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Effects of 3.5–23.0 T static magnetic fields on mice: A safety study
NeuroImage 199 (2019) 273–280
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Effects of 3.5–23.0 T static magnetic fields on mice: A safety study Xiaofei Tian a, b, 1, Dongmei Wang a, c, 1, Shuang Feng a, c, Lei Zhang a, Xinmiao Ji a, Ze Wang a, d, Qingyou Lu a, d, e, Chuanying Xi a, Li Pi a, Xin Zhang a, b, c, * a High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, PR China b Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui, 230601, PR China c Science Island Branch of Graduate School, University of Science and Technology of China, Hefei, Anhui, 230026, PR China d Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, 230026, PR China e Anhui Province Key Laboratory of Condensed Matter Physics at Extreme Conditions, Hefei, Anhui, 230031, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Static magnetic field (SMF) Magnetic resonance imaging (MRI) Ultra-high field (UHF) Safety Mice
People are exposed to various magnetic fields, including the high static/steady magnetic field (SMF) of MRI, which has been increased to 9.4 T in preclinical investigations. However, relevant safety studies about high SMF are deficient. Here we examined whether 3.5–23.0 T SMF exposure for 2 h has severe long-term effects on mice using 112 C57BL/6J mice. The food/water consumption, blood glucose levels, blood routine, blood biochemistry, as well as organ weight and HE stains were all examined. The food consumption and body weight were slightly decreased for 23.0 T-exposed mice (14.6%, P < 0.01, and 1.75–5.57%, P < 0.05, respectively), but not the other groups. While total bilirubin (TBIL), white blood cells, platelet and lymphocyte numbers were affected by some magnetic conditions, most of them were still within normal reference range. Although 13.5 T magnetic fields with the highest gradient (117.2 T/m) caused spleen weight increase, the blood count and biochemistry results were still within the control reference range. Moreover, the highest field 23.0 T with no gradient did not cause organ weight or blood biochemistry abnormality, which indicates that field gradient is a key parameter. Collectively, these data suggest 3.5–23.0 T static magnetic field exposure for 2 h do not have severe long-term effects on mice.
1. Introduction Since higher magnetic field intensity could improve image quality and acquisition capability of MRI (magnetic resonance imaging), the magnetic field intensity used on MRI for humans has increased from 1.5 and 3.0 T in most hospitals to 9.4 T in preclinical stage (Neuner et al., 2013, 2014; U gurbil, 2018). For example, the 9.4 T MRI has been used to do quantification of sodium concentration distribution studies in human brain, which were not feasible with MRIs with lower magnetic fields (Thulborn et al., 2017). Moreover, not only 11.7 T MRI has been manufactured and tested on human tissue (Beaujoin et al., 2018; Vedrine et al., 2014), people have also pushed the limit of static/static magnetic field (SMF) to as high as 21.1 T, which has been used on rodents to get high resolution brain images (Nagel et al., 2016; Schepkin et al., 2011). Correspondingly, the resolution has been improved from ~1 mm for 1.5–3.0 T MRI, ~100 μm for 9.4 T MRI to ~18 μm for 21.1 T MRI. The increased MRI imaging resolution is essential for early diagnosis of many
diseases, such as small internal lesions and cancer. However, as the main core component of MRI, the biosafety issues of high SMFs are still under investigated. Although there are some sporadic cellular experiments, animal studies about the safety issues of high SMFs above 9.4 T are very limited. Recently, Wang et al reported that 2-12 T SMF treatment for 28 days did not generate significant defects on C57BL/6 mice (Wang et al., 2019). In fact, Budinger and Bird have written two very comprehensive reviews about the technology and application potentials of high field MRIs up to 20 T (Budinger et al., 2016; Budinger and Bird, 2018). They concluded that there are no foreseen barriers of brain MRI and MRS (magnetic resonance spectroscopy) at magnetic fields up to 20 T and suggested that people should try to develop high field MRI and MRS up to 20 T. They mentioned that no deleterious effects have been observed on rodents at 21.1 T by NHMFL (National High Magnetic Field Laboratory) investigators, except for some temporary effects on the vestibular apparatus (Budinger et al., 2016; Budinger and Bird, 2018). Howeber, there
* Corresponding author. High Magnetic Field Laboratory, Chinese Academy of Sciences, Shushanhu Road #350, Hefei, Anhui, 230031, PR China. E-mail address: xinzhang@hmfl.ac.cn (X. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.neuroimage.2019.05.070 Received 10 February 2019; Received in revised form 3 May 2019; Accepted 27 May 2019 Available online 31 May 2019 1053-8119/© 2019 Elsevier Inc. All rights reserved.
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force on those diamagnetic mice. Accordingly, the mice in the bottom part of the magnet were in “hypergravity” conditions. Therefore, although there are two groups of mice for 21.9 T, 13.5 T and 6.7 T, they experienced magnetic force of different directions, which make them in “hypogravity” and “hypergravity” conditions respectively. Only static magnetic fields were examined in this study and no radiofrequency was involved. On the day of exposure, we randomly divided the 112 mice into three groups, control group, sham control group and magnetic field exposed group (Fig. 1B). For each specific magnetic field exposure condition, we have 6 mice that were tested on 6 independent days. All mice were placed in individual nonmagnetic stainless-steel tube and then stacked them into the inner cylinder in turn before they were placed into the water-cooled magnet or left outside the magnet as illustrated (Fig. 1) and as previously described (Tian et al., 2018). The magnetic field exposure time is 2 h and 10 min in total including increasing field for 5 min (dB/dt is about 0.077 T/s), constant at 23.0 T for 2 h and reducing field for 5 min for each experiment. After the mice were treated with control, sham control or magnetic field exposure, the mice were fed normally by sterilized food and autoclaved water for another three weeks. The food and water consumption and body weight were measured daily during the whole experiment. Blood glucose levels were measured every two days.
was no experimental data available. In fact, although there are some in vitro cellular studies about biological effect studies of SMFs above 9.4 T (Nakahara et al., 2002; Qian et al., 2009, 2012, 2013; Sun et al., 2015), there are very limited studies about any biological effect studies of SMFs above 20 T, which is not only limited by the availability of Ultra high field (UHF) magnets, but also by the compatibility of biological samples. Until now, other than the unicellular organism Paramecium (Guevorkian and Valles, 2006) and human cells (Zhang et al., 2017), there was only one preliminary experimental study about whole animals in high SMFs above 20 T, which was done by our group (Tian et al., 2018). We used 16 tumor-bearing nude mice to examine the effects of 9-h exposure to 3.7–24.5 T SMFs. The small sample size was due to limited machine time. Our results show that while most indicators are normal, the mice liver was affected to some extent (Tian et al., 2018). Since 9-h is much longer than what is needed for an MRI examination, here in this study, we reduced it to 2 h and also increased the sample size to 112 mice to systematically investigate the safety issue of ultra-high static magnetic fields on normal mice. Using 112 mice and a water-cooled magnet that provides a 23.0 T SMF in the center, and descending magnetic field intensities with various gradient off the center, we investigated the effects of 3.5–23.0 T SMFs on mice. After comprehensive examination of these mice, we did not find deleterious effects of these high SMFs, except for some spleen weight enlargement by the highest gradient magnetic field exposure, as well as some changes on white blood cell count in some magnetic field exposed conditions. However, most of the changes are either statistically not significant from the sham control or still within the normal reference range, which was defined by the results from sixteen mice in the control group.
