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The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells
ARTICLE IN PRESS
Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells W. Fana, J. Dinga,*, W. Duanb, Y.M. Zhub a
Department of Materials Science, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Lower Kent Ridge Road, Singapore 119260, Singapore b Department of Biochemistry, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Available online 3 May 2004
Abstract The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.3870.06 compared to 1.9170.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field. r 2004 Elsevier B.V. All rights reserved. PACS: 87.19.La; 87.50.Mn Keywords: PC12 cells; Pulsed magnetic field; Nerve growth factor; Neurite outgrowth
Today, kinds of electrical appliance and equipments have been largely used in our society. It is of great interest to study the effects of the increased level of the electromagnetic field (EMF) in the environment on biological system [1], especially when it is related to health issues. One of the focus points of research in this field is to investigate the interaction between EMF and biological system and its possible consequence on human health. Recently, several studies have demonstrated that EMF can promote tissue regeneration, such as *Corresponding author. Tel.: +65-874-4317; fax: +65-7763604. E-mail address: [email protected] (J. Ding).
bone repair and neural regeneration [2]. On the other side, a high level of EMF may cause damage on human brain [3], showing that the strength of EMF may have a significant effect on biological systems. PC12 cells were established from rat pheochromocytoma cells by Greene and Tischler in 1976. They can react reversibly to the addition of the nerve growth factor (NGF) by differentiating and growing neurites [4]. This makes it a commonly used model for studies on neuronal differentiation and apoptosis. The possible influence of EMF on neurite out-growth in PC12 cells has been investigated. Some results show that a weak electromagnetic field (mT) could promote the neurite
0304-8853/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2004.04.076
ARTICLE IN PRESS 326
W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
outgrowth in PC12 cells [5,6], while inhibition of neurite out-growth was also observed [7]. One possible reason of the inconsistency of the published data is that the effect of EMF is strongly depended on the experimental condition, such as field strength, frequency of the pulsed field, orientation of electromagnetic field, exposure duration and NGF concentration. A systematic investigation is needed to clarify the effect of EMF on neurite out-growth in PC12 cells, especially the effect of the strength of magnetic field on neurite outgrowth. The aim of the present work was to investigate the influences of the strength of 50 Hz pulsed magnetic field on neurite outgrowth in PC12 cells, as a part of our on-going systematic study. Rat pheochromocytoma PC12 cells were cultured in 25 cm2 flask (Falcon, Biomed) in 5 ml medium at 37 C in a humidified atmosphere containing 5% CO2 and 95% air. The growth medium comprised of Dulbecco’s modified Eagle medium (DMEM, Invitrogen, #12100046) supplemented with 10% horse serum (Invitrogen, #16050122) and 5% fetal bovine serum (Invitrogen, #10270106). When confluent, the cells were detached by washing with PBS-1 mM EDTA solution. The suspension was centrifuged and the cells were replated at 2 104 cells/cm2 on collagencoated 24-well dishes (Falcon, #356408). For the induction of differentiation, cells were plated 2 days prior to the addition of the differentiation medium. The differentiation medium comprised of the DMEM medium, 1% horse serum and 0.5% FBS supplemented with different NGF concentration (7S, Sigma, # N0513) where needed. Dishes containing NGF were placed in the exposure system, which was housed in a 5% CO2 incubator maintained at 37 C. The control dishes were always placed in the same incubator inside a metal shielding box that could reduce the influence of the magnetic field generated by the exposure system to less than 1%. The differentiation medium with NGF was refreshed once every 2 days. The final cells were assayed after 96 h culture. Magnetic exposure system was composed of a rectangle copper coil of 1000 turns. The magnetic coil was placed into the incubator. In order to avoid contamination in the humidified condition,
