Bioelectromagnetics Supplement 1:161-178 (1992)
Microelectrode Measurements of Low Frequency Electric Field Effects in Cells and Tissues Kenneth
J. McLeod
Musculo-Skeletal Research Laboratory, Department of Orthopaedics, State University of New York, Stony Brook, New York The average intensities of electric fields induced into tissue can be calculated if the morphology and conductivities ofthe tissue are known, and such valucs provide one estimate of dosage for a given field exposure level. However, the rnicroanatoniical structures of living tissue, which include gap junctions, tight junctions, highly charged cell coats, and extracellular matrices, as well as complex cell shapes, precludes a detailed characterization of the field and current distribution near the cells which are actually responding to the electric fields. This suggests that a more useful electric field dose metric may be one based on an induced physical effect on the cells. Electric fields have at least three distinct physical effects on cells: the normal plasma membrane potential will be altered; the ionic currents and ion distributions at the extracellular surface will be modified; and mechanical forces will be imposed at the cell surface. Each of these effects can, in principle, be measured through the application of specific microelectrode techniques. Here, the feasibility of using various intracellular and extracellular recording methods to obtain dosimetric values, as well as the contribution these measurements could make to our understanding of electric field interactions with biological tissue, are discussed. o 1992 Wiky-l.i\b. Inc.
Key words: microelectrodes, transmembrane potentials, ionic currents, electromechanical forces, atomic force microscopy
INTRODUCTION Low frequency electromagnetic fields have been shown capable of altering the growth and phenotypic expression of cells under both culture conditions and within living tissue [Polk and Postow, 19861. Combined with recent epidemiological reports [Tomenius, 1986; Savitz et al., 19881, these observations have led to concerns as to whether inadvertent residential or occupational exposure could be detrimental to the population. In order to determine the extent to which low frequency fields could be interacting with cellular processes in people it will first be necessary to establish definitions of dosage. Dosimetric definitions would permit comparison of the experimental results obtained by different research groups using various
Address reprint requests to Kenneth J. McLeod, Musculo-Skeletal Research Laboratory, Department of Orthopaedics, S.U.N.Y., Stony Brook, NY 11794-81 81.
0 1992 Wiley-Liss, Inc.
162
McLeod
experimental protocols, and as well, would provide the ability to scale these experimental exposures to the conditions associated with human exposure. A fundamental problem in low frequency electromagnetic field dosimetry is that it has not yet been determined whether the reported biological responses are due to the magnetic field, the electric field, or perhaps specific combinations of these two components of the field. However, while magnetic fields may have either independent or synergistic effects on biological tissue [Marron et al., 19881, electric fields are definitely capable of initiating biological responses [McLeod et al., 1987; Fitzsimmons et al., 19891. For the case of pure electric field exposures the solution to the dosimetry problem seems obvious, the response of the cells in a tissue is expected to correlate with the electric field intensity in the tissue. One might expect to obtain a useful estimate of the electric field distribution in a tissue through calculation, but such calculations are not at all straightforward, particularly when the electric fields arise through induction by a time changing magnetic flux. Even if tissues could be assumed to be of uniform conductivity and permittivity, when the gross anatomical structure is considered, the intricate three dimensional geometry invariably demands a complex numerical solution [Polk and Song, 19901. Moreover, when the microanatomy of the tissue is included, it becomes clear that any meaningful calculation of the electric field distributions near cells is impractical. MICROANATOMICAL INFLUENCES ON ELECTRIC FIELD DISTRIBUTIONS
Three morphologic characteristics of biological cells and ensembles of cells ensure that any attempt to calculate electric field distributions at the cellular level will provide questionable results. These include the existence of intercellular junctions (tight junctions and gap junctions), the charged nature of the pericellular space (glycocalyx), and the complex geometries (size and shape) that individual cells can attain. Though the cells of some major tissues remain anatomically disconnected under normal physiologic conditions (e.g., skeletal muscle cells), most cell types have been found to connect to their neighboring cells through at least one of three different types of junctions; desmosomes, tight junctions, and gap junctions [Larsen and Wert, 19881. Desmosomes are a type of adhering junction which enable cells to form structural units. While these junctions can encircle a cell, they consist of a coarse filamentous structure which would have electrical properties similar to that of the extracellular matrix, and therefore, would not be expected to have any unique effect on electric field distributions [Weiss, 19831. Tight junctions, however, would be expected to affect field distributions. These junctions form between adjacent cells and are sufficiently narrow as to eliminate the intercellular space, effectively producing a continuous sheet of cells and providing tissues with the ability to operate as a selective barrier [Gonzalez-Mariscal et al., 19901. For any protein or ion to pass across this sheet of cells (e.g., epithelium or endothelium) it is necessary for the cells to actively transport the molecules across the cell membrane [Milton and Knutson, 19901. The transverse electrical conductivity of an ensemble of cells forming tight junctions approaches that of the cell plasma membrane, significantly limiting conduction pathways, and correspondingly, affecting electric field distributions.