2.3. Blood biochemistry analysis Blood samples were obtained by removing the mice eyeballs at the end of the experiment. 200 μL blood samples with 0.15% (M/V) EDTAK2⋅2H2O were used for blood routine examination by an automatic hematology analyzer (Sysmex, Japan). Meanwhile, the heart, liver, spleen, lungs and kidneys were weighed and fixed with 4% formaldehyde (#10010018, Sinopharm Chemical Reagent) for 24 h after blood sampling. The serum was collected by centrifugation after the blood sample placed at room temperature for 2 h. The serum was stored at 80 C or dry ice until they were sent to Nanjing Biomedical Research Institute of Nanjing University for blood biochemistry analysis by an automatic analyzer (HITACHI 7020, Japan) as soon as possible. Biochemical indicators to be tested included aspartic transaminase (AST), alanine
2. Materials and methods 2.1. Construction of the ultra-high magnetic mice exposure system A water-cooled magnet (WM2) was used to generate 3.5–23 T SMFs in Chinese High Magnetic Field Laboratory (CHMFL, Hefei, China). The magnet cavity is 50 mm in diameter and 1300 mm long. In order to explore the biological safety of strong static magnetic field, we designed and constructed two identical sets of the devices with accurate temperature and gas control, which were suitable for the WM2 magnet. The device contains a removable inner cylinder which is 41 mm diameter and 700 mm long. Each mouse was housed in a nonmagnetic stainless-steel tube (38.5 mm diameter, 80 mm long) and eight mice were placed into the inner cylinder in turn before each experiment. To make sure the mice get enough oxygen, a pump was used to circulate the air inside the cylinder at 450 L/min. Meanwhile, the air duct was placed into the ice water to prevent air from overheating caused by the pump. The temperature inside the mice tube was controlled at 22–24 C by thermal conduction from temperature-controlled water that flowed through the space of the devices. 2.2. Mice exposure to magnetic fields, sham control and control conditions The animal protocols were approved by the ethical and humane committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences and strictly followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). One hundred and twelve 8-week-old male C57BL/6J mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Each cage contained five mice and all mice were kept in the clean condition with food and water available ad libitum. The magnet provides a 23.0 T SMF in the center and descending SMF intensities with various gradient off the-center (Fig. S1). As a result, the mice in the upper part of the magnet were in “hypogravity” conditions, because of the magnetic field gradient generates an upward magnetic
Fig. 1. Magnetic field exposure conditions. (A) 112 mice were divided into three groups. (B) Illustration of mice position and magnetic field exposure conditions. Mice were placed in individual tubes. Red arrows show magnetic field direction. 274
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aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), total bilirubin (TBIL), blood urea nitrogen (BUN), serum creatinine (CREA), calcium (Ca), phosphorus (P), iron ion (Fe), total cholesterol (CHOL), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU).
differences between the “sham” group and SMF group. Statistical analysis was performed by using SPSS statistical analysis software (SPSS 19.0; SPSS, Chicago, IL) and P value less than 0.05 was considered statistically significant.
2.4. Histologic examination
The data and/or code used in the study are available in the public domain or upon direct request and comply with the requirements of the funders and institutional ethics approval.
2.6. Data and code availability statement
After mice were sacrificed on day 21, the heart, liver, spleen, lungs and kidneys tissues were fixed in 4% formaldehyde for 24 h. Then the tissues were embedded in paraffin after formaldehyde removal, dehydration, cleaning and waxing. The paraffin-embedded specimens were sliced at 5 μm thickness and then they would be stained by means of routine HE staining.
3. Results We exposed the mice to control, sham control or magnetic fields as illustrated (Fig. 1). We used two set of controls. One is the regular “control”, in which the mice were placed in the tube but not placed in the magnet. The other one is the “sham control”, in which the mice were placed in the tube and placed in the magnet with water running. This fully mimics the bore vibration and noise conditions in the magnetic field exposed group. Therefore, we can compare and analyze whether the observed effects are caused by bore vibration and noise, or by magnetic fields themselves. In general, the food and water consumptions were not significantly
2.5. Statistical analysis The normality of the data was analyzed by Shapiro–Wilk test and the homogeneity of variance was assessed with levene test. The significance of the differences in all data among the “sham” group and SMF group was evaluated by unpaired t-test. If the data didn't comply with the normal distribution, the Mann-Whitney U test was used to compare the
Fig. 2. Food and water consumption, as well as body weight and blood glucose of mice exposed to control, sham control or magnetic fields. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Data are mean standard deviations. Statistical analysis was performed for all data. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. “**”, P < 0.01. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05). 275
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Table 1 Liver and kidney function parameters. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Red bold font illustrates data that are statistically significant than the sham control. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
affected by magnetic field exposure, except for groups 4 and 5 for food consumption, and groups 1 and 2 for water consumption (Fig. 2A). The food consumption for group 4 (21.9 T) is reduced by 6.1% and for group 5 (23.0 T) is reduced by 14.6% (Fig. 2A, left). The water consumption for groups 1 and 2 were both increased, for 23.1% and 12.5% respectively (Fig. 2A, right). We also monitored the body weight every day (Fig. 2B and Fig. S2) and measured blood glucose every two days for each mouse (Fig. 2C). For groups 1, 2 and 8, the average mice body weight was decreased between control and sham control groups, which is likely due to the machine vibrating since they were located at both ends of the magnet (Fig. S2). To examine the magnetic field-induced effects per se, we compared between the sham control and the magnetic field exposed groups. We found that