the coil was airproofed into a plastic box. The coil was connected to a function generator (Arb/ Function generator). A pulsed signal with a frequency of 50 Hz was applied with field strengths of 0.23 and 1.32 mT, respectively. The magnetic field (B) was adjusted by varying the coil current. The assay procedure for neurite outgrowth used in this work was as described by Greene and Tischler [4]. The cells were examined in randomized areas at the center of each dish because it has the lowest intensities of induced electric field [7]. Neurite orientation with respect to the magnetic field was assayed with the aid of the labeling on the culture dish. Neurite outgrowth was evaluated from computer-captured images of each cell. Images were acquired by using a Zeiss phase-contrast microscopy (Axiovert 25, Zeiss) equipped with a digital camera (Nikon coolpix 995). The neurite length and direction were analysed with Leica image measurement software (Qwin 2.0). The known measurement between two points on a hemocytometer grid had been done and used for measurement calibration. All cells or cell clusters with each microscope field (minimum of 100 cells per dish) were assayed in a blinded fashion. The neurite length was recorded in micron unit (mm). Statistical analyses were performed using the Student’s t-test, in which difference were considered as significant for P o 0.05. Data were expressed as mean7S.E. Three experiments were performed for each of the two exposure tests. In one run of experiments, four dishes were exposed to the pulsed magnetic field and four were non-exposed which were placed inside the shielded box. Neurite orientation with respect to the magnetic field was noted by identification labeling at the end of the rectangular dish. In this paper, 30 ng/ml of NGF concentration was used in all experiments. The number of neurites per cell, the ratio of total number of neurite outgrowth to the total neurite-bearing cell number, and the average length of neurites are shown in Table 1. In table (a), it is found that, exposure to lower pulsed magnetic field (0.23 mT) had significantly stimulated neurite outgrowth in the numbers of neurite per cell. There were more neurite outgrowth in
ARTICLE IN PRESS W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
327
Table 1 Effect of 50 Hz pulsed magnetic fields on neurite outgrowth in PC12 cells Magnetic field (mT)
Control 0.23 1.32
(b) Average neurite length (mm)
Mean7S.E.
Change (%)
Significance
Mean7S.E.a
Change (%)
Significance
1.9170.07 2.3870.06 1.9970.12
24.6 4.2
0.004 0.555
50.9171.5 52.4372.22 42.7771.06
3 16
0.57 0.002b
Control, n ¼ 6 (from 24 dishes in 6 experiments); 0.23 and 1.32 mT, n ¼ 3 (from 12 dishes in 3 experiments). Significant by the Student’s t-test, where Po0:05:
each cell than the control mean (2.3870.06 compared to 1.9170.07 neurites/cell, P ¼ 0:004). However, Exposure to higher pulsed magnetic field (1.32 mT) caused small non-significant increase in the numbers of neurites for each cell (1.9970.12, P ¼ 0:56). The comparison for average neurite lengths of PC12 cells with exposed to different field intensities demonstrated that, exposure to the higher magnetic field (1.32 mT) caused significantly inhibition in average neurite length (42.7771.06 mm compared to 50.9171.50 mm, P ¼ 0:002). In contrast, exposure to the lower magnetic field had small non-significant increase on the average neurite length. Fig. 1 showed the distribution curves of neurite outgrowth. It could be seen clearly that there were more neurites growth in the lower range of average neurite lengths exposed to 1.32 mT than that of the control group and there were the similar curves between the control group and the group exposed to 0.23 mT. Directivity of outgrowth can be clearly shown by gathering the contributions of neurite outgrowths in all three experiments in one exposure test. As it was known, if all of the neurites had an optional outgrowth, the resultant pattern should be perfect radial. Fig. 2 showed the polar distribution of neurites outgrowth of PC12 cells, whereas the pulsed magnetic field was applied along the 0–180 axis and each neurite was indicated by a dot in terms of its length and angle. It can be seen, in the case of Fig. 2a (control group) and Fig. 2b (0.23 mT), all neurites was nearly radial outgrowth. It indicated that exposure to the lower magnetic field, the neurite outgrowth had not been induced by the direction of magnetic field. However, in the case of Fig. 2c (exposed to
neurite length distribution (%)
a b
(a) Neurite numbers per cell
20 control 0.23 mT 1.32 mT
15 10 5 0 0
50
100
150
200
250
average neurit length (µm)
Fig. 1. The distribution curves of neurite outgrowth of PC12 cells. White dots (J) represent the control group and black dots(m, ’) represent the exposed groups.