Microelectrode Measurements of Field Effects
163
Similarly, gap junctions may affect field distributions. Gap junctions are a type of communicating junction that forms between cells [Brink and Dewey, 19811, permitting the easy passage of small molecules (those less than 1,000-1,500 daltons) from one cell interior to another [Schwarzmann et al., 19811. These junctions allow metabolic cooperation between cells but also confer electrical coupling between large groups of cells. Gap junctions are formed by a group of several proteins that go from one cell to the other and are arranged to form a channel. Individually, these structures, or connexons, have conductivities of only about 100 picosiemens [Neyton and Trautmann, 1985; Veenstra and DeHaan, 19861, but the gap junctional areas of cells may contain anywhere from tens to thousands of connexons. As a result, the net coupling conductivity of two cells may vary from a few thousand picoSiemens to several microSiemens. In addition, the cells are capable of modulating the state of these connexons, so gap junction conductivities are not static values but can change dynamically depending on the condition of the cell [Fraser et al., 1987; Maldonado et al., 19881. Gap junctions, therefore, provide a dynamically varying conductivity path through ensembles of cells which may in some cases dominate the overall conductivity of the tissue. Moreover, this type of junction is ubiquitous, permitting cell communication not only between genotypically similar cells but between almost any group of apposing cells [Segal and Duling, 1986; Wade et al., 1986; Hobbie et al., 1987; Beny and Gribi, 1989; Ushiyama, 19891. A second aspect of cell morphology which may critically affect the electric field distribution in tissue is the variation in the properties of the extracellular matrix. The extracellular space of most tissues contains substantial amounts of uncharged fibrous proteins (predominantly collagen, but also fibronectin and laminin) embedded in a gel of hydroscopic proteoglycans [Hynes, 19791. While some tissues (e.g., skin and bone) consist mostly of this extracellular matrix, other cells (neurons in the brain and circulating cells in the blood) are associated with almost no such material. However, all cells maintain, immediately adjacent to the cell membrane, a cell coat or glycocalyx which is a region rich in carbohydrates [Alberts et al., 19831. Though the role of the glycocalyx is not well understood, this structure has a distinct electrical consequence. Attached to the carbohydrate side chains are sialic acid residues which give the surface of the cell a net negative charge. The magnitude of this charge distribution is dependent on the thickness of the glycocalyx which can vary from very thin (300 A) on circulating cells, to quite thick (10,000 A) in some epithelial tissues. This charge density has been estimated to be between .01 and . I coulomb/m2depending on the cell type and the thickness of the coat [Vargas et al., 1989; Ohshima and Kondo, 19911. The effects of this charged layer is to significantly alter ionic distributions [Chew, 19841, which, in addition to altering important metabolic factors such as the pH at the surface of the cell, alters the conductivity and dielectric properties near the cell surface. The final morphologic consideration which precludes accurate calculation of fields and currents in tissues is the enormous variation in cellular size and shape. Consider, for example, the cells involved in the healing of a bone fracture [Brand and Rubin, 19871, an area of intense investigation from the perspective of using exogenous induced fields to enhance the healing process [Brighton and McCluskey, 19861. A primary event is the invasion of platelets to inhibit hemorrhage. These are circulating cell fragments which are of discoid shape and are extremely small in size, being only about 2 to 4 pm in diameter when they begin to invade the site
164
McLeod
of a broken blood vessel. On contact with the subendothelial tissue, the platelets transform into a spherical shape, pseudopodia begin to form, and the platelets aggregate into a large hemostatic plug. In contrast, the muscle cells which are torn during a fracture are densely packed spindle shaped cells that are at the opposite extreme in size. Depending on the specific muscle body affected, these cells can be up to 100 pm in diameter and tens of centimeters i n length. Finally, the bone cells which will eventually remodel the healed bone are averaged sized cells (10 pn i n diameter) which are sparsely distributed within the bone matrix. But osteocytes can have up to 40 or more osteocytic processes which interconnect the cell to neighboring cells up to 100 pn distant. While it may not be possible to determine field intensity at the cell surfaces in a tissue, there exists the possibility that average field intensities may accurately reflect cellular