Neuromuscular Disruption with
Ultrashort Pulses
Karl H. Schoenbach, Ravindra
P. Joshi, Juergen F. Kolb
Center for Bioelectrics
Old Dominion University, Norfolk, VA
and
James A. Ross
Brooks City-Base
San Antonio, TX
NTAR VI 15 - 17 November 2004
Graylyn Conference Center, Winston-Salem, NC, USA
Experiments: Stunning of Aquatic
Nuisance Species
Charging Time Constant for Stunning of
Brine Shrimp: ≈10 Microseconds
[Corresponds to Optimum in Efficiency]
K.H. Schoenbach, F.E. Peterkin, R.W. Alden, and S. Beebe, “The effect of pulsed electric fields on
biological cells: experiments and applications,” IEEE Trans. Plasma Sci. 25, 284 (1997)
Charging Time Constant for Hydrazoan
is Less than One Microsecond
E n e r g y D e n s i t y 3( )m J / c m
800
5 m in . S tu n n in g
700
600
500
400
300
200
Salt Water Hydrazoan
100
0
1 0 -8
1 0 -7
1 0 -6
1 0 -5
P u ls e W id t h ( s e c )
A. Abou-Ghazala, S. Katsuki, K.H. Schoenbach, F.C. Dobbs, and K.R. Moreira, “Bacterial Decontamination
of Water by Means of Pulsed Corona Discharges,” IEEE Trans. Plasma Science 30, 1449 (2002).
The Effect of a DC Electric Field on
the Charge Distribution in the Cell
++ + +- +
+- +
+ -++
+++ + -
++++++++++++++ -
ρ
E
A dc or slowly varying electric field
applied to the cell causes accumulation of
charges at the outer membrane.
⇒ increase in voltage across outer
membrane
⇒ voltage gating – electroporation
of plasma membrane
Minimum External Electric Field
Hypothesis: any electrically stimulated
membrane effect is a threshold effect:
The voltage across a membrane needs to reach a critical value,
Vcr, in excess of the resting voltage, to obtain the effect.
Voltage Gating: ≈ 20 mV
Electroporation: ≈ 1 V
⇒Minimum external electric field for a cell with Φ = 10 µm:
Voltage Gating: ≈ 20 V/cm
Electroporation: ≈ 1 kV/cm
Required Pulse Duration
Charging time constant:
Membrane
Cytoplasm
τc = acm(ρo/2 + ρc)
a: cell radius
cm: membrane capacitance
ρc: cytoplasm resistivity
ρ0: medium resistivity
Typical values:
cm: 1 µF/cm2
ρc: 100 Ωcm
τc is on the order of 100 ns
Electric Field - Pulse Duration Range for
Voltage Induced Membrane Effects
The amplitude of the
applied electric field pulse
required to charge the
membrane to the critical
voltage for electroporation
(or voltage gating) is:
Ecr = Vcr/{fa[1–exp(-τ/τc)]}
The temperature rise
caused by the electrical
pulse is:
∆T ~ E2 τ
From Suspension to Tissue:
Calculation of Charging Time Constants for Tissue from
Measured Complex Dielectric Constants using Fourier Transform
K.R. Foster, “Thermal and Nonthermal Mechanisms of Interaction of Radio-Frequency
Energy with Biological Systems,” IEEE Trans. Plasma Science 28, 15 (2000)
Equivalent Circuit of Muscle Tissue
Muscle Tissue
(1cm×1cm ×1cm)
D.C. Salter, “Ouantifying skin disease and healing in vivo using electrical impedance measurement,” in Non-invasive
physiological measurements, vol. 1., P. Rolfe (ed.), Academic Press, New York, 1979.
Impulse Response of the Muscle
Voltage (V/V0)
1
α
β
0.1
0.01
1E-9
1E-8
1E-7
1E-6
Time (s)
V0: charging voltage
1E-5
1E-4
1E-3
α
β
100
10
10
1
1
1E-8
1E-7
1E-6
1E-5
Energy Density (A.U.)
Electric Field (E/Estim)
Strength-Duration Curves for
Voltage Gating
0.1
Time (s)
Estim= 20 V/cm ≅ 20 mV across membrane of Φ = 10 µm cell
Measured Strength Duration Curve
100 µs
Walter A. Rogers et al., IEEE Trans. Plasma Science 32, 1587 (2004)
How come that there is such a large
difference between our results and
published time constants?
ODU:
Computed:
Experimental:
approximately 1 µs
500 ns to 10 µs
Literature:*
approximately 0.2 ms for for nerve stimulation
approximately 2 ms for muscle stimulation
*(J.P. Reilly, “Applied Bioelectricity,” Springer Verlag, 1998)
Strength-Duration Curve for an Electrically
Excitable Tissue – Capacitive Component
Walter A. Rogers et al., IEEE Trans. Plasma
Science 32, 1587 (2004)
Displacement currents indicate large
capacitance in series to tissue.
