Growth and differentiation of PC6 cells: the effects of pulsed electromagnetic fields (PEMF).

Previous studies in our laboratory showed that neurite outgrowth in vitro and nerve regeneration in vivo were stimulated by 2 Hz, 0.3 mT (3 G) pulsed electromagnetic fields (PEMF). To learn more about the effects of PEMF on nerve cells, we exposed PC6 cells, a standard neuronal-like cell model, to the same pulsed electromagnetic fields for 2 h/day for 2 days and asked whether two different cell processes, proliferation and differentiation, were affected. The cells were also treated with a differentiating agent, nerve growth factor (NGF), to further define any interactive effects. We found that proliferation was unaffected by either PEMF or NGF alone or in combination. Differentiation, expressed as neurite outgrowth, was strongly upregulated with NGF, but this NGF response was significantly depressed in cells treated with PEMF.

Studies of the interactions between melatonin and 2 Hz, 0.3 mT PEMF on the proliferation and invasion of human breast cancer cells.

Interactions between the hormone melatonin at pharmacological concentrations (10(-3) M) and 2 Hz, 0.3 mT pulsed electromagnetic fields (PEMF) on the proliferation and invasion of human breast cancer cells were studied in vitro. Three types of human breast cancer cells were used in this study: MDA-MB-435, MDA-MB-231, and MCF-7. Results showed that cellular growth of MDA-MB-231 cells, which were reported to be lowly metastatic, and MCF-7 cells, which were reported to be nonmetastatic, were both significantly reduced by melatonin regardless of the presence of the field. Results also showed that MDA-MB-435 and MDA-MB-231 cells were invasive, with MDA-MB-231 cells being more invasive than the MDA-MB-435 cells for both unexposed and experimental-PEMF groups. In addition, invasion studies showed that MCF-7 cells were not invasive and that melatonin did not have any effects on the invasion of these cells, with or without the PEMF. It is also suggested that since metastasis requires growth and invasion into tissue, anti-invasion agents can be used in conjunction with melatonin to prevent formation of secondary metastases. The overall studies suggest that PEMF at 2 Hz, 0.3 mT does not influence cancer metastasis; while having clinical merit in the healing of soft tissue injury, this field has shown no influence on cancer cells as 60 Hz power line fields have.

EMF signals and ion/ligand binding kinetics: prediction of bioeffective waveform parameters.

The kinetics of an electromagnetic field (EMF) target pathway are used to estimate frequency windows for EMF bioeffects. Ion/ligand binding is characterized via first order kinetics from which a specific electrical impedance can be derived. The resistance/capacitance properties of the binding pathway impedance, determined by the kinetics of the rate-determining step, define the frequency range over which the target pathway is most sensitive to external EMF. Applied signals may thus be configured such that their spectral content closely matches that of the target, using evaluation of the signal to thermal noise ratio to optimize waveform parameters. Using the approach proposed in this study, a pulsed radio frequency (PRF) waveform, currently employed clinically for soft tissue repair, was returned by modulation of burst duration, producing significant bioeffects at substantially reduced signal amplitude. Application is made to Ca2+/Calmodulin-dependent myosin phosphorylation, for which the binding time constants may be estimated from reported kinetics, neurite outgrowth from embryonic chick dorsal root explants and bone repair in a fracture model. The results showed that the retuned signal produced increased phosphorylation rates, neurite outgrowth and biomechanical strength that were indistinguishable from those produced by the clinical signal, but with a tenfold reduction in peak signal amplitude, approximately 800-fold reduction in average amplitude and approximately 10(6)-fold reduction in average power.

Electromagnetic fields influence NGF activity and levels following sciatic nerve transection.

