CHARACTERIZATION OF THE ENCAPSULATION
PROCESS OF DEEP BRAIN STIMULATION ELECTRODES
USING IMPEDANCE SPECTROSCOPY IN A RODENT MODEL
K. Badstübner
1*
, T. Kröger
2*
, E. Mix
1
, U. Gimsa
3
, R. Benecke
1
and J. Gimsa
2**
1
Department of Neurology, University of Rostock, Gehlsheimer Str. 20, 18147 Rostock, Germany
2
Chair of Biophysics, Institute of Biology, University of Rostock, Gertrudenstr. 11A, 18157 Rostock, Germany
3
Research Unit Behavioural Physiology, Leibniz Institute for Farm Animal Biology,
Wilhelm-Stahl-Allee 2,18196 Dummerstorf, Germany
* These authors contributed equally to the work.
** Corresponding author.
Keywords: EIS, Intracerebral electrodes, Basal ganglia, Subthalamic nucleus, Rat brain, Chronic instrumentation,
6-OHDA, Parkinson’s disease.
Abstract: Deep brain stimulation (DBS) is effective for the treatment of patients with Parkinson’s disease (PD),
especially in advanced stages which are refractory to conventional therapy. Despite of the regular use in
clinical therapy, rodent models for basic research into DBS are not routinely available. The main reason is
the geometry difference from rodents to humans, imposing larger problems in the transfer of the stimulation
conditions than from primates to humans. For rodents, the development of miniaturized mobile stimulators
and stimulation parameters, as well as improved electrode materials and geometry are desirable. The
impedance of custom made, cylindrical (contact diameter 200 µm, length 100 µm), platinum/iridium
electrodes has been measured in vivo for two weeks to characterize the influence of electrochemical
processes and of the adherent cell growth at the electrode surface. During the encapsulation process, the real
part of the electrode impedance at 10 kHz doubled with respect to its initial value after a characteristic
decrease by approximately one third at the second day. An outlook is given on further investigations with
different electrode designs for long-term DBS.
1 INTRODUCTION
Parkinson’s disease (PD) is a widespread
degenerative disorder of the central nervous system
that affects motor function, speech, cognition and
vegetative functions. The cardinal symptoms such as
tremor, rigidity, bradykinesia and postural instability
result mainly from the death of dopaminergic cells
in the substantia nigra pars compacta and the
subsequent lack of dopaminergic inputs into the
striatum. This causes an alteration of the activity
pattern in the basal ganglia (Braak and Braak, 2000).
Deep brain stimulation (DBS) is a novel therapeutic
option for PD as well as an increasing number of
neuropsychiatric disorders. Before DBS became a
therapeutic intervention, electric stimulation of basal
ganglia had been used to guide neurosurgeons to the
precise position for a surgical lesion, the ultimate
therapy of a late-stage PD. The main advantage of
DBS over surgical lesions is the reversibility and
possibility to modulate stimulation parameters
(Benabid et al., 1987). The small volume of the
target region for DBS in the human brain requires a
highly specific adaption of the electrodes which
need to be thoroughly tested in animal models,
including different materials and geometries. So far,
DBS data of animal models of PD are scarce. During
in vivo stimulation, the properties of the DBS
electrodes are changing as a function of time caused
by electrochemical processes at the surface of the
implant and the subsequent tissue response (Gimsa
et al., 2005). The tissue response is a foreign
substance reaction. Its intensity depends on the
material (Grill and Mortimer, 1994) and is correlated
with the thickness of the adventitia finally
125
Badstübner K., Kröger T., Mix E., Gimsa U., Benecke R. and Gimsa J..
CHARACTERIZATION OF THE ENCAPSULATION PROCESS OF DEEP BRAIN STIMULATION ELECTRODES USING IMPEDANCE SPECTROS-
COPY IN A RODENT MODEL.
DOI: 10.5220/0003790301250130
In Proceedings of the International Conference on Bio-inspired Systems and Signal Processing (BIOSIGNALS-2012), pages 125-130
ISBN: 978-989-8425-89-8
Copyright
c
2012 SCITEPRESS (Science and Technology Publications, Lda.)
encapsulating the implant (Wintermantel et al.,
2002). Adventitia formation causes a steady change
in the impedance of the electrodes leading to
changes in the attenuation of the stimulating signal.
As a result, the efficiency of the surrounding tissue
stimulation is changing (Lempka et al., 2009 and
2011; Grill and Mortimer, 1994). One opportunity to
minimize this problem is to choose an appropriate
electrode material. Previous investigations of our
group (Gimsa et al. 2006; Henning et al., 2007) have
shown that the use of stainless steel electrodes is not
appropriate because of the corrosion and erosion
processes intensified by electrolytic electrode
processes. Electrochemically induced alterations are
negligible for inert platinum electrodes, even though
electrode processes may still influence the
surrounding tissue (Gimsa et al., 2005). For an
optimal adjustment of the DBS signal, the kinetics of
the electrode impedance alterations caused by the
adventitia formation must be taken into account
(Lempka et al., 2009 and 2011).