Importance of Contacts
S. Grimnes and Φ.G.
Martinsen, Bioimpedance and
Bioelectricity, Academic
Press, San Diego, 2000,.
Contact
Influence of contacts is decreasing with increasing
frequency ⇒ SHORTER PULSES.
From Short Pulses to Ultrashort Pulses:
Considering Cell Substructures
HL-60 (leukemia cell)
Transient Electric Fields Inside the Cell
++ + +- +
+- +
+ -++
+++ + -
→
j
- (+)
- (+)
-(+)
-(+)
(-) +
(-)+
(-) +
(-)+
→
j
++++++++++++++ -
Monopolar, external electric fields
with duration less than the plasma
membrane charging time constant
cause accumulation of charges
at the outer membrane and,
temporarily, at the membranes
of cell substructures.
→
E
Monopolar External Pulse ⇒ Bipolar Pulse Across Membrane-Bound Substructure
Pulse Conditions for Intracellular
Electromanipulation
1. pulse risetime should be as short as possible to maximize
discharge current (voltage across organelle)
2. Pulse duration should be determined by charging time
time of target organelle (which is always less than the
plasma membrane charging time)
⇒ NANOSECOND PULSES
⇒ but, short pulse operation requires intense electric
fields: typically 10s to 100s of kV/cm;
⇒ energy is low because of ultrashort pulse duration
“Strength-Duration Curves”
∆T
By applying pulses
short compared to
the charging time of
the outer membrane
we are able to
electroporate not
only the plasma
membrane but also
organelles.
Cytoplasm
Nucleus
Acridine Orange (AO)
before
after
Normalized Intensity, log
10I(t)/I0
The effect on the nucleus (of HL60 cells)
control (18 cells)
60 ns (18 cells)
10 ns (18 cells)
1
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Time, t (s)
N. Chen, KH Schoenbach, JF Kolb, RJ Swanson, AL
Garner, J Yang, RP Joshi, and SJ Beebe, ”Leukemic
Cell Intracellular Responses to Nanosecond Electric
Fields,” Biochem. Biophys. Res. Comm. (BBRC)
317, 421 (2004).
Using PI Uptake as Indicator for Plasma
Membrane Integrity:
The plasma membrane does not seem to be affected by 10 ns
pulses and shows delayed uptake of PI for 60 ns pulses.
before
30 min after
10 ns
Cytoplasm
65 kV/c m
Nucleus
60 ns
26 kV/c m
Propidium Iodide (PI)
Ultrashort Pulses Mobilize Calcium From
Intracellular and Extracellular Sources
Jody A. White, Peter F.
Blackmore,
Karl
H.
Schoenbach, and Stephen
J. Beebe, “Stimulation of
Capacitive Calcium Entry
in
HL-60
Cells
by
Nanosecond
Pulsed
Electric Fields (nsPEF),” J
Biol Chem. 2004 Mar 16
[Epub ahead of print]
Calcium Release and Subsequent Immobilization
of Mammalian Cells
calcium responses to 300 ns pulse application
E. Stephen Buescher and Karl H. Schoenbach, “The Effects of Submicrosecond, High Intensity
Pulsed Electric Fields on Living Cells – Intracellular Electromanipulation,” IEEE Trans. on
Dielectrics and Electrical Insulation 10, 788 (2003)
Effect of Pulse Number and Repetition Rate
Experiments with hydrazoans
showed that with increasing
pulse repetition rate up to 50 Hz
the stunning efficiency increased
and stayed flat for higher
repetition rates.
This indicates recovery times of
approximately 20 ms.
A. Abou-Ghazala and K.H. Schoenbach,
“Biofouling Prevention with Pulsed Electric
Fields,” IEEE Trans. Plasma Science 28, 115
(2000).
Instrumentation:
Basic Pulsed Power Circuits
capacitive storage
discharge circuit
transmission line type
pulse generator
J. Mankowski and M. Kristiansen, “A review of short pulse generator
technology,” IEEE Trans. Plasma Science 28, 102 (2000)
Blumlein Pulse Generator
Pulse Duration:
Propagation time of an
electromagnetic wave
in the transmission
line
Pulse Amplitude: Full
applied voltage
Holds for a load being twice as
large as the transmission line
impedance.
Blumlein - Pulse Forming
Network (PFN)
Pulse Shape
2
Voltage, V (kV)
0
-2
-4
-6
-8
-10
-12
-500
0
500
1000
1500
Time, t (ns)
2000
2500
Pulse Generator with Pulse
Delivery System
Research Plan
Old Dominion University
• Design and Construction of Nanosecond Pulse
Generators and Pulse Delivery Systems
• Study of Contact Effects (including plasma
formation)
• Modeling of Nanosecond Pulse Effects on Tissues
Brooks City-Base
• Study of Neuromuscular Stimulation with
Nanosecond Pulses Using Animal Model
The Center for Bioelectrics
Old Dominion University – Eastern Virginia Medical School
Laboratories
Offices and Conference Room