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Effect of magnetic fields on damaged mice sciatic nerves.

Abstract

This article deals with the cutting edge science of bioelectromagnetism. It speaks to a resonance in biological systems. The resonance deals with a connection between biochemistry and biophysics. This means that all matter is electromagnetic in nature at the same time that all space has gravity. Putting these forces together, this research shows that the human body is affected by very, very weak magnetic fields. These magnetic forces are millions of times weaker than the earth's steady magnetic field. MRI or magnetic resonance imaging uses a magnetic force about 20,000 times stronger than the Earth's. Thus, life must be based in a subtle field of forces dictated by an unknown source of utter perfection. This paper illustrates the use of such weak and low frequency magnetic fields that appear physiologic. Indeed, these forces have been shown to be useful in the regeneration of mouse sciatic nerves. The studies are well replicated.

The leg nerves of mice were cut into pieces about one centimeter in length and then placed in growth media, in culture. Some were treated with a picotesla range magnetic field and some were not, but all were placed in between two electrical coils that produced a uniform magnetic field. The device is called the Jacobson Resonator, which is being considered for approval by the FDA and was prototyped by NASA subcontractors at the Stennis Space Center in Mississippi. The study showed that weak and low frequency magnetic fields maintained the integrity of cellular and subcellular structures over several weeks, whereas the untreated samples degenerated and decayed. The treated samples were not only maintained, they were regenerated. Particular attention should be paid to the fact that the myelin sheath was regenerated and grew much thicker than it had been. Myelin is critical to nerve conduction and is prominently lost in neurological disorders such as multiple sclerosis.

The implication of growing and maintaining the integrity of nerves with weak magnetic forces is enormous. We may have here a non-invasive painless and safe way of regenerating nerves that are diseased and/or damaged and have lost their function. Furthermore, if Jacobson Resonance is indeed correct, in that gravity and electromagnetism may be quantified in humans, then a diverse new modality to treat disease may have been initiated. Following up the in vitro study of mice sciatic nerves, it has been demonstrated in an in vivo pilot study at Cornell University Medical College that neuromuscular damage secondary to a neurotoxin administration can be reversed with the Jacobson Resonator, with restored function in damaged mice. Indeed, clinical and basic science studies at universities around the world have shown that Jacobson Resonance, utilizing magnetic fields millions of times weaker than the earth's, and with frequencies comparable to brain waves, is efficacious in the treatment of chronic pain, neurological disorders, cardiac arrhythmias, wound healing, and the promotion of immune function.

The importance of the effects described in this paper is critical. Indeed, a new and revolutionary modality is under careful scrutiny at research facilities around the world, and it now cries for all to participate in the search to understand the wondrous subtlety of life.

Introduction

Nerve regeneration is possible, provided that the damage occurs outside the central nervous system (CNS) and the neuron cell body is intact. (1, 2, 3) Todorov et at. demonstrated axonal reconnection in the earthworm medial giant axon by electric fields of 0.1 Hz to 100 MHz and amplitude of 80 to 200 V. (4) Recent study by Kerns et al. has shown that electric currents accelerate the rate and the direction of growth of cultured neurites of chick ganglia, and disaggregated frog neural tube. (5) Borgens and Bonhert showed increased growth of mammalian spinal axons in response to DC voltage gradients. (6) In vivo evidence of electrical stimulation of nerve regeneration has been presented by Kerns et al.5 Rusovan and Kanje concluded that continuous exposure for 4 h/day for a period of 7 days to 0.2 mT (1 milli Tesla = 10 Gauss) and 0.4 mT sinusoidal magnetic fields enhanced the regeneration of crushed rat sciatic nerves. (7) From their study, Kerns et al. concluded that DC electrical stimulation enhanced regeneration of transected and crushed rat sciatic nerve. (5)

