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Sensors and Materials, Vol. 29, No. 12 (2017) 1689–1702
MYU Tokyo
S & M 1462

Long-Term Analysis of In Vivo Characteristics
of Recording Electrode
Using Electrochemical Impedance Spectroscopy
Mariko Kuwabara,1* Hiroyuki Tashiro,2 Yasuo Terasawa,1,3 Yukari Nakano,1,3
Yurina Yoshimura,4 Koji Osawa,5 Toshihiko Noda,1 Takashi Tokuda,1 and Jun Ohta1
1

Graduate School of Materials Science, Nara Institute of Science & Technology
8916-5 Takayama-cho, Ikoma, Nara 630-0192, Japan2
Department of Health Sciences, Faculty of Medical Sciences, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
3
Artificial Vision Institute, Research and Development Division, Nidek Co., Ltd.
13-2 Hama-cho, Gamagori, Aichi 443-0036, Japan
4
Department of Health Sciences, Graduate School of Medical Sciences, Kyushu University
3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
5
Artificial Retina Development Office, Development Division, Nidek Co., Ltd.
34-14 Maehama Hiroishi-cho, Gamagori, Aichi 443-0038, Japan(Received May 1, 2017; accepted September 14, 2017)

Keywords: electrochemical impedance spectroscopy (EIS), equivalent circuit, emulation, visual evoked
potential (VEP), long-term recording

Implanted electrodes are widely used for in vitro and in vivo neural recording; however, theestablished properties of these electrodes can change over time. Here, we characterize the longterm changes in the electrode surface during in vivo recording and investigate the effect of thesechanges on visual evoked potentials (VEPs) using electrochemical impedance spectroscopy (EIS)
spectra. VEP emulation was simulated on the basis of EIS measurements to identify the factorsthat contribute to time-dependent changes in the VEP. The VEP measurements revealed a timedependent decrease in the latency for the first negative peak, while the amplitude was stable. EIS
measurements revealed a stable impedance. An equivalent circuit analysis confirmed a timedependent increase in solution resistance. However, VEP emulation indicated that the VEPchanges were not related to changes in the recording electrode properties, but rather, to changes
in vital reactions. Our results suggest that time-dependent VEP measurement requires EIS andemulation analyses to identify changes in the characteristics of the interface between the recording
electrode surface and the surrounding tissue and the effects of these changes on electrical
recordings.

1.

Introduction

Microelectrode arrays (MEAs) and implanted electrodes are routinely used for in vitro and invivo recording of neuronal electric potentials.(1,2) Notably, implanted electrodes are also used intherapeutic devices such as cochlear implants and deep brain stimulators for both nerve stimulationand nerve response recording.(3,4) Electrode impedance can affect the recorded nerve activity*

Corresponding author: e-mail: kuwabara.mariko.kd8@ms.naist.jp
http://dx.doi.org/10.18494/SAM.2017.1635
ISSN 0914-4935 © MYU K.K.

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Sensors and Materials, Vol. 29, No. 12 (2017)

