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Calcium release induction in living cells by nanosecond electric pulses

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Calcium release induction in living cells by nanosecond electric pulses

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Content CALCIUM RELEASE INDUCTION IN LIVING CELLS BY NANOSECOND
ELECTRIC PULSES
by
Sarah Salemi
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirement for the Degree
MASTER OF SCIENCE
(PHYSICS)
May 2004
Copyright 2004 Sarah Salemi
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UMI Number: 1421792
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Dedication
To my beloved parents, and husband for their everlasting love and support.
ii
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Acknowledgements
I would like to thank my advisor and thesis committee chairman, Professor
Martin A. Gundersen, for his invaluable attention, guidance, and support
throughout my graduate studies. I would also like to extend my great appreciation
to the other thesis committee members, Professor Feinberg and Professor Hass. In
addition, I would like to thank Paul T. Vernier, and Dr. Laura Marcu for their
particular influence on my way of thinking. And finally, I would like to pay my
sincere thanks to my colleagues for their generous help throughout my graduate
career.
This work was supported by grants from the Air Force Office of Scientific
Research, the Army Research Office, and the Whitaker Foundation.
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Table of contents
DEDICATION.............................................................................................................................................ii
ACKNOWLEDGEMENTS.................................................................................................................... iii
LIST OF FIGURES................................................................................................................................... v
ABSTRACT...............................................................................................................................................vii
CHAPTER 1 INTRODUCTION........................................................................................................1
CHAPTER 2 ELECTRICAL PROPERTIES OF BIOLOGICAL CELLS.............................. 4
2.1 Physical model of A C E LL..............................................................................................................4
2.2 APPLYING EXTERNAL ELECTRIC FIELD...................................................................................6
CHAPTER 3 EXPERIMENTAL ENVIRONMENT...................................................................10
3.1 CELL CUTURE................................................................................................................................. 10
3.2 PULSE GENERATOR........................................................................................................................ 11
3.3 LOADING SL ID E ..............................................................................................................................12
3.4 IMAGING M E T H O D ........................................................................................................................13
CHAPTER 4 CALCIUM SIGNALING IN CELLS.................................................................... 16
4.1 INTRACELLULAR IONIZED CALCIUM DISTRIBUTION........................................................16
CHAPTER 5 RESULTS AND CONCLUSIONS......................................................................... 18
5.1 ELECTROPORATION B Y MICROSECOND PULSES................................................................19
5.2 CALCIUM RELEASE B Y NANOSECOND PU LSES................................................................... 21
REFERENCES ..................................................................................................................................... 28
iv
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List of Figures
Figure 1 An approximate physical model of a biological cell.................................... 5
Figure 2 Cross section and equivalent circuit of and living cell in suspension 6
Figure 3 Pore formation in plasma cell membrane after electroporation pulses were
applied............................................................................................................... 8
Figure 4 Voltages across the cytoplasmic membrane and mitochondrial membrane
after a pulse delivered. For a short pulse ( >10 ns) the mitochondrial
membrane charges faster than the cytoplasmic membrane in a few
nanosecond after the pulse delivered. With a pulse amplitude around 2
MV/m, a few nanosecond time delay can decrease to 1 nanosecond..........9
Figure 5 Micropulser and the shape of 30ns pulse......................................................12
Figure 6 Design schematic of the Microchamber......................................................13
Figure 7 Microchamber cross section..........................................................................13
Figure 8 Real-time electroperturbation microscopy system..................................... 15
Figure 9 Calcium distribution in the cell..................................................................... 17
Figure 10 Real-Time imaging of electroporation with microsecond pulses. After
cells were shocked with 5, 100 ps, 0.5 MV/m pulses, within 5s
propidium iodide enters a cell from the anodic pole................................20
Figure 11 Real-Time imaging of stained cells with Calcium Green and Propidium
Iodide after exposing to nanosecond pulses (10, 30ns, 2.5 MV/m).