Methods and Materials

In this study, the effect of pico Tesla (pT) magnetic fields on the excised mice sciatic nerve segments in vitro was investigated. Molecules targeted in this study were selected on the basis of their roles in nerve repair, growth, and regeneration (Table I). The magnetic fields used in this study were generated by two 18" diameter coils constructed of 30 gauge copper wire connected in series (Helmholtz configuration), placed 9" apart. The coils were driven by a frequency and amplitude adjustable sinusoidal AC power supply (Hewlett-Packard Model 3325A synthesizer-function generator) and connected in series to an attenuator to obtain the pre-elected pT range field in the space between coils. For exposing the nerve segments contained in the growth medium to the magnetic fields, the flask was placed in between the two coils, and centered on the imaginary line connecting the centers of two coils, to ensure an even distribution of the magnetic field. In calculating the intensity of the externally applied magnetic field the Jacobson's equation, m[c.sup.2] = Blvq was used, where m is the mass of the particle, B is the magnetic field intensity, c is the velocity of electromagnetic field, independent of its inertial frame of reference, q represents a unit charge of 1 coulomb (by defining electromotive force as energy per unit charge,) v is the velocity of the carrier in which the particle exists, and l is its length. (8-14) The frequency f was calculated from the ion Cyclotron resonance equation, f = qB / 2pm using the B value obtained from the Jacobson's equation. (10, 14, 15, 16) Note that m is the mass of charge-bearing particles.

Two sets of experiments were conducted. The experimental groups were selected to determine (a) the effect of increased time of exposure on the dimensional and structural change of the exposed nerves, (b) the effect of multiple versus single exposure on the same, and (c) a frequency and amplitude window that promotes greater effect on the growth and structure of the nerve segments.

In the first experiment four segments of the sciatic nerves, 1.5 cm in length and 1.0 mm in width, were surgically excised under aseptic conditions from mice under ether anesthesia. Following excision, the nerves were washed with Hank's balanced salt solution (HBSS) containing 3 g/1 bovine serum albumin (BSA), then immersed in 0.025% trypsinized HBSS and placed in Dulbecco's modified Eagle's medium (DMEM) to stop the trypsin action. Finally, the nerve segments were placed individually in flasks containing 15 ml of DMEM with high glucose, L-glutamine, 110 mg/l sodium pyruvate, and pyridoline hydrochloride. The flasks were stored in a 5% C[O.sub.2] humidified incubator at 37[degrees] C. The growth medium was changed three times a week. A set of two nerve segments was exposed to 14 magnetic field settings, 1-4, 6, 7, 9-12 and 14-17 (Table I) for 2.5 min per one field setting for a total of 35 min each day for 5 days. The other set served as the unexposed control. Controlled cultures of excised nerve segments were removed from the incubator and placed in between the coils (having the coils turned off). Following the exposure to the magnetic field, the length and the width of the exposed and the control nerve segments were measured using a caliper and a ruler.

In the second set of experiments, both sciatic nerves of 12 mice were excised aseptically to yield a total of 24 nerve segments. The growth medium consisted of minimum essential medium (MEM) with 2mM L-glutamine, Earl's salts, a mixture of 90% sodium bicarbonate and 10% fetal calf serum. Six nerve segments served as the Control (C1-C6) and were not exposed to magnetic fields. The remaining 18 nerves were divided into three experimental groups of 6 nerve segments each and exposed to magnetic fields daily for 15 days. Group E1-E6 nerve segment were exposed for 3 minutes per setting to 14 settings. Settings used were 1-4, 6, 7, 9-12 and 14-17 (Table I) for a total of 42 minutes per day. E7-E11 were exposed to settings 1 through 20 for 3 minutes per setting for a total of 60 minutes per day. E12-E18 were exposed to individual settings each for 40 minutes per day. The nerve segments with their corresponding settings were as follows: E12-18, E13-12, E14-10, E15-7, E16-15, E17-9, E18-6. After fifteen days, the nerve segments were taken for dimensional studies under phase-contrast microscope. All of the eighteen nerve segments in the three experimental groups were cut in half; one set of halves was prepared for light and electron microscopy. Nerve segments were washed with 0.1 M sodium cacodylate, fixed in 2.5% glutaraldehyde, 4% paraformaldehyde, 0.02% picric acid in cacodylate buffer overnight at 4[degrees]C and washed three times with the cacodylate buffer followed by fixation in a solution containing 1% osmium tetroxide and 1.5% potassium ferricyanide for 60 min at room temperature. The nerve segments were washed again three times with the cacodylate buffer and then immersed in 50% ethanol and 3% uranyl acetate for 90 min followed by dehydration through a graded ethanol series, and were left overnight in absolute ethanol--Spurr's resin solution (1:1), followed by Spurr's resin alone for 8 hrs at room temperature and then overnight in a 70[degrees] C oven prior to sectioning by the aid of an ultramicrotome for light and electron microscope observations.