wave pattern; thus, the development of such devices and treatment strategies must consider suchelectrode properties.(5)The development of novel implantable neural devices and treatment strategies can also involvethe use of in vivo implanted electrodes for chronic recordings. We developed a prosthetic to restore
lost vision through the electrical stimulation of the remaining optic nerve in patients with blindness
owing to retinitis pigmentosa or age-related macular degeneration.(6) To evaluate the safety of ourdevice, brain-implanted recording electrodes were used for the chronic recording of visual evoked
potentials (VEPs).(7) The assessment of chronic VEP recordings can also be applied to the practicalresearch of ophthalmological medicines, such as the protective medication of the optic nerve.(8)Other studies involving evoked potential recordings include that by Wang et al. who chronicallyrecorded evoked potentials using an MEA.(9) Notably, Guarino et al. reported that anesthesia
conditions and the recording electrode position can affect the recording of evoked potentials,(10)and similarly, Charng et al. reported the effects of anesthesia on wireless VEP measurement.(11)A time-dependent and functional evaluation of the optic nerve using chronic VEP measurement
requires an understanding of the factors that affect the VEP. During chronic measurement of
evoked potentials under a fixed anesthetic depth, the acquired waveform may change in response
to several factors. For example, such variations may be related to changes in not only stimulation
parameters, but also vital reactions, recording electrode properties, and the surrounding tissue
environment of the recording electrode.(2)To accurately evaluate time-dependent functional changes of the optic nerve, we developed
recording electrodes that minimize brain tissue damage,(7) and successfully obtained chronic
VEP recordings using our electrodes. However, we observed a time-dependent decrease in thefirst negative wave (N1) latency of the VEP.(7) Identifying the causes of VEP changes using
only an evoked potential analysis can lead to false positive results. To identify changes in thecharacteristics of the interface between the electrode and the surrounding tissue, an analysis of thetime-dependent recording electrode characteristics using electrochemical impedance spectroscopy(EIS) may be useful.(12) EIS is a nonsteady assay for determining electrode reaction mechanisms
using a frequency response analysis that involves sine wave potential. With this analysis, thefrequency response of the electrode impedance is expressed in a Bode plot or Cole–Cole plot.
Bode plots can be used to express the magnitude of the impedance and phase shift against thelogarithm of the frequency. Cole–Cole plots can be used to present the locus of the impedance
response against each frequency in the complex plane.(13) Nonetheless, these plots are difficult to
use to determine the behavior of the electrode.
Equivalent circuit analyses using minimal simplified elements are useful for expressing
electrode behavior. Equivalent circuit analysis yields parameters that reflect physical activity,
and thus are useful for EIS interpretation. In a previous study, an impedance of 1 kHz was used
to evaluate chronic changes in the properties of a recording electrode.(14) In this study, we foundthat electrode frequency impedance is related to the capacitive property of the electrode, but not
to other characteristics, such as the reactivity of the electrode or the solution resistance.(5) Various
parameters of equivalent circuit analysis can be used to understand the surface interface behaviorof an electrode.(15) Equivalent circuit analysis has been applied in several in vivo and in vitro
studies.(16–18) For example, short-term changes in the properties of in vivo recording electrodes
have been reported.(5) However, long-term changes in the properties of in vivo recording electrodes
are unknown. In addition, modulation of the evoked potentials due to such changes in theSensors and Materials, Vol. 29, No. 12 (2017)

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properties of the recording electrode surface interface has not been investigated using equivalentcircuit analyses. As aforementioned, such time-dependent changes in chronically recorded evoked
potentials may occur in response to physiological stimulation or recording electrode characteristics.
Electrical circuit models for cortical recordings have been proposed.(19,20) Emulation
analyses of such models and simulation of neuron activity well describe potential waveforms andamplitudes.(21) A simple method can also be used to simulate electroencephalography (EEG)
data.(22,23) Although the effect of a change in electrode interface activity on time-dependent
evoked potentials is unknown, these methods for emulation and simulation may validate models
that predict a relationship between the evoked potentials and the environment surrounding therecording electrode. Although chronic evoked potentials have been recorded using the implanted
electrodes,(9–11) there are no reports using chronic VEP recordings and EIS measurement,
equivalent circuit analysis, and VEP emulation in a single study.
In this study, we investigate whether the changes in the VEP result are related to the invivo electrode properties, the tissue environment surrounding the recording electrode, or otherfactors. To investigate the recording electrode properties, we used EIS. Clarifying the cause of
VEP variation will allow for a distinction between changes in the VEP, which result from thedegeneration of either the optic nerve or the recording electrode.

2.

Materials and Methods

2.1

Electrode design

We developed recording electrodes that were designed to minimize mechanical stress to brain
tissue.(7) The electrodes were implanted into three Dutch rabbits (weight 2.6 kg, age 19 weeks),
hereafter referred to as ‘rabbit A’, ‘rabbit B’, and ‘rabbit C’. Platinum (Pt) ball-tip screw electrodes
were implanted into rabbit A and rabbit B. The Pt ball-tip screw electrode had the structure of aplastic screw and was composed of polymethylmethacrylate with Pt balls on the tip [Fig. 1(a)].(7)Smooth-surface Pt balls were constructed of Pt wire (351265, Nilaco, Tokyo, Japan). The lengthof the electrode was 17.5 mm, the diameter of the screw was 2.6 mm, and the diameter of thePt balls was 0.7 mm. The threaded insert made of stainless steel (Ensat 302000025.500, KKV,
Amberg, Germany) was used to adjust the insertion depth of the electrode into the brain to enable
the optimization of the VEP. Hereafter, the platinum ball-tip screw electrode is referred to as therecording SE. The platinum/iridium (Pt/Ir) ball-tip planar MEA that was implanted into rabbit C
had a sheet structure with Pt/Ir ball electrodes arranged in a 3 × 3 lattice pattern [Fig. 1(b)].(7) The
MEA was composed of a Pt/Ir wire (9 twist lines; 967223, Nilaco), silicone sheets (0.1 t and 0.2 t,
Unique Medical, Tokyo, Japan), a silicone tube (986902, AS ONE, Tokyo, Japan), silicone adhesive(KE-41-T, Shin-Etsu Chemical, Tokyo, Japan), and a connector (HR25-9TR-12P, Hirose Electric,
Tokyo, Japan). The sheet size was approximately 8.3 × 12 mm2. The diameter of the Pt/Ir ball
was approximately 0.7 mm. Hereafter, the Pt/Ir ball-tip planar MEA is referred to as the recording
MEA. The recording SEs were implanted 6 mm anterior and 6 mm lateral to the lambda suture.
The recording MEA was inserted under the right skull. Indifferent electrodes (M2 stainlesssteel anchor screw AM2-8, Unique Medical) were placed 14 mm anterior to the lambda suture.
Additional details of the recording electrodes have been published.(7)1692