Intensification of Calcium Green fluorescence indicates the raise of
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intracellular calcium. The absence of electroporation was shown by
observing no Propidium Iodide influx...................................................22
Figure 12 Intracellular calcium concentration of electroperturbed cells loaded
with Chelating agents, channel blockers, and transport inhibitors.
Normalized calcium green fluorescence intensity for shocked cells with
10 nanosecond pulses increases with the presence of chelating agent
(EGTA), calcium channel blockers (La3 +, Gd3 +, Verapamil), the
mitochondrial permeability transition inhibitor (Cycloscporin A) and
the mitochondrial Ca2 + channel blocker (Ruthenium Red).
Only the fluorescence of calcium green in cells with thapsigargin
which is the ER calcium pump inhibitor, increased less than the
fluorescence of control cells......................................................................24
Figure 13 Sodium flux entry into the cells after nanosecond and microsecond
pulses applied.(a) An evidence for sodium entry in electroporated cells
(b) No sodium entry in electroperturbed cells...........................................25
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ABSTRACT
Ultra-short (30 ns), high-field (2.5 MV/m), electric pulses raise the
concentration of cytosolic calcium in the cells. Here I report a collaboration for the
study of real-time imaging of the calcium release induction in the shocked Jurkat T
cells with nanosecond, megavolt-per-meter electric pulses project which was
founded and developed by a biophotonics group in the Electrophysics Department
of the University of Southern California. The cytosolic calcium increase is one of
the apoptosis indicators among the cells. Nanoelectropulses induce release of
calcium from intracellular compartments within seconds of pulse exposure. Ultra-
short, high field pulses effects on the cells are different from those associated with
electroporation. The longer pulses apply a few tens of kilovolts-per-meter electric
field for a few tens of microseconds. These pulses open pores in the cytoplasmic
membrane while because the duration of the nanosecond pulses is less than the
plasma membrane charging time they apply electric field across intracellular
structures without porating the cells.
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Chapter 1
Introduction
Ever since scientific fields such as physics, optics and electrical engineering
came together with biology and diagnostics, there has been a lot of concentration in
finding new physical techniques for healing methods to be more efficient and
economical. Currently, the treatment methods for disorders such as cancer are based
on eliminating or killing directly the unhealthy cells, as a result, many of the
healthy cells are injured unintentionally during the processes. Also, many of the
present healing methods have several serious side effects, so many patients would
face with a lot of pain and harms because of them.
1
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Recently a great deal of interest has been centered at an innovative therapeutic
technique which is based on the induction of apoptosis in unhealthy cells by using
ultra-short, high electric field pulses [6], This technique has the potential to be more
efficient and less harmful than existing healing methods. Apoptosis (“normal” or
“programmed” death of living cells) is the physiological process by which
unwanted or useless cells are eliminated during development or other normal
biological processes.
In this research we study the response of blood cancer cells (Human Jurkat T
lymphocytes) to megawatt electric fields. Although the fields are strong, they are
delivered in extremely short (nanosecond) pulses, so that the total energy
transferred to the cells is much less than a standard electroporation dose which
produces long living pores in the cells membrane [4, 5], The responses are fast and
measurable. In contrast to the mechanical disruption associated with
electroporation, the electroperturbation developed by ultra-fast, high field pulses
lead to responses that are not the results of physical damage to the external
membrane, but are instead consequences of intracellular disturbances of
physiological equilibria.
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In recent investigations many apoptosis symptoms such as caspase activation,
phosphatidylserine translocation [22] and DNA fragmentation and many others
have been observed in the cells shocked with ultra-short pulses. Knowledge of the
effects of ultra-short, high field pulses on different cells will potentially lead us to a
new era of selective non-chemical healing methods for many diseases. But, only
recently scientific observations are giving us some clues about the biological effect
of ultra-short pulses. Today a lot is still unknown and research is in progress.
