Proceedings of the International Conference on Non-Ionizing Radiation at UNITEN (ICNIR 2003)
Electromagnetic Fields and Our Health
20th –
22nd October 2003
Effects of Whole Body Exposure to 50Hz Electromagnetic Fields
on the Intramicrovascular Leukocyte Adhesion in Mice
A. Ushiyama, C. Ohkubo
Department of Environmental Health, National Institute of Public Health
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8638, Japan
Tel/Fax: +81-3-3446-6740, e-mail:[email protected]
ABSTRACT
Whole body exposure effects to threshold levels of 50 Hz electromagnetic fields (3.0, 10.0,
30.0 mT for acute exposure and 0.3, 1.0, 3.0 mT for subchronic exposure) on
intramicrovascular behavior of leukocytes in the cutaneous microcirculation was evaluated by
a dorsal skinfold chamber technique in mice under conscious conditions. The results
indicated that the exposure intensity at 3.0 mT is a threshold level for increasing leukocyte
adhesion to the endothelial walls.
KEYWORDS
50 Hz EMF exposure, leukocyte adhesion, microcirculation
INTRODUCITON
Effects of 50Hz electromagnetic fields (EMF) exposure on leukocyte are mainly performed in
vitro system, however, little information of these is available in vivo experiments. In order
to investigate the acute and subchronic exposure effects of threshold levels of 50Hz EMF on
leukocyte behavior in vivo system, we measured the behavior of intra-microvascular
leukocytes in the cutaneous microcirculation in mice by use of a dorsal skinfold chamber
(DSC) technique (Fig 1, 2).
Fig. 1. Mouse Having A DSC
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Proceedings of the International Conference on Non-Ionizing Radiation at UNITEN (ICNIR 2003)
Electromagnetic Fields and Our Health
20th –
22nd October 2003
1mm
100mm
Fig. 2. Microvasculature Within A DSC
Low Magnification(Upper, Bar Indicates 1mm),
High Magnification(Lower, Bar Indicates 100µ m)
MATERIALS AND METHODS
Male mice (BALB/c) having the DSC were subjected to intravital-microscopic study. We
have developed the DSC with non-metal materials of Duracon resin, which could not be
physically affected by EMF exposure. For visualization of intra-microvascular leukocytes,
fluorescent dye (rhodamine 6G; 0.3mg/kg, iv) was injected. The numbers of leukocyte
rolling or adhering to the venular walls were measured by confocal laser microscopy and
recorded into VCR and analyzed from the images (Fig 3, 4). The magnetic flux densities
used for the acute exposure (30 minutes) were controlled at 3, 10, 30 mT at the center of
animal body (n=10 each), respectively. For subchronic exposure study, mice were divided
into 4 groups(n=10 each), i.e., exposure group with 50 Hz EMF at 0.3, 1.0 and 3.0 mT and
control group with sham exposure. The 50Hz EMF exposure was intermittently performed
everyday from 16:00 to 12:00(20hours/day) for 15 days (Fig 5). Plasma cytokine (IL-1ß,
TNF-a) concentration was measured by ELISA.
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Proceedings of the International Conference on Non-Ionizing Radiation at UNITEN (ICNIR 2003)
Electromagnetic Fields and Our Health
20th –
22nd October 2003
EB-CCD
( Hmamatsu)
Dye: 0.02% Rhodamine 6G
VTR
Monitor
Dye was injected from tail vein.
CSU10
( Yokokawa)
YAG laser
( CrystaLaser)
Fig. 3. Experimental Set-Up For Visualization Of Leukocytes
Leukocytes labeled with rhodamine 6G are visualized under confocal laser microscopy
system with EB-CCD and CSU10
Fig. 4. Video Image of Intramicrovascular Leukocyte
arrow shows labeled leukocyte.
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Proceedings of the International Conference on Non-Ionizing Radiation at UNITEN (ICNIR 2003)
Electromagnetic Fields and Our Health
20th –
22nd October 2003
[day]
-3
dorsal skin fold chamber implantation to BALB/c
mice (male, 8 -10Wks, each group n= 10)
0
beginning of EMF exposure (50 Hz, 20 hr / day)
8
measurement and analysis of leukocyteendothelial interaction under the intravital
microscopy with confocal unit
15
: EMF exposure
(Day0 ~ Day15)
end of EMF exposure
Fig. 5. Experimental Protocol
Control
30.0 mT
10.0 m T
3.0 mT
25
p<0.05
20
15
20
20
15
15
10
15
10
10
10
5
5
5
5
0
0
0
0
pre
post
pre
pre
post
post
pre
post
Fig. 6. Changes In Rolling Leukocyte Counts Due To Acute Exposure (30min) Of 50 Hz
Statistically significant difference (p<0.05) was noticed between pre- and post-exposure at 30.0 mT
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Proceedings of the International Conference on Non-Ionizing Radiation at UNITEN (ICNIR 2003)
Electromagnetic Fields and Our Health
20th –
22nd October 2003
RESULTS AND DISCUSSION
Acute Effects: A tendency to increase the adherent cell count of leukocytes due to the 50 Hz
EMF exposure toward higher magnetic field intensity was recognized. Following the
exposure at 30 mT, the counts of adherent cell was significantly higher than those obtained
before exposure (p<0.05, Fig 6).
Subchronic Effects: Following subchronic exposure to 50Hz EMF at 3.0 mT, statistically
significant increases (p<0.05) in adherent leukocyte counts were noticed, however, there was
no statistically significant changes between before and after exposures in any other groups
(Fig 7,8). No noticeable changes in IL-1ßand TNF-a concentration were observed in any
groups.
Subchronic Effects: Following subchronic exposure to 50Hz EMF at 3.0 mT, statistically
significant increases(p<0.05) in adherent leukocyte counts were noticed, however, there was
no statistically significant changes between before and after exposures in any other groups
(Fig 7,8). No noticeable changes in IL-1ßand TNF-a concentration were observed in any
groups.
The results indicated that 50 Hz EMF exposure may influence cell to cell interaction between
venular endothelial cells and leukocytes. Previous observations using human monocyte in
in vitro system indicated that changes in cytokine profile of monocyte were induced by
exposures of 50 Hz EMF [1,2]. We hypothesize that 50 Hz EMF exposure effects on
leukocyte and endothelial cell interaction due to change in cytokine levels, however, IL-1ß
and TNF-a may not be involved in this phenomenon (Fig 9). Further studies will be
required.
REFFERENCES
1) Cossarizza A, et al.: Exposure to low frequency pulsed electromagnetic fields increases
interleukin-1 and interleukin-6 production by human peripheral blood mononuclear cells.
Exp. Cell. Res., 204: 385-387, 1993
2) Jonai H, Villanueva MBG, Yasuda A: Cytokine profile of human peripheral blood
mononuclear cells exposed to 50 Hz EMF. Industrial Health, 34: 359-368, 1996
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