Sensors and Materials, Vol. 29, No. 12 (2017)

silicone tube

Pt ball-tip
screw electrodesilicone adhesive

17.5 mmthreaded
insertskull

silicone sheet

Pt/Ir balls

connector

0.7 mm

8.3 mmPt balls
dura
mater
0.7 mm
26 mm

(a)

12 mm

(b)

Fig. 1. Schematic view of the recording electrodes. (a) Pt ball-tip screw electrode. This electrode was fabricatedfrom a plastic screw made of polymethylmethacrylate and Pt balls on the tip. The insertion depth of electrode into
the brain was adjusted using the threaded insert to optimize the VEP recording. (b) Pt/Ir ball-tip planar MEA. This
electrode had a silicone sheet structure with Pt/Ir ball electrodes arranged in a 3 × 3 lattice pattern.

2.2

VEP recording

Each rabbit was fasted for 2 h before beginning the experiment. A rabbit was then placed in arecovery box that was covered with black-out curtains and connected to an anesthesia apparatus(NS-3000, Acoma Medical, Tokyo, Japan). Next, oxygen at a flow rate of 4 L/min was introduced
into the recovery box. The laboratory lights were turned off, and the rabbit was adapted tothe dark for 15 min. Subsequently, vaporized isoflurane gas (Forane, AbbVie, North Chicago,USA) was introduced into the recovery box until a 2.5% concentration was reached in the box
while confirming with a medical monitor (BP-608EV, Nihon Colin, Tokyo, Japan). Once a
sufficient anesthetic depth was reached, the anesthesia was switched to mask anesthesia at a 2.4%
anesthetic concentration with an oxygen flow rate of 1 L/min. The pupil of the stimulated eye wasdilated using tropicamide (Mydrin-P, Santen, Osaka, Japan). Local anesthesia (oxybuprocainehydrochloride; Benoxil, Santen) and a drying agent (Scopisol, Senju, Osaka, Japan) were thenapplied. The stimulated eye was opened using an eye-opening device 5 min after applying
local anesthesia. The contralateral eye was covered with gauze after the application of a drying
agent to prevent dryness. The luminance of the room was maintained at 0.2–0.3 lx. During theexperiment, the anesthetic depth was controlled by adjusting the 2.4% end tidal concentration of
the isoflurane gas based on the medical monitor information. The experiment began after theisoflurane concentration of the expired gas was stable for 5 min. Light stimuli with a 20 J lightintensity and 3 s repetition period from a flash stimulator (SLS-3100, Nihon Kohden, Tokyo,
Japan) were adapted at a distance of 30 cm from the eyes. The actual measured values of the
flash stimuli were as follows: 3.74 cd·s/cm2 for the time-integrated luminance, 29.5 cd/cm2 for the
peak luminance, and 75 ms for the half-value width with respect to the peak luminance. Evoked
potentials were recorded using a biological signal recording analysis system (PowerLab/8 SP,1693

Sensors and Materials, Vol. 29, No. 12 (2017)

ML135, AD Instrument, Sydney, Australia). Recording was carried out at frequencies of 1–100
Hz and 32 counts on average. The latency and amplitude measured using the peak-to-troughmethod for N1 were evaluated.(24) VEPs were recorded over a 32-week period after the electrodeimplantation.
2.3