2 MATERIALS AND METHODS
2.1 Animal Treatment
A number of 30, adult, male Wistar Han rats (240-
260 g) were obtained from Charles River
Laboratory, Sulzfeld, Germany) and housed under
temperature-controlled conditions in a 12 h light-
dark cycle with conventional rodent chow and water
provided ad libitum. The rats were subject to the
following treatments:
anesthesia
6-OHDA-lesioning (28 rats)
electrode implantation (6 rats)
impedance measurement (2 rats)
The study was carried out in accordance with
European Community Council directive 86/609/EEC
for the care of laboratory animals and was approved
by Rostock’s Animal Care Committee (LALLF M-
V/TSD/7221.3-1.2-043/06).
2.1.1 Anesthesia
The rats were anesthetized by ketamine-
hydrochloride (10 mg per 100 g body weight, i.p.,
Ketanest S
®
, Pfizer, Karlsruhe, Germany) and
xylazine (0,5 mg per 100 g body weight, i.p.,
Rompun
®
, Pfizer). Before surgery, the eyes of the
rats were medicated with Vidisic (Bausch and
Lomb, Berlin Germany). After surgery, the wound
was sutured and the rats received 0.1 ml
novaminsulfone (Ratiopharm, Ulm, Germany) and
4 ml saline subcutaneously. Rats were exposed to
red light (Petra, Burgau, Germany) until
normalization of vital functions.
2.1.2 6-Hydroxydopamine (6-OHDA)
Lesioning
The 6-OHDA (Sigma, Deisenhofen, Germany)
lesioning was performed by stereotactic surgery in
adaption to Kirik et al. (1998). The lesions of the
right medial forebrain bundle of rats were made by
injection of 26 µg 6-OHDA in 4 µl saline with 1 g/l
ascorbic acid delivered over 4 min via a 5 µl
hamilton microsyringe (Postnova Analytics,
Landsberg/Lech, Germany). The coordinates relative
to bregma were: anterior-posterior
(AP) = -2.3 mm, medial-lateral (ML) = 1.5 mm and
dorsal-ventral (DV) = -8.5 mm (Paxinos and
Watson, 1998).
2.1.3 Electrode Implantation
Electrodes were implanted into the subthalamic
nucleus (STN), which is the most common target
region for treatment of PD patients. The surgical
procedure was performed using a stereotactic frame
(Stoelting, Wood Dale, IL, USA) modified
according to Harnack et al. (2004). To support the
surgical procedure, a cold light source (KL 1500
LCD, Schott, Mainz, Germany) was used in
combination with a stereo-microscope (Leica,
Wetzlar, Germany). The skull was opened by a
dental rose-head bur (Kaniedenta, Germany). The
coordinates relative to bregma were: anterior-
posterior (AP) = -3.5 mm, medial-lateral
(ML) = 2.4 mm and dorsal-ventral (DV) = -7.6 mm
(Paxinos and Watson, 1998). A dental drill was used
to bore an additional hole in the skull for an anchor
screw. The electrode was fixed to the skull by an
adhesive-glue bridge (Technovit 5071, Heraeus,
Germany) to the anchor screw.
Counter-electrode wires were implanted into the
neck of the rats.
2.2 Stimulation- and Counter-
electrodes
For impedance measurements, we designed unipolar
platinum-iridium (Pt/Ir) microelectrodes which were
covered with polyesterimide insulation and custom-
made by Polyfil (Zug, Switzerland; Figure 1).
BIOSIGNALS 2012 - International Conference on Bio-inspired Systems and Signal Processing
126
Figure 1: Photograph (a) and scheme (b) of the unipolar
Pt/Ir electrode. The electrode pole was a round wire made
from Pt90Ir10 with a diameter of 200 µm. The length of
the non-insulated tip of the electrode pole was 100 µm.
The insulation consisted of polyesterimide 180 with a
thickness of 25 µm (Nowak et al., 2011).
Dental wires made of biocompatible, nickelfree steel
alloy (18% Cr, 18% Mn, 2% Mo, 1% N, remander
iron) of 1 mm diameter and 10 mm length were used
as counter electrodes (see: Figure 4) in combination
with the unipolar DBS electrodes (Figure 1).
Electrochemical electrode effects were negligible
at the counter electrode due to the low current
density at its large surface.
