Sensory transduction of weak electromagnetic fields: role of glutamate neurotransmission mediated by NMDA receptors.

Subliminal electromagnetic fields (EMFs) triggered nonlinear evoked potentials in awake but not anesthetized animals, and increased glucose metabolism in the hindbrain. Field detection occurred somewhere in the head and possibly was an unrecognized function of sensory neurons in facial skin, which synapse in the trigeminal nucleus and project to the thalamus via glutamate-dependent pathways. If so, anesthetic agents that antagonize glutamate neurotransmission would be expected to degrade EMF-evoked potentials (EEPs) to a greater extent than agents having different pharmacological effects. We tested the hypothesis using ketamine which blocks N-methyl-d-aspartate (NMDA) receptors (NMDARs), and xylazine which is an α2-adrenoreceptor agonist. Electroencephalograms (EEGs) of rats were examined using recurrence analysis to observe EEPs in the presence and absence of ketamine and/or xylazine anesthesia. Auditory evoked potentials (AEPs) served as positive controls. The frequency of observation of evoked potentials in a given condition (wake or anesthesia) was compared with that due to chance using the Fisher's exact test. EEPs were observed in awake rats but not while they were under anesthesia produced using a cocktail of xylazine and ketamine. In another experiment each rat was measured while awake and while under anesthesia produced using either xylazine or ketamine. EEPs and AEPs were detected during wake and under xylazine (P<0.05 in each of the four measurements). In contrast, neither EEPs nor AEPs were observed when anesthesia was produced partly or wholly using ketamine. The duration and latency of the EEPs was unaltered by xylazine anesthesia. The afferent signal triggered by the transduction of weak EMFs was likely mediated by NMDAR-mediated glutamate neurotransmission.

Evidence of a nonlinear human magnetic sense.

Human subjects respond to low-intensity electric and magnetic fields. If the ability to do so were a form of sensory transduction, one would expect that fields could trigger evoked potentials, as do other sensory stimuli. We tested this hypothesis by examining electroencephalograms from 17 subjects for the presence of evoked potentials caused by the onset and by the offset of 2 G, 60 Hz (a field strength comparable to that in the general environment). Both linear (time averaging) and nonlinear (recurrence analysis) methods of data analysis were employed to permit an assessment of the dynamical nature of the stimulus/response relationship. Using the method of recurrence analysis, magnetosensory evoked potentials (MEPs) in the signals from occipital derivations were found in 16 of the subjects (P<0.05 for each subject). The potentials occurred 109-454 ms after stimulus application, depending on the subject, and were triggered by onset of the field, offset of the field, or both. Using the method of time averaging, no MEPs were detected. MEPs in the signals from the central and parietal electrodes were found in most subjects using recurrence analysis, but no MEPs were detected using time averaging. The occurrence of MEPs in response to a weak magnetic field suggested the existence of a human magnetic sense. In distinction to the evoked potentials ordinarily studied, MEPs were nonlinearly related to the stimulus as evidenced by the need to employ a nonlinear method to detect the responses.

Nonlinear dynamical law governs magnetic field induced changes in lymphoid phenotype.

The results of many different types of animal and human studies dealing with the biological effects of exposure to low frequency electromagnetic fields (EMFs) have consistently been both positive and negative. We addressed the question of why this pattern had occurred so commonly in biological studies involving exposure to EMFs and hypothesized that it stemmed from the prevalent use of a linear model to characterize what are inherently nonlinear input-output relationships. The hypothesis was tested by analyzing biological data using a novel statistical procedure that could be adjusted to detect either nonlinear or linear effects. The reliability of the procedure was established using positive and negative controls and by comparison with the results obtained from sampling a known nonlinear system. In four independent experiments, male and female mice were exposed continuously to 0.1 or 0.5 mT, 60 Hz, for 175 days, and the effect on 20 immune parameters was measured using flow cytometry and functional assays. In each experiment, EMF exposure resulted in statistically significant changes in lymphoid phenotype when and only when the response of the animals to the fields was analyzed as if it were governed by nonlinear laws. Our results suggest that the pattern of inconsistency in the EMF bioeffects studies is an artifact resulting from an incorrect choice of the conceptual model for the relation between the field and the biological effect it causally determines.

Coincident nonlinear changes in the endocrine and immune systems due to low-frequency magnetic fields.

