Supplemental information:
Electrochemical Activation and Inhibition of Neuromuscular Systems through
Modulation of Ion Concentrations with Ion-Selective Membranes
Yong-Ak Song1,2, Rohat Melik1,2, Amr Rabie3, 4, Ahmed M.S. Ibrahim3, David Moses5,
Ara Tan6, *Jongyoon Han1,2 , *Samuel J. Lin3
1Department
of Electrical Engineering and Computer Sciences, Massachusetts Institute
of Technology, Cambridge, MA
2Department
of Biological Engineering, Massachusetts Institute of Technology,
Cambridge, MA
3Divisions
of Plastic Surgery and Otolaryngology, Beth Israel Deaconess Medical
Center, and Harvard Medical School, Boston, MA
4
Department of Otolaryngology, Ain Shams University, Cairo, Egypt
5 Department
6
of Bioengineering, Rice University, Houston, TX
Department of Chemical Engineering, University of Minnesota, Twin Cities, MN
* Co-corresponding Authors:
Jongyoon Han (jyhan@mit.edu) and Samuel J. Lin (sjlin@bidmc.harvard.edu)
S-1
Strategy to overcome the limited storage capability of ISM
The ability of ion-selective membranes to change the ion concentration depends on
both the amount of ions adjacent to the nerve and the reservoir capacity of the ISM to
store specific ion species. To maximize the amount of ions stored in the ISM, we plan to
optimize the geometry of the ISM in terms of width and thickness as well as the amount
of ionophores in the ISM. The porosity and the pore size of the ISM are other important
parameters to take into consideration. A potential solution to address this issue of
limited ion storage capacity in the membrane is designing a stimulation device, where
ion-selective membrane material is used as a ‘filter’ rather than ‘storage’ of the
particular ion (see Figure S-1). The electrodes on the both side of the membrane can
used to ‘pump’ a particular ion species away from the nerve.
electrode layer
ion-selective
membrane
+
nerve
f iber
+ Ca2+ ions
+
+
ion depletion
current id
+
+
+
electrode layer
Figure S-1. Schematics of the new membrane device
S-2
-
Experiment with ion-selective pipette tip electrodes
In a separate control experiment with a conventional glass pipette tip filled with a Ca 2+
ion-selective membrane at the tip end and a 100mM CaCl2 solution inside the glass
pipette (see the experimental setup with a glass pipette tip-based ISM in Figure S-2),
we also observed a continuous decrease of the electrical threshold value from 20A to
10A (50% decrease; Figure S-3). As a negative control experiment, we performed the
same stimulation test with a plasticized amorphous polymer matrix such as PVC
(polyvinyl chloride) membrane in a glass pipette tip without Ca2+ ion-specific ionophore
added and confirmed that the electrical threshold value remained the same (see Figure
S-4). Slightly above the reduced stimulation threshold, the muscle twitch force
amplitude was attenuated by approximately 90%, gradually increasing with increased
stimulation current afterwards. This experiment reproduced the same trend of
stimulation threshold reduction as in Figure S-5. This observation is a qualitatively
different behavior from the common “all-or-none” electrical stimulation characteristics.
This result clearly implies that the activity of muscle in terms of force can be controlled
with a higher degree of resolution and dynamic range, compared with pure electrical
stimulation. We speculate that changing the ion concentration in small axons is more
rapid than in large axons due to the smaller size. Therefore, the effect of Ca 2+ ion
depletion, which resulted in a graded response, might lower the threshold value of small
axons more effectively than that of large axons. Further investigation is required to
definitely determine whether our system achieves the graded response by reversing the
relative threshold for large and small axons in the nerve fiber. Even under a constant
perfusion of Ringer’s solution onto the nerve at the site of stimulation with a flow rate of
S-3
0.5L/min, which served to emulate the in vivo ion homeostatic conditions, we could
lower the electrical threshold from is = 5.6 to 4.4A (see Figure S-5). With a constant
perfusion of Ringer’s solution on the stimulation site with the depletion current turned off,
the original nerve excitability state was restored, both in terms of the current stimulation
threshold and the characteristically sharp transition between an “all-or-none” force
generation.
