Frequency-dependent interference by magnetic fields of nerve growth factor-induced neurite outgrowth in PC-12 cells

We have shown that 50 Hz sinusoidal magnetic fields within the 5-10 micro Tesla (μT) rms range cause an intensity-dependent reduction in nerve growth factor (NGF) stimulation of neurite outgrowth (NO) in PC-12 cells. Here we report on the frequency dependence of this response over the 15-70 Hz range at 5 Hz intervals. Primed PC-12 cells were plated in collagen-coated, 60 mm plastic petri dishes with or without 5 ng/ml NGF and were exposed to sinusoidal magnetic fields for 22 h in a CO₂ incubator at 37 °C. One 1,000-turn coil, 20 cm in diameter, generated vertically oriented magnetic fields. The dishes were stacked on the center axis of the coil to provide a range of intensities between 3.5 and 9.0 μT rms. The flux density of the ambient DC magnetic field was 37 μT vertical and 19 μT horizontal. The assay consisted of counting over 100 cells in the central portion (radius ≤0.3 cm) of each dish and scoring cells positive for NO. Sham exposure of cells treated identically with NGF demonstrated no difference in the percentage of cells with NO between exposed and magnetically shielded locations within the incubator. Analysis of variance demonstrated flux density-dependent reductions in NGF-stimulated NO over the 35-70 Hz frequency range, whereas frequencies between 15 Hz and 30 Hz produced no obvious reduction. The results also demonstrated a relative maximal sensitivity of cells at 40 Hz with a possible additional sensitivity region at or above 70 Hz. These findings suggest a biological influence of perpendicular AC/DC magnetic fields different from those identified by the ion parametric resonance model, which uses strictly parallel AC/DC fields. © 1995 Wiley-Liss, Inc.     

A Study of the Effects of Flux Density and Frequency of Pulsed Electromagnetic Field on Neurite Outgrowth in PC12 Cells

The aim of this study was to investigate the influence of pulsed electromagnetic fields with various flux densities and frequencies on neurite outgrowth in PC12 rat pheochromocytoma cells. We have studied the percentage of neurite-bearing cells, average length of neurites and directivity of neurite outgrowth in PC12 cells cultured for 96 hours in the presence of nerve growth factor (NGF). PC12 cells were exposed to 50 Hz pulsed electromagnetic fields with a flux density of 1.37 mT, 0.19 mT and 0.016 mT respectively. The field was generated through a Helmholtz coil pair housed in one incubator and the control samples were placed in another identical incubator. It was found that exposure to both a relatively high flux density (1.37 mT) and a medium flux density (0.19 mT) inhibited the percentage of neurite-bearing cells and promoted neurite length significantly. Exposure to high flux density (1.37 mT) also resulted in nearly 20% enhancement of neurite directivity along the field direction. However, exposure to low flux density field (0.016 mT) had no detectable effect on neurite outgrowth. We also studied the effect of frequency at the constant flux density of 1.37 mT. In the range from 1 ∼ 100 Hz, only 50 and 70 Hz pulse frequencies had significant effects on neurite outgrowth. Our study has shown that neurite outgrowth in PC12 cells is sensitive to flux density and frequency of pulsed electromagnetic field.     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

Empirical test of an ion parametric resonance model for magnetic field interactions with PC-12 cells

A companion paper describes a predictive ion parametric resonance (IPR) model of magnetic field interactions with biological systems based on a selective relation between the ratio of the flux density of the static magnetic field to the AC magnetic field and the charge-to-mass ratio of ions of biological relevance. Previous studies demonstrated that nerve growth factor (NGF)-stimulated neurite outgrowth (NO) in PC-12 cells can be inhibited by exposure to magnetic fields as a function of either magnetic field flux density or AC magnetic field frequency. The present work examines whether the PC-12 cell response to magnetic fields is consistent with the quasiperiodic, resonance-based predictions of the IPR model. We tested changes in each of the experimentally controllable variables [flux densities of the parallel components of the AC magnetic field (B_ac) and the static magnetic field (B_dc) and the frequency of the AC magnetic field] over a range of exposure conditions sufficient to determine whether the IPR model is applicable. A multiple-coil exposure system independently controlled each of these critical quantities. The perpendicular static magnetic field was controlled to less than 2 mG for all tests. The first set of tests examined the NO response in cells exposed to 45 Hz B_ac from 77 to 468 mG(rms) at a B_dc of 366 mG. Next, we examined an off-resonance condition using 20 mG B_dc with a 45 Hz AC field across a range of B_ac between 7.9 and 21 mG(rms). Finally, we changed the AC frequency to 25 Hz, with a corresponding change in B_dc to 203 mG (to tune for the same set of ions as in the first test) and a B_ac range from 78 to 181 mG(rms). In all cases the observed responses were consistent with predictions of the IPR model. These experimental results are the first to support in detail the validity of the fundamental relationships embodied in the IPR model. © 1994 Wiley-Liss, Inc.     