In this thesis I will try to contribute to the study of the Ultra-short Pulsed
System Electroperturbation Technology (UPSET) effect on cytosolic calcium in
cancer cells which is one of the initial indicators of apoptosis in cells. Although the
apoptosis induction in cells is the ultimate target of our research [15], but here my
only purpose is to study one of the apoptotic symptoms after exposing the cells to
the ultra-short pulses.
3
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Chapter 2
Electrical properties of biological cells
In this chapter some basic and some more advanced facts about electric and
dielectric properties of living cells are introduced. The effects of applying electric
fields on cell functions are also presented.
2.1 Physical model of a cell
Although a real model of a living cell could be very complex, the effects of
electric fields on the cells can be explained by very simple models. One such
physical model of a cell with a membrane bound organelle is shown in Fig 1. The
present model, termed the single-shell model, is suitable for studying electrical
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properties of mammalian erythrocytes which do not contain the nucleus or
cytoplasmic organelle [2],
The function of the cell membrane is to isolate the cellular structural units.
The outer plasma membrane of a mammalian cell consists of a highly insulating
lipid bilayer approximately 5 nm in width. The cell membrane, however, is a leaky
dielectric where embedded proteins act as channels for ions.
»»
Cytoplasm
Figure 1 An approximate physical model of a biological cell.
This kind of cell configuration can be modeled by an equivalent electrical
circuit similar to what is shown in Fig.2. Rs and Cs respectively denote resistance
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Suspension
Cm
Re
Suspension Cell
Fig 2 Cross section and equivalent electrical circuit of a living cell in
suspension [9]
and capacitance of the suspension. Cm is the cell membrane’s capacitance and the
cytoplasmic resistance is shown by Rc.
2.2 Applying external electric field
As long as we consider the living cell as a spherical dielectric shell in a
conductive material, by the integration of Laplace equation the voltage induced
across the membrane (Vm) by ultra-short pulses with ultra-fast rise time, is as
follows:
Vm(t) = 3/2 E a cos f i [ 1 - e ' (t/ r » }] (1)
Where
(xmy l = (Rm Cm y ‘ + 2a,ae [aCm (oi+2ae)]'1 (2)
6
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E is the external electric field, a is the cell radius,^? is the position angle relative to
the electric field, t is the time after the initiation of the pulse, and zm is the
membrane charging time constant and ae and oL are conductivities of extracellular
and intracellular media, respectively [1,8].
Generally, the electrical pulses do not affect the intracellular membranes and
structures. However, if the pulse duration becomes very short (in orders of
nanosecond, which is less than the plasma membrane charging time), the electric
field can penetrate the outer membrane and affect subcellular structures [11]. This
allows modulation of cell functions without permanent damage (pore formation) to
the outer cell membrane. Vm higher than 0.3V, produced by external electric fields
from tens to hundreds of kilovolts-per-meter, make current-carrying pores in the
membrane through electroporation (Fig3) [14]. In this case the delivered energy to
the cytoplasmic membrane should be large enough (up to lOOkV/m for more than
lps) to induce the production of pores without killing the cells. In the best situation
of the exposure method there is a balance between pulse amplitude and frequency,
the conductivity, osmolality, and specific ionic composition of the medium and the
type and the physiological condition of the cell [20]. Creation of temporary
membrane pores with small electric pulses is a common tool in biotechnology for
7
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inserting drug and nucleic acid into the cells. If the external field is delivered in
short pulses (microseconds to milliseconds), cells can survive the disruption.
E
High Voltage
Cell Membrane Electro Pore
Fig 3 Pore formation in plasma cell membrane after electroporation pulses
were applied
The electric field which penetrates inside the cell develops a voltage across the
intracellular membranes (such as the nucleus and mitochondria membrane and
other organelles). For electric fields greater than 1 MV/m, the potentials across the
intracellular membranes can cause depolarization and pore formation [10, 16]
(Fig.4). The ultra-short, high field pulses can even reach into the nucleus,
mitochondria and other intracellular compartments and affect the nucleoplasm and
mitochondrial matrix. The extended electric field inside the cell causes cell
electroperturbations which can have significant consequences for the cell such as
triggering the apoptotic cascade [16, 6, 17, 7, 18].