The other set of halve was processed for cell proliferation studies. The nerve segments were fixed in buffered 2 - 4% paraformaldehyde at 4[degrees] C overnight. After thorough washing PBS they were dehydrated through a graded ethanol series followed by immersion in a 1:1 ethanol-xylene solution for 30 min, followed by placement in a 1:1 xylene-paraffin solution for 30 min at 56[degrees] C. Next, the nerve segments were washed three times with paraffin for 30 min, 60 min, and 30 min in that order at 56[degrees] C. Finally, the nerve segments were embedded using prewarmed forceps. Melted paraffin was poured into the molds and was allowed to cool until a film of solidified paraffin covered the top. 8 mm sections were cut using an ultramicrotome and stained with MIB-1 stain, which is a diagnostic marker for abnormal cell proliferation. (17, 18)

Three control and three experimental nerve segments were randomly chosen for DNA analysis. DNA was extracted using Nucleon resin as described by the manufacturers (Amersham Pharmacia Biotech). Gel electrophoresis was performed on the extracted DNA samples, using 0.8% agarose gel and 2.0% agarose gel at 100 V for 60 min. (19)

Results

In the first experiment, 14 magnetic field settings 1-4, 6, 7, 9-12 and 14-17 (Table I) were used. The initial dimensions of both the control and experimental nerve segments were 15 mm in length and 1 mm in width. At the end of the experiment the ends of the exposed nerve segments showed significantly more dendritic growth than the control. Control nerve segments remained at their initial length, whereas the exposed nerve segments appeared to grow in length. The final dimensions of exposed nerve segments were 20 mm in length and 1.5 mm in width; a 33% increase in length and a 50% increase in width.

The various magnetic field settings used in a subsequent second set of experiments are listed in Table I. The response of the nerve segments in the second set of experiments to magnetic fields was similar to the first. The initial dimensions of the nerve segments in the second experiment for the control E1-E6, E7-E11 and E12-E18 were 5.2 [+ or -] 1.7, 5.2 [+ or -] 1.0, 5.4 [+ or -] 0.5, and 5.1 [+ or -] 0.9 and the final dimensions were 5.0 control [+ or -] 1.5, 5.5 [+ or -] 1.1, 5.8 [+ or -] 0.8, and 5.7 [+ or -] 1.4, respectively, indicating an increase in the length of exposed nerve segments. The light and electron microscope observations (Figs. 1 and 2) of the cross-sections of exposed and control nerve segments show a typical response to magnetic fields. The light microscope observations, revealed a normal and regular distribution of the axons in the exposed halves (Fig. 1A) of the nerve segments. In contrast, the axons (A) in the control nerve segments (Fig. 1B) were fewer in number, had an irregular and abnormal shape, and a vary narrow band of myelination (NM). Under the electron microscope, as shown in Fig. 2A, the exposed nerve segments exhibited myelin sheaths (MS) with a normal, well defined circular shape, axons (A) with a normal distribution of microtubules and microfilaments, Schwann cells (SC) with normal configuration, and mitochondria (MT) with condensed conformation indicative of anabolic activity (Fig. 2). In contrast, nerve segments in the control group, as shown in Fig. 2B showed fragmented and disintegrated myelin sheath (MS) suggestive of lack of myelin synthesis, highly vacuolated Schwann calls (SC), and mitochondria (MT) with inactive and unorthodox conformation. Results of the electrophoresis of DNA extracted from control and experimental nerves showed a similar single band of DNA in 0.8% and 2.0% agarose minigel, which suggests that the magnetic fields used in these experiments did not cause DNA degradation. Both the exposed and control nerves also stained negative for the MIB-1 marker, implying that the magnetic fields used in this experiment did not lead to uncontrolled cell proliferation.