EIS analysis

The EIS analysis was performed on rabbits A and B in which the recording SE was implanted.
The EIS spectra using the three-electrode method were measured using a potentiostat equippedwith a frequency response analyzer (FRA) (Autolab PGSTAT32, Metrohm, Herisau, Switzerland).
The working electrode (WE) terminal was connected to the Pt-constructed recording SE, thecounter electrode (CE) terminal was connected to the stainless steel-constructed indifferent
electrode, and the reference electrode (RE) terminal was connected to the silver/silver chloride (Ag/
AgCl)-constructed ear clip electrode. The measurement conditions were a frequency of 1–1000 Hz
and an applied voltage of 1 mVrms with a single sine constant voltage. In vitro EIS was measured
before implantation in the 0.01 mol/L phosphate-buffered saline (164-18541, Wako Chemicals,
Osaka, Japan) with 0.2 g/L albumin (020-01862, Kishida Chemical, Osaka, Japan) at 37 ℃.
2.4

Equivalent circuit analysis

The EIS measurements were analyzed using electrochemical analysis software (NOVA,
Metrohm). The circuit shown in Fig. 2 was used for the equivalent circuit analyses and waspreviously published as an electrode interface equivalent circuit.(25) The solution resistance(Rsol) was the bulk resistance of the solution.(25) The charge transfer resistance (Rct) indicated the
difficulty of the charge transfer between the electrode and the electrolyte solution interface.(5) The
constant phase element (CPE) approximated an electric double-layer capacitor with a nonlinear
characteristic. A deviation from an ideal capacitance behavior corresponds to a frequencydependent phase angle and the CPE represents the nonideal behavior as a complex impedance
having the special characteristic of a phase angle that is independent of frequency. The capacitancedispersion at solid electrodes strongly depends on the electrode surface state, e.g., its surface
roughness, the degree of crystallinity, and anion adsorption.(26) The impedance of the CPE (ZCPE)
is defined as Eq. (1). The CPE functions as a complete capacitor when n = 1 and as a complete
resistor when n = 0.(5) The index Y0 equals the admittance of the CPE at the angular frequency ω =1 rad/s.

ZCPE
Rsol

Rct

Fig. 2. Equivalent circuit model for the recording electrode (Rsol: solution resistance, Rct: charge transfer
resistance, and ZCPE: impedance of constant phase element).

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Sensors and Materials, Vol. 29, No. 12 (2017)

ZCPE =

1
Y0 ( jω)n(1)

The equivalent circuit was analyzed using a frequency range of 5–1000 Hz, which is thefrequency component of the N1 response.(27)
2.5

VEP emulation

The evoked potential was emulated using circuit simulation software (LTspice, Linear
Technology, Milpitas, CA, USA). The equivalent circuit of the electrode interface is depicted in Fig. 3.
To simulate brain tissue in the circuits, we used a parallel circuit with bulk tissue resistance (1480
Ω) and tissue capacitance (2.99 × 10−7 F).(20) The CPE in the equivalent circuit can approximate
capacitor activity. The input impedance of the bioamplifier (ML135, AD Instrument) consistedof a parallel circuit with an input resistor (200 MΩ) and an input capacitor (200 pF). The signal
current source as the neural response was defined as Eq. (2).(22)−(t − tc )2
I(t) = √
exp
2σ2
2πσ
1



(2)

Here, tc represents the start time for the activation of the neurons and σ represents the duration of
the activation. I(t) is expressed in units of A, and tc and σ are expressed in units of s. The values oftc and σ were calculated on the basis of the VEP waveform at 8 weeks after electrode implantation.
In our study, tc refers to the actual measured N1 latency and σ is calculated on the basis of theactually measured duration of the N1 peak. The amplitude of I(t), as shown in Eq. (2), is correctedwith the conformation of the emulated evoked-potential Vin illustrated in Fig. 3 to fit the actual

Electrode interface
component
Rsol
Tissuecomponent
Vin

Cdl
Bioamplifier
input
component

Rct

Vout

I
Current
sourceCtissue

Rtissue

Cbioamp

Rbioamp

Fig. 3. An equivalent circuit to emulate the first negative wave of the visual evoked potential (Rtissue: tissue
resistance, Ctissue: tissue capacitance, Rsol: solution resistance, Rct: charge transfer resistance, Cdl: double-layer
capacitance, Rbioamp: input resistance of bioamplifier, and Cbioamp: input capacitance of bioamplifier). The current
source I emulates an electrical source model for the evoked potential, as denoted in Eq. (3). Vin represents theelectrical potential on the surface of the recording electrode. Vout is input voltage to the bioamplifier after electrodeinterface modulation.