ELF in vitro exposure systems for inducing uniform electric and magnetic fields in cell culture media.

Many in vitro experiments on the biological effects of extremely low frequency (ELF) electromagnetic fields utilize a uniform external magnetic flux density (B) to expose biological materials. A significant number of researchers do not measure or estimate the resulting electric field strength (E) or current density (J) in the sample medium. The magnitude and spatial distribution of the induced E field are highly dependent on the sample geometry and its relative orientation with respect to the magnetic field. We have studied the E fields induced in several of the most frequently used laboratory culture dishes and flasks under various exposure conditions. Measurements and calculations of the E field distributions in the aqueous sample volume in the containers were performed, and a set of simple, quantitative tables was developed. These tables allow a biological researcher to determine, in a straightforward fashion, the magnitudes and distributions of the electric fields that are induced in the aqueous sample when it is subjected to a uniform, sinusoidal magnetic field of known strength and frequency. In addition, we present a novel exposure technique based on a standard organ culture dish containing two circular, concentric annular rings. Exposure of the organ culture dish to a uniform magnetic field induces different average electric fields in the liquid medium in the inner and outer rings. Results of experiments with this system, which were reported in a separate paper, have shown the dominant role of the magnetically induced E field in producing specific biological effects on cells, in vitro. These results emphasize the need to report data about the induced E field in ELF in-vitro studies, involving magnetic field exposures. Our data tables on E and J in standard containers provide simple means to enable determination of these parameters.     

Morphological and electrophysiological changes produced by electrical stimulation in cultured neuroblastoma cells.

Electric fields, which were equivalent to those generated by medical devices, were applied to cultured neuroblastoma cells (mouse and human) to test for morphological damage and to establish damage thresholds. Each of two methods of applying fields permitted flow of electrical current and minimized exposure of cells to electrode-breakdown products. One method consisted of a pair of parallel wires in a Petri dish by which current was delivered within a fixed volume of flowing tissue-culture media. With the other method, the cells were held in a confined geometrical chamber and current was applied via agar bridges. Under a given set of stimulation parameters, damage was found to be variable from cell to cell. By changing the strength of the electric field (frequency and duration of stimulation held constant), thresholds of several V/cm were found above which cell damage could be reliably produced. Depending on the intensity of the field, damage took the form of cell lysis or damage to neurites. Intracellular recordings from the mouse neuroblastoma cells revealed a correlation between a decline in resting transmembrane potential and stimulus intensity. Human neuroblastoma cells were less susceptible to damage than were the mouse neuroblastoma cells, given the same strength of applied electric fields.     

Double blind test of magnetic field effects on neurite outgrowth

Previous work reported that nerve growth factor-stimulated neurite outgrowth in PC-12 cells could be altered by exposure to parallel alternating current (AC) and direct current (DC) magnetic fields under a variety of exposure conditions, producing results that are consistent with the predictions of the ion parametric resonance (IPR) model. The credibility of these results, considered extraordinary by some scientists, could be strengthened if the cell response were found to persist under alternate assay conditions. We replaced part of our standard assay procedure with a double blind procedure. This new procedure obscured 1) whether a particular set of dishes of cells was exposed or not, and 2) which individual dish was in which exposure system. The goal was to determine whether the previously observed responses of PC-12 cells to magnetic fields would be sufficiently robust to decode the imposed blinding, thereby removing any question of experimenter bias in reported results. We placed three coded dishes of cells in each of two otherwise identical exposure systems, one not energized and one energized to produce exposure conditions predicted to maximally suppress neurite outgrowth (B_dc of 36.6 μT, parallel 45 Hz AC of 23.8 μT rms). Each of the six dishes were recoded before assay to further obscure the exposure identity of any individual dish. The combined results of four distinct runs of these double blind experiments unequivocally demonstrated that 1) there was a clear, distinctive, repeatable consistency with the actual energization of the exposure systems and location of each dish, and with the predictions of the IPR model; 2) only the explicitly stated experimental variables influenced the experiment; and 3) the reported response of the cells was very improbably due to chance (P = .000024). Bioelectromagnetics 19:204–209, 1998. © 1998 Wiley-Liss, Inc.