[FIGURES 1-2 OMITTED]

Discussion

According to Clegg, the ligand-receptor association is followed by cytoskeletal-mediated events and by the production of a second messenger (cAMP or cGMP) through activation of the cyclase in the membrane itself, or in its proximity. (20) The microtrabecular reticulum establishes connections with membrane receptors and other structures in the cytoplasm. (21) These reticulum-receptor connections result in an excellent means of intracellular communication. The microtrabecular reticulum consists of actin filaments and ATP molecules. The mechanism of magnetic resonance in the growth and regeneration of damaged nerve segments may be visualized as follows: The magnetic component of an electromagnetic field will pass through the cell membrane while the electric component is sharply attenuated due to the high impedance of the phospholipid domain. One may concede that magnetic fields produce piezoelectricity, converting electromagnetic oscillations to mechanical vibrations (i.e., photon-phonon transductions), which causes amplifications of weak currents at the membrane surfaces, as well as GAP junctions. Many molecules recognized as piezo-electric have [alpha]-helices, ordered polypeptide structures and sets of ordered dipoles fulfilling the requirement for cooperative systems. Thus, the amplification of weak triggers by a factor of about [10.sup.12] may occur through mediation of the magnetic component of electromagnetic field by piezo-electric structures. It has been suggested that this may contribute to increase energy ([DELTA]E) to activate cyclase system that in turn may act as ATP to provide energy for cellular function. Amplifications secondary to photon-photon transductions can thus compensate for configurational entropy from the excised nerve segments thus enabling the use of free energy ([DELTA]E) derived from ATP driven metabolic engine to promote cellular activity for growth and regeneration of damaged cells. It may be of interest to point out that magnetic fields in the pico Tesla (pT = 1 x [10.sup.-12] T = 1 x [10.sup.-8] Gauss) range have been used in the treatment of various neurologic disorders. Sandyk exposed 20 Parkinsonian patients to a 2 Hz, 7x[10.sup.-8] Gauss field and observed an improvement in the motor as well as the non-motor aspects of the disease. (22) Sandyk also demonstrated a beneficial effect of magnetic fields on patients with multiple sclerosis. (23) Anninos et al. showed an attenuation of seizure activity in epileptic patients exposed to similar ELF-pT range magnetic fields. (24) Weak magnetic fields in the pT range have been linked to [alpha]- and [delta]-brain waves by Cohen and electrical currents on the order of 1 mA have been implicated in the normal functioning of the human body. (25)

Conclusion

Our observations demonstrate an in vitro stimulation of growth and repair of damaged nerve segments and suggest a possible therapeutic use of magnetic fields in neurological disorders. Pico-Tesla magnetic resonance sustained the cellular structures of the new organelles intact during the course of the experiment. The further experiment in vivo will have to be performed to evaluate the structure-function relationship. The effect of the Jacobson magnetic resonance was seen in greater dendritic growth, maintenance of structural integrity of axons and neurons, as compared to the degeneration in the unexposed nerve segments, strictly limited to the experimental period of exposure. It is to be tested next with an in vivo neuropathy model in conjunction with clinical studies (double blinded).

Acknowledgments.