OBJECTIVE: The characteristic biological effects of low-frequency electromagnetic fields (EMFs) appear to be functional changes in the central nervous, endocrine and immune systems. For unapparent reasons, however, the results of similar studies have often differed markedly from one another. We recognized that it had generally been assumed, in the studies, that EMF effects would exhibit a dose-effect relationship, which is a basic property of linear systems. Prompted by recent developments in the theory on nonlinear systems, we hypothesized that there was a nonlinear relationship between EMFs and the effects they produced in the endocrine and immune systems. METHODS: We developed a novel analytical method that could be used to distinguish between linear and nonlinear effects, and we employed it to examine the effect of EMFs on the endocrine and immune systems. RESULTS: Mice exposed to 5 G, 60 Hz for 1-175 days in 7 independent experiments reliably exhibited changes in serum corticosterone and lymphoid phenotype when the data were analyzed while allowing that the field exposure and the resulting effects could be nonlinearly related. When the analysis was restricted to linear relationships, no effects due to the field were found. CONCLUSIONS: The results indicated that transduction of EMFs resulted in changes in both the endocrine and immune systems, and that the laws governing the changes in each system were not the type that govern conventional dose-effect relationships. Evidence based on mathematical modeling was found suggesting that the coincident changes could have been causally related.

Nonlinear determinism in the immune system. In vivo influence of electromagnetic fields on different functions of murine lymphocyte subpopulations.

Animal studies of the effects of low-frequency electromagnetic fields (EMFs) on the immune system appear inconsistent, and recent evidence indicates that inconspicuous experimental problems are not responsible. We hypothesized that the inconsistencies resulted from use of linear methods and models to study inherently nonlinear input-output relationships. Using a novel analytical method, we found that exposure of mice to 5 G, 60 Hz, for 1-105 days in 6 independent experiments consistently affected a broad panel of immune variables when and only when the reaction of the immune system was modeled to allow the possibility of nonlinearity in the relationship between the field and the immune variables. It was possible to mimic the pattern observed in the immune data by sampling from a known chaotic system, suggesting the possibility that the observed pattern was the result of intrinsic nonlinear regulatory mechanisms in the immune system. Overall, the results suggested that lymphoid sub-populations were vulnerable to the physiological consequences of EMF transduction, that it may never be possible to predict specific changes in particular immune-system variables, and that the underlying behavior of the immune system (that which occurs in the absence of specific inputs) may be governed by laws that manifest extreme sensitivity to prior states.

Nonlinear response of the immune system to power-frequency magnetic fields.

Studies of the effects of power-frequency electromagnetic fields (EMFs) on the immune and other body systems produced positive and negative results, and this pattern was usually interpreted to indicate the absence of real effects. However, if the biological effects of EMFs were governed by nonlinear laws, deterministic responses to fields could occur that were both real and inconsistent, thereby leading to both types of results. The hypothesis of real inconsistent effects due to EMFs was tested by exposing mice to 1 G, 60 Hz for 1-105 days and observing the effect on 20 immune parameters, using flow cytometry and functional assays. The data were evaluated by means of a novel statistical procedure that avoided averaging away oppositely directed changes in different animals, which we perceived to be the problem in some of the earlier EMF studies. The reliability of the procedure was shown using appropriate controls. In three independent experiments involving exposure for 21 or more days, the field altered lymphoid phenotype even though the changes in individual immune parameters were inconsistent. When the data were evaluated using traditional linear statistical methods, no significant difference in any immune parameter was found. We were able to mimic the results by sampling from known chaotic systems, suggesting that deterministic chaos could explain the effect of fields on the immune system. We conclude that exposure to power-frequency fields produced changes in the immune system that were both real and inconsistent.

Gap junctions in human synovial cells and tissue.

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.

Resting potential of excitable neuroblastoma cells in weak magnetic fields.

The mechanism by which static and low-frequency magnetic fields are transduced into biological signals responsible for reported effects on brain electrical activity is not yet ascertained. To test the hypothesis that fields can cause a subthreshold change in the resting membrane potential of excitable cells, we measured changes in transmembrane current under voltage clamp produced in SH-SY5Y neuroblastoma cells, using the patch-clamp method in the whole-cell configuration. In separate experiments, cells were exposed to static fields of 1, 5, and 75 G, to time-varying fields of 1 and 5 G, and to combined static and time-varying fields tuned for resonance of Na+, K+, Ca2+, or H+. To increase sensitivity, measurements were made on cells connected by gap junctions. For each cell, the effect of the field was evaluated on the basis of 100 trials consisting of a 5-s exposure immediately followed by a 5-s control period. In each experiment, the field had no discernible effect on the transmembrane current in the vicinity of zero current (- 50 mV voltage clamp). The sensitivity of the measuring system was such that we would have detected a current corresponding to a change in membrane potential as small as 38 microV. Consequently, if sensitivity of mammalian cells to magnetic fields is mediated by subthreshold changes in membrane potential, as in sensory transduction of sound, light, and other stimuli, then the ion channels responsible for the putative changes are probably present only in specialized sensory neurons or neuroepithelial cells. A change in transmembrane potential in response to magnetic fields is not a general property of excitable cells in culture.