only the mice in group 5 showed significant weight gain reduction after magnetic field exposure (Fig. 2B, right), which means that 23 T exposure for 2 h may affect the mice weight gain, but 21.9 T or below may not (Fig. 2B left and Fig. S2). The decreased body weight is likely due to the reduced food intake (Fig. 2A). We also measured their blood glucose every two days and found no significant difference after magnetic field exposure (Fig. 2C). To analyze magnetic field exposure-induced physiological consequences, we further measured multiple biochemistry parameters to reflect the liver and kidney functions (Table 1). For all these 8 indicators, AST (aspartic transaminase) and TBIL (total bilirubin) are the only two that showed alterations. In general, the AST levels were increased and TBIL were decreased by magnetic field exposure, although only two
Table 2 Metabolic parameters of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data and they do not have statistical significance (P > 0.05). Glu (mmol/L) P (mmol/L) Ca (mmol/L) Fe (μmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L) CHOL (mmol/L)
5.53 1.10 (2.98–7.07) 3.72 0.32 (3.22–4.35) 2.38 0.07 (2.28–2.58) 16.20 4.51 (9.10–24.90) 0.36 0.06 (0.29–0.51) 1.92 0.21 (1.48–2.26) 0.56 0.12 (0.36–0.82) 2.65 0.29 (2.04–3.13) Mean SD (Range) Control
6.02 1.02 3.48 0.67 2.32 0.08 17.26 4.45 0.39 0.05 1.86 0.14 0.69 0.27 2.62 0.19 Sham Group 1
6.66 1.01 3.80 0.43 2.38 0.08 20.82 5.82 0.33 0.05 1.71 0.35 0.68 0.48 2.37 0.45 3.5T
5.89 1.30 3.41 0.38 2.31 0.08 16.13 4.07 0.40 0.11 1.76 0.31 0.67 0.24 2.49 0.46 Sham Group 2
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5.19 2.01 3.78 0.48 2.34 0.09 16.58 7.81 0.40 0.05 1.86 0.13 0.56 0.27 2.61 0.16 6.7T
6.93 0.85 3.32 0.46 2.09 0.57 19.00 4.58 0.41 0.09 1.91 0.11 0.68 0.21 2.70 0.24 Sham Group 3
5.55 1.53 3.95 0.75 2.40 0.12 19.58 9.92 0.37 0.08 1.93 0.16 0.71 0.45 2.69 0.26 13.5T
6.46 0.83 3.52 0.33 2.29 0.04 18.78 6.46 0.36 0.11 1.75 0.31 0.71 0.36 2.47 0.50 Sham Group 4
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Table 3 Blood routine examination of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Red bold font illustrates data that are statistically significant than the sham control. There were six mice in sham control and SMFtreated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
by multiple high magnetic field exposure conditions, especially by magnetic fields with higher gradients. For example, group 3 and 7 are both 13.5 T with the highest magnetic field gradient (117.2 T/m) as well as BB’ (1582.2 T2/m) increased the spleen weight by 44.6% and 28.5%, respectively. However, it is interesting that the changes only happened to mice that were exposed to gradient magnetic fields, but not to the mice exposed to 23.0 T with no gradient. Moreover, the HE stain of the organs did not show obvious abnormalities for all these mice (Fig. 4 and Figs. S3–9).
groups (groups 2 and 8) showed statistical significance for AST and only one group (group 3) showed statistical significance for TBIL (Table 1). All the other parameters were not much affected. Moreover, we also tested several metabolic parameters, including blood glucose, cholesterol, Fe and Calcium etc, and did not find any alterations caused by high SMF exposure (Table 2). It should be mentioned that mice in group 5, which were exposed to the strongest magnetic field (23.0 T) with no gradient, did not show any statistically significant changes in these biochemistry parameters related to liver and kidney functions or metabolism. To get a more comprehensive understanding about the high magnetic field effects, we also did the blood routine examination. We found that high SMFs did not affect red blood cell numbers and its related parameter (Table 3 and Table S1), including HGB (hemoglobin) (Table 3). Although some magnetic conditions affected platelet, white blood cells and lymphocyte numbers, all the values after magnetic field exposure are still within the control reference range (Table 3). At the end of the experiment, we also measured the weight of mice organs for all these 112 mice, including their heart, liver, spleen, lung and kidney (Fig. 3). Among them, spleen weight was the only one that was affected by magnetic fields (Fig. 3). The spleen weight was increased
6.02 1.39 4.03 0.53 2.42 0.14 19.18 7.48 0.36 0.06 1.82 0.31 0.51 0.41 2.51 0.43 21.9T Group 4
6.26 0.61 3.41 0.32 2.33 0.06 20.28 4.76 0.38 0.08 1.81 0.25 0.66 0.28 2.56 0.35 Sham Group 5
6.86 1.24 3.71 0.22 2.37 0.04 24.12 13.4 0.35 0.09 1.84 0.18 0.46 0.13 2.51 0.28 23.0T
7.01 1.50 3.40 0.72 2.34 0.11 17.33 3.73 0.38 0.11 1.78 0.38 0.57 0.17 2.51 0.57 Sham Group 6
4. Discussion Our results show that 3.5–23.0 T static magnetic field exposure for 2 h do not have severe long-term impact on mice. The changes in most parameters are statistically not significant. For a few parameters, although the changes are statistically significant, most of them are still within the control reference range, such as white blood cells. None of the changes are substantially off the control reference range. Although we did not do an actual MRI examination and the effect of radiofrequency (RF) magnetic field was not evaluated in this study, our results indicate that the
6.43 1.63 3.86 0.59 2.35 0.05 25.36 14.53 0.40 0.07 1.88 0.25 0.49 0.18 2.61 0.31 21.9T
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6.52 1.31 3.76 0.88 2.34 0.04 19.04 2.28 0.38 0.07 1.91 0.19 0.86 0.34 2.69 0.26 Sham Group 7
6.24 0.99 3.72 0.32 2.36 0.08 22.87 9.48 0.31 0.04 1.81 0.26 0.52 0.25 2.42 0.35 13.5T
7.11 1.81 3.32 0.68 2.34 0.14 18.60 4.10 0.37 0.06 1.89 0.18 0.56 0.24 2.61 0.25 Sham Group 8
6.36 1.11 3.63 0.23 2.36 0.05 22.88 8.64 0.39 0.07 2.02 0.19 0.61 0.44 2.77 0.27 6.7T
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Fig. 3. Relative organ weight of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Grey dashed lines show the average organ weight of 16 control mice. There were six mice in sham control and SMF-treated group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05, “**”, P < 0.01. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
static magnetic field part in the range of 3.5–23.0 T is relatively safe, at least at mice level, especially if we consider that our exposure time is 2 h, which is much longer than the actual MRI examination time. We expect to see even less effects if we further reduce the exposure time to 1 h or to a few minutes. Although it has been shown that the orientation of red blood cells can be influenced even by 1 T static magnetic field and almost 100% of them could be oriented when exposed to 4 T (Higashi et al., 1993), we did not observe any red blood cell defects. This indicates that the orientation change of red blood cells does not affect their key parameters, including its number and HGB content. A previous in vitro cellular study investigated the effects of a 4.75 T homogeneous SMF and found that it does not affect the physiologic behavior of normal lymphomonocytes (Aldinucci et al., 2003). Here we found that in mice, 3.5–23.0 T SMFs generally decreased lymphocyte numbers, although still within the normal control range. It should be noted that the highest magnetic field intensity of 23.0 T in group 5 reduced the food consumption and body weight gain but did not have other significant effects. Moreover, although still within the normal reference range, there are some small but statistically significant effects on blood cell count for several SMF exposed groups. However, we did not find direct relationship between these changes with SMF intensity or gradient. It is likely that these mild effects are influenced by a combination of multiple factors, including magnetic field intensity, gradient and gradient direction. In addition, the generally elevated AST and decreased TBIL may indicate some liver function abnormalities, but they