8
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1.0
o 'S’
> | 0.9
4 ) 3
C 0 5
s s
Cytoplasmic
Mitochondrial
> _
V-
0.7
10 20 0
Time after Leading Pulse Edge (ns)
Fig 4 Voltages across the cytoplasmic membrane and mitochondrial
membrane after a pulse delivered. For a short pulse (>10 ns) the
mitochondrial membrane charges faster than the cytoplasmic
membrane in a few nanoseconds after the pulse delivered. With
a pulse amplitude around 2 MV/m, a few nanoseconds time delay
can decrease to 1 nanosecond. [21]
The vertical axis shows the normalized values of Log V.
9
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Chapter 3
Experimental Environment
In this chapter, the equipment used for the experiments is described.
3.1 Cell Culture
The type of cells we used for our experiments was Human Jurkat T
lymphocytes. These cells were grown in medium (RPMI 1640) which contains 10%
heat-inactivated fetal bovine serum (FBS), 2mM L-glutamine, 50 U/mL penicillin,
and 50 pg/mL streptomycin. These compounds provide nutrition and necessary
materials for cell to grow. To reach to the useful concentration of the cells (3 x 105
cell/mL), they were kept in the incubator with 37 °C temperature, 5% carbon
dioxide and humid atmosphere for 48 hours.
10
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3.2 Pulse Generator
The pulse generator is designed by Matthew Behrend for pulsing cells on a
microscope slide with intense pulsed electric fields for cell electroperturbation [12]
(Fig 5). It is very flexible in applying different electroperturbation pulse parameters
(i.e. rise time, amplitude and width). It is also capable of producing pulses with 25-
MHz maximum repetition rate. The micropulser was designed for both
miniaturization and flexibility for any pulse width as a single-MOSFET output
stage pulse generator.
The load for the micropulser is a microscope glass slide with attached gold
electrodes that form channels 100 pm wide, 10 pm deep, and more then 1 cm in
length (microchamber). Cells are pipetted into the channels while they are
suspended in liquid growth medium. The one such channel plus the cell suspension
within it present an electrical load of 37 ohms in parallel withl4 pF.
Real-time observation with an inverted fluorescence microscope is possible by
mounting the loaded micropulser with the slide on the stage of the microscope.
11
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D river circuit
a n d v "
divide
T tk Run: 5.00GS/S sample (Q0E
M O S F E T
2.220ns
M TYOmVtr IS Aug 2001
15.13:41
E n e rg y
s to r a g e
c a p a c ito r
Platinum i
slid e
Fig 5 Micropulser and the shape of 30 ns pulse
3.3 Loading Slide
The microchamber is transparent to the visible and ultraviolet light and it does
not fluoresce in the infrared to ultraviolet range. These properties of the
microchamber are important for the fluorescence microscopy of living cells [19].
The microchamber is constructed with microelectronic fabrication technology
[19]. A standard glass microscope slide was used as the substrate of the chamber.
After the microscope slide was etched, a thin gold film (100-250 nm) was deposited
on the sidewalls of the created channel to shape electrodes (Fig.7). The electrodes
were chosen to be gold to prevent any reaction between the conductive surfaces and
biological buffers or growth media. Since the lymphocytes cells that were used to
12
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be shocked in the channel have diameters up to 10 pm, the depth of the channel was
at least 10 pm. For applying fields up to 4 MV/m with the pulse generator (Fig.5),
which was mounted along with the microchamber on the stage of an inverted
microscope in a standard slide holder to the cells, the interelectrode distance was
100 pm or less (Fig.6). Also, in order to produce a uniform electric field, the
channel sidewalls were made smooth and perpendicular to the chamber floor. In
attempt to provide more cells for experimental observation four separated channels
were patterned on a glass slide (Fig 6).