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Sensors and Materials, Vol. 29, No. 12 (2017)

VEP that was measured at 8 weeks after implantation. Thereafter, the signal current source in theemulation is represented as Eq. (3).
−7

I(t) = −4.34 × 10

−(t − 0.0265)2
× exp
10−5



(3)

For this emulation, the equivalent circuit analysis results of rabbit A were used. The values
for Rct and the capacitor-approximated CPE were based on the value measured at 8 weeks after
electrode implantation. The regulation value for solution resistance was on the basis of the value
measured at 8 weeks after electrode implantation. This value was compared with the maximum
value at 32 weeks after implantation. In addition, emulation was attempted using an extremely
high value of solution resistance (10 MΩ). The simulation values for VEP emulation are shown in
Table 1. The results obtained from the circuit simulation software were filtered using a range of
1–100 Hz.
The animal experiment was performed in compliance with the guidelines of the Association
for Research in Vision and Ophthalmology regarding the use of animals in ophthalmic andvisual research. Research approval was also obtained from the Animal Experimentation Ethics
Committee at Nidek.

3.

Results

Figure 4 shows the typical VEPs obtained at 8 weeks after the electrode implantation. The
parameters recorded from each rabbit all showed similar values. Figures 5 and 6 show timedependent changes in the amplitude and latency of N1 from the VEPs in Fig. 4. A significant
correlation was observed between latency and the electrode implantation period from week 6 to
32 (rabbit A: correlation coefficient r = −0.85, test of non-correlations, p < 0.01, t = 4.26; rabbit B:r = −0.82, p < 0.01, t = 3.73; rabbit C: r = −0.92, p < 0.01, t = 6.24). These data indicate that latency
was reduced over time. Moreover, amplitude was stable and there was no correlation between
amplitude and the week 12 to 32 period of electrode implantation (rabbit A: correlation coefficientr = 0.01, test of non-correlations, p > 0.05, t = 0.02; rabbit B: r = −0.26, p > 0.05, t = 0.54; rabbit C:r = 0.28, p > 0.05, t = 0.57).

Table 1
Simulation values of visual evoked potential emulation parameter.
Parameter
Description
Simulation value
Tissue resistance
1480 Ω
Rtissue
Tissue capacitance  299 nF
Ctissue
Solution resistance (8 weeks;
1415 Ω;Rsol (8 weeks;maximum;
maximum in 32 weeks;
1757 Ω;extremely high value)
extremely high value)     10 MΩ
Charge transfer resistance    1.1 TΩ
Rct
Double-layer capacitance  217 nF
Cdl (approximated CPE)
Input resistance of bioamplifier    200 MΩ
Rbioamp
Input capacitance of bioamplifier  200 pF
Cbioamp1696

Sensors and Materials, Vol. 29, No. 12 (2017)

Latency t N1
0
Amplitude V N1
200 µV
30 ms

Flash

Fig. 4. A typical waveform of the visual evoked potential at 8 weeks after electrode implantation. Flashstimulation was applied at time zero.

1000

N1 amplitude V N1 (μV)

N1 latency t N1 (ms)

50
40
30
20
10
0

0

8

16

24

32

Weeks after implantation T (weeks)

Fig. 5. Changes in the N1 latency of the visualevoked potential over time.(7) The solid line wasobtained using a least-squares method with datacollected at 6–32 weeks after electrode implantation.
The latency period appeared to decrease with time.

750
500
250
0

0

8

16

24

32

Weeks after implantation T (weeks)

Fig. 6. Changes in the N1 amplitude of the visualevoked potential over time.(7) The solid line wasobtained using a least-squares method with datacollected at 12–32 weeks after electrode implantation.
Except for a few weeks after electrode implantation,over the 32-week period, the N1 amplitude wasstable.

The electrochemical impedances were stable over time at each frequency after week 6
of electrode implantation (Fig. 7). The Cole–Cole plot and Bode plot at week 8 of electrodeimplantation are shown in Figs. 8 and 9, respectively. The results from the equivalent circuit
simulation were consistent with the experimental findings (Figs. 8 and 9). The equivalent circuit
analysis was based on the equivalent circuit illustrated in Fig. 2 and the results of the analysis
are shown in Figs. 10–13. A significant correlation was found between Rsol and the week 4 to 32
period of electrode implantation (rabbit A: correlation coefficient r = 0.68, test of non-correlations,p < 0.05, t = 2.63; rabbit B: r = 0.65, p < 0.05, t = 2.43). These results indicate that Rsol increased
over time (Fig. 10). In addition, in vitro Rsol was significantly lower than the in vivo value. Rct
gradually increased during the few weeks after electrode implantation and then reached infinity (Fig.
11). The CPE admittance was stable at 8 weeks after electrode implantation (Fig. 12). The value of

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Electrical impedance Z (Ω)