responses. Unfortunately, experimental results do not appear to confirm this hypothesis. Our recent in vitro investigations can be used to illustrate how different cellular responses can arise even in a preparation where physical factors have been carefully controlled to ensure that electromagnetic fields in the bulk media are well defined (Fig. 1 ) . In this preparation, a uniform magnetic field is used to induce an electric field into square well culture dishes in which cloned rat osteosarcoma cells (ROS 17/23) are growing in monolayer on the bottom of the dishes. Despite the electromagnetic field exposure being constant in all experimental trials (30Hz, 1.8 mT flux inducing a 5 pV/cm RMS electric field), and observations showing the cells at all densities to be in the same biological state, a cellular response to field exposure is clearly evident at intermediate cell densities but disappears as the cell density is increased or decreased. How the physical changes in the cell population alter the interaction of the electromagnetic fields with the cells is not yet known, but it is clear that even the accurate calculations of average field intensities that can be made for a well controlled in vitro experiment are inadequate to describe the effective dose of an electric field exposure. PHYSICAL RESPONSES TO ELECTRIC FIELDS
Review of tissue microanatomy leads to the conclusion that detailed calculations of the electric field intensities and current densities near the cells, which are the actual entities responding to the fields, are not practical. And, the experimental results reported above indicate that a correlation between cellular response and average field intensity will not always exist. These observations identify the need for an alternative approach to establishing dosimetric definitions and one possibility is to base dosage estimates on measurements of the physical effects of the field exposure. The dosimetry issue then becomes a question of identifying an appropriate physical effect at the cellular level which can be measured. The ideal dosimetric parameter would relate directly to the mechanism of field interaction with the cell. Unfortunately, a viable interaction mechanism has not yet been established, so one is left to consider the various physical responses of cells to low frequency electric fields and try to identify the most appropriate physical parameter which may reflect dosage. At the low frequencies and intensities associated with reported field effects on living tissue there exist three important processes which are associated with measurable parameters (Fig. 2). First, current flow will result in charge redistribution at boundaries between materials of differing con-
Microelectrode Measurements of Field Effects
165
Rat osteosarcoma cells
ROS 17/2.8
Micromole p-nitrophenol per 106 cells
---------------
per minute 0
30 Hz exposure inducing 5 pV/cm rms electric field
-
2 30
5 60
Cell Number per cm2
150
1 300
(thousands)
Fig. I.Changes in alkaline phosphotase activity of rat osteoblast-like cells (ROS 1712.8) in response to uniform 30 Hz magnetic flux of 1.8 mT inducing an average electric field intensity of 5 Vlcm i n square culture dishes. Field exposure causes a significant enhancement of enzyme activity at the intermediate cell densities evaluated, but caused no enhancement at the lower cell density used, and an insignificant increase at the highest cell density. At 3 lo4 cells/cm', the cells are sparsely distributed, at lo5cells/cm' the cells form a confluent monolayer, and at 3 1O-r cells/cmz the cells are densely packed.
ductivities with a corresponding development of transboundary potentials. Second, surface current flow will result in the translation of surface bound charges and cause changes in ion distributions at surfaces, resulting in the modification of diffusion gradients and currents. Finally, Coulombic and polarization forces will be imposed on any structure supporting either net charge or dipoles. Transmembrane Potential Changes The most obvious and investigated effect of electric fields on cells and tissues is the development of large electric fields across relatively nonconducting membranes. This effect can be demonstrated by calculating the perturbation of the transmembrane potential of an individual spherical cell in a uniform electric field. Assuming the frequencies of interest are those where the displacement current across the membrane can be considered to be negligible, that is, for frequencies f,, where
166
McLeod
Fig. 2. Three physical responses predominate when biological tissue is exposed to low frequency electric fields. The transmembrane potential of the cells is perturbed, conduction and displacement currents are established in the extracellular space, and electromechanical forces are developed within the extracellular matrix and cell coat (glycocalyx) and transmitted to the cell surface, and perhaps to the interior of the cell.