1 This article was prepared by a group consisting of both United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

The growth of PC-12 neurites is biased towards the anode of an applied electrical field

We have exposed cultures of PC12 cells to uniform DC electric fields following the addition of NGF. The success of these experiments relied upon the design of new chambers enabling fields to be applied to mammalian cell cultures. After 48 h of field application, the distribution of neurite outgrowths was biased towards the anode. More neurites faced the anode than would be expected if growth was uniform. The magnitude of this bias was strongly correlated with field strenght, with a threshold value of about 1 mV/mm. At field strengths above 30 mV/mm, the neurites growing towards the cathode were shorter than those growing towards the anode or perpendicular to the field. This response was not correlated with field strenght. This report confirms that mammalian neurons respond to electrical fields and supports the notion that neurites are influenced by endogenous electrical fields during development. As far as we are aware, this is the only report the documents a response towards the anode. 1994 John Wiley & Sons, Inc.     

Measurement of magnetically induced electric fields in conductive media near a 60 Hz current-carrying wire.

Electric fields induced in a conductive body by the magnetic field of a current-carrying wire were analyzed theoretically and experimentally to assess the dosimetric importance of highly nonuniform, field-exposure conditions. Experimentation revealed that a 60-Hz magnetic field was inversely proportional to the radius of a wire bundle carrying 100 A within a 0.5-m2 test area. A miniaturized electric field probe was used to measure the electric fields induced in 5-cm-deep, saline-filled models. In the theoretical analysis, numerical estimates of induced fields were made by a spreadsheet method. The theoretical calculations and the measured values of induced electric fields were generally in good agreement. The induced fields were in a plane perpendicular to a vertically incident magnetic field; the maximally induced fields were in areas nearest the wire bundle. The strength of the induced field increased with model size: from 96 microV/cm in a 10 x 10 cm model to 176 microV/cm in a 40 x 40 cm model. The strength of the field induced in a 20 x 20 cm model decreased with increasing model-to-wire spacing: from 132 microV/cm for a 1-cm spacing (2-mT maximum, incident field) to 50 microV/cm for a 6-cm spacing (0.33-mT maximum). The results indicate that increases in local values of nonuniformly incident fields produce relatively small increases in induced electric fields. This finding may be important in dosimetric consideration of circumstances, such as use of electric blankets, in which fields of low average strength are accompanied by intense local fields.     

Electric field-induced astrocyte alignment directs neurite outgrowth

The extension and directionality of neurite outgrowth are key to achieving successful target connections during both CNS development and during the re-establishment of connections lost after neural trauma. The degree of axonal elongation depends, in large part, on the spatial arrangement of astrocytic processes rich in growth-promoting proteins. Because astrocytes in culture align their processes on exposure to an electrical field of physiological strength, we sought to determine the extent to which aligned astrocytes affect neurite outgrowth. To this end, dorsal root ganglia cells were seeded onto cultured rat astrocytes that were pre-aligned by exposure to an electric field of physiological strength (500 mV mm(-1)). Using confocal microscopy and digital image analysis, we found that neurite outgrowth at 24 hours and at 48 hours is enhanced significantly and directed consistently along the aligned astrocyte processes. Moreover, this directed neurite outgrowth is maintained when grown on fixed, aligned astrocytes. Collectively, these results indicate that endogenous electric fields present within the developing CNS might act to align astrocyte processes, which can promote and direct neurite growth. Furthermore, these results demonstrate a simple method to produce an aligned cellular substrate, which might be used to direct regenerating neurites.     

Addition of magnetic field capability to existing extremely-low-frequency electric field exposure systems

Magnetic field systems were added to existing electric field exposure apparatuses for exposing cell suspensions in vitro and small animals in vivo. Two horizontally oriented, rectangular coils, stacked one directly above the other, have opposite electric currents. This configuration minimizes leakage fields and allows sham- and field-exposure systems to be placed in the same room or incubator. For the in vitro system, copper plates formed the loop-pair, with up to 900 A supplied by a 180:1 transformer. Electric fields were supplied via electrodes at the ends of cell-culture tubes, eight of which can be accommodated by each exposure system. Two complete systems are situated in an incubator to allow simultaneous sham and field exposure up to 1 mT. For the in vivo system, four pairs of 0.8 x 2.7-m coils made of copper bus bar are employed. This arrangement is energized from the power grid via a 30:1 transformer; horizontal magnetic flux densities up to 1 mT can be generated. Pairs of electrode plates spaced 30.5 cm apart provide electric field exposure of up to 130 kV/m. Four systems with a capacity of 48 rats each are located in one room. For both the in vitro and in vivo systems, magnetic exposure fields are uniform to within +/- 2.5%, and sham levels are at least 2,500-fold lower than exposure levels. Potential confounding factors, such as heating and vibration, were examined and found to be minimal.     