610

1.0 Hz51012.7 Hz

104

112.9 Hz
1000 Hz

103

0

8

16

24

32

Imaginary part of impedance -Z" (Ω)

Sensors and Materials, Vol. 29, No. 12 (2017)

× 5
1x10

Actual measured value

× 4
5x10

Result of simulation

0

Weeks after implantation T (weeks)

Impedance Z (Ω)×
1x10

Simulation
result of Z

× 4
5x10

101

102

Frequency f (Hz)

90

60

30

Simulation
result of α

Actualmeasuredvalue of Z

0

Actualmeasuredvalue of α

Phase α (°)

5

103

0

Fig. 9. Result of electrochemical impedance
spectroscopy (Bode plot) at 8 weeks after electrodeimplantation. The plotted points indicate actual
measured values and the lines represent thesimulation results from the equivalent circuit analysis.

× 4
2x10

× 4
3x10

Real part of impedance Z' (Ω)

Fig. 8. Result of electrochemical impedance
spectroscopy (Cole–Cole plot) at 8 weeks after
electrode implantation. The plotted points indicate
actual measured values and the line represents thesimulation result from the equivalent circuit analysis.

Solution resistance R sol (Ω)

Fig. 7. Change in electrical impedance of theconstructed electrodes over time.(7) The solid lines
were obtained using a least-squares method with datacollected at 6–32 weeks after electrode implantation.
Except for a few weeks after electrode implantation,over the 32-week period, the electrical impedanceswere stable.

× 3
5x10

× 3
2x10

× 3
1x10

0
in-8vitro

0

8

16

24

32

Weeks after implantation T (weeks)

Fig. 10. Time-dependent changes in in vivo solution
resistance Rsol. ‘In vitro’ denotes the value measured
in phosphate-buffered saline before implantation. The
solid line was obtained using a least-squares methodwith data collected at 4–32 weeks after electrodeimplantation. A positive correlation between Rsol andtime is apparent.

n in Eq. (1) was also stable and close to 1 (Fig. 13). The in vivo Rct and CPE values stabilized at alevel close to the in vitro values after several weeks of electrode implantation.
The results of the VEP emulation are shown in Figs. 14–16. The values of the parameters, whichwere from rabbit A, are shown in Table 1. Figures 14 and 15 show the results of the VEP emulation
using the regulation value from week 8 for Rsol and the maximum value during the 32-week period
for Rsol, respectively. Figure 16 shows the results of the VEP emulation using significantly higher
Rsol values than those obtained in the actual animal experiment. The emulation results based on

1698

Admittance magnitude of CPE Y0 (nS)

Charge transfer resistance R ct (Ω)

Sensors and Materials, Vol. 29, No. 12 (2017)

1012

108

104

in-8
vitro

0

8

16

24

104

103

102

101

32

Weeks after implantation T (weeks)

Fig. 11. Time-dependent changes in in vivo chargetransfer resistance Rct. ‘In vitro’ denotes the value
measured in phosphate-buffered saline before
implantation. A trend of a gradual increase wasobserved during the few weeks after electrodeimplantation. The value of Rct then reached infinity.

0.0

Input current
I (µA)

in-8vitro

0

8

16

24

32

Weeks after implantation T (weeks)

Fig. 13. Time-dependent changes in the exponent
term n of the in vivo CPE. ‘In vitro’ denotes thevalue measured in phosphate-buffered saline before
implantation. The solid line was obtained using aleast-squares method with data collected at 4–32
weeks after electrode implantation. Except for a few
weeks after implantation, over the 32-week period,
the values were stable and close to 1.

8

16

24

32

Fig. 12. Time-dependent changes in the magnitudeof the in vivo admittance Y0 of the CPE. ‘In vitro’
denotes the value measured in phosphate-bufferedsaline before implantation. The solid line wasobtained using a least-squares method with datacollected at 4–32 weeks after electrode implantation.
Over the 32-week period, the values were stable,
except for a few weeks after electrode implantation.

Input voltage
Vin (µV)

0.5

0Weeks after implantation T (weeks)

Output voltage
Vout (µV)

Exponent term of CPE n

1.0

in-8
vitro

0.0

I

-0.2
-0.4
0

Vin

-300
-600
0

Vout

-300
-600
0

26.5

50

100

150

Times after emulation start temulation (ms)Fig. 14. Result of visual evoked potential emulation
with the standard (8-week) value for solution
resistance in rabbit A. The input current I is denotedin Eq. (3). The input voltage Vin and the output
voltage Vout are as described in Fig. 2. Using thestandard solution resistance, very similar wave
patterns were observed between Vin and Vout.

the Rsol values that were obtained in the actual animal experiment slightly differed in both latency
and amplitude between the signal source potential Vin and the output potential Vout (input potential
to the bioamplifier), as shown in Fig. 3. The emulation result based on the high Rsol values had alonger latency and a decreased amplitude.

