J. P. Blanchard

Magnetic fields at resonant conditions for the hydrogen ion affect neurite outgrowth in PC-12 cells: A test of the ion parametric resonance model

PC-12 cells primed with nerve growth factor (NGF) were exposed to sinusoidal extremely-low-frequency (ELF) magnetic fields (MFs) selected to test the predictions of the ion parametric resonance (IPR) model under resonance conditions for a single ion (hydrogen). We examined the field effects on the neurite outgrowth (NO) induced by NGF using three different combinations of flux densities of the parallel components of the AC MF (Bac) and the static MF (Bdc). The first test examined the NO response in cells exposed to 45 Hz at a Bdc of 2.96 microT with resonant conditions for H+ according to the model. The Bac values ranged from 0.29 to 4.11 microT root-mean-square (rms). In the second test, the MF effects at off-resonance conditions (i.e., no biologically significant ion at resonance) were examined using the frequency of 45 Hz with a Bdc of 1.97 microT and covering a Bac range between 0.79 and 2.05 microT rms. In the third test, the AC frequency was changed to 30 Hz with the subsequent change in Bdc to 1.97 microT to tune for H+ as in the first test. The Bac values ranged from 0.79 to 2.05 microT rms. After a 23 h incubation and exposure to the MF in the presence of NGF (5 ng/ml), the NO was analyzed using a stereoscopic microscope. The results showed that the NGF stimulation of neurite outgrowth (NSNO) was affected by MF combinations over most of the Bac exposure range generally consistent with the predictions of the IPR model. However, for a distinct range of Bac where the IPR model predicted maximal ionic influence, the observed pattern of NSNO contrasted sharply with those predictions. The symmetry of this response suggests that values of Bac within this distinct range may trigger alternate or additional cellular mechanisms that lead to an apparent lack of response to the MF stimulus.

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 (Bdc of 36.6 microT, parallel 45 Hz AC of 23.8 microT 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).

Experimental determination of hydrogen bandwidth for the ion parametric resonance model

The ion parametric resonance (IPR) model predicts that distinct patterns of field-induced biological responses will occur at particular magnetic field combinations which establish ion resonances. An important characteristic of resonance is the bandwidth response of the system, in part because it determines the required tolerances of the test system. Initial development of the IPR model used literature data to estimate the bandwidth for any ion resonance to be -/+10% of its exact resonance. Because the charge-to-mass ratio of hydrogen is much larger than any other biologically significant ion, hydrogen resonance provides a unique test case by which a single ionic bandwidth can be clearly measured. Of particular relevance is work by Trillo et al. that demonstrated a hydrogen-only, resonance-based IPR response of neurite outgrowth in PC-12 cells. The work reported here considers the response of nerve-growth-factor-stimulated PC-12 cells exposed to magnetic fields tuned at or near hydrogen resonance. This work was designed to test directly the IPR model hypothesis of a -/+10% ionic bandwidth. Consistent with the work of Trillo et al., resonance conditions were established using a 2.97 microT static magnetic field, and the AC frequency and field strength were varied to prove different aspects of the resonance. With this static field 45 Hz was the resonance frequency identified for hydrogen, 42.5 and 47.5 Hz were near-resonance frequencies, and 40 and 50 Hz bounded the assumed -/+10% hydrogen resonance bandwidth. We repeated each test three times. The cell responses at 45 Hz exposures agreed with the IPR model predictions and replicated results obtained by Trillo et al. Cells exposed to 42.5 and 47.5 Hz (near resonance) magnetic fields responded in the same general pattern as those exposed to 45 Hz fields, but neurite outgrowth was less than that observed at resonance. Measured results for cells exposed to either 40 Hz or 50 Hz fields were indistinguishable from off-resonance responses, consistent with the hypothesized bandwidth. These results confirm that the response bandwidth for the hydrogen ion is no greater than -/+10%, and give further support to the resonance-based predictions of the IPR model.

Nonbinomial distributions of relative neurite outgrowth in PC-12 cells

Previously we reported the results of a series of experimental tests using PC-12 cells to examine the biological effect of prescribed combinations of both nerve growth factor and magnetic fields. Because our assay of the PC-12 cells is based on binary classification of the cells following treatment, our data might be expected to have a binomial distribution. However, our data consistently show a smaller variability than that predicted by the binomial distribution model. In this paper, we examine some possible reasons for this reduction in variability in our results.