Gold film
Electrode
>10 pn,
< 100 jtm
Transparent
Fig 6 Design schematic of
the Microchamber
Fig7 Microchamber cross section
3.4 Imaging Method
Real-Time observations of living cells were made possible by a Zeiss Axiovert
200 fluorescence microscope (Fig.8). Images were captured and analyzed with a La
Vision Pico Star HR12 and an AxioCam MRm camera. To observe the intracellular
13
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calcium, cells were stained with Calcium Green (CG; Molecular Probes; Xex= 480
nm, A ,c m =535 nm) at lpM in growth medium for lhour at 37 °C, washed in
Phosphate Buffered Saline (PBS), and resuspended in growth medium. When CG
binds to calcium, this indicator exhibit an increase in fluorescence emission
intensity. To observe the intracellular sodium, cells were stained with Sodium
Green (Molecular Probes; Xex = 480 nm, 7c m =535 nm) at 2 pM in growth medium
at 37 °C for 30 minutes. To visualize the cells membrane poration, propidium
iodide (PI; Molecular Probes; 7e x = 480 nm, =580 nm) was added to the
suspending medium. PI is impermeant to live cells, but stains electroporated cells
with red fluorescence, binding tightly to the nucleic acids in the cell. Once the dye
is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold.
14
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HAL Lamp
i H -
TP95104
Oscilloscope
hps35«a Timer
Synthesizer
HP8238
Output Power
xhr dc Power
Supply
Micro Pulser
Mercury
Lamp
- f ~ -
Microscope
c h a m b e r
3
♦ I ;* i .
CCD C am era
. r t t " Fll,er ,
■ - s t f i Sw itch for C a m e ra s ,
# ■ # ... i
P C I
Pico S tar
C a m e ra
Fig. 8 Real-time electroperturbation microscopy system.
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Chapter 4
Calcium Signaling in Cells
By the time the research on calcium and apoptosis accelerated [3], ionized
calcium [Ca2 + ] had been recognized as a fundamental intracellular messenger. Thus,
a lot of studies have been focused on the signaling mechanisms that connect the
variation of calcium to the induction of apoptosis. Although a great deal of work
needs to be done in this area, many links which couple calcium to the different
pathways of apoptosis have been discovered.
4.1 Intracellular Ionized Calcium Distribution
The cytoplasmic calcium concentration ([Ca2 + ]c ) is about 100 nM, which is
very low relative to the extracellular concentration ([Ca2 + ]Ec)- The calcium
concentration in the nuclear matrix ([Ca2 + ]n ) and in the mitochondrial matrix
16
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([Ca2 + ]m ) is similar to the cytoplasmic concentration. But there are other
intracellular compartments (like endoplasmic reticulum (ER)) which have a large
concentration of [Ca2 + ] compared to the cytoplasmic calcium. The cell uses these
compartments as calcium storage for changing the [Ca2 + ]c, [Ca2 + ]m and [Ca2 + ]„
which represent some of the most common intracellular signals. Also, calcium can
enter the cell through the plasma membrane Ca2 + channels which can be opened or
closed based on the intracellular calcium concentration and electrical gradients of
membrane. In Fig. 9 the calcium distribution inside the cell has been shown
schematically.
Mltochondria'^v [Ca2 + ]: 500 pM
[Ca2 +l:100nM
ER
fCa2 +l: 100-1000 uM
Fig.9 Calcium distribution in the cell
17
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Chapter 5
Results and Conclusions
In the many of current studies, scientists concerned with harmful effects of
external electric fields on biological systems usually narrow their studies to either
low-power (miliwatt) DC or AC stimuli or medium-power (watt to kilowatt) pulsed
fields used for electroporation. Electroporation is the formation of microscopic,
current-carrying pores in a lipid bilayer exposed to a large transmembrane potential.
The pores are long lived, often surviving in the membrane for up to several minutes
and providing pathways for the movement of ions, drugs, and even DNA fragments
into the cells. These properties have made electroporation a common tool in
biotechnology, and the medical applications of electroporation are now being
realized.