Pulsed electromagnetic fields (PEMF) have been shown to increase the rate of nerve regeneration. Transient post-transection loss of target-derived nerve growth factor (NGF) is one mechanism proposed to signal induction of early nerve regenerative events. We tested the hypothesis that PEMF alter levels of NGF activity and protein in injured nerve and/or dorsal root ganglia (DRG) during the first stages of regeneration (6-72 hr). Rats with a transection injury to the midthigh portion of the sciatic nerve on one side were exposed to PEMF or sham control PEMF for 4 hr/day for different time periods. NGF-like activity was determined in DRG, in 5-mm nerve segments proximal and distal to the transection site and in a corresponding 5-mm segment of the contralateral nonoperated nerve. NGF-like activity of coded tissue samples was measured in a blinded fashion using the chick DRG sensory neuron bioassay. Overall, PEMF caused a significant decrease in NGF-like activity in nerve tissue (P < 0.02, repeated measures analysis of variance, ANOVA) with decreases evident in proximal, distal, and contralateral nonoperated nerve. Unexpectedly, transection was also found to cause a significant (P=0.001) 2-fold increase in DRG NGF-like activity between 6 and 24 hr postinjury in contralateral but not ipsilateral DRG. PEMF also reduced NGF-like activity in DRG, although this decrease did not reach statistical significance. Assessment of the same nerve and DRG samples using ELISA and NGF-specific antibodies confirmed an overall significant (P < 0.001) decrease in NGF levels in PEMF-treated nerve tissue, while no decrease was detected in DRG or in nerve samples harvested from PEMF-treated uninjured rats. These findings demonstrate that PEMF can affect growth factor activity and levels, and raise the possibility that PEMF might promote nerve regeneration by amplifying the early postinjury decline in NGF activity.

Effects of pulsed magnetic fields on neurite outgrowth from chick embryo dorsal root ganglia.

We have previously shown that neurite outgrowth from 6-day chick embryo dorsal root ganglia (DRG) in vitro was stimulated when nerve growth factor (NGF) and pulsed magnetic fields (PMF) are used in combination. 392 DRGs were studied in a field excited by a commercial PMF generator. We have now analyzed an additional 416 DRGs exposed to very similar PMF's produced by an arbitrary wavefrom generator and power amplifier. We reproduced our previous findings that combination of NGF and bursts of asymmetric, 220 microsecond-wide, 4.0 mT-peak pulses induced significantly (p < 0.05) greater outgrowth than NGF alone, that fields without NGF do not significantly alter outgrowth, and that, unlike NGF alone, 4.0 mT fields and NGF can induce asymmetric outgrowth. The asymmetry does not seem to have a preferred orientation with respect to the induced electric field. Analysis of the data for the entire 808 DRGs confirms these findings. Importantly, we find similar results for pulse bursts repeated at 15 or 25 Hz.

Acute treatment with pulsed electromagnetic fields and its effect on fast axonal transport in normal and regenerating nerve.

The mechanism whereby low-frequency electromagnetic fields accelerate axonal regrowth and regeneration of peripheral nerve after crush lesion is not known. One candidate is an alteration in axonal transport. In this study we exposed unoperated rats for 15 min/day, and rats that had undergone a crush lesion of the sciatic nerve, for 1 hr/day for 2 days, to 2-Hz pulsed electromagnetic fields. To label fast transported proteins, [3H]-proline was microinjected into the spinal cord, and the sciatic nerves were removed 2, 3.5, and 5 hr later. The rates of fast axonal transport were obtained for animals in all groups by counting sequential 2-mm segments of nerves. The following transport rates were found: in unoperated normal sciatic nerve not exposed to PEMF, 373 +/- 14 mm/day; in unoperated normal nerve exposed to PEMF, 383 +/- 14 mm/day; in sham crush nerves not exposed to PEMF, 379 +/- 19 mm/day; in sham crush nerve exposed to PEMF, 385 +/- 17 mm/day; in crushed nerves not exposed to PEMF, 393 +/- 16 mm/day. and in crushed nerves exposed to PEMF, 392 +/- 15 mm/day. The results of these experiments indicate that 1) a crush injury to the sciatic nerve does not alter the rate of fast axonal transport, and 2) low-frequency pulsed electromagnetic fields do not alter fast axonal transport rates in operated (crush) or unoperated sciatic nerves.

Gait-stance duration as a measure of injury and recovery in the rat sciatic nerve model.