Effect of soft-tissue trauma on the early periosteal response of bone to injury.

OBJECTIVE: To determine whether the periosteal response to skeletal trauma is impaired when muscle is also injured, thereby providing a possible explanation for why fractures with extensive soft-tissue damage may take longer to heal. METHODS: A bone defect was made in the tibia of male Fisher rats, and the proliferative response, osteoblast concentration, and callus formation that occurred within 7 days were measured in the presence and absence of simultaneously administered model soft-tissue injury (removal of 10% of the anterior tibialis muscle from a region within 2 to 3 mm of the bone defect). Measurements were made by using autoradiography, quantitative histology, and morphometry. RESULTS: Addition of the muscle injury increased proliferation in the cambium and in the fibrous periosteum on day 1, but had no effect thereafter; proliferation of fibroblasts in the loose connective tissue above the periosteum was not affected. Addition of the muscle injury resulted in increased osteoblast levels 2 to 5 days after injury but had no effect on the amount of callus produced. CONCLUSION: The inflammatory milieu created by the muscle injury unexpectedly resulted in an increased periosteal response to skeletal trauma, suggesting that inflammatory mediators generated in response to wounding of soft tissues are unlikely to account for delayed fracture healing. These findings may indicate that surgical trauma associated with internal fixation by using plates and screws may not be as deleterious to the fracture-healing response as previously thought.

Programmed cell death in post-traumatic bone callus.

Some osteoblasts in the expanded population of periosteal cells that occurs following bone injury are removed from the callus by apoptosis. Our objective was to study whether the consequences of activation of the death program could include feedback control of the healing response. Transforming growth factor beta and interleukin-1beta were delivered together continuously to a standardized tibial defect in rats for 3 days using implanted micro-osmotic pumps. The bones were recovered at 1, 2, 3, 5, 7, 10 and 14 days after injury (n = 6 in each treated and control group) and concentrations of proliferating cells, osteoblasts and apoptotic bodies were determined. The injury-induced apoptotic component of the healing response was shifted in time due to the combined cytokines, compared with vehicle only, with the result that the peak in the concentration of apoptotic bodies occurred 2-3 days earlier in the treated animals. Neither osteoprogenitor proliferation nor osteoblast concentration was affected by addition of the cytokines. The results suggested that activation of apoptosis during injury repair was not necessarily a passive consequence of the cellular response to injury. Programmed cell death could therefore have an active role in modulating bone repair.

Regulation of osteoblast levels during bone healing.

OBJECTIVE: To confirm the occurrence of programmed cell death of osteoblasts during bone healing and to evaluate the role of interleukin-1beta (IL-1beta) in regulating osteoblast concentration. STUDY DESIGN: Electron microscopic study of the response of rats to a controlled bone injury, and a randomized controlled study of the effect of IL-1beta administered continuously for three days. METHODS: A standardized defect (1.1 millimeter in diameter, 0.5 millimeter deep) was created unilaterally on the anteromedial surface of the tibia. In some animals, the injury site was recovered five days after operation and processed for ultrastructural evaluation of osteoblasts in the callus. In another group, IL-1beta was delivered to the bone defect using micro-osmotic pumps (0.5 nanograms/hour); control rats received vehicle only. The bones were recovered one to fourteen days after injury, and concentrations of proliferating cells, osteoblasts, and apoptotic bodies were determined. The amount of callus that formed in the defect was measured. RESULTS: Osteoblasts in the callus exhibited ultrastructural changes characteristic of cells undergoing apoptosis, including condensation of chromatin, membrane blebbing, formation of apoptotic bodies, and phagocytosis by nearby osteoblasts. Addition of IL-1beta significantly increased the number of osteoblasts at the injury site and significantly decreased the number of apoptotic bodies in relation to the number of osteoblasts. The amount of callus in the bone defect was not affected by IL-1beta treatment. CONCLUSION: The role of programmed cell death of osteoblasts as a normal concomitant of bone healing was confirmed. Evidence was found suggesting that IL-1beta mediated the appearance and disappearance of osteoblasts, possibly by affecting the rates of differentiation and apoptosis, respectively. Understanding these mechanisms conceivably could lead to the ability to control osteoblast levels at an injury site.