1.32 mT), there was an asymmetrical growth pattern, which more neurite expended outgrowth parallel to the direction of magnetic field than that of perpendicular to the magnetic field. It should be noticed that the stack at the center of the circle indicated that most of the outgrowths were nearly radial. The control and exposure data were compared in another way to indicate the directional outgrowth of neurites, shown in Table 2. All neurites were labeled with the angles and divided into four quadrants with an x2y-axis rotated 45 . The lengths of all neurites were analysed in each quadrant, and then combined with the average neurite length of two quadrants to be Dp and Dt ; in which Dp contained neurite outgrowths in the direction parallel to the magnetic field, and Dt contained neurite outgrowths expending perpendicular to the magnetic field. As discussed before, if all neurites had the same outgrowth, Dp should be equal to Dt : For the control data, there was no
ARTICLE IN PRESS W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
328
90 250 200 150 100 50 50 100 150 200 250
120 150
30
180
0
210
330 240
300 270
(a)
90
90
60
250 200 150 100 50 50 100 150 200 250
120
60
150
30
180
330
210 240
(b)
300 270
250 200 150 100 50 50 100 150 200 250
(c)
120
60
150
30 90 120 60 15 0 30 18 0 0 36 0 0 21 0 33 24 027 0 030
180
0
330
210 240
300 270
Fig. 2. Polar plots of neurite outgrowth of PC12 cell culture with different exposure conditions. (a) Control; (b) 0.23 mT; (c) 1.32 mT.
Table 2 Comparison of the directional neurite outgrowth of PC12 cells exposure to 50 Hz pulsed magnetic fields in different intensities Magnetic field (mT)
Dp (mm)
Control 0.23 1.32
50.9171.5 55.7773.43 49.4871.43 45.7671.02 39.6171.05
Dt (mm)
Significance 0.14 0.006
significant difference between Dp and Dt : For exposure, it was observed, exposure to the higher pulsed field, the average length parallel to the direction of magnetic field was significantly longer than that of perpendicular to the magnetic field. As was shown before, exposed to the higher field had caused significantly inhibition in the average neurite length, we found exposed to the higher field not only decrease the outgrowth parallel to the magnetic field, but also tends to quickly decrease the outgrowth perpendicular to the magnetic field, which the mean difference was 15.5% from the data parallel to the magnetic field. However, exposure to the lower magnetic field, there was no significant change between Dp and Dt : The mechanism underlying these effects has been not yet known. Blackman et al. [7] have hypothesized that specialized chemical structures uniquely sensitive to magnetic fields may be involved in cellular responses to magnetic fields; these structures would form the basis for the transduction of magnetic fields into a physical chemical change. Some reports have given evidences that Ca2+ channels may play an important
role in the cell responses to EMF. The Ca2+ level is increased under magnetic field at 100 mT [8], neurite outgrowth is triggered by Ca2+ entry through Ca2+ channels [9], and melatonin inhibits the influx of Ca2+ through the voltage-sensentive channels [10]. In conclusion, we have found that the neurite outgrowth is strongly dependent on field strength. A promotion was observed under a weak field, while inhibition and directional outgrowth was evident under a relatively stronger field. Our work has shown again that biological systems can be very sensitive to the strength of electromagnetic field.
References [1] R. Pool, Science 249 (4975) (1990) 1378. [2] A.L. Hulbert, J.C. Emtcalfe, R. Hesketh, FASEB 12 (1998) 395. [3] K. Iramina, T. Maeno, Y. Kowatari, S. Ueno, IEEE Trans. Magn. 38 (2002) 3347. [4] K.K. Teng, J.M. Angelastro, M.E. Cummingham, S.E. Farinelli, Lloyd A. Greene, Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 1, 1998, p. 244. [5] H. Takatsuki, A. Yoshikoshi, A. Sakanishi, Colloids Surf. B Biointerfaces 26 (2002) 379. [6] C.F. Blackman, J.P. Blanchard, S.G. Benane, D.E. House, Biochem. Biophys. Res. Commum. 220 (1996) 807. [7] C.F. Blackman, S.G. Benane, D.E. House, FASEB 7 (1993) 801. [8] L.A. Greene, S.E. Farinelli, M.E. Cunningham, D.S. Park, Culturing Nerve Cells, London, 1992. [9] G. Rusanescu, H. Qi, S.M. Thomas, J.S. Brugge, S. Halegoua, Neuron 15 (1995) 1415. [10] O. Slanar, H. Zemkova, J. Vanecek, Biol. Signals 6 (1997) 284.
Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells W. Fana, J. Dinga,*, W. Duanb, Y.M. Zhub a
Department of Materials Science, Faculty of Science, National University of Singapore, 10 Kent Ridge Crescent, Lower Kent Ridge Road, Singapore 119260, Singapore b Department of Biochemistry, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Available online 3 May 2004
Abstract The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.3870.06 compared to 1.9170.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field. r 2004 Elsevier B.V. All rights reserved. PACS: 87.19.La; 87.50.Mn Keywords: PC12 cells; Pulsed magnetic field; Nerve growth factor; Neurite outgrowth
Today, kinds of electrical appliance and equipments have been largely used in our society. It is of great interest to study the effects of the increased level of the electromagnetic field (EMF) in the environment on biological system [1], especially when it is related to health issues. One of the focus points of research in this field is to investigate the interaction between EMF and biological system and its possible consequence on human health. Recently, several studies have demonstrated that EMF can promote tissue regeneration, such as *Corresponding author. Tel.: +65-874-4317; fax: +65-7763604. E-mail address: [email protected] (J. Ding).
bone repair and neural regeneration [2]. On the other side, a high level of EMF may cause damage on human brain [3], showing that the strength of EMF may have a significant effect on biological systems. PC12 cells were established from rat pheochromocytoma cells by Greene and Tischler in 1976. They can react reversibly to the addition of the nerve growth factor (NGF) by differentiating and growing neurites [4]. This makes it a commonly used model for studies on neuronal differentiation and apoptosis. The possible influence of EMF on neurite out-growth in PC12 cells has been investigated. Some results show that a weak electromagnetic field (mT) could promote the neurite
0304-8853/$ - see front matter r 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jmmm.2004.04.076
ARTICLE IN PRESS 326
W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
outgrowth in PC12 cells [5,6], while inhibition of neurite out-growth was also observed [7]. One possible reason of the inconsistency of the published data is that the effect of EMF is strongly depended on the experimental condition, such as field strength, frequency of the pulsed field, orientation of electromagnetic field, exposure duration and NGF concentration. A systematic investigation is needed to clarify the effect of EMF on neurite out-growth in PC12 cells, especially the effect of the strength of magnetic field on neurite outgrowth. The aim of the present work was to investigate the influences of the strength of 50 Hz pulsed magnetic field on neurite outgrowth in PC12 cells, as a part of our on-going systematic study. Rat pheochromocytoma PC12 cells were cultured in 25 cm2 flask (Falcon, Biomed) in 5 ml medium at 37 C in a humidified atmosphere containing 5% CO2 and 95% air. The growth medium comprised of Dulbecco’s modified Eagle medium (DMEM, Invitrogen, #12100046) supplemented with 10% horse serum (Invitrogen, #16050122) and 5% fetal bovine serum (Invitrogen, #10270106). When confluent, the cells were detached by washing with PBS-1 mM EDTA solution. The suspension was centrifuged and the cells were replated at 2 104 cells/cm2 on collagencoated 24-well dishes (Falcon, #356408). For the induction of differentiation, cells were plated 2 days prior to the addition of the differentiation medium. The differentiation medium comprised of the DMEM medium, 1% horse serum and 0.5% FBS supplemented with different NGF concentration (7S, Sigma, # N0513) where needed. Dishes containing NGF were placed in the exposure system, which was housed in a 5% CO2 incubator maintained at 37 C. The control dishes were always placed in the same incubator inside a metal shielding box that could reduce the influence of the magnetic field generated by the exposure system to less than 1%. The differentiation medium with NGF was refreshed once every 2 days. The final cells were assayed after 96 h culture. Magnetic exposure system was composed of a rectangle copper coil of 1000 turns. The magnetic coil was placed into the incubator. In order to avoid contamination in the humidified condition,