are mostly still within the normal control range and the liver tissues also have normal appearance in their HE stains. These results indicated that the liver alteration and the other blood cell count changes are all minimal. It should be noted that dB/dt is a very important factor that contributes to the symptoms in high magnetic fields, such as dizziness and nausea. In our study, the mice were placed in the magnet before we ramped up the field over a duration of 5 min and the dB/dt for the highest magnetic field condition is 0.077 T/s. This is way below the limit set by ICNIRP (International Commission on Non-Ionizing Radiation Protection), which recommends that the change of the magnetic flux density dB should not exceed 2 T during any 3-s period (International Commission on Non-Ionizing Radiation Protection, 2014). In addition, it is well known that high SMF exposure alters behavioral reactions of rodents during and after exposure (Houpt et al., 2003, 2011). In fact, we have a separate study that systematically investigated the transient and long-term effects of these 3.5–23.0 T high static magnetic fields on mice behavior (Khan et al., unpublished data). We have previously shown that 27 T SMF with no gradient could affect the orientation of spindles in the cell after 4-h exposure (Zhang et al., 2017), which may be an important mechanism for the anti-tumor effects of static magnetic fields (Tian et al., 2018). We also showed that the tumor growth of nude mice bearing GIST-T1 tumor could be reduced by 3.7–24.5 T high SMFs (Tian et al., 2018). Therefore, the high magnetic fields may have clinical potentials in cancer patient care. We found that 9-h exposure showed some liver damage in our previous study, which did
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Fig. 4. HE stain of heart, liver, spleen, lung, and kidney in control, sham control, and 23.0 T high SMF treatment groups. Representative images are shown. Scale bar: 50 μm.
exposed to the highest field intensity, 23.0 T, with no gradient. Therefore, further studies are needed to evaluate the effects of gradient high magnetic field at mice level. Overall, our study indicates that 3.5–23.0 T high static magnetic field exposure for 2 h is relatively safe on mice, which did not generate severe effects on the vital parameters or key organs. Considering the reported cellular studies about high magnetic fields on bone system (Hammer et al., 2008; Kotani et al., 2002; Qian et al., 2009), neural system (Eguchi et al., 2003), reproductive and developmental systems etc (Emura et al., 2001, 2003; Pan and Liu, 2004), detailed examination on other systems that were not examined here, including bones, neural system and reproductive system, should be investigated at mice level in the future to get a more comprehensive assessment of the safety issues of high static magnetic fields.
not show in this present 2-h exposure study. The exposure time is likely a key issue. However, to get a more complete assessment of the safety issue of high magnetic fields, we need to do more analysis for more aspects including more examination of brain function, etc. In fact, we are working on this as a separate study and found that there are no long-term effects of these magnetic field exposure on mice stress levels, social activities as well as memories (Khan et al., unpublished data). Lastly, it has been known for a long time that the magnetic field gradient is a key factor that contributes to magnetic field-induced bioeffects (Zablotskii et al., 2018). Although for most vital parameters test in this study, there was no direct relationship between the field gradient and the effects, the spleen weight increase was most obvious in groups 3 and 7, which are both 13.5 T with the highest magnetic field gradient (117.2 T/m). Moreover, the spleen weight changes only happened to mice that were exposed to gradient magnetic fields, but not the mice 279
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Acknowledgements We would like to thank the staff members in the High Magnetic Field Laboratory, Chinese Academy of Sciences for their technical assistance, and Shu-tong Maggie Wang for cartoon illustration. This work was supported by the National Key R&D Program of China (Grant No. 2016YFA0400900 and 2017YFA0402903), National Natural Science Foundation of China (Grant No U1532151 and 51627901), the Major/ Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (2016FXCX004), and the CASHIPS Director’s Fund (YZJJ201704 and KP-2017-26). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.neuroimage.2019.05.070. Notes The authors declare no competing financial interest. References Aldinucci, C., Garcia, J.B., Palmi, M., Sgaragli, G., Benocci, A., Meini, A., Pessina, F., Rossi, C., Bonechi, C., Pessina, G.P., 2003. The effect of strong static magnetic field on lymphocytes. Bioelectromagnetics 24, 109–117. https://doi.org/10.1002/ bem.10071. Beaujoin, J., Palomero-Gallagher, N., Boumezbeur, F., Axer, M., Bernard, J., Poupon, F., Schmitz, D., Mangin, J.F., Poupon, C., 2018. Post-mortem inference of the human hippocampal connectivity and microstructure using ultra-high field diffusion MRI at 11.7 T. Brain Struct. Funct. 223, 2157–2179. https://doi.org/10.1007/s00429-0181617-1. Budinger, T.F., Bird, M.D., Frydman, L., 2016. Toward 20 T magnetic resonance for human brain studies: opportunities for discovery and neuroscience rationale. Magn. Reson. Mater. Phy. 29, 617–639. https://doi.org/10.1007/s10334-016-0561-4. Budinger, T.F., Bird, M.D., 2018. MRI and MRS of the human brain at magnetic fields of 14T to 20T: technical feasibility, safety, and neuroscience horizons. Neuroimage 168, 509–531. https://doi.org/10.1016/j.neuroimage.2017.01.067. Eguchi, Y., Ogiue-Ikeda, M., Ueno, S., 2003. Control of orientation of rat Schwann cells using an 8-T static magnetic field. Neurosci. Lett. 351, 130–132. https://doi.org/ 10.1016/s0304-3940(03)00719-5. Emura, R., Ashida, N., Higashi, T., Takeuchi, T., 2001. Orientation of bull sperms in static magnetic fields. Bioelectromagnetics 22, 60–65. https://doi.org/10.1002/1521186x(200101)22:1<60::aid-bem7>3.0.co;2-a. Emura, R., Takeuchi, T., Nakaoka, Y., Higashi, T., 2003. Analysis of anisotropic diamagnetic susceptibility of a bull sperm. Bioelectromagnetics 24, 347–355. https:// doi.org/10.1002/bem.10109. Guevorkian, K., Valles, J.M., 2006. Swimming paramecium in magnetically simulated enhanced, reduced, and inverted gravity environments. Proc. Natl. Acad. Sci. U. S. A 103, 13051–13056. https://doi.org/10.1073/pnas.0601839103. Hammer, B.E., Kidder, L.S., Williams, P.C., Xu, W.W., 2008. Magnetic levitation of MC3T3 osteoblast cells as a ground-based simulation of microgravity. Microgravity Sci. Technol. 21, 311–318. https://doi.org/10.1007/s12217-008-9092-6. Higashi, T., Yamagishi, A., Takeuchi, T., Kawaguchi, N., Sagawa, S., Onishi, S., Date, M., 1993. Orientation of erythrocytes in a strong static magnetic field. Blood 82, 1328–1334. PubMed:8353291. Houpt, T.A., Pittman, D.W., Barranco, J.M., Brooks, E.H., Smith, J.C., 2003. Behavioural effects of high-strength static magnetic fields on rats. J. Neurosci. 23, 1489–1505. https://doi.org/10.1523/JNEUROSCI.23-04-01498.2003. Houpt, T.A., Carella, L., Gonzalez, D., Janowitz, I., Mueller, A., Mueller, K., Neth, B., Smith, J.C., 2011. Behavioral effects on rats of motion within a high static magnetic
