An Environment for Studying Effects of Electromagnetic Isolation or Specific Electromagnetic Fields on Nociception in Mice.

L.D. Keenliside, A.W. Thomas, F.S. Prato.

Imaging Division, Bioelectromagnetics, Lawson Health Research Institute, Dept. of Nuclear Medicine, St. Josephs Health Care; and Biophysics, Schulich School of Medicine and Dentistry, University of Western Ontario, 268 Grosvenor St., London, Ontario, Canada N6A 4V2 Tel: 519-646-6100 ext. 61068, Fax: 519-646-6399 Email: lynnk@lawsonimaging.ca

Abstract
We have shown in mice (Prato et al., 2005) that exposure to a magnetically shielded environment for 1 hour per day for 10 consecutive days induces a decrease in nociception (i.e. induces analgesia) that peaks at day 5. We have further shown (Koziak et al., 2006) that the induced analgesia can be abolished if during the shielding there is simultaneous exposure to visible light (400-750 nm; 1.35 1018 photons/s/m2). We have also shown (Robertson et al., 2005) that the reintroduction of the time varying component of the ambient magnetic field will abolish the analgesic effect. Here, we report the difficulties and solutions explored to provide the environment for these experiments.

Procedure
The mu-metal boxes attenuated ambient static and fluctuating magnetic fields by a factor of 100 or more. The copper boxes provided shielding for RF and electrical fields, working as Faraday shields. The sham box was made with an outer shell of fiberglass and provided shielding from ambient light only. All enclosures were lined with black, opaque PVC plastic. Initially, we attempted to introduce light using compact florescent lamps mounted in a supporting case under the ports of the enclosures. These lamps, while more efficient than incandescent bulbs, produced too much heat which raised the internal temperature several degrees. We then changed to 4 arrays of 9 white LEDs (light emitting diodes) 5600 mcd, powered with a DC source. (figure 2) A spectrum from 440 to 650 nm was produced using a combination of blue white and incandescent white LEDs. Each array was placed in a PVC coupling and mounted under the bottom ports of the enclosure. Light was directed into the centre of the enclosure and evenly distributed initially with mirrored reflectors, then later with light pipes made from acrylic rods. The overall result produced a strong blue peak at 460 nm and a lesser broad peak at 570 nm and approximates intensities typical of office or lab room lighting (figure 3). Adjustable current regulated DC power supplies were made to provide adjustable light intensities. Three different light intensities were tested with integral number of photons/s/m2 of: 2.2 1018 (100%), 1.45 1018 (66%) and 0.726 1018 (33%). Additional arrays of red, green, yellow and blue LEDs were made with peaks equal or greater than respective peaks in the white light arrays and likewise made to work with the adjustable power supplies.

Next, an array of 4 coils comprised of 150 turns #28 magnet wire were constructed on polycarbonate forms to fit inside the enclosures in a Merritt-like configuration. Two acrylic strips notched to hold the forms accurately in place retain these 31.4cm by 18.8cm forms. The forms also provide support for a base to hold the mouse cage in position. The coils were connected to our custom-made signal generator (Cnp Generator Ver2) to produce complex magnetic waveforms of our choosing. This included the ability to digitally record a sample of the earths magnetic field to replay in a repeating loop. Intensity of the field was adjustable from 50nT to 200uT.

Specifications
Ambient field attenuation- >100:1 Re-introduced field0 to 200uT Light intensity (max)2.5 1018 photons/s/m2 Wavelength (white)440-650nm Other coloursRed, Yellow, Green, Blue Dimensions33cm by 39cm by 20cm

Results

Objective
To develop an environment for mice where we can eliminate the ambient magnetic field, re-introduce an electromagnetic field of our choosing as well as control the internal lighting colour and intensity (figure 1).

Fig.3. Example plots of white LED light

Methods
Initial experiments in the effects of magnetic shielding on nociception in mice at Lawson Research Institute were begun in 1997 with the use of two mu-metal enclosures (Magnetic Shielding Corporation Bensenville IL), one copper enclosure (constructed in-house) and one sham enclosure (in-house). In all cases, the 33cm wide by 39cm deep by 20cm high enclosures were constructed to be physically identical. Shielded portholes in all 4 corners of the top and bottom provide ventilation and instrumentation access. A hinged door with latch provides access at one end. Only the external material for the shell varied. As experiments evolved and became more complex, the enclosures were modified to accommodate the needs of the investigators. These needs included; 1) the introduction of light without affecting the magnetic field or internal temperature. 2) the introduction of specific magnetic fields without introducing audible noise or heat to the environment.

Fig. 1. Finished Enclosure

References
Koziak AM et al. Bioelectromagnetics, 27:1-6, 2006. Prato FS et al. Bioelectromagnetics, 26:109-117, 2005. Robertson JA et al. Reintroduced Time-Varying Geomagnetic Field Attenuates Analgesia Due to Repeated Magnetic Field Shielding. Bioelectromagnetics 2005 P-B-197 & Platform Presentation

Final Testing
Measurement of the ambient magnetic field, internal magnetic field and mapping the homogeneity of the reintroduced field was done with a Bartington Mag03MS1000 (Oxford, England) magnetometer. The volume within the mouse cage has a uniform field within 7%. Light levels were measured at the centre of the enclosure with a LightSpex, McMahan Research, (Chapel Hill NC) spectroradiometer equipped with a fibre optic probe. Internal temperature was measured with a Fluke 179 (Mississauga, ON) multimeter and thermocouple. A rise of <1C was noted after 2 hours of operation with the access door closed. Sound levels measured with a 1" condenser type 4131 microphone made by Brel & Kjr (Copenhagen, Denmark). No signal was detected over ambient noise while coils were energized with 60Hz sinusoidal waveform at maximum field (200uT).

Acknowledgements
Thanks to John Robertson for assistance in qualifying the enclosure. This project was funded by St. Josephs Health Care (London) Foundation, Lawson Health Research Institute, Canadian Institutes of Health Research (CIHR), Ontario Research and Development Challenge Fund (ORDCF), Canada Foundation for Innovation (CFI) and Ontario Innovation Trust (OIT).

Fig. 2. Led Light Sources, regulator and light pipe