the coil was airproofed into a plastic box. The coil was connected to a function generator (Arb/ Function generator). A pulsed signal with a frequency of 50 Hz was applied with field strengths of 0.23 and 1.32 mT, respectively. The magnetic field (B) was adjusted by varying the coil current. The assay procedure for neurite outgrowth used in this work was as described by Greene and Tischler [4]. The cells were examined in randomized areas at the center of each dish because it has the lowest intensities of induced electric field [7]. Neurite orientation with respect to the magnetic field was assayed with the aid of the labeling on the culture dish. Neurite outgrowth was evaluated from computer-captured images of each cell. Images were acquired by using a Zeiss phase-contrast microscopy (Axiovert 25, Zeiss) equipped with a digital camera (Nikon coolpix 995). The neurite length and direction were analysed with Leica image measurement software (Qwin 2.0). The known measurement between two points on a hemocytometer grid had been done and used for measurement calibration. All cells or cell clusters with each microscope field (minimum of 100 cells per dish) were assayed in a blinded fashion. The neurite length was recorded in micron unit (mm). Statistical analyses were performed using the Student’s t-test, in which difference were considered as significant for P o 0.05. Data were expressed as mean7S.E. Three experiments were performed for each of the two exposure tests. In one run of experiments, four dishes were exposed to the pulsed magnetic field and four were non-exposed which were placed inside the shielded box. Neurite orientation with respect to the magnetic field was noted by identification labeling at the end of the rectangular dish. In this paper, 30 ng/ml of NGF concentration was used in all experiments. The number of neurites per cell, the ratio of total number of neurite outgrowth to the total neurite-bearing cell number, and the average length of neurites are shown in Table 1. In table (a), it is found that, exposure to lower pulsed magnetic field (0.23 mT) had significantly stimulated neurite outgrowth in the numbers of neurite per cell. There were more neurite outgrowth in
ARTICLE IN PRESS W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
327
Table 1 Effect of 50 Hz pulsed magnetic fields on neurite outgrowth in PC12 cells Magnetic field (mT)
Control 0.23 1.32
(b) Average neurite length (mm)
Mean7S.E.
Change (%)
Significance
Mean7S.E.a
Change (%)
Significance
1.9170.07 2.3870.06 1.9970.12
24.6 4.2
0.004 0.555
50.9171.5 52.4372.22 42.7771.06
3 16
0.57 0.002b
Control, n ¼ 6 (from 24 dishes in 6 experiments); 0.23 and 1.32 mT, n ¼ 3 (from 12 dishes in 3 experiments). Significant by the Student’s t-test, where Po0:05:
each cell than the control mean (2.3870.06 compared to 1.9170.07 neurites/cell, P ¼ 0:004). However, Exposure to higher pulsed magnetic field (1.32 mT) caused small non-significant increase in the numbers of neurites for each cell (1.9970.12, P ¼ 0:56). The comparison for average neurite lengths of PC12 cells with exposed to different field intensities demonstrated that, exposure to the higher magnetic field (1.32 mT) caused significantly inhibition in average neurite length (42.7771.06 mm compared to 50.9171.50 mm, P ¼ 0:002). In contrast, exposure to the lower magnetic field had small non-significant increase on the average neurite length. Fig. 1 showed the distribution curves of neurite outgrowth. It could be seen clearly that there were more neurites growth in the lower range of average neurite lengths exposed to 1.32 mT than that of the control group and there were the similar curves between the control group and the group exposed to 0.23 mT. Directivity of outgrowth can be clearly shown by gathering the contributions of neurite outgrowths in all three experiments in one exposure test. As it was known, if all of the neurites had an optional outgrowth, the resultant pattern should be perfect radial. Fig. 2 showed the polar distribution of neurites outgrowth of PC12 cells, whereas the pulsed magnetic field was applied along the 0–180 axis and each neurite was indicated by a dot in terms of its length and angle. It can be seen, in the case of Fig. 2a (control group) and Fig. 2b (0.23 mT), all neurites was nearly radial outgrowth. It indicated that exposure to the lower magnetic field, the neurite outgrowth had not been induced by the direction of magnetic field. However, in the case of Fig. 2c (exposed to
neurite length distribution (%)
a b
(a) Neurite numbers per cell
20 control 0.23 mT 1.32 mT
15 10 5 0 0
50
100
150
200
250
average neurit length (µm)
Fig. 1. The distribution curves of neurite outgrowth of PC12 cells. White dots (J) represent the control group and black dots(m, ’) represent the exposed groups.