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Effects of 3.5–23.0 T static magnetic fields on mice: A safety study Xiaofei Tian a, b, 1, Dongmei Wang a, c, 1, Shuang Feng a, c, Lei Zhang a, Xinmiao Ji a, Ze Wang a, d, Qingyou Lu a, d, e, Chuanying Xi a, Li Pi a, Xin Zhang a, b, c, * a High Magnetic Field Laboratory, Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, 230031, PR China b Institutes of Physical Science and Information Technology, Anhui University, Hefei, Anhui, 230601, PR China c Science Island Branch of Graduate School, University of Science and Technology of China, Hefei, Anhui, 230026, PR China d Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, 230026, PR China e Anhui Province Key Laboratory of Condensed Matter Physics at Extreme Conditions, Hefei, Anhui, 230031, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Static magnetic field (SMF) Magnetic resonance imaging (MRI) Ultra-high field (UHF) Safety Mice
People are exposed to various magnetic fields, including the high static/steady magnetic field (SMF) of MRI, which has been increased to 9.4 T in preclinical investigations. However, relevant safety studies about high SMF are deficient. Here we examined whether 3.5–23.0 T SMF exposure for 2 h has severe long-term effects on mice using 112 C57BL/6J mice. The food/water consumption, blood glucose levels, blood routine, blood biochemistry, as well as organ weight and HE stains were all examined. The food consumption and body weight were slightly decreased for 23.0 T-exposed mice (14.6%, P < 0.01, and 1.75–5.57%, P < 0.05, respectively), but not the other groups. While total bilirubin (TBIL), white blood cells, platelet and lymphocyte numbers were affected by some magnetic conditions, most of them were still within normal reference range. Although 13.5 T magnetic fields with the highest gradient (117.2 T/m) caused spleen weight increase, the blood count and biochemistry results were still within the control reference range. Moreover, the highest field 23.0 T with no gradient did not cause organ weight or blood biochemistry abnormality, which indicates that field gradient is a key parameter. Collectively, these data suggest 3.5–23.0 T static magnetic field exposure for 2 h do not have severe long-term effects on mice.
1. Introduction Since higher magnetic field intensity could improve image quality and acquisition capability of MRI (magnetic resonance imaging), the magnetic field intensity used on MRI for humans has increased from 1.5 and 3.0 T in most hospitals to 9.4 T in preclinical stage (Neuner et al., 2013, 2014; U gurbil, 2018). For example, the 9.4 T MRI has been used to do quantification of sodium concentration distribution studies in human brain, which were not feasible with MRIs with lower magnetic fields (Thulborn et al., 2017). Moreover, not only 11.7 T MRI has been manufactured and tested on human tissue (Beaujoin et al., 2018; Vedrine et al., 2014), people have also pushed the limit of static/static magnetic field (SMF) to as high as 21.1 T, which has been used on rodents to get high resolution brain images (Nagel et al., 2016; Schepkin et al., 2011). Correspondingly, the resolution has been improved from ~1 mm for 1.5–3.0 T MRI, ~100 μm for 9.4 T MRI to ~18 μm for 21.1 T MRI. The increased MRI imaging resolution is essential for early diagnosis of many
diseases, such as small internal lesions and cancer. However, as the main core component of MRI, the biosafety issues of high SMFs are still under investigated. Although there are some sporadic cellular experiments, animal studies about the safety issues of high SMFs above 9.4 T are very limited. Recently, Wang et al reported that 2-12 T SMF treatment for 28 days did not generate significant defects on C57BL/6 mice (Wang et al., 2019). In fact, Budinger and Bird have written two very comprehensive reviews about the technology and application potentials of high field MRIs up to 20 T (Budinger et al., 2016; Budinger and Bird, 2018). They concluded that there are no foreseen barriers of brain MRI and MRS (magnetic resonance spectroscopy) at magnetic fields up to 20 T and suggested that people should try to develop high field MRI and MRS up to 20 T. They mentioned that no deleterious effects have been observed on rodents at 21.1 T by NHMFL (National High Magnetic Field Laboratory) investigators, except for some temporary effects on the vestibular apparatus (Budinger et al., 2016; Budinger and Bird, 2018). Howeber, there
* Corresponding author. High Magnetic Field Laboratory, Chinese Academy of Sciences, Shushanhu Road #350, Hefei, Anhui, 230031, PR China. E-mail address: xinzhang@hmfl.ac.cn (X. Zhang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.neuroimage.2019.05.070 Received 10 February 2019; Received in revised form 3 May 2019; Accepted 27 May 2019 Available online 31 May 2019 1053-8119/© 2019 Elsevier Inc. All rights reserved.
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force on those diamagnetic mice. Accordingly, the mice in the bottom part of the magnet were in “hypergravity” conditions. Therefore, although there are two groups of mice for 21.9 T, 13.5 T and 6.7 T, they experienced magnetic force of different directions, which make them in “hypogravity” and “hypergravity” conditions respectively. Only static magnetic fields were examined in this study and no radiofrequency was involved. On the day of exposure, we randomly divided the 112 mice into three groups, control group, sham control group and magnetic field exposed group (Fig. 1B). For each specific magnetic field exposure condition, we have 6 mice that were tested on 6 independent days. All mice were placed in individual nonmagnetic stainless-steel tube and then stacked them into the inner cylinder in turn before they were placed into the water-cooled magnet or left outside the magnet as illustrated (Fig. 1) and as previously described (Tian et al., 2018). The magnetic field exposure time is 2 h and 10 min in total including increasing field for 5 min (dB/dt is about 0.077 T/s), constant at 23.0 T for 2 h and reducing field for 5 min for each experiment. After the mice were treated with control, sham control or magnetic field exposure, the mice were fed normally by sterilized food and autoclaved water for another three weeks. The food and water consumption and body weight were measured daily during the whole experiment. Blood glucose levels were measured every two days.