The rat sciatic nerve is a well-established animal model for the study of peripheral nerve crush injury. Footprint analysis is the most widely used non-invasive method of measuring functional recovery after injury in this model. However, this method has significant limitations due to inability to obtain clear reproducible prints, especially when the injury is severe, and variation of these prints with gait velocity. In the case of contracture or toe loss, footprint analysis is unreliable. We describe a new technique, gait-stance duration, which is capable of non-invasively quantitating functional recovery in the rat model. This method is not dependent on accurate foot positioning during gait. It utilizes video recording of the animal walking and measures the time each hind foot is in contact with the floor by counting the number of frames that pass. By pairing consecutive steps, it minimizes variation due to changes in velocity and, by calculating a ratio of injured/uninjured hind feet, comparisons to normal gait can be made. This method shows recovery patterns similar to footprint analysis with small inter-animal variability. We believe it has significant advantages over footprint analysis for the measurement of functional recovery in the crushed sciatic nerve rat model.

Improved footprint analysis using video recording to assess functional recovery following injury to the rat sciatic nerve.

Footprint analysis is a non-invasive method to quantitate functional recovery after crush injury in the rat sciatic nerve model. Traditional methods of producing the footprints for measurement are limited by inability to reliably produce clear prints when the injury is severe. We describe the use of video technique with image analysis to record and measure these prints. Video had fewer unmeasurable prints than ink. For the 1-5 and 2-4 toe spreads, there was good correlation of video measurements with ink method and better repeatability using video as compared with ink. However, the print length parameter determined by video had poorer repeatability and poorly correlated with that measured by ink. Therefore, calculation of a Sciatic Function Index by video is not appropriate. Since the print length also varies with gait velocity, we believe that a ratio of injured:uninjured hindfoot 1-5 toe spreads as measured by video is a more reliable and repeatable measure of functional recovery in this model.

Maturation of the central nervous system: comparison of equine and other species.

This review covers the development and maturation of the cerebellum of the horse and compares this developmental sequence with that of man, mouse and chicken. These comparisons attempt to correlate morphological and neurochemical features, developmental time and functional performance necessary for survival at birth. Although there is great disparity between these 3 species, the basic anatomical structures of the cerebellum are present as are specific cellular groups, synapses and neurochemical markers. In addition to this structural homogeneity, other attributes of the cerebellum are its easily identified cellular populations and its well ordered pattern of growth and differentiation. The cerebella of the developing chick and mouse have been studied in great detail as they are amenable to experimental manipulations. The pattern of cellular differentiation appears to be reproducible from species to species and differs primarily as it relates to gestational age and functional requirements at birth. For instance, most of the large neurones of the cerebellar cortex differentiate early with small neurones and neuroglia differentiating later. Neurogenesis of the cerebellar cortex is fairly complete in the newborn foal and chick hatchling, but not in the human or rodent newborn.

Prospects on clinical applications of electrical stimulation for nerve regeneration.

Regenerative capability is limited in higher vertebrates but present in organ systems such as skin, liver, bone, and to some extent, the nervous system. Peripheral nerves in particular have a relatively high potential for regeneration following injury. However, delay in regrowth or growth, blockage, or misdirection at the injury site, and growth to inappropriate end organs may compromise successful regeneration, leading to poor clinical results. Recent studies indicate that low-intensity electrical stimulation is equivalent to various growth factors, offering avenues to improve these outcomes. We present a review of studies using electric and electromagnetic fields that provide evidence for the enhancement of regeneration following nerve injury. Electric and electromagnetic fields (EMFs) have been used to heal fracture non-unions. This technology emerged as a consequence of basic studies [Yasuda, 1953; Fukada and Yasuda, 1957] demonstrating the piezoelectric properties of (dry) bone. The principle for using electrical stimulation for bone healing originated from the work of Bassett and Becker [1962], who described asymmetric voltage waveforms from mechanically deformed live bone. These changes were presumed to occur in bone during normal physical activity as a result of mechanical forces, and it was postulated that these forces were linked to modifications in bone structure. Endogenous currents present in normal tissue and those that occur after injury were proposed to modify bone structure [Bassett, 1989]. These investigators proposed that tissue integrity and function could be restored by applying electrical and/or mechanical energy to the area of injury. They successfully applied electrical currents to nonhealing fractures (using surgically implanted electrodes or pulsed currents using surface electrodes) to aid endogenous currents in the healing process.(ABSTRACT TRUNCATED AT 250 WORDS)

Design and characterization of a system for exposure of cultured cells to extremely low frequency electric and magnetic fields over a wide range of field strengths.