1.32 mT), there was an asymmetrical growth pattern, which more neurite expended outgrowth parallel to the direction of magnetic field than that of perpendicular to the magnetic field. It should be noticed that the stack at the center of the circle indicated that most of the outgrowths were nearly radial. The control and exposure data were compared in another way to indicate the directional outgrowth of neurites, shown in Table 2. All neurites were labeled with the angles and divided into four quadrants with an x2y-axis rotated 45 . The lengths of all neurites were analysed in each quadrant, and then combined with the average neurite length of two quadrants to be Dp and Dt ; in which Dp contained neurite outgrowths in the direction parallel to the magnetic field, and Dt contained neurite outgrowths expending perpendicular to the magnetic field. As discussed before, if all neurites had the same outgrowth, Dp should be equal to Dt : For the control data, there was no
ARTICLE IN PRESS W. Fan et al. / Journal of Magnetism and Magnetic Materials 282 (2004) 325–328
328
90 250 200 150 100 50 50 100 150 200 250
120 150
30
180
0
210
330 240
300 270
(a)
90
90
60
250 200 150 100 50 50 100 150 200 250
120
60
150
30
180
330
210 240
(b)
300 270
250 200 150 100 50 50 100 150 200 250
(c)
120
60
150
30 90 120 60 15 0 30 18 0 0 36 0 0 21 0 33 24 027 0 030
180
0
330
210 240
300 270
Fig. 2. Polar plots of neurite outgrowth of PC12 cell culture with different exposure conditions. (a) Control; (b) 0.23 mT; (c) 1.32 mT.
Table 2 Comparison of the directional neurite outgrowth of PC12 cells exposure to 50 Hz pulsed magnetic fields in different intensities Magnetic field (mT)
Dp (mm)
Control 0.23 1.32
50.9171.5 55.7773.43 49.4871.43 45.7671.02 39.6171.05
Dt (mm)
Significance 0.14 0.006
significant difference between Dp and Dt : For exposure, it was observed, exposure to the higher pulsed field, the average length parallel to the direction of magnetic field was significantly longer than that of perpendicular to the magnetic field. As was shown before, exposed to the higher field had caused significantly inhibition in the average neurite length, we found exposed to the higher field not only decrease the outgrowth parallel to the magnetic field, but also tends to quickly decrease the outgrowth perpendicular to the magnetic field, which the mean difference was 15.5% from the data parallel to the magnetic field. However, exposure to the lower magnetic field, there was no significant change between Dp and Dt : The mechanism underlying these effects has been not yet known. Blackman et al. [7] have hypothesized that specialized chemical structures uniquely sensitive to magnetic fields may be involved in cellular responses to magnetic fields; these structures would form the basis for the transduction of magnetic fields into a physical chemical change. Some reports have given evidences that Ca2+ channels may play an important
role in the cell responses to EMF. The Ca2+ level is increased under magnetic field at 100 mT [8], neurite outgrowth is triggered by Ca2+ entry through Ca2+ channels [9], and melatonin inhibits the influx of Ca2+ through the voltage-sensentive channels [10]. In conclusion, we have found that the neurite outgrowth is strongly dependent on field strength. A promotion was observed under a weak field, while inhibition and directional outgrowth was evident under a relatively stronger field. Our work has shown again that biological systems can be very sensitive to the strength of electromagnetic field.
References [1] R. Pool, Science 249 (4975) (1990) 1378. [2] A.L. Hulbert, J.C. Emtcalfe, R. Hesketh, FASEB 12 (1998) 395. [3] K. Iramina, T. Maeno, Y. Kowatari, S. Ueno, IEEE Trans. Magn. 38 (2002) 3347. [4] K.K. Teng, J.M. Angelastro, M.E. Cummingham, S.E. Farinelli, Lloyd A. Greene, Cell Biology: A Laboratory Handbook, 2nd Edition, Vol. 1, 1998, p. 244. [5] H. Takatsuki, A. Yoshikoshi, A. Sakanishi, Colloids Surf. B Biointerfaces 26 (2002) 379. [6] C.F. Blackman, J.P. Blanchard, S.G. Benane, D.E. House, Biochem. Biophys. Res. Commum. 220 (1996) 807. [7] C.F. Blackman, S.G. Benane, D.E. House, FASEB 7 (1993) 801. [8] L.A. Greene, S.E. Farinelli, M.E. Cunningham, D.S. Park, Culturing Nerve Cells, London, 1992. [9] G. Rusanescu, H. Qi, S.M. Thomas, J.S. Brugge, S. Halegoua, Neuron 15 (1995) 1415. [10] O. Slanar, H. Zemkova, J. Vanecek, Biol. Signals 6 (1997) 284.