Intracellular signaling mechanisms of interleukin-1beta in synovial fibroblasts.

The possibility that membrane depolarization of synovial fibroblasts caused by interleukin-1beta (IL-1beta) was mediated by protein kinase C (PKC) and Ca2+ influx was studied using inhibitor and activator analysis. The effect of IL-1beta was blocked by bisindolylmaleimide I, an inhibitor of PKC, and by the Ca2+ channel blockers nifedipine and verapamil. In other experiments, PKC was activated using phorbol 12-myristate 13-acetate, and Ca2+ influx was increased by means of a Ca2+ ionophore. Simultaneous application of phorbol ester and Ca2+ ionophore in the absence of IL-1beta mimicked the depolarization caused by IL-1beta. The results were consistent with the hypothesis that, under the conditions studied, activation of PKC and Ca2+ influx are necessary and sufficient processes in the transduction of IL-1beta by synovial cells leading to membrane depolarization. The essential role of protein phosphorylation and Ca2+ influx in the early electrophysiological response of synovial fibroblasts to IL-1beta was therefore established. The role of IL-1beta-induced depolarization in regulating protein expression by the cells remains to be determined, but the results reported here, taken together with observations that protein phosphorylation and Ca2+ influx also mediate the effect of IL-1beta on protease production (1, 2), suggest that electrophysiological changes are actually part of the pathway for expression of proteases in response to IL-1beta.

Low-frequency electromagnetic fields alter the replication cycle of MS2 bacteriophage.

The effect of exposure to 60-Hz electromagnetic fields (EMFs) on RNA coliphage MS2 replication was studied. EMF exposure commenced when the bacterial cultures were inoculated with the phage (t = 0). In 12 experiments in which the strength of the field was 5 G, a significant delay in phage yield was found in the EMF-exposed cultures 45-65 min after inoculation, compared with control cultures. However, the EMF did not alter the final phage concentration. Experiments at 25 G (N = 5) suggested that the stronger field resulted in both impeded phage replication and increased phage yield. No differences between test groups were found in experiments involving sham-EMF exposure, thereby indicating that the results obtained with the EMFs were not due to systematic error. It appears that MS2, which codes for only four proteins, is the simplest biological system in which an EMF-induced effect has been demonstrated. The MS2 system is, therefore, conducive to follow-up studies aimed at understanding the level and nature of the underlying interaction process, and perhaps to biophysical modeling of the interaction process.

Repair of fascial defects in dogs using carbon fibers.

Hernia repair may involve the use of an implant to augment or replace autologous tissue, but the best material for use in this application has not been established. We developed a dog model to evaluate the mechanical strength of fascial defects repaired using carbon fibers, compared with the strength of similar defects repaired using polypropylene mesh (Marlex). Unrepaired defects were included as an additional control. Bilateral defects (1 cm square) were made in the fascia of the back, and the ultimate mechanical strength and stiffness at the repair sites were measured 3-12 months after operation. Defects repaired with carbon fibers were significantly stronger 12 months after operation compared with defects repaired with polypropylene mesh and compared with unrepaired defects. It is concluded that carbon fibers are biocompatible and significantly increase mechanical strength at the repair site. A randomized clinical trial involving patients undergoing hernia repair seems justified to determine whether carbon fibers are superior to standard therapy.

Interleukin-1 beta switches electrophysiological states of synovial fibroblasts.

The role of electro-physiological events in signal transduction of interleukin-1 beta (IL-1 beta) was investigated in rabbit synovial fibroblasts using the perforated-patch method. Aggregated synovial fibroblasts using the perforated-patch method. Aggregated synovial fibroblasts occurred in two different electrophysiological states having membrane potentials (Vm) of -63 +/- 4 (n = 71) and -27 +/- 10 mV (n = 55) (high and low Vm, respectively). IL-1 beta affected the cells with high Vm; it switched the state of the cell from high to low Vm. This effect was strongly dependent on the external potential applied to the cell membrane. Low Vm (-30 mV) alone without IL-1 beta did not switch the state of the cells. Thus a synergistic effect involving the cytokine and cell Vm in switching the electrophysiological state of the cell was shown, indicating that electrophysiological changes are involved in signal transduction. Gap junctions between aggregated cells were necessary for the cells to have a high Vm and to respond to IL-1 beta. Gap junction resistance between adjacent cells was estimated as 300 +/- 100 M omega. Our findings suggest that the electrophysiological behavior of synovial fibroblasts is tightly connected to a signaling or intracellular mediator system that is triggered by IL-1 beta.