2.3 Electric Impedance Spectroscopy
(EIS)
Electric impedance spectroscopy (EIS) is a common
measureing technique for determining the electrical
properties of tissues (Foster and Schwan, 1989). It is
used in a wide range of applications, such as breast
cancer detection (Kerner et al., 2002), the
monitoring of the lung volume (Adler et al., 1997)
and in material sciences. EIS is nondestructive and
therefore suitable for the characterization of the DBS
electrodes during the encapsulation process.
Equipment
The EIS measurements were conducted with an
impedance spectrometer Sciospec ISX3 (Sciospec
Scientific Instruments, Pausitz, Germany) and a test
fixture HP16047D (Hewlett-Packard, Japan)
connected to a personal computer with Sciospec-
measuring software. The two connectors of the test
fixture were connected to the DBS and the counter
electrodes.
Measurement
To characterize the electrode properties during
encapsulation, the impedance was recorded in the
frequency range from 100 Hz to 10 MHz over a
period of two weeks after implantation. Frequency
range, amplitude, number of points and the
averaging of the impedance spectrometer were
programmed by the measuring-software (Sciospec).
401 frequency points were logged which were
distributed equidistantly over a logarithmic
frequency scale. The measuring voltage (peak to
peak) was 12.5 mV
PP
. The measuring-software
logged the measuring data of the impedance
spectrometer (real and imaginary parts of the
impedance vs. frequency), saving them to a file.
Before each measurement, the impedance
spectrometer was calibrated by open, short and load
measurements. Each measurement was repeated
three times to improve the statistical significance.
The measurements were repeated every day for one
week and every second day during the second week.
The stimulation pulse usually applied in DBS has
a frequency of 130 Hz and a pulse width of 60 µs.
Because of the steep slopes of the needle-shaped
pulse, the signal is rich in high harmonic frequencies
(Gimsa et al. 2005). For this reason, we measured
the impedance within the wide frequency range from
100 Hz to 10 MHz, which is beyond the range of up
to 10 kHz reported by Lempka et al. (2009).
Impedance theory
The electrical impedance describes the magnitude
ratio between the applied AC voltage and the
resulting current flowing with a certain phase shift.
Mathematically speaking, the impedance Z* is a
complex number with the unit [], which is
composed of a real (Z´) and an orthogonal imaginary
part (Z´´) marked by the complex unit j =
1
:
Z* = Re(Z*) +
j
·Im(Z*) = Z´+
j
Z´´ (1)
For interpretation of the measuring data, an
equivalent circuit model is required to be fitted to
the measuring data. The aim was to model
electrochemical processes and adherent cell growths
by combinations of resistors, capacitors and constant
phase elements (Lempka et al., 2009).
Data analysis
The logged data were transferred to Matlab (The
MathWorks™, Version 7.9.0.529) to calculate
means and standard deviations. For their graphic
representation, they were finally copied to Sigma
Plot 11.0 (Systat Software, 11.0, Build 11.2.0.5).
2.4 Electron Microscopy Study of
Electrode Encapsulation by Tissue
Concentric bipolar microelectrodes with an inner
CHARACTERIZATION OF THE ENCAPSULATION PROCESS OF DEEP BRAIN STIMULATION ELECTRODES
USING IMPEDANCE SPECTROSCOPY IN A RODENT MODEL
127
pole diameter of 75 µm and an outer pole diameter
of 250 µm (CB CSG75; FHC, Bowdoinham, ME)
were placed into the STN of two anesthetized rats.
The rats were stimulated for 3 h with biphasic
constant-current pulses with a repetition frequency
of 130 Hz and pulse duration of 60 µs at 250 µA
with a stimulus generator (Multichannel Systems,
Reutlingen, Germany). The electrodes were removed
and one electrode was incubated in trypsin solution
(Trypsin-EDTA (1x) in HBSS W/O CA&MG
W/EDTA.4NA, Gibco, UK) at 37°C for 1 h. The
other electrode was postfixed overnight in 4%
glutaraldehyde (Merck, Germany) in PBS.
Electrodes were washed, fixed in 4%
glutaraldehyde in PBS, washed again, postfixed in
1% osmium tetroxide, dehydrated in acetone, and
subjected to critical-point drying (EMITECH K850,
Ashford, Kent, UK). The samples were sputtered
with colloidal gold using a Sputter Coater (BAL-
TEC SCD 004, Schalksmühle, Germany) before
examination in the SEM (DSM 960 A, Zeiss,
Oberkochen, Germany).
3 RESULTS
3.1 Stimulation- and Counter
Electrode Implantation
As a first test, the localization of the electrode tip in
the target region was verified by ink injection via a
5 µl hamilton microsyringe of approximately the
same size as the implanted stimulation electrode
(Figure 2).