1699

I

-0.2
-0.4
0

Vin

-300
-600
0

Vout

-300
-600
0

26.5

50

100

150

Time after emulation start temulation (ms)Fig. 15. Result of visual evoked potential emulation
with the maximum solution resistance in 32 weeks
in rabbit A. The input current I is denoted in Eq.
(3). The input voltage Vin and the output voltage Voutare as described in Fig. 2. By using the maximum
solution resistance, very similar wave patterns wereobserved between Vin and Vout.

4.

0.0

I

-0.2
-0.4
0

Input voltage
Vin (µV)

Input current
I (µA)

0.0-300

Output voltage
Vout (µV)

Output voltage
Vout (µV)

Input voltage
Vin (µV)

Input current
I (µA)

Sensors and Materials, Vol. 29, No. 12 (2017)

-300

Vin

-600
0

Vout

-600
0

26.5

50

100

150

Time after emulation start temulation (ms)Fig. 16. Result of visual evoked potential emulation
with the significantly higher solution resistance inrabbit A. The input current I is denoted in Eq. (3).
The input voltage Vin and the output voltage Voutare as described in Fig. 2. Increasing latency anddecreasing amplitude were observed in the Vout wave
compared with that for the Vin wave.

Discussion

We used equivalent circuit analysis to demonstrate that Rsol values are altered in response to
the tissue surrounding a recording electrode. The in vivo Rsol was increased compared with thein vitro value due to electrode contact with living tissue. The time-dependent increase in Rsol
values was related to electrode surface condition changes that occurred in response to surgicaldamage, such as the formation of a fibrosis encapsulation layer around the electrode.(5) Thus, we
assumed that the initial Rct value decreased a few weeks after electrode implantation because oftissue fluid and blood infiltration; subsequently, Rct stabilized at a high value after the amountof tissue fluid infiltration decreased. The CPE approximates an electric double-layer capacitor
and represents both the nonlinearity that results from the adsorption of proteins and the effectsof surface roughness, in contrast to pure capacitance.(12) The values of n in Eq. (1) were close to
1. This finding suggests that the primary property of the CPE used in this study was capacitive.
For the implanted electrode, the capacitance is dependent on the medium surrounding theelectrode interface. The CPE admittance values were stable and almost unchanged comparedwith the in vitro values; thus, the effect of the medium surrounding the electrode interface wasminimal. In addition, the Rct values after several weeks of electrode implantation were like thoseobserved under the in vitro condition; thus, the in vivo electrode reactivity characteristics wereconsidered stable. The inflammation and fibrosis of the tissue surrounding the electrodes werelikely results of wound healing. Electrode implantation activates the host innate immune system
to induce a foreign body reaction. This reaction involves protein adsorption on the electrodesurface, inflammatory cell infiltration, macrophage fusion into foreign body giant cells, fibroblast
activation, and ultimately fibrous encapsulation. Such a foreign body reaction at the interfaceof the electrode continues for 2–4 weeks after implantation and affects the life of the implanted
device.(28) After the foreign body reaction subsides, the electrode characteristics stabilize.
The time-dependent increase in solution resistance suggests a continuation of tissue
encapsulation around the electrode surface. As mechanical stress, such as pressure and rubbing,