S-4
a)
1. Ion depletion
depletion current
id ~ 100nA-1µA
(subthreshold value)
- - - +
+
+
+
+ +
id
+
+ + ++ + K
+ Ca2+
- +
cathode (-)
sciatic nerve
Ca2+ ion-selective
membrane
2. Electrical stimulation
stimulus current is
conducted into nerve under
continuous ion depletion
-
+
is - - + is
-
f orce
transducer
+
+ id +
+ + ++
+ - +
muscle
I
II
V
V
i
i: stimulus current isolator
V: voltage supply f or ISE
Ion depletion
depletion current
id ~ 100nA-1µA
(subthreshold value)
K+ ion-selective
membrane
- - - + +
+
+ + + + +
id
+ K+
+
+
+
+ Ca2+
cathode (-)
Ion enrichment
enrichment current
ie ~ 100nA-1µA
conducted into nerve
+ -- +
+
++ - +
ie +
+
+
+ +
anode (+)
I: location for electrical stimulation under Ca2+ ion depletion
II: location for nerve blocking under K+ ion depletion or enrichment
b)
silver wire between the nerve and the ion-selective
membrane f or ion depletion and nerve stimulation
sciatic nerve of a f rog
gastrocnemius muscle
ion-selective membrane at the tip of a
pipette (cathode integrated inside tubing)
Figure S-2. Functional electrochemical stimulation of a frog’s sciatic nerve using an ion-selective
pipette tip. a) Experimental setup for electrochemical stimulation of frog sciatic nerves with ionselective pipette tip. The operation modes for Ca2+ ion-depleted excitation are shown
schematically in the inset at position I. Note that the ion depletion current id is at least by one
order of magnitude lower than the electrical threshold value is required for electrical stimulation.
For nerve signal blocking, a K+ ion-selective electrode is positioned further down from the
stimulation site at position II. Both the K+ ion depletion and enrichment modes are shown in the
inset schematically. b) A photograph of the experimental setup.
S-5
is = 12-20 µA in 1 µA step (stimulus current)
id= 1 µA (depletion current)
t p= 300 µs (pulse width)
f =1 Hz (pulse f requency)
Muscle contractile f orce [mN]
10
Stimulation with
Ca 2+ ion depletion
8
Stimulation without
Ca 2+ ion depletion
6
4
2
0
10
12
14
16
18
20
Stimulus current [A]
Figure S-3. Influence of the depletion time on the threshold value. By continuously depleting the
Ca2+ ions, the minimum electrical current required to elicit a muscle contraction was lowered
from 20 A down to is=10 A at a pulse width of tp=300 s, and a pulse frequency of f=1 Hz
(threshold lowered from 20 A to 18 A after td=18 s, 16A after 1 min 11 s, 14A after 4 min 30
s, 10 A after 5 min). At the same time, the muscle twitch amplitude was modulated by almost
90% of the twitch amplitude achieved at 20 A.
S-6
a)
b)
14µA
14µA
12µA
Muscle contractile f orce [mN]
Muscle contractile f orce [mN]
12µA
40
30
20
10
40
30
20
10
0
0
0
stimulation time [s]
200
0
stimulation time [s]
200
Threshold
value :12.4µA
Threshold
value :12.2µA
Figure S-4. Negative control experiment with a PVC membrane without adding Ca2+ ionophore
in the glass pipette tip. a) before ion depletion. b) after ion depletion at id=1µA for t=5 min. For
the stimulation, we applied a pulse train of monophasic stimuli starting from an electrical current
of is=12A, at a pulse width of tp=1ms and a pulse frequency of f=1 Hz. The threshold value
changed only minimally from 12.2 µA to 12.4 µA.
S-7
b)
5.6 A
(threshold bef ore Ca2+ depletion)
15
5
Stimulus [A]
Stimulus [A]
a)
5
20
Contractile f orce [mN]
20
Contractile f orce [mN]
4.4 A
(threshold af ter Ca2+ ion depletion)
5.6 A
15
15
10
5
0
time [s]
dynamic range of control 
resolution of control 
15
10
5
150
0
time [s]
300
Figure S-5. Comparison of excitability a) without and b) with depletion of the Ca2+ ions for 5 min
at id = 100nA under a continuous perfusion of Ringer’s solution. The maximum muscle twitch
amplitude increased by ~2.5 times at the same stimulus current is=5.6µA after the depletion of
Ca2+ ions. For the stimulation, we applied a pulse train of monophasic stimuli starting from an
electrical current of is=4 A, at a pulse width of tp=1ms and a pulse frequency of f=1 Hz. The
stimulus pulse height was gradually increased in order to characterize the stimuli-force
response.