f,
Microelectrode Measurements of Low Frequency Electric Field Effects in Cells and Tissues Kenneth
J. McLeod
Musculo-Skeletal Research Laboratory, Department of Orthopaedics, State University of New York, Stony Brook, New York The average intensities of electric fields induced into tissue can be calculated if the morphology and conductivities ofthe tissue are known, and such valucs provide one estimate of dosage for a given field exposure level. However, the rnicroanatoniical structures of living tissue, which include gap junctions, tight junctions, highly charged cell coats, and extracellular matrices, as well as complex cell shapes, precludes a detailed characterization of the field and current distribution near the cells which are actually responding to the electric fields. This suggests that a more useful electric field dose metric may be one based on an induced physical effect on the cells. Electric fields have at least three distinct physical effects on cells: the normal plasma membrane potential will be altered; the ionic currents and ion distributions at the extracellular surface will be modified; and mechanical forces will be imposed at the cell surface. Each of these effects can, in principle, be measured through the application of specific microelectrode techniques. Here, the feasibility of using various intracellular and extracellular recording methods to obtain dosimetric values, as well as the contribution these measurements could make to our understanding of electric field interactions with biological tissue, are discussed. o 1992 Wiky-l.i\b. Inc.
Key words: microelectrodes, transmembrane potentials, ionic currents, electromechanical forces, atomic force microscopy
INTRODUCTION Low frequency electromagnetic fields have been shown capable of altering the growth and phenotypic expression of cells under both culture conditions and within living tissue [Polk and Postow, 19861. Combined with recent epidemiological reports [Tomenius, 1986; Savitz et al., 19881, these observations have led to concerns as to whether inadvertent residential or occupational exposure could be detrimental to the population. In order to determine the extent to which low frequency fields could be interacting with cellular processes in people it will first be necessary to establish definitions of dosage. Dosimetric definitions would permit comparison of the experimental results obtained by different research groups using various
Address reprint requests to Kenneth J. McLeod, Musculo-Skeletal Research Laboratory, Department of Orthopaedics, S.U.N.Y., Stony Brook, NY 11794-81 81.
0 1992 Wiley-Liss, Inc.
162
McLeod
experimental protocols, and as well, would provide the ability to scale these experimental exposures to the conditions associated with human exposure. A fundamental problem in low frequency electromagnetic field dosimetry is that it has not yet been determined whether the reported biological responses are due to the magnetic field, the electric field, or perhaps specific combinations of these two components of the field. However, while magnetic fields may have either independent or synergistic effects on biological tissue [Marron et al., 19881, electric fields are definitely capable of initiating biological responses [McLeod et al., 1987; Fitzsimmons et al., 19891. For the case of pure electric field exposures the solution to the dosimetry problem seems obvious, the response of the cells in a tissue is expected to correlate with the electric field intensity in the tissue. One might expect to obtain a useful estimate of the electric field distribution in a tissue through calculation, but such calculations are not at all straightforward, particularly when the electric fields arise through induction by a time changing magnetic flux. Even if tissues could be assumed to be of uniform conductivity and permittivity, when the gross anatomical structure is considered, the intricate three dimensional geometry invariably demands a complex numerical solution [Polk and Song, 19901. Moreover, when the microanatomy of the tissue is included, it becomes clear that any meaningful calculation of the electric field distributions near cells is impractical. MICROANATOMICAL INFLUENCES ON ELECTRIC FIELD DISTRIBUTIONS
Three morphologic characteristics of biological cells and ensembles of cells ensure that any attempt to calculate electric field distributions at the cellular level will provide questionable results. These include the existence of intercellular junctions (tight junctions and gap junctions), the charged nature of the pericellular space (glycocalyx), and the complex geometries (size and shape) that individual cells can attain. Though the cells of some major tissues remain anatomically disconnected under normal physiologic conditions (e.g., skeletal muscle cells), most cell types have been found to connect to their neighboring cells through at least one of three different types of junctions; desmosomes, tight junctions, and gap junctions [Larsen and Wert, 19881. Desmosomes are a type of adhering junction which enable cells to form structural units. While these junctions can encircle a cell, they consist of a coarse filamentous structure which would have electrical properties similar to that of the extracellular matrix, and therefore, would not be expected to have any unique effect on electric field distributions [Weiss, 19831. Tight junctions, however, would be expected to affect field distributions. These junctions form between adjacent cells and are sufficiently narrow as to eliminate the intercellular space, effectively producing a continuous sheet of cells and providing tissues with the ability to operate as a selective barrier [Gonzalez-Mariscal et al., 19901. For any protein or ion to pass across this sheet of cells (e.g., epithelium or endothelium) it is necessary for the cells to actively transport the molecules across the cell membrane [Milton and Knutson, 19901. The transverse electrical conductivity of an ensemble of cells forming tight junctions approaches that of the cell plasma membrane, significantly limiting conduction pathways, and correspondingly, affecting electric field distributions.