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accumulates over time with chronic implantation, granulation tissue composed of fibers andcollagen encapsulated the electrode. Stable CPE admittance indicated that protein adsorption did
not occur after the foreign body reaction subsided. The continuous increase in Rsol suggests thatthe connective tissue thickened and grew outward, despite the stabilization of the electrode-surface
Rct or CPE characteristics. In the equivalent circuit analysis of EIS, the Rct and CPE values differ
for the electrode material. The electrode materials do not affect Rsol, as this characteristic reflects
changes in the tissue surrounding the electrode. Recording electrode composed of biocompatible
and capacitive characteristic materials other than Pt show a similar electrode surface interface
behavior, such as the EIS time change.
The stability of the recording electrode characteristics was confirmed on the basis of thestability of the N1 amplitude of the VEP. The observed time-dependent decrease in the VEP
latency is consistent with the results of previous reports.(29) We emulated the evoked potentials
to determine whether changes in the recording electrode characteristics affected the evoked
potentials. The difference between the emulation reflecting the recording electrode characteristicchange and the actual VEP change revealed the factor that changed the VEP. Thus, theoretically,
VEP emulation can be used to determine the influence of other factors on VEP modulation, such ascell type, electrode interface, or bioamp and input impedance. We found that increases in solution
resistance that are within the range of values obtained in actual animal experiments slightly affectthe size and latency of evoked potentials. Thus, the time-dependent decrease in VEP latency was
not due to changes in the recording electrode characteristics. Moreover, regardless of whether
we used a different type of electrode (where the electrode materials were not Pt), the N1 latency
decreased. An evaluation of the N1 latency is useful for VEP assessment because N1 is a reliable
reaction in cortex layer Ⅳ.(30) Our findings with a different electrode confirmed that a decreasing
N1 latency occurs during chronic VEP measurement and that this decreased N1 latency is not due
to changes in the characteristics of electrode interface, but to biological reactions. Moreover, using
an electrode made of a material other than Pt would likely yield comparable results.
The specific reasons for the observed decrease in VEP latency are unknown; however, threepossibilities should be considered. First, glial cells, which support neuronal signaling, might
have affected our results. When brain tissue is damaged, astrocytes are stimulated to protect
neurons via Ca2+ signaling, resulting in wound healing.(31) In addition, astrocytes’ Ca2+ isimportant for the regulation of synaptic function.(31) We hypothesize that electrode implantationinduced nerve damage, and subsequently, astrocytic neuronal protection and wound healing,altering synaptic transmission. As the result, animal’s reactions improve gradually. Hence, N1
latency chronologically decreases. Second, our observations could have been due to electrodeimplantation-induced mechanical pressure on the brain, which decreased the distance to thesignal source. This reduction in distance to the signal source can decrease latency.(32) Third,
the anesthesia we used may have affected the recorded evoked potentials.(33) That is, the rabbits
may have become accustomed to the isoflurane anesthesia due to repeated exposure. Although
we measured the anesthetic concentrations in the exhaled gas, anesthesia acclimation could havedecreased the depth of anesthesia. Monitoring of the bispectral index via EEG analysis, which isused to assess the level of consciousness during anesthesia, can be used to control the anesthesia
depth and would resolve this issue. The emulation of the evoked potentials using an extremely
large solution resistance increased latency and decreased amplitude. This finding suggests that
greater damage to the brain following electrode implantation increases the foreign body reactions

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around the electrode, resulting in a marked increase in solution resistance. This idea is consistent
with reports suggesting that conventional recording electrodes can only be used for the shortterm recording of evoked potentials.(2,7) Our emulation approach can estimate recording electrodedegradation. The time constant τ of the equivalent circuit for the electrode surface [the electrodeinterface component as shown in Fig. (3)] is based on Rsol, Rct, and Cdl, as represented in Eq. (4).

τ=

R sol × Rct × Cdl
R sol + Rct(4)

Equation (4) shows that large increases in solution resistance increase τ. Hence, our analysis
indicates that solution resistance and the time constants chronologically increase as the reaction
slows. A marked increase in solution resistance can degrade VEP recording; therefore, extreme
elevations of solution resistance should be avoided for the long-term recording of evoked potentials.

5.

Conclusions

We used EIS and equivalent circuit analysis to determine the long-term changes in the propertiesof recording electrodes. Our recording electrodes performed exceptionally well for long-term
recording, as we did not observe an N1 amplitude reduction or an N1 latency extension over time.
In a previous study, an N1 amplitude reduction and N1 latency extension occurred in response toforeign body reactions around the electrodes.(2) Our VEP emulation results support these findingsof changes in the VEP. Although we observed the decreased N1 latency, our emulation results
suggest that this decrease in latency was not related to the electrode characteristics, but rather, tobiological reactions in the electrode-implanted tissue. Nonetheless, our approach for equivalentcircuit analysis and evoked potential emulation improved our understanding of the interaction
between the electrode interface characteristics and the stimulation response. Our approach using
VEP emulation was useful for determining whether electrode characteristics or biological reaction
modulates the VEP and can be used further to identify the factors that affect time-dependentchanges in the VEP. Moreover, our technique can be used for the long-term evaluation of novel
electrodes in neural prostheses. In future studies, we will investigate the effects of glial cells andanesthesia depth on evoked potentials.

Acknowledgments
The authors thank Masayoshi Nakatani and Chisato Taki of Nidek Co., Ltd. for their assistancewith the animal experiments. This study was supported by the New Energy and Industrial
Technology Development Organization (NEDO) of Japan.

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