Figure 2: Rat brain fixed in formalin, (a): top view of a rat
brain with a puncture resembling an electrode canal (1),
(b): sagittal section of one hemisphere with the puncture
(1), (c): hemisphere with tissue removed to the injection
canal, (d): enlarged photo of (c) with ink injection canal
(2), striatum (3), and basal ganglia (4).
Chronic instrumentation was established and
applied to freely moving PD -rats (Figure 3). For
this, a commercial rat jacket (Lomir Biomedical,
Quebec, Canada) with a backpack was used. All
electronic components (Figure 2) were located in the
backpack. This allowed for a stimulation of up to six
weeks when the batteries were changed every
second day.
For the EIS measurements, counter electrodes were
pierced through the intact scalp close behind the cut
for the implantation of the stimulation electrode. An
example of implantation of a counter electrode using
dental wire of biocompatible steel alloy is shown in
Figure 4.
Figure 3: Photograph of a rat that was anesthetized for
implantation of chronic instrumentation consisting of: (1):
Pt/Ir electrode, (2): counter-electrode, (3): plug-connector
electrodes, (4): battery, (5): DBS-stimulator and (6):
carrying bag.
Figure 4: Implanted dental wire of biocompatible steel
alloy used as counter electrode for the EIS measurement.
3.2 Impedance Measurement
Our data showed the general tendency of an
impedance increase during the encapsulation
process. The measuring results are shown in
Figure 6. After 12 days, the value of Z´ (Figure 6a)
has almost doubled compared to its initial value at
10 kHz. In parallel, also the absolute value of Z´´
(Figure 6b) increased. The results match with
findings of Lempka et al. (2009) for the monkey
brain. The main reason for the impedance increase is
the formation of the adventitia as a foreign substance
BIOSIGNALS 2012 - International Conference on Bio-inspired Systems and Signal Processing
128
reaction. Indeed, the adventitia covered the electrode
surface as shown by Lempka et al. (2009). Our own
electron-microcopy study showed that stimulation
electrodes get overgrown by cells within 3h post
implantation (Figure 5).
Figure 5: Scanning electron-microscopical images of elec-
trodes that had been implanted and used for 3 h of stimu-
lation. (a): Electrode cleaned by trypsination prior to
electron microscopy. (b): Electrode fixed with adhering tissue.
Figure 6: In vivo impedance measurements with a unipolar
Pt/Ir electrode in combination with a steel alloy counter-
electrode ((a) Z´; (b) Z´´).
Our data showed an impedance decrease at the
second day after implantation followed by a
significant increase from the third day onwards.
Interestingly, the same impedance behavior has
already been reported by Lempka et al. (2009, p. 6,
Figure 4). However, no explanation has been given
for this phenomenon.
4 CONCLUSIONS
AND PERSPECTIVES
Pilot experiments in the rat model have shown that
the impedance of a unipolar DBS electrode is
significantly increasing with time after implantation.
In future experiments, different biocompatible
electrode materials such as titanium, diverse
titanium alloys, platinum, gold, silver and stainless
steel will be tested. For these tests, an array of
various counter electrodes will be pierced into the
necks of a number of rats.
In addition, bipolar electrodes possessing modified
configurations such as shifted tips will be used to
test the effects of non-axial symmetric field
distributions.
The equivalent circuit model will be improved
for a better understanding of the measuring data in
order to extract encapsulation parameters. These
investigations aim at clarifying the phenomenon of
the impedance drop at the second day after
implantation.
Potential effects at the electrode-tissue-interface
will be analyzed by histological, immunochemical
and electron-microscopical methods.
ACKNOWLEDGEMENTS
K.B. is grateful for a stipend of the German
Research Foundation (DFG, Research Training
Group 1505/1 “welisa”). T.K. acknowledges
financing by a project of the Federal Ministery of
Economics and Technology (BMWi, V230-630-08-
TVMV-S-031). Part of the work was conducted
within a project financed by the Federal Ministry of
Education and Research (BMBF, FKZ 01EZ0911).
The authors are grateful to Dr. J. Henning for help
with the electron microscopy study and would like
to thank the staff of the electron microscopy center
at the University of Rostock's Medical Faculty for
outstanding technical support.
Real
log( f ) / Hz
4567
Z´ / kOhm
0
2
4
6
8
10
12
14
Day 1
Day 2
Day 4
Day 5
Day 6
Day 8
Day 12
Imaginary
log ( f ) / Hz
4567
Z´´ / Ohm
-10
-8
-6
-4
-2
0
(a)
(b)
Day 1
Day 2
Day 4
Day 5
Day 6
Day 8
Day 12
CHARACTERIZATION OF THE ENCAPSULATION PROCESS OF DEEP BRAIN STIMULATION ELECTRODES
USING IMPEDANCE SPECTROSCOPY IN A RODENT MODEL
129
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