Microelectrode Measurements of Field Effects
163
Similarly, gap junctions may affect field distributions. Gap junctions are a type of communicating junction that forms between cells [Brink and Dewey, 19811, permitting the easy passage of small molecules (those less than 1,000-1,500 daltons) from one cell interior to another [Schwarzmann et al., 19811. These junctions allow metabolic cooperation between cells but also confer electrical coupling between large groups of cells. Gap junctions are formed by a group of several proteins that go from one cell to the other and are arranged to form a channel. Individually, these structures, or connexons, have conductivities of only about 100 picosiemens [Neyton and Trautmann, 1985; Veenstra and DeHaan, 19861, but the gap junctional areas of cells may contain anywhere from tens to thousands of connexons. As a result, the net coupling conductivity of two cells may vary from a few thousand picoSiemens to several microSiemens. In addition, the cells are capable of modulating the state of these connexons, so gap junction conductivities are not static values but can change dynamically depending on the condition of the cell [Fraser et al., 1987; Maldonado et al., 19881. Gap junctions, therefore, provide a dynamically varying conductivity path through ensembles of cells which may in some cases dominate the overall conductivity of the tissue. Moreover, this type of junction is ubiquitous, permitting cell communication not only between genotypically similar cells but between almost any group of apposing cells [Segal and Duling, 1986; Wade et al., 1986; Hobbie et al., 1987; Beny and Gribi, 1989; Ushiyama, 19891. A second aspect of cell morphology which may critically affect the electric field distribution in tissue is the variation in the properties of the extracellular matrix. The extracellular space of most tissues contains substantial amounts of uncharged fibrous proteins (predominantly collagen, but also fibronectin and laminin) embedded in a gel of hydroscopic proteoglycans [Hynes, 19791. While some tissues (e.g., skin and bone) consist mostly of this extracellular matrix, other cells (neurons in the brain and circulating cells in the blood) are associated with almost no such material. However, all cells maintain, immediately adjacent to the cell membrane, a cell coat or glycocalyx which is a region rich in carbohydrates [Alberts et al., 19831. Though the role of the glycocalyx is not well understood, this structure has a distinct electrical consequence. Attached to the carbohydrate side chains are sialic acid residues which give the surface of the cell a net negative charge. The magnitude of this charge distribution is dependent on the thickness of the glycocalyx which can vary from very thin (300 A) on circulating cells, to quite thick (10,000 A) in some epithelial tissues. This charge density has been estimated to be between .01 and . I coulomb/m2depending on the cell type and the thickness of the coat [Vargas et al., 1989; Ohshima and Kondo, 19911. The effects of this charged layer is to significantly alter ionic distributions [Chew, 19841, which, in addition to altering important metabolic factors such as the pH at the surface of the cell, alters the conductivity and dielectric properties near the cell surface. The final morphologic consideration which precludes accurate calculation of fields and currents in tissues is the enormous variation in cellular size and shape. Consider, for example, the cells involved in the healing of a bone fracture [Brand and Rubin, 19871, an area of intense investigation from the perspective of using exogenous induced fields to enhance the healing process [Brighton and McCluskey, 19861. A primary event is the invasion of platelets to inhibit hemorrhage. These are circulating cell fragments which are of discoid shape and are extremely small in size, being only about 2 to 4 pm in diameter when they begin to invade the site
164
McLeod
of a broken blood vessel. On contact with the subendothelial tissue, the platelets transform into a spherical shape, pseudopodia begin to form, and the platelets aggregate into a large hemostatic plug. In contrast, the muscle cells which are torn during a fracture are densely packed spindle shaped cells that are at the opposite extreme in size. Depending on the specific muscle body affected, these cells can be up to 100 pm in diameter and tens of centimeters i n length. Finally, the bone cells which will eventually remodel the healed bone are averaged sized cells (10 pn i n diameter) which are sparsely distributed within the bone matrix. But osteocytes can have up to 40 or more osteocytic processes which interconnect the cell to neighboring cells up to 100 pn distant. While it may not be possible to determine field intensity at the cell surfaces in a tissue, there exists the possibility that average field intensities may accurately reflect cellular