A system is described that is capable of producing extremely low frequency (ELF) magnetic fields for relatively short-term exposure of cultured mammalian cells. The system utilizes a ferromagnetic core to contain and direct the magnetic field of a 1,000 turn solenoidal coil and can produce a range of flux densities and induced electric fields much higher than those produced by Helmholtz coils. The system can generate magnetic fields from the microtesla (microT) range up to 0.14 T with induced electric field strengths on the order of 1.0 V/m. The induced electric field can be accurately varied by changing the sample chamber configuration without changing the exposure magnetic field. This gives the system the ability to separate the bioeffects of magnetic and induced electric fields. In the frequency range of 4-100 Hz and magnetic flux density range of 0.005-0.14 T, the maximum total harmonic distortion of the induced electric field is typically less than 1.0%. The temperature of the samples is held constant to within 0.4 degrees C by constant perfusion of warmed culture medium through the sample chamber.

Pulsed electromagnetic fields stimulate nerve regeneration in vitro and in vivo.

The influence of non-invasive, low level, pulsed electromagnetic fields (PEMF) on regeneration was tested on in vitro and in vivo models. Cultures of dorsal root ganglia were exposed to 2 Hz PEMF, amplitude of 0.05 mTesla while rats after a 'crush' lesion of sciatic nerves were exposed to 2 Hz PEMF, amplitude of 0.3 mTesla. In in vitro experiments, relative to controls, cultures treated with PEMF exhibited a significant increase in neurite outgrowth with dense labeling of neurons and neurites on autoradiographs after incorporation of [3H]proline into new proteins. In vivo exposure of rats to PEMF for 3, 4 or 6 days after lesioning produced a 22% increase in the regeneration rate relative to controls with no effect on the initial delay period. When rats were exposed to PEMF before lesioning without further treatment, the same degree of stimulation of axonal sprouting was obtained. Reduction of the amplitude from 0.3 mTesla to 0.06 mTesla eliminated this pre-exposure response. Alterations in the distribution of new proteins synthesized 2 weeks after PEMF treatment provide additional evidence for its influence at the whole body and cellular levels.

Stimulation of rat sciatic nerve regeneration with pulsed electromagnetic fields.

The effects of pulsed electromagnetic fields (PEMF) on rat sciatic nerve regeneration after a crush lesion were determined. The rats were placed between a pair of Helmholtz coils and exposed to PEMF of frequency 2 Hz and magnetic flux density of 0.3 mT. A 4 h/day treatment for 3-6 days increased the rate of nerve regeneration by 22%. This stimulatory effect was independent of the orientation of the coils. Exposure times of 1 h/day-10 h/day were equally effective in stimulating nerve regeneration. Rats exposed to PEMF for 4 h/day for 7 days before crush, followed by 3 days after crush without PEMF, also showed significantly increased regeneration. This pre-exposure 'conditioning' effect suggests that PEMF influences regeneration indirectly.

Sources of trophic factors that induce limb regeneration and prevent amputation-induced neuronal death.

Segments of 2-, 4-, 6- and 8-day neural tube, or of 15-day peripheral nerve were implanted longitudinally into limb stumps of 4-day chick embryos whose right-wing buds were amputated at the future elbow region. Stumps of amputated limbs (ALs) implanted with 7-day heart or without implant served as controls. Effects of progressively older neural tube implants (NTIs) upon ALs and host spinal cord neurons were analyzed by area measurements of the peripheral limb field (PLF) and NTI and by cell counts of the host lateral motor column (LMC). Nine days postamputation, 2- and 4-day NTIs contained many neurons and induced epimorphic regeneration in more than one-fourth of the embryos. Six-day NTIs contained few neurons and induced only tissue regeneration. Eight-day NTIs and peripheral nerve containing only non-neuronal cells were as ineffective as controls in stimulating regeneration, although peripheral nerve did cause a significant increase in the peripheral field. The NTIs of all ages and implants of peripheral nerve were equally effective in protecting LMC neurons from amputation-induced cell death in the host spinal cord. The results may indicate that neurons of the implant induce limb regeneration and non-neuronal cells of the implant protect against LMC neuronal death.