was no experimental data available. In fact, although there are some in vitro cellular studies about biological effect studies of SMFs above 9.4 T (Nakahara et al., 2002; Qian et al., 2009, 2012, 2013; Sun et al., 2015), there are very limited studies about any biological effect studies of SMFs above 20 T, which is not only limited by the availability of Ultra high field (UHF) magnets, but also by the compatibility of biological samples. Until now, other than the unicellular organism Paramecium (Guevorkian and Valles, 2006) and human cells (Zhang et al., 2017), there was only one preliminary experimental study about whole animals in high SMFs above 20 T, which was done by our group (Tian et al., 2018). We used 16 tumor-bearing nude mice to examine the effects of 9-h exposure to 3.7–24.5 T SMFs. The small sample size was due to limited machine time. Our results show that while most indicators are normal, the mice liver was affected to some extent (Tian et al., 2018). Since 9-h is much longer than what is needed for an MRI examination, here in this study, we reduced it to 2 h and also increased the sample size to 112 mice to systematically investigate the safety issue of ultra-high static magnetic fields on normal mice. Using 112 mice and a water-cooled magnet that provides a 23.0 T SMF in the center, and descending magnetic field intensities with various gradient off the center, we investigated the effects of 3.5–23.0 T SMFs on mice. After comprehensive examination of these mice, we did not find deleterious effects of these high SMFs, except for some spleen weight enlargement by the highest gradient magnetic field exposure, as well as some changes on white blood cell count in some magnetic field exposed conditions. However, most of the changes are either statistically not significant from the sham control or still within the normal reference range, which was defined by the results from sixteen mice in the control group.
2.3. Blood biochemistry analysis Blood samples were obtained by removing the mice eyeballs at the end of the experiment. 200 μL blood samples with 0.15% (M/V) EDTAK2⋅2H2O were used for blood routine examination by an automatic hematology analyzer (Sysmex, Japan). Meanwhile, the heart, liver, spleen, lungs and kidneys were weighed and fixed with 4% formaldehyde (#10010018, Sinopharm Chemical Reagent) for 24 h after blood sampling. The serum was collected by centrifugation after the blood sample placed at room temperature for 2 h. The serum was stored at 80 C or dry ice until they were sent to Nanjing Biomedical Research Institute of Nanjing University for blood biochemistry analysis by an automatic analyzer (HITACHI 7020, Japan) as soon as possible. Biochemical indicators to be tested included aspartic transaminase (AST), alanine
2. Materials and methods 2.1. Construction of the ultra-high magnetic mice exposure system A water-cooled magnet (WM2) was used to generate 3.5–23 T SMFs in Chinese High Magnetic Field Laboratory (CHMFL, Hefei, China). The magnet cavity is 50 mm in diameter and 1300 mm long. In order to explore the biological safety of strong static magnetic field, we designed and constructed two identical sets of the devices with accurate temperature and gas control, which were suitable for the WM2 magnet. The device contains a removable inner cylinder which is 41 mm diameter and 700 mm long. Each mouse was housed in a nonmagnetic stainless-steel tube (38.5 mm diameter, 80 mm long) and eight mice were placed into the inner cylinder in turn before each experiment. To make sure the mice get enough oxygen, a pump was used to circulate the air inside the cylinder at 450 L/min. Meanwhile, the air duct was placed into the ice water to prevent air from overheating caused by the pump. The temperature inside the mice tube was controlled at 22–24 C by thermal conduction from temperature-controlled water that flowed through the space of the devices. 2.2. Mice exposure to magnetic fields, sham control and control conditions The animal protocols were approved by the ethical and humane committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences and strictly followed the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). One hundred and twelve 8-week-old male C57BL/6J mice were purchased from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Each cage contained five mice and all mice were kept in the clean condition with food and water available ad libitum. The magnet provides a 23.0 T SMF in the center and descending SMF intensities with various gradient off the-center (Fig. S1). As a result, the mice in the upper part of the magnet were in “hypogravity” conditions, because of the magnetic field gradient generates an upward magnetic
Fig. 1. Magnetic field exposure conditions. (A) 112 mice were divided into three groups. (B) Illustration of mice position and magnetic field exposure conditions. Mice were placed in individual tubes. Red arrows show magnetic field direction. 274
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aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), total bilirubin (TBIL), blood urea nitrogen (BUN), serum creatinine (CREA), calcium (Ca), phosphorus (P), iron ion (Fe), total cholesterol (CHOL), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and blood glucose (GLU).
differences between the “sham” group and SMF group. Statistical analysis was performed by using SPSS statistical analysis software (SPSS 19.0; SPSS, Chicago, IL) and P value less than 0.05 was considered statistically significant.
2.4. Histologic examination
The data and/or code used in the study are available in the public domain or upon direct request and comply with the requirements of the funders and institutional ethics approval.
2.6. Data and code availability statement
After mice were sacrificed on day 21, the heart, liver, spleen, lungs and kidneys tissues were fixed in 4% formaldehyde for 24 h. Then the tissues were embedded in paraffin after formaldehyde removal, dehydration, cleaning and waxing. The paraffin-embedded specimens were sliced at 5 μm thickness and then they would be stained by means of routine HE staining.