responses. Unfortunately, experimental results do not appear to confirm this hypothesis. Our recent in vitro investigations can be used to illustrate how different cellular responses can arise even in a preparation where physical factors have been carefully controlled to ensure that electromagnetic fields in the bulk media are well defined (Fig. 1 ) . In this preparation, a uniform magnetic field is used to induce an electric field into square well culture dishes in which cloned rat osteosarcoma cells (ROS 17/23) are growing in monolayer on the bottom of the dishes. Despite the electromagnetic field exposure being constant in all experimental trials (30Hz, 1.8 mT flux inducing a 5 pV/cm RMS electric field), and observations showing the cells at all densities to be in the same biological state, a cellular response to field exposure is clearly evident at intermediate cell densities but disappears as the cell density is increased or decreased. How the physical changes in the cell population alter the interaction of the electromagnetic fields with the cells is not yet known, but it is clear that even the accurate calculations of average field intensities that can be made for a well controlled in vitro experiment are inadequate to describe the effective dose of an electric field exposure. PHYSICAL RESPONSES TO ELECTRIC FIELDS
Review of tissue microanatomy leads to the conclusion that detailed calculations of the electric field intensities and current densities near the cells, which are the actual entities responding to the fields, are not practical. And, the experimental results reported above indicate that a correlation between cellular response and average field intensity will not always exist. These observations identify the need for an alternative approach to establishing dosimetric definitions and one possibility is to base dosage estimates on measurements of the physical effects of the field exposure. The dosimetry issue then becomes a question of identifying an appropriate physical effect at the cellular level which can be measured. The ideal dosimetric parameter would relate directly to the mechanism of field interaction with the cell. Unfortunately, a viable interaction mechanism has not yet been established, so one is left to consider the various physical responses of cells to low frequency electric fields and try to identify the most appropriate physical parameter which may reflect dosage. At the low frequencies and intensities associated with reported field effects on living tissue there exist three important processes which are associated with measurable parameters (Fig. 2). First, current flow will result in charge redistribution at boundaries between materials of differing con-
Microelectrode Measurements of Field Effects
165
Rat osteosarcoma cells
ROS 17/2.8
Micromole p-nitrophenol per 106 cells
---------------
per minute 0
30 Hz exposure inducing 5 pV/cm rms electric field
-
2 30
5 60
Cell Number per cm2
150
1 300
(thousands)
Fig. I.Changes in alkaline phosphotase activity of rat osteoblast-like cells (ROS 1712.8) in response to uniform 30 Hz magnetic flux of 1.8 mT inducing an average electric field intensity of 5 Vlcm i n square culture dishes. Field exposure causes a significant enhancement of enzyme activity at the intermediate cell densities evaluated, but caused no enhancement at the lower cell density used, and an insignificant increase at the highest cell density. At 3 lo4 cells/cm', the cells are sparsely distributed, at lo5cells/cm' the cells form a confluent monolayer, and at 3 1O-r cells/cmz the cells are densely packed.
ductivities with a corresponding development of transboundary potentials. Second, surface current flow will result in the translation of surface bound charges and cause changes in ion distributions at surfaces, resulting in the modification of diffusion gradients and currents. Finally, Coulombic and polarization forces will be imposed on any structure supporting either net charge or dipoles. Transmembrane Potential Changes The most obvious and investigated effect of electric fields on cells and tissues is the development of large electric fields across relatively nonconducting membranes. This effect can be demonstrated by calculating the perturbation of the transmembrane potential of an individual spherical cell in a uniform electric field. Assuming the frequencies of interest are those where the displacement current across the membrane can be considered to be negligible, that is, for frequencies f,, where
166
McLeod
Fig. 2. Three physical responses predominate when biological tissue is exposed to low frequency electric fields. The transmembrane potential of the cells is perturbed, conduction and displacement currents are established in the extracellular space, and electromechanical forces are developed within the extracellular matrix and cell coat (glycocalyx) and transmitted to the cell surface, and perhaps to the interior of the cell.
f,