S-8
a)
b)
Threshold: 30 µA, required f or CAP
Threshold: 6 µA required f or CAP
Figure S-6. Comparison of compound action potentials in standard Ringer’s and Ca2+ ion–
depleted Ringer’s solutions. a) Compound action potential of a demyelinated sciatic nerve after
25 min. in Ringer’s solution. b) the same demyelinated nerve in Ringer’s solution with lower
Ca2+ ion concentration (0.3mM CaCl2) for 10 min. The electrical threshold value was significantly
reduced from is=30 µA down to is=6 µA.
S-9
sciatic nerve
a)
ion-selective membrane
cross-sectional view A
Epineurium
A
-
force
transducer
gastrocnemius
muscle
id
+
-
nerve fibers
+
id: ion depletion current
+
ion-selective
membrane
-
sciatic nerve
+
tripolar electrodes
for stimulation
b)
18 µA
16 µA
Muscle contractile f orce [mN]
4
is =16-30 µA in 0.2 µA step
id= 1 µA (depletion current)
tp= 1ms
f =1 Hz
Signal blocking
3
2
Stimulation with
Na + ion depletion
1
Stimulation without
Na + ion depletion
0
50
100
150
stimulation time [s]
Figure S-7. Nerve block caused by Na+ ion depletion using ISMs. a) Experimental setup for a
nerve conduction block by Na+ ion depletion. b) After depleting Na+ ions at a constant ion
depletion current of id= 1µA (the depletion voltage was 1.4V) for t = 5 min. distal to the site of
electrical stimulation, we observed a decreasing muscle twitch force until it became
undetectable with the force transducer above is=17.6µA. Even after exceeding the upper limit of
electrical stimulus up to is=30 µA, the nerve could not be stimulated electrically. At higher twitch
amplitude, ~100mN, significantly higher blocking current ib =50-100 µA was required to initiate a
blocking effect.
S-10
b)
10
100
Contractile f orce [mN]
Contractile f orce [mN]
100
11 A
(with K+ ion depletion)
20
Stimulus [A]
10
c)
80
60
40
20
0
time [s]
500
11 A
(af ter enriching K+ ions)
20
10
100
reduced amplitude
80
60
40
20
0
time [s]
500
Contractile f orce [mN]
11 A
(without K + ion depletion)
20
Stimulus [A]
Stimulus [A]
a)
80
60
40
complete signal blocking
20
0
time [s]
300
Figure S-8. Effect of K+ ion depletion and enrichment on the signal propagation along the sciatic
nerve using K+ ion selective pipette tip. a) before depletion and b) after depleting K+ ions for 5
min. at id = 100 nA on the sciatic nerve. For the stimulation, we applied a pulse train of
monophasic stimuli starting from an electrical current of is=10 A, at a pulse width of tp=1ms and
a pulse frequency of f=1 Hz. The muscle twitch amplitude was decreased by ~50% at the same
stimulus. c) After reversing the polarity and enriching K+ ions for 5 min. at id = 100nA, no
response could be obtained even at significantly higher current pulses (is > 18A). This blocking
effect, however, was reversible. After applying Ringer’s solution on the stimulation site and
waiting for 10 min., the nerve was responsive again.
S-11
a)
b)
120
4
Conractile f orce [mN]
pulse current [A]
stimulus current pulses at is =4 A, tp= 300 µs and f=1 Hz
3
2
1
contractile f orce [mN]
0
unfused tetany
30
Tetany was
blocked after 4 min.
of continuous K+ ion
depletion at id =1µA
100
80
60
40
20
20
5
10
6
7
8
9
10
12
Depletion time [min]
0
K+ ion depletion started at t = 5 min.
0
5
10
15
20
[s]
Figure S-9. Initiation of a tetany-like muscle contraction by Ca2+ ion depletion and subsequent
nerve conduction blocking by K+ ion depletion. a) Continuous depletion of Ca2+ ions at id = 1µA
caused a tetany-like muscle twitching. Stimulation created higher mean force at low electrical
stimulus current. Controlled depletion time may be required to stay above the finding of tetanic
contraction. b) After depleting K+ ions with a K+ ion-selective membrane in a micropipette tip for
4 minutes at id=1 µA, we observed a blocking of the muscle tetanic motion. There was no
contractile force measured after 4 min. of continuous K+ ion depletion. This finding may simulate
the blockage of unwanted spasticity of the neuromuscular unit in a pathologic state.