18
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Instead here we describe the response of mammalian cells to megawatt electric
field which is delivered by the extremely short pulses (nanosecond), so that the total
energy transferred to the cells is much less than a standard electroporation.
5.1 Electroporation by Microsecond pulses
In order to demonstrate the behavior of cells which were exposed to the
microsecond electric pulses, five 100 ps, 0.5 MV/m pulses were delivered to the
Jurkat T lymphoblasts in the growth medium. The medium had 5pg/mL propidium
iodide (fluorochrome). In non-shocked cells the cell membranes are not permeable
to propidium iodide, so the fluorochrome stays in the medium. As it is shown in
Fig. 10, within seconds after the pulses applied to the cells a flux of propidium
iodide runs into the cell from a point near the anode pole. This event is a
demonstration of pore formation in the cytoplasmic membrane by microsecond
pulses with suitable amplitude (100 ps, 0.5MV/m).
19
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20 25 30 35
Time After Pulse ( s ) --------*
(5 pulses, 0.5 MV/m, 100 |is)
Fig. 10 Real-Time imaging of electroporation with microsecond pulses. After
the cells were shocked with five 100 ps, 0.5 MV/m pulses, within 5
seconds propidium iodide enters the cell from the anodic pole.
20
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5.2 Calcium release by nanosecond pulses
In order to observe the effect of nanosecond pulses on the cytosolic calcium
concentration inside the cells, a calcium sensitive fluorochrome (Calcium Green)
was used to stain the cells. Also, 5pg/mL propidium iodide was added to the
growth medium to investigate possible damage to the cell’s membrane by the
nanosecond pulses. Most of the staining procedures were prepared by Tom Vernier.
As is shown in Fig. 11, after applying ten 30ns, 2.5 MV/m pulses to the cells, no
influx of propidium iodide into the cells was observed. This result conforms that
the nanosecond pulses do not have pore-forming effects on the cells membrane.
Instead the same cells show a significant intensification of Calcium Green
fluorescence in a few seconds after pulse exposure (Fig. 11). This Calcium Green
fluorescence increase indicates a rise in calcium ion concentration all over the cell.
Although the nanosecond pulses induce a potential around IV across the cell
membrane (which is enough for the membrane breakdown), no permanent pore
formation has been observed in the cells membrane after exposing to the pulses (no
propidium iodide influx into the cell). However there is a possibility that
nanosecond pulses
21
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BrfcjM
fluid
L j>
r
r
Gildum
G ram
Pniptsiiim
lo d k fs
0 15 30
Time After Pulse (s) ->
(10 pulses, 2.5 N V/m , 30 ns)
Fig. 11 Real-Time imaging of stained cells with Calcium Green and Propidium
Iodide after exposing to nanosecond pulses (10,30ns, 2.5 MV/m).
Intensification of Calcium Green fluorescence indicates the raise of
intracellular calcium. The absence of electroporation was shown by
observing no Propidium Iodide influx.
22
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opened small (nanometer) pores on the cell membrane for a very short
(nanosecond) time which do not allow the entry of the large and slowly diffusing
propidium iodide molecules. Since there is a very large calcium concentration
gradient between cytoplasm and the medium (in the order of a thousand times) the
calcium ions can pass through these small pores to the inside of the cell and cause
the observed increase in intracellular calcium concentration.
To verify the validity of this hypothesis, a calcium chelat agent EGTA was
added to the growth medium. Calcium entry from the medium into the cells should
be reduced by EGTA in 10-fold excess. Also, to investigate the role of voltage-
dependent calcium channels, which are placed in the plasma membrane, several
calcium channel-blockers La3 + , Gd3 + were separately added to the cells suspension.
Calcium entry from the external medium through calcium channels should decrease
with the presence of these calcium channel-blockers. In all these cases no
significant Calcium Green fluorescence intensification reduction was observed
(Fig. 12). All these observations support the hypothesis that the source of
intracellular calcium increase by nanosecond pulses is not from outside of the cell.