3. Results We exposed the mice to control, sham control or magnetic fields as illustrated (Fig. 1). We used two set of controls. One is the regular “control”, in which the mice were placed in the tube but not placed in the magnet. The other one is the “sham control”, in which the mice were placed in the tube and placed in the magnet with water running. This fully mimics the bore vibration and noise conditions in the magnetic field exposed group. Therefore, we can compare and analyze whether the observed effects are caused by bore vibration and noise, or by magnetic fields themselves. In general, the food and water consumptions were not significantly
2.5. Statistical analysis The normality of the data was analyzed by Shapiro–Wilk test and the homogeneity of variance was assessed with levene test. The significance of the differences in all data among the “sham” group and SMF group was evaluated by unpaired t-test. If the data didn't comply with the normal distribution, the Mann-Whitney U test was used to compare the
Fig. 2. Food and water consumption, as well as body weight and blood glucose of mice exposed to control, sham control or magnetic fields. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Data are mean standard deviations. Statistical analysis was performed for all data. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. “**”, P < 0.01. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05). 275
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Table 1 Liver and kidney function parameters. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Red bold font illustrates data that are statistically significant than the sham control. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
affected by magnetic field exposure, except for groups 4 and 5 for food consumption, and groups 1 and 2 for water consumption (Fig. 2A). The food consumption for group 4 (21.9 T) is reduced by 6.1% and for group 5 (23.0 T) is reduced by 14.6% (Fig. 2A, left). The water consumption for groups 1 and 2 were both increased, for 23.1% and 12.5% respectively (Fig. 2A, right). We also monitored the body weight every day (Fig. 2B and Fig. S2) and measured blood glucose every two days for each mouse (Fig. 2C). For groups 1, 2 and 8, the average mice body weight was decreased between control and sham control groups, which is likely due to the machine vibrating since they were located at both ends of the magnet (Fig. S2). To examine the magnetic field-induced effects per se, we compared between the sham control and the magnetic field exposed groups. We found that
only the mice in group 5 showed significant weight gain reduction after magnetic field exposure (Fig. 2B, right), which means that 23 T exposure for 2 h may affect the mice weight gain, but 21.9 T or below may not (Fig. 2B left and Fig. S2). The decreased body weight is likely due to the reduced food intake (Fig. 2A). We also measured their blood glucose every two days and found no significant difference after magnetic field exposure (Fig. 2C). To analyze magnetic field exposure-induced physiological consequences, we further measured multiple biochemistry parameters to reflect the liver and kidney functions (Table 1). For all these 8 indicators, AST (aspartic transaminase) and TBIL (total bilirubin) are the only two that showed alterations. In general, the AST levels were increased and TBIL were decreased by magnetic field exposure, although only two
Table 2 Metabolic parameters of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. There were six mice in sham control and SMF-treated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data and they do not have statistical significance (P > 0.05). Glu (mmol/L) P (mmol/L) Ca (mmol/L) Fe (μmol/L) LDL-C (mmol/L) HDL-C (mmol/L) TG (mmol/L) CHOL (mmol/L)
5.53 1.10 (2.98–7.07) 3.72 0.32 (3.22–4.35) 2.38 0.07 (2.28–2.58) 16.20 4.51 (9.10–24.90) 0.36 0.06 (0.29–0.51) 1.92 0.21 (1.48–2.26) 0.56 0.12 (0.36–0.82) 2.65 0.29 (2.04–3.13) Mean SD (Range) Control
6.02 1.02 3.48 0.67 2.32 0.08 17.26 4.45 0.39 0.05 1.86 0.14 0.69 0.27 2.62 0.19 Sham Group 1
6.66 1.01 3.80 0.43 2.38 0.08 20.82 5.82 0.33 0.05 1.71 0.35 0.68 0.48 2.37 0.45 3.5T
5.89 1.30 3.41 0.38 2.31 0.08 16.13 4.07 0.40 0.11 1.76 0.31 0.67 0.24 2.49 0.46 Sham Group 2
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5.19 2.01 3.78 0.48 2.34 0.09 16.58 7.81 0.40 0.05 1.86 0.13 0.56 0.27 2.61 0.16 6.7T
6.93 0.85 3.32 0.46 2.09 0.57 19.00 4.58 0.41 0.09 1.91 0.11 0.68 0.21 2.70 0.24 Sham Group 3
5.55 1.53 3.95 0.75 2.40 0.12 19.58 9.92 0.37 0.08 1.93 0.16 0.71 0.45 2.69 0.26 13.5T
6.46 0.83 3.52 0.33 2.29 0.04 18.78 6.46 0.36 0.11 1.75 0.31 0.71 0.36 2.47 0.50 Sham Group 4
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Table 3 Blood routine examination of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Red bold font illustrates data that are statistically significant than the sham control. There were six mice in sham control and SMFtreated group and sixteen mice in control group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
by multiple high magnetic field exposure conditions, especially by magnetic fields with higher gradients. For example, group 3 and 7 are both 13.5 T with the highest magnetic field gradient (117.2 T/m) as well as BB’ (1582.2 T2/m) increased the spleen weight by 44.6% and 28.5%, respectively. However, it is interesting that the changes only happened to mice that were exposed to gradient magnetic fields, but not to the mice exposed to 23.0 T with no gradient. Moreover, the HE stain of the organs did not show obvious abnormalities for all these mice (Fig. 4 and Figs. S3–9).
groups (groups 2 and 8) showed statistical significance for AST and only one group (group 3) showed statistical significance for TBIL (Table 1). All the other parameters were not much affected. Moreover, we also tested several metabolic parameters, including blood glucose, cholesterol, Fe and Calcium etc, and did not find any alterations caused by high SMF exposure (Table 2). It should be mentioned that mice in group 5, which were exposed to the strongest magnetic field (23.0 T) with no gradient, did not show any statistically significant changes in these biochemistry parameters related to liver and kidney functions or metabolism. To get a more comprehensive understanding about the high magnetic field effects, we also did the blood routine examination. We found that high SMFs did not affect red blood cell numbers and its related parameter (Table 3 and Table S1), including HGB (hemoglobin) (Table 3). Although some magnetic conditions affected platelet, white blood cells and lymphocyte numbers, all the values after magnetic field exposure are still within the control reference range (Table 3). At the end of the experiment, we also measured the weight of mice organs for all these 112 mice, including their heart, liver, spleen, lung and kidney (Fig. 3). Among them, spleen weight was the only one that was affected by magnetic fields (Fig. 3). The spleen weight was increased
6.02 1.39 4.03 0.53 2.42 0.14 19.18 7.48 0.36 0.06 1.82 0.31 0.51 0.41 2.51 0.43 21.9T Group 4
6.26 0.61 3.41 0.32 2.33 0.06 20.28 4.76 0.38 0.08 1.81 0.25 0.66 0.28 2.56 0.35 Sham Group 5
6.86 1.24 3.71 0.22 2.37 0.04 24.12 13.4 0.35 0.09 1.84 0.18 0.46 0.13 2.51 0.28 23.0T
7.01 1.50 3.40 0.72 2.34 0.11 17.33 3.73 0.38 0.11 1.78 0.38 0.57 0.17 2.51 0.57 Sham Group 6
4. Discussion Our results show that 3.5–23.0 T static magnetic field exposure for 2 h do not have severe long-term impact on mice. The changes in most parameters are statistically not significant. For a few parameters, although the changes are statistically significant, most of them are still within the control reference range, such as white blood cells. None of the changes are substantially off the control reference range. Although we did not do an actual MRI examination and the effect of radiofrequency (RF) magnetic field was not evaluated in this study, our results indicate that the
6.43 1.63 3.86 0.59 2.35 0.05 25.36 14.53 0.40 0.07 1.88 0.25 0.49 0.18 2.61 0.31 21.9T
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6.52 1.31 3.76 0.88 2.34 0.04 19.04 2.28 0.38 0.07 1.91 0.19 0.86 0.34 2.69 0.26 Sham Group 7
6.24 0.99 3.72 0.32 2.36 0.08 22.87 9.48 0.31 0.04 1.81 0.26 0.52 0.25 2.42 0.35 13.5T
7.11 1.81 3.32 0.68 2.34 0.14 18.60 4.10 0.37 0.06 1.89 0.18 0.56 0.24 2.61 0.25 Sham Group 8
6.36 1.11 3.63 0.23 2.36 0.05 22.88 8.64 0.39 0.07 2.02 0.19 0.61 0.44 2.77 0.27 6.7T
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Fig. 3. Relative organ weight of mice exposed to control, sham control or magnetic fields. Comparisons were made between each sham control and the corresponding magnetic field exposed groups. Grey dashed lines show the average organ weight of 16 control mice. There were six mice in sham control and SMF-treated group. Values show mean standard deviation. We did statistical analysis for all data. For those that have statistical significance, we label them as “*”, P < 0.05, “**”, P < 0.01. To increase the readability, we did not label the rest data that has no statistical significance (P > 0.05).