S-12
a)
b)
is = 4-20 µA in 0.2 µA step
tp= 1ms
f =1 Hz
14 µA
12 µA
15.2 µA
12 µA
30
20
with
ion concentration
modulation
without
Ion concentration
modulation
20
Graded blocking
at id=100nA
10
0
50
100
stimulation time [s]
Muscle contractile f orce [mN]
Muscle contractile f orce [mN]
is = 4-20 µA in 0.2 µA step
t p= 1ms
f =1 Hz
with
ion concentration
modulation
15
without
Ion concentration
modulation
10
Complete
blocking at
id=1µA
5
0
150
100
200
300
stimulation time [s]
Figure S-10. Nerve conduction blocking with cation depletion using Nafion membrane. a) On the
sciatic nerve, we could measure a decrease of the twitch amplitude by ~80% at the same
stimulus. b) After depleting cations for 5 min. at id = 1µA with Nafion membrane, no response
could be obtained even at significantly higher current pulses (is > 15.2A). This blocking effect,
however, was reversible. After bathing the nerve in a Ringer’s solution and waiting for 10 min.,
the nerve was responsive again. The configuration of the electrode is shown in Figure S-7a.
S-13
20µA
24µA
4µA
contractile force [mN]
5
4
3
threshold at 16.8µA
2
1
0
50
100
150
200
Stimulation time [s]
Figure S-11. After blocking, the nerve was put into Ringer’s bath for 10 min and became
electrically excitable again. An increase of the threshold value from previous is=12.2 µA to 16.8
µA was due to the fact that the nerve was not positioned exactly to the same position after
bathing in Ringer’s solution. (stimulation parameters: tp=1ms, f=1Hz)
S-14
Estimation of energy expenditure
To estimate the energy expenditure of our stimulation device, we analyzed a case with
a 50% reduction of the threshold from isb=10µA to isa=5µA. The condition for achieving
energy savings would be;
𝑖𝑑 2 𝑅1 𝑡𝑑 + (𝑖𝑠𝑎 )2 𝑅2 𝑡𝑝 ≤ (𝑖𝑠𝑏 )2 𝑅2 𝑡𝑝
id:
ion depletion current applied prior to stimulation
isa:
stimulation current after ion depletion
isb:
stimulation current before ion depletion
R1:
electrical resistance of the nerve fiber over 200µm gap between two opposed
center electrodes
R2:
electrical resistance of the nerve fiber over 10mm distance between cathode and
anode, R2 >> R1
td:
ion depletion time
tp:
total pulsing time for stimulation
If we use id=1µA for td=60s and assume the least optimum case with R1=R2, the total
pulsing time tp to reach the energy break-even point is 0.8s. Since we apply a pulse
width of 1ms, this scenario means that more than 800 single pulses are required for our
ion depletion–based method to be more energy-efficient than the conventional FES.
However, if we apply id=100nA for ion depletion which seemed to be sufficient, as
shown in Figure 4c, the energy break-even point is already reached after tp = 8ms.
Indeed, after 8 single pulses, our method is more energy-efficient that the current FES.
In addition, if we further lower down the ion depletion current id to 10nA which is
possible due to the small gap distance of 200µm between two opposed microfabricated
electrodes (in fact, we could push the gap size even further down by using a
S-15
photolithography-based microfabrication technique, to ~5µm, and decrease the ion
depletion current id, the break-even point would be reached at tp=80µs. So, even with a
shorter pulse width of 300µs, our method would be more energy-efficient than the
current FES method.
S-16
Imaging of Ca2+ ion concentration modulation
We performed direct imaging of the Ca2+ ion concentration change inside the nerve fiber
using confocal microscopy and a fluorescent Ca2+ indicator dye, fluo-4 NW, and
observed the Ca2+ ion concentration change as a function of ion depletion time id by
measuring the fluorescence intensity of the fluorescent dye (see Methods section). First,
we immersed a sciatic nerve into a Ca2+ indicator dye solution prepared according to
the protocol for non-adherent cells of Molecular Probes inc. for 2 hours prior to imaging
and then positioned the nerve between two 10mm long ITO electrodes (see Figure S12a). The gap between the electrodes was 300µm and the cathode was covered with a
~20µm thick Ca2+ ion-selective membrane. The probenecid concentration used was
10mM. Then, we applied ion-depletion current with a source meter (Keithley 2612)
between the electrodes for 1-3 min in 1 min intervals and recorded confocal images with
a 10x objective from the nerve through the transparent ITO electrodes after each ion
depletion time. The confocal imaging started below the ISM in the glass substrate (z=0
µm) and z height was increased at an interval of 6.17µm toward the nerve specimen.