We thank Leona Cohen-Gould, Linda Berg Friedman, and Yarka Chvojka for help in light and electron microscopy and Shalini Arora for assistance in the preparation of the manuscript.

References

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(2.) Hackenbrock, C. R. (1968). Ultrastructural basis for metabolically linked mechanical activity in mitochondria. J. Cell Biol., 37, 345-369.

(3.) Vander, A. J., Sherman, J. H., and Luciano, D. S. (1994). Human Physiology (6th ed.). New York, NY: McGraw-Hill.

(4.) Todorov, A. T. et al. (1992). Electric-field-induced reconnection of severed axons. Brain Res., 582, 329-334.

(5.) Kern, J. M. et al. (1991). Electrical stimulation of nerve regeneration in the rat: The early effects evaluated by a vibrating probe and electron microscopy. Neurosci., 40, 93-97.

(6.) Borgens, R. B., and Bonhert, D. M. (1997). The response of mammalian spinal axons to an applied DC voltage gradient. Exp. Neurol., 45, 376-389.

(7.) Rusovan, A. and Kanje, M. (1991). Stimulation of regeneration of the rat sciatic nerve by 50 Hz sinusoidal magnetic fields. Exp. Neurol., 112, 312-316.

(8.) Jacobson, J. I. (1986). Mc2 = Bvlq: Gravitational and EM potential in dual resonance. Ind. J. Theor. Phys., 34, 231-239.

(9.) Jacobson, J. I. (1987). A testable theoretical model for the mechanism of magneto therapy. Pan Minervba Medica, 29, 263-270.

(10.) Jacobson, J. I. (1991). A look at possible mechanism and potential of magnetotherapy. J. Theor. Biol., 149, 97-120.

(11.) Jacobson, J .I. (1992). Exploring the potential of magneto-recrystallization of genes and associated structures with respect to nerve regeneration and cancer. Int. J. Neurosci., 64, 153-165.

(12.) Jacobson, J. I. (1994). BCEC--Gravitational circuits and Jacobson resonance: The basis from which to evaluate potential hazard and therapeutic benefit from extrinsic magnetic fields. In Europ. J. Surgery Supplement, 574, (pp. 137-147). Scandinavian University Press.

(13.) Jacobson, J. I. (1996). Speculations on the influence of electromagnetism on genomic and associated structures. J. In. Med. Res. 24, 1-11.

(14.) Jacobson, J. I. and Yamanashi, W. S. (1994). A possible physical mechanism in the treatment of neurologic disorders with externally applied pico Tesla magnetic fields. Physiol. Chem. Phys. and Med., 26, 287-297.

(15.) Jacobson, J. I. and Yamanashi, W. S. (1995). An initial physical mechanism in the treatment of neurologic disorders with externally applied pico Tesla magnetic fields. Neurol. Res., 17, 144-148.

(16.) Liboff, A. R. (1985). Cyclotron resonance in membrane transport. In A. Chiabrera, C. Nicolini, and H. P. Schwan (Eds.), Interactions between electromagnetic fields and cells (pp. 281-296). New York, NY: Plenum.

(17.) Lopez, F. et al. (1994). The labeling of proliferating cells by Ki67 and MIB-1 antibodies depends on the binding of a nuclear protein to the DNA. Exp. Cell Res., 210, 145-53.

(18.) Willey, R. L. (1971). Microtechniques: A laboratory guide. New York, NY: Macmillan.

(19.) Maniatis, T., Fritsch, E. F., and Sambrook, J. (1989). Molecular cloning: A laboratory manual (2nd ed.). New York, NY: Cold Spring Harbor Press.

(20.) Clegg, J. S. (1983). Intracellular water, metabolism and cell architecture, Part 2. In H. Frolich and F. Kremer (Eds.) Coherent excitations in biological systems (pp. 162-177). Springer, New York.

(21.) Del Giudice, E. et al. (1995). In H. Frolich (Ed.). Structures, correlations and electromagnetic interactions in living matter: Theory and applications (pp.144-148). New York: Marcel Dekker, Inc.

