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