Studies of Schwann cell proliferation. III. Evidence for the surface localization of the neurite mitogen

In the preceding paper (Salzer et al., 1980, J. Cell Biol. 84:753-- 766), evidence was presented that a neurite membrane fraction could be used to stimulate Schwann cell proliferation in culture. In this study, we present evidence that the mitogenic signal by which intact neurites or neurite membranes stimulate Schwann cell proliferation is located at the neurite surface. This conclusion is based on the following observations: (a) stimulation of Schwann cell proliferation by neurons requires direct contact between neurites and Schwann cells, separation of the two cells by a permeable collagen diaphragm 6 microns thick prevents Schwann cell proliferation; (b) treatment of intact neurites with trypsin before preparation of neurite membranes abolishes the ability of these membranes to be mitogenic for Schwann cells; and (c) the mitogenic activity of neurite homogenates is exclusively localized in the particulate rather than the soluble fraction of the homogenate. The mitogenic component on the neurite surface is heat labile, and is inactivated by aldehyde fixation. Preliminary data suggest that the mitogenic effect of neurite on Schwann cells is not mediated by 3',5'- cyclic AMP.     

Time-varying and static magnetic fields act in combination to alter calcium signal transduction in the lymphocyte.

We have tested the hypothesis that extremely low frequency (ELF) time-varying magnetic fields act in combination with static magnetic fields to alter calcium signalling in the lymphocyte. Results indicate that a 60-min exposure of thymic lymphocytes at 37 +/- 0.05 degrees C to a 16 Hz, 421 mG (42.1 microT) magnetic field simultaneously with a colinear static magnetic field of 234 mG (23.4 microT) (a.c./d.c. field intensity ratio = 1.8) inhibits calcium influx triggered by the mitogen Concanavalin A. Significantly, resting lymphocytes do not respond to the fields, thus, only mitogen-activated cells undergoing calcium signalling exhibit a field response. These results indicate that signal transduction involving calcium is an important biological constraint which operates to mediate this field interaction. Additional split field exposures show that the presence of the a.c. field or the d.c. field alone does not produce an effect. This is consistent with a proposed parametric resonance theory of interaction of low intensity magnetic fields with biological systems (L.L. Lednev (1991) Bioelectromagnetics 12, 71-75), which predicts the occurrence of biological effects at specific values for the frequency and field intensity of the ELF and static magnetic fields.     

Preferential outgrowth of central nervous system neurites on astrocytes and Schwann cells as compared with nonglial cells in vitro

I have compared central nervous system (CNS) neurite outgrowth on glial and nonglial cells. Monolayers of glial cells (astrocytes and Schwann cells) or nonglial cells (e.g., fibroblasts) were prepared and were shown to be greater than 95% pure as judged by cell type-specific markers. These monolayers were then tested for their ability to support neurite outgrowth from various CNS explants. While CNS neurites grew vigorously on the glial cells, most showed little growth on nonglial cell monolayers. Neurites grew singly or in fine fascicles on the glial cells at rates greater than 0.5 mm/d. The neurite outgrowth on astrocytes was investigated in detail. Scanning and transmission electron microscopy showed that the neurites were closely apposed to the astrocyte surface and that the growth cones were well spread with long filopodia. There was no evidence of significant numbers of explant- derived cells migrating onto the monolayers. Two types of experiments indicated that factors associated with the astrocyte surface were primarily responsible for the vigorous neurite outgrowth seen on these cells: (a) Conditioned media from either astrocytes or fibroblasts had no effect on the pattern of outgrowth on fibroblasts and astrocytes, and conditioned media factors from either cell type did not promote neurite outgrowth when bound to polylysine-coated dishes. (b) When growing CNS neurites encountered a boundary between astrocytes and fibroblasts, they stayed on the astrocytes and did not encroach onto the fibroblasts. These experiments strongly suggest that molecules specific to the surfaces of astrocytes make these cells particularly attractive substrates for CNS neurite outgrowth, and they raise the possibility that similar molecules on embryonic glial cells may play a role in guiding axonal growth during normal CNS development.     

High-intensity electrostatic-field exposure system for cultured biological cells.

