microchannel outlet (200 μm diameter) demonstrates
focal delivery.

confirm absence of EMG artifact in the recorded nerve signals. The
bipolar recording configuration, using an electrode partially inside themicrofluidic channel and a surface electrode, obtained neural activities
in response to increasing stimulation pulses similar to recordings previously reported using the same set-up (Yu et al., 2014), (Xue et al.,2015). Furthermore, stimulation of the nerve through LACE was successfully demonstrated in muscle activity recorded with needle electrodes in the rat hind leg.
Although cuff electrodes have been used successfully in animals andhumans (Graczyk et al., 2016), (Guiraud et al., 2016), there has been
limited success in recording nerve fibers with high sensitivity and stimulating them with high selectivity. Some peripheral interfaces achieve
higher acute performance by sacrificing the long-term health of thenerve. Microscale drug delivery, as demonstrated using the LACE, may
offer an alternative strategy. We demonstrated the capability of LACE todeliver controlled boluses of drug focally to biochemically manipulate
the electrode-tissue interface. Histological analysis and 2 P-SHG imaging suggest that collagenase lysed the epineurium layer focally withminimal diffusion from the microfluidic outlet. This is in comparison toprevious work (Rydevik et al., 1985), where an uncontrolled large bolusof 1 ml of collagenase was applied around the tibial nerve to study theeffect of collagenase on nerve tissue.
Following the focal lysing of epineurium which has now been demonstrated, LACE provides an ideal setup to potentially take advantageof the phenomenon of collateral sprouting of nerve fibers. Applicationof neurotrophic factors, such as NGF and methylcobalamin, has beenshown to enhance collateral sprouting from an intact nerve (McCallister
et al., 2001). Collateral sprouting of nerve fibers following microsurgical removal of the epineurium layer from the nerve has been

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