Electromagnetic fields enhance chemically-induced hyperploidy in mammalian oocytes.

Epidemiological studies suggest that exposure to electromagnetic fields (EMFs) in the environment may be associated with mutagenic changes, but the relation between EMF exposure and aneuploidy has not previously been studied. Environmental EMFs apparently lack the energy necessary to function as aneugens, but the possibility exists that EMFs could influence the incidence of aneuploidy synergistically because EMFs can activate the neuroendocrine system, and ovulation and oocyte meiotic maturation are under neurohormonal control. This hypothesis was tested by examining the effect of EMF exposure on the occurrence of hyperploidy in mouse oocytes induced by vinblastine sulphate (VBS), which was employed as a surrogate for aneugens in the environment. The incidence of hyperploidy in metaphase II oocytes of individual mice following superovulation was determined, and statistical methods were developed to assess whether EMF exposure during oogenesis in the presence of VBS altered the rate of hyperploidy. A significant effect of EMF exposure on VBS-induced hyperploidy was found (P < 0.05). The data suggested that the EMF primarily affected the mice that exhibited a high incidence of VBS-induced hyperploidy. Exposure had no effect on the number of oocytes ovulated nor on the occurrence of hypoploidy. The results support the hypothesis that EMF exposure can promote the occurrence of aneuploidy caused by an aneugen via a mechanism involving the neuroendocrine system.

Electromagnetic fields can affect osteogenesis by increasing the rate of differentiation.

Electromagnetic fields of various kinds can alter osteogenesis in animals with osteotomies and patients with nonunions, but the underlying cellular mechanisms are unknown. The aims of this study were to determine whether I gauss at 60 Hz affected periosteal proliferation and differentiation in either the normal rat tibia or 1 to 14 days after a surgically induced defect. In the injured rats, using histologic study, autoradiography, and morphometry, it was found that exposure for 1 or 3 days had no effect on proliferation but that it produced an increase in osteoblasts 3 days after the injury. Proliferation and differentiation were unaffected by exposure in the absence of injury. The results suggest that the primary effect of the fields was to promote differentiation but not proliferation. Because fields can stimulate proliferation of osteoblastlike cells in vitro, the results of this study may indicate the presence of an in vivo factor that antagonizes the tendency of fields to increase mitotic activity.

Low-level EMFs are transduced like other stimuli.

The aims of this study were to test the theory that transduction of low-level electromagnetic fields (EMFs) is mediated like other stimuli, and to determine the false-negative rate of the method used to assess the occurrence of transduction (intra-subject comparison of stimulus and non-stimulus states (ICOS)). A light stimulus was chosen as a basis of comparison because light could be applied and removed at precise time points, similar to the manner in which EMFs were controlled. Subjects exposed to a weak light stimulus during 2-second epochs exhibited alterations in brain electrical activity that were similar to those previously observed in subjects exposed to EMFs. The false-negative rate of the ICOS method was 61%, since it registered an effect in only 39% of the subjects (11/28) whereas all subjects were actually aware of the light. In a second group of subjects that were exposed to 0.8 G (1.5 or 10 Hz), 58% (11/19) exhibited similar alterations in brain activity, as determined using ICOS. Previous measurements in the same subjects using a different method showed that the EMFs actually affected brain electrical activity in all subjects; consequently, the false-negative rate was 42% when an EMF was used as the stimulus. The results suggested that the post-transduction brain electrical processes in human subjects were similar in the cases of EMF and light stimuli, as hypothesized, and that the high negative rate of the ICOS method (here and in previous studies) was composed partly or entirely of false-negative results.

Bone injury response. An animal model for testing theories of regulation.

Therapeutic treatment of bone disease and attempts to accelerate normal healing require knowledge of the soluble factors that control bone repair and the specific effects that they produce. To facilitate study of this regulatory system, an animal model involving creation of a hole in the cortex of the rat tibia was developed. Proliferation, differentiation, and callus formation at the injury site were measured more precisely than in previous animal models by means of autoradiographic, histologic, histochemical, and morphometric methods. Several novel features of bone healing were observed, including the following: (1) synthesis of bone matrix in the defect occurred only after a cambial compartment was established by regeneration of the fibrous periosteum and (2) at least 3 kinds of osteoblasts could be distinguished depending on when and where they deposited calcifiable matrix. The model is well suited to evaluating the use of interventional strategies that involve chemical or electrical agents because the cellular parameters of interest can be measured precisely.