We describe a new system for exposing cultured biological cells that have been plated on coverslips to strong electrostatic fields at magnitudes greater than 10(3) V/cm. Techniques are described that make use of mineral oil to render insignificant electrical conduction currents (total leakage current is less than 1.0 nA or less than 0.1 nA/coverslip), joule heating (less than 10(-6) W), or current-induced magnetic fields (less than 10(-13) T) in regions inhabited by cells. The mineral oil also eliminates a reduction in the strength of the applied field, which otherwise can occur from increased electrode-to-medium impedance at the site of application. Thus the applied field is reliably specified in the vicinity of a cell membrane. Control and electrostatic field chambers are housed in a grounded metal incubator. Cylindrical mu-metal shields can be used to reduce background magnetic fields in each chamber from 40 microT static and approximately 1 microT ac to, respectively, less than 3 microT static and approximately 100 nT ac. Contamination of cells by impurity atoms that may leach from electrodes was measured by atomic-absorption spectrophotometry and found to be negligible. Stray magnetic- and electric-field components within the incubator were measured, as were background fields around the laboratory.     

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set.
In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells *** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.     

An integrated ELF magnetic-field generator and incubator for long-term in vitro studies.

Long-term studies of the effects of low-frequency magnetic fields on cells in culture require an incubator that is free of contaminating magnetic fields and that provides automatic control of exposure duration, uniform applied fields, a uniform and well-controlled environmental temperature, and high reliability of the total system. We describe a dual-incubator system in which the enclosed solenoid of either unit can provide a sham exposure while the other generates a magnetic induction of up to 500 microT. Each solenoid-incubator unit contains an enclosed rack that can hold as many as 140 petri dishes for culturing cells; each unit is heated radiantly by circulating warm water. Field-exposure conditions and temperatures are continuously monitored and controlled by a microcomputer.     

A scheme for incorporating DC magnetic fields into epidemiological studies of EMF exposure

Experimental data on calcium-ion release in chicken brain tissue suggest that biological effects of electric and magnetic fields (EMFs) are concentrated near certain “active combinations” of DC magnetic field strength and “effective” AC magnetic field frequencies. We hypothesize that active AC/DC combinations may exist and suggest that epidemiologic data, coupled with DC magnetic field measurements, may be used to identify critical exposure conditions. An empirical model is used to calculate these multiple active combinations at any given DC magnetic field strength and to define a rating system that incorporates the proximity of AC magnetic field frequencies generated by electric power lines to the new, computed effective frequencies. Such an exposure score may be useful in investigating correlations of EMF exposure with disease incidence. For 60 Hz and 50 Hz, the highest EMF exposure scores occurred at DC field strengths of 506 mG and 422 mG, respectively. The exposure score contains a factor which may be adjusted to reflect the importance of harmonics of the AC magnetic field as well as of the fundamental frequency. Using this factor, we consider two important special cases consistent with chick brain data: 1) we consider active pairs associated with all detectable harmonics (up to 660 Hz) without regard to relative intensity of the harmonics, and 2) we use the relative intensities of the AC field frequencies to adjust their contribution to the exposure score. © 1993 Wiley-Liss. Inc.     

Development and evaluation of intermediate frequency magnetic field exposure system for studies of in vitro biological effects

We have developed an intermediate frequency (IF) magnetic field exposure system for in vitro studies. Since there are no previous studies on exposure to heating-frequency magnetic fields generated from an induction heating (IH) cook top, there is a strong need for such an exposure system and for biological studies of IF magnetic fields. This system mainly consists of a magnetic-field-generating coil housed inside an incubator, inside which cultured cells can be exposed to magnetic field. Two systems were prepared to allow the experiment to be conducted in a double-blind manner. The level of the generated magnetic field was set to 532 microT rms in the exposure space, 23 kHz, 80 times the value in the International Commission on Non-ionizing Radiation Protection (ICNIRP) guidelines, with a spatial field uniformity better than 3.8%. The waveforms were nearly sinusoidal. It was also confirmed that the parasitic electric field was 157 V/m rms and the induced electric field was 1.9 V/m rms. The temperature was maintained at 36.5 +/- 0.5 degrees C for 2 h. Furthermore, leaked magnetic flux density was 0.7 microT rms or lower at extremely low frequency (ELF) and IF in the stopped system when the other system was being operated, and the environmental magnetic flux density was 0.1 microT rms or lower at the center of the coils. As a result, it was confirmed that this system could be successfully used to evaluate the biological effects of exposure to IF magnetic fields.