Another way to support this hypothesis is to look for the change in the
cytoplasmic sodium concentration. Since there is a sodium concentration gradient
23
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y # s ■ ? / / / /
s / f /
Intracellular calcium concentration of electroperturbed cells loaded
with chelating agents, channel blockers, and transport inhibitors.
Normalized calcium green fluorescence intensity for shocked cells with
10 nanosecond pulses increases with the presence of chelating agent
(EGTA), calcium channel blockers (La3 +, Gd3 +, Verapamil), the
mitochondrial permeability transition inhibitor (Cycloscporin A) and
the mitochondrial Ca2 + channel blocker (Ruthenium Red).
Only the fluorescence of calcium green in cells with thapsigargin
which is the ER calcium pump inhibitor, increased less than the
fluorescence of control cells.
between outside and inside of the cells (in the order of 10), if nanosecond pulses
cause nanopore formation and consequently allow penetration of the calcium ions
into the cells, sodium ions might also be expected to enter the cells along with the
calcium ions.
24
O
Fig. 12
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To visualize the possible penetration of sodium ions through the nanopore
openings, Sodium Green loaded cells were exposed separately to the
electroporation pulses (Microseconds) and UPSET pulses (Nanoseconds). In the
former case a measurable sodium influx produced while in the latter case no
significant sodium entry was observed (Fig. 13)[22],
Electroporation
1 0 Pulses
th i* m
Electroperturbation
1#
1 Pulse
2 Pulses 5 Pulses Pulses
■ A ,.— ..............» ...............i..............i............
1# »
Fig.13 Sodium flux entry into the
cells after nanosecond and
microsecond pulses applied.
(a) An evidence for sodium
entry in electroporated cells
(b) No sodium entry in
electroperturbed cells.
25
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The fact that the mitochondrial Ca2 + does not have a significant contribution to
intracellular calcium rise by nanosecond pulses was verified by the mitochondrial
permeability transition inhibitor, cyclosporin A, and the mitochondrial Ca2 +
uniporter and voltage-dependent calcium channel blocker, ruthenium red (Fig. 12).
After using different chelating agents, calcium channel blockers and transition
inhibitors, it is clear that neither medium nor mitochondria are the sources of
calcium rise in the electroperturbed cells. But there are still other calcium
compartments in the cells, one of the best known of which is endoplasmic
reticulum (ER). To verify the dependence of calcium raise in electroperturbed cells
to the operation of the calcium stores of the ER, thapsigargin, which inhibits uptake
of calcium into compartments of the endoplasmic reticulum, was loaded with the
stained cells. As it is shown in Fig. 12 the fluorescence increase of calcium green in
latter cells is lower than the fluorescence increase in control cells. This shows that
pulse-induced intracellular calcium increase is dependent on the operation of
calcium stores of the ER.
In all of our experiments we have exposed cells between two parallel
electrodes. This simplified pulse delivery system is just appropriate for
experimental conditions. If this technology desired to be more practical, it is
26
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necessitate to develop a system of pulse delivery devices, that can be used for
different types of cells (such as attached cells to the substrates; Glioma) and tissues
in living organisms.
In conclusion, since the intracellular calcium signals have a numerous role in
cell functions at all levels, more knowledge and better understanding of its
responses to different stimuli, will give us a precious opportunity to control many
cellular malfunctions. By gathering and analyzing all the data in this study, our
challenge was to understand, characterize and identify those responses. As
scientists we know that the present study is only a pioneering effort and a lot of
biological and electrical investigations are required before our observations can be
placed in a secure biological frame work.
27
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Creator Salemi, Sarah (author)

Core Title Calcium release induction in living cells by nanosecond electric pulses

School Graduate School

Degree Master of Science

Degree Program Physics

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,biophysics, general,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Gundersen, Martin A. (committee chair), Feinberg, Jack (committee member), Hass (Prof.) (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-316127

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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