Conductivity values of tissue culture medium from 20 degrees C to 40 degrees C.

Few studies are available that relate conductivity and temperature in solutions commonly used in tissue culture media. The purpose of this paper is to provide equations relating conductivity and temperature (in the range 20 degrees C-40 degrees C) for five solutions: 0.9% saline, MEM (Minimum Essential Media), horse serum, MEM with 1% horse serum, and MEM with 10% horse serum.

Triethanolamine, tris, hepes, and cytosine arabinoside show neuritogenic activity in cultured chick embryo ganglia.

Neuritogenesis, which occurs to a slight extent in chick embryo ganglia maintained under standard conditions and which is maximally stimulated by nerve growth factor, also was enhanced by presence in the medium of buffers (triethanolamine, Tris, and Hepes) and cytosine arabinoside and by the passage of direct electric current. The major effect of the buffers probably was to remove protons from cell membranes, that of the current to produce accelerated movement of ions through membranes of the ganglionic cells, and that of cytosine arabinoside to decrease the numbers of nonneural cells by inhibiting DNA synthesis. The buffers were neuritogenically ineffective on nerve growth factor-sensitive PC12 pheochromocytoma cells in culture. Media from ganglia in which triethanolamine or passage of electric current had elicited outgrowth of neurites produced no observable effect on PC12 cells under our experimental conditions. Current data fit the hypothesis that, whereas nerve growth factor exerts direct neuritogenic effects on neurons, the other treatments affect neural-nonneural interactions, possibly by way of gap junctions or changes in direct physical contact, so as to disinhibit inherent neural neuritogenic potential and/or to stimulate it.

Responses of host motor and sensory neurons to a neural tube implant in amputated chick limbs.

The right wingbuds of stage 23-25 chick embryos were amputated at the future elbow region and a segment of 2-day neural tube was implanted longitudinally into the limb stump of experimental embryos to induce limb regeneration. Control embryos had no implant in the amputated limb stump. To analyze effects of the neural tube implant (NTI) upon the host nervous system, quantitative determinations were made of the peripheral limb field (PLF), dorsal root ganglia (DRG) and spinal cord lateral motor column (LMC) of the amputated side for comparison with similar determinations of the unamputated side in all embryos. The PLF was estimated by determining the area of the skeletal elements of the amputated and unamputated limb of each embryo. The size of the DRG was estimated by determining the sectional-profile area of a pair of ganglia; the LMC was determined by counting the neurons on both sides of a single spinal cord segment. The PLF was less on the amputated than on the unamputated side but was significantly greater in amputated limbs that received a NTI. The size of the DRG was positively correlated with the size of the PLF in all groups, indicating the DRG was not directly affected by the NTI but did respond to target structures. The number of neurons in the LMC was not positively correlated with the PLF and was not reduced by limb amputation in embryos with a NTI. The data suggest that the NTI may have protected host spinal cord cells from the induced-cell death expected to follow limb amputation; perhaps the implant produces a neuronal survival factor.

The threshold quantity of nerve required to induce limb regeneration in the chick embryo.

Wingbuds of 4-day chick embryos were amputated at the future elbow joint and a segment of embryonic neural tube was implanted longitudinally in the stump. The cross-sectional area and number of nerve bundles were determined in limbs in which limb regeneration occurred and compared with similar measurements in control limbs without neural tube implant. The number of nerve bundles in regenerated limbs was not significantly greater than in control limbs which did not regenerate. However, the cross-sectional area of nerve bundles was significantly greater in the limbs that had undergone regeneration. The data support the view that the amount of axoplasm available at the amputation (surface) site is the essential factor in determining the success or failure of regeneration.