(22.) Sandyk, R. (1992). Magnetic fields in the therapy of Parkinsonism. Int. J. Neurosci., 66, 209-235.

(23.) Sandyk, R. (1992). Successful treatment of multiple sclerosis with magnetic fields. Int. J. Neurosci., 66, 237-250.

(24.) Anninos, P. A., Tsagas, N., and Sandyk, R. (1991). Magnetic stimulation in the treatment of partial seizures. Int. J. Neurosci. 60, 141-171.

(25.) Cohen, D. (1972). Magnetoencephalography: detection of the brain's electrical activity with a superconducting magnetometer. Science, 175(664).

Jerry I. Jacobson (1), William S. Yamanashi (2), Bennet Brown (3), Palak Parekh (4), Deborah Shin (4), and Brij B. Saxena (4)

(1.) Department of Medical Physics and Neuromagnetics, National Medical and Research Institute, 9960 Central Park Boulevard, Boca Raton, FL 33428, USA

(2.) Department of Medicine, Cardiovascular Section, University of Oklahoma Health Sciences Center, VA Medical Center, 151-F, Research Services, 921 NE 13th Street, Oklahoma City, OK 73104, USA

(3.) Department of Biological Sciences, Fairleigh Dickinson University, Teaneck, NJ 07666, USA

(4.) Department of Obstetrics/Gynecology, Division of Reproductive Endocrinology, Cornell University Medical College, 1300 York Avenue, Room A-267, New York, NY 10021, USA Research Site: Cornell Medical College in New York City

Correspondence and requests for materials should be addressed to Dr. Jerry Jacobson (Dr.J.I.Jacobson@worldnet.att.net)
Table I. Magnetic Field settings used for molecules critical
in nerve growth and repair.

Setting Intensity, B Freq., F
numbers Critical Molecules (Gauss) (Hz)

 1 Spectrin, Brain Specific Fodrin 1.0 x 10-5 0.15

 2 Neurofilaments, L-70kb, 2.5 x 10-6 71.0
 Hemoglobin, MAP-70kd

 3 interferon, Platelet Derived 1.3 x 10-6 36.0
 Growth Factor (PDGF)

 4 Nerve Growth Factor (NGF), 9.97 x 10-7 27.9
 Kinesine

 5 Motor Proteins 9.0 x 10-7 25.2

 6 Microtubule Associated Protein 8.25 x 10-7 23.0
 (MAP) 2a, 2b

 7 Calmodulin, Spectrin, 7.0 x 10-7 19.0
 Brain Specific Fodrin

 8 IgE 6.2 x 10-7 17.4

 9 Neurofilaments, Calmodulin 5.7 x 10-7 16.0

 10 IgG, Epinephrine 4.6 x 10-7 12.8

 11 Tubulin dimer 3.4 x 10-7 3.6

 12 IgM (900KD), Homeoboxes 2.7 x 10-7 7.6

 13 Neurofilaments L-70KD 2.1 x 10-7 5.6

 14 MAP, G-actin, Calcium ion, 1.75 x 10-7 5.4
 Tubulin lobular monomer

 15 Potassium Bone Growth Factor BGF 1.5 x 10-7 4.1

 16 GAP, Homeoboxes, Iron 1.26 x 10-7 3.5

 17 Interferon, Platelet Derived 9.0 x 10-8 2.5
 Growth Factor (PDGF)

 18 NGF 7.5 x 10-8 2.1

 19 Calmodulin, Profilin 5.0 x 10-8 1.4

 20 ATP, Epinephrine, Serotonin 3.4 x 10-8 0.952
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Author:Jacobson, Jerry I.; Yamanashi, William S.; Brown, Bennet; Parekh, Palak; Shin, Deborah; Saxena, Brij
Publication:Frontier Perspectives
Date:Mar 22, 2000
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