For the analysis of intensity values, we used ImageJ software and averaged the
intensity values over the entire area of ISM (cathode) in each image. As shown in
Figure S-12b, the fluorescence intensity decreased gradually as a function of ion
depletion time td in the nerve fiber (80µm ≤ z ≤300µm), while the fluorescence intensity
inside the ~20µm thick membrane (z =60-80µm) increased due to a storage of ions in
the pores of the membrane. A typical frog’s sciatic nerve has a diameter of ~1mm, and
fluorescence intensity signal could be detected up to z=300µm. As the comparison of
two confocal images taken at z=111µm before and after ion depletion shows in Figure
S-17
S-12c, Ca2+ ion was depleted around the axons after td=3 min depletion at id=1µA. A
fast confocal imaging of Ca2+ ion concentration near ISM during electrical stimulation
would potentially reveal more information about the role of Ca2+ ion for electrical
stimulation.
a)
sciatic nerve
ITO electrode
covered with ISM
ITO electrode
+
-
z
confocal microscope
Fluorescence Intensity (A. U.)
b)
ISM Nerve
160
140
Ca2+ ion enrichment in ISM
120
100
Series1
td=0 min
80
Series2
td=1 min, id=1µA
Ca2+ ion
depletion
in nerve fiber
60
40
Series3
td=2 min, id=1µA
Series4
td=3 min, id=1µA
20
0
0
100
200
300
400
z height (µm)
c)
I
II
II
I
50 µm
Confocal image
before ion depletion
(td=0, z=117µm)
Confocal image
after ion depletion
at id=1µA for td=3 min (z=117µm)
Figure S-12. Confocal imaging of the sciatic nerve before and after Ca2+ ion depletion. a)
Experimental setup for confocal imaging of a frog’s sciatic nerve using transparent ITO
electrodes with z height starting just below the glass substrate. b) Measurement of the
fluorescence intensity with Ca2+ ion reporter dye showed a gradual depletion of Ca2+ ions in the
sciatic nerve bundle when ion depletion current id =1µA was applied from td=0 to 3 min (z >
80µm). In the membrane with a thickness of ~20µm (z=60-80 µm), however, the fluorescence
S-18
intensity increased due to the stored Ca2+ ions in the membrane. c) The areas marked with
circles I and II show the ion depletion through ISM. The confocal image taken at z=117µm
showed a decreased fluorescence intensity of Ca2+ reporter dye around the axons after applying
ion depletion current id=1µA for 3 min.
S-19
Preparation of nerves and gastrocnemius muscles The frog was handled along the
middle abdominal area for transport to the workspace and was beheaded with scissors.
The frog’s central nervous system was obliterated by inserting a pithing needle along
the spinal cord. Using forceps and dissecting scissors, the visceral area of the frog was
dissected for the sciatic nerve. The nerve could be found emerging from the vertebral
column as two spindles of creamy, white-colored cylinders with one on each side of the
column. Portions of the upper torso of the frog were then removed along with the other
organs and the lower region of the frog was deskinned. With blunt-tip forceps, a length
of thread was inserted to secure one of the two sciatic nerves. The surrounding tissues
and short branches were dissected further to free the nerve. To dissect the nerve along
the hip and thigh areas, an incision was made along the frog’s dorsal area to allow
passage of the nerve from the abdominal to the dorsal side of the frog. After exposing
the nerve along these areas, the nerve was dissected down through the thigh just above
the knee joint. The same procedure was employed in dissecting the other sciatic nerve.
During experiments, the nerves were kept moist with Amphibian Ringer’s solution
(Connecticut Valley Biological Supply) for preservation. For experiments, we preserved
the epineurium and perineurium around each nerve. To measure contractile force, we
tied a ligature around the lower part of the gastrocnemius muscle and dissected the
muscle away from the foot leaving as much of the Achilles tendon as possible. Next,
we removed the muscle by dissecting the superior portion of the knee joint with the
sciatic nerve attached to the muscle.
S-20