static magnetic field part in the range of 3.5–23.0 T is relatively safe, at least at mice level, especially if we consider that our exposure time is 2 h, which is much longer than the actual MRI examination time. We expect to see even less effects if we further reduce the exposure time to 1 h or to a few minutes. Although it has been shown that the orientation of red blood cells can be influenced even by 1 T static magnetic field and almost 100% of them could be oriented when exposed to 4 T (Higashi et al., 1993), we did not observe any red blood cell defects. This indicates that the orientation change of red blood cells does not affect their key parameters, including its number and HGB content. A previous in vitro cellular study investigated the effects of a 4.75 T homogeneous SMF and found that it does not affect the physiologic behavior of normal lymphomonocytes (Aldinucci et al., 2003). Here we found that in mice, 3.5–23.0 T SMFs generally decreased lymphocyte numbers, although still within the normal control range. It should be noted that the highest magnetic field intensity of 23.0 T in group 5 reduced the food consumption and body weight gain but did not have other significant effects. Moreover, although still within the normal reference range, there are some small but statistically significant effects on blood cell count for several SMF exposed groups. However, we did not find direct relationship between these changes with SMF intensity or gradient. It is likely that these mild effects are influenced by a combination of multiple factors, including magnetic field intensity, gradient and gradient direction. In addition, the generally elevated AST and decreased TBIL may indicate some liver function abnormalities, but they
are mostly still within the normal control range and the liver tissues also have normal appearance in their HE stains. These results indicated that the liver alteration and the other blood cell count changes are all minimal. It should be noted that dB/dt is a very important factor that contributes to the symptoms in high magnetic fields, such as dizziness and nausea. In our study, the mice were placed in the magnet before we ramped up the field over a duration of 5 min and the dB/dt for the highest magnetic field condition is 0.077 T/s. This is way below the limit set by ICNIRP (International Commission on Non-Ionizing Radiation Protection), which recommends that the change of the magnetic flux density dB should not exceed 2 T during any 3-s period (International Commission on Non-Ionizing Radiation Protection, 2014). In addition, it is well known that high SMF exposure alters behavioral reactions of rodents during and after exposure (Houpt et al., 2003, 2011). In fact, we have a separate study that systematically investigated the transient and long-term effects of these 3.5–23.0 T high static magnetic fields on mice behavior (Khan et al., unpublished data). We have previously shown that 27 T SMF with no gradient could affect the orientation of spindles in the cell after 4-h exposure (Zhang et al., 2017), which may be an important mechanism for the anti-tumor effects of static magnetic fields (Tian et al., 2018). We also showed that the tumor growth of nude mice bearing GIST-T1 tumor could be reduced by 3.7–24.5 T high SMFs (Tian et al., 2018). Therefore, the high magnetic fields may have clinical potentials in cancer patient care. We found that 9-h exposure showed some liver damage in our previous study, which did
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Fig. 4. HE stain of heart, liver, spleen, lung, and kidney in control, sham control, and 23.0 T high SMF treatment groups. Representative images are shown. Scale bar: 50 μm.
exposed to the highest field intensity, 23.0 T, with no gradient. Therefore, further studies are needed to evaluate the effects of gradient high magnetic field at mice level. Overall, our study indicates that 3.5–23.0 T high static magnetic field exposure for 2 h is relatively safe on mice, which did not generate severe effects on the vital parameters or key organs. Considering the reported cellular studies about high magnetic fields on bone system (Hammer et al., 2008; Kotani et al., 2002; Qian et al., 2009), neural system (Eguchi et al., 2003), reproductive and developmental systems etc (Emura et al., 2001, 2003; Pan and Liu, 2004), detailed examination on other systems that were not examined here, including bones, neural system and reproductive system, should be investigated at mice level in the future to get a more comprehensive assessment of the safety issues of high static magnetic fields.
not show in this present 2-h exposure study. The exposure time is likely a key issue. However, to get a more complete assessment of the safety issue of high magnetic fields, we need to do more analysis for more aspects including more examination of brain function, etc. In fact, we are working on this as a separate study and found that there are no long-term effects of these magnetic field exposure on mice stress levels, social activities as well as memories (Khan et al., unpublished data). Lastly, it has been known for a long time that the magnetic field gradient is a key factor that contributes to magnetic field-induced bioeffects (Zablotskii et al., 2018). Although for most vital parameters test in this study, there was no direct relationship between the field gradient and the effects, the spleen weight increase was most obvious in groups 3 and 7, which are both 13.5 T with the highest magnetic field gradient (117.2 T/m). Moreover, the spleen weight changes only happened to mice that were exposed to gradient magnetic fields, but not the mice 279
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Acknowledgements We would like to thank the staff members in the High Magnetic Field Laboratory, Chinese Academy of Sciences for their technical assistance, and Shu-tong Maggie Wang for cartoon illustration. This work was supported by the National Key R&D Program of China (Grant No. 2016YFA0400900 and 2017YFA0402903), National Natural Science Foundation of China (Grant No U1532151 and 51627901), the Major/ Innovative Program of Development Foundation of Hefei Center for Physical Science and Technology (2016FXCX004), and the CASHIPS Director’s Fund (YZJJ201704 and KP-2017-26). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.neuroimage.2019.05.070. Notes The authors declare no competing financial interest. References Aldinucci, C., Garcia, J.B., Palmi, M., Sgaragli, G., Benocci, A., Meini, A., Pessina, F., Rossi, C., Bonechi, C., Pessina, G.P., 2003. The effect of strong static magnetic field on lymphocytes. Bioelectromagnetics 24, 109–117. https://doi.org/10.1002/ bem.10071. Beaujoin, J., Palomero-Gallagher, N., Boumezbeur, F., Axer, M., Bernard, J., Poupon, F., Schmitz, D., Mangin, J.F., Poupon, C., 2018. Post-mortem inference of the human hippocampal connectivity and microstructure using ultra-high field diffusion MRI at 11.7 T. Brain Struct. Funct. 223, 2157–2179. https://doi.org/10.1007/s00429-0181617-1. Budinger, T.F., Bird, M.D., Frydman, L., 2016. Toward 20 T magnetic resonance for human brain studies: opportunities for discovery and neuroscience rationale. Magn. Reson. Mater. Phy. 29, 617–639. https://doi.org/10.1007/s10334-016-0561-4. Budinger, T.F., Bird, M.D., 2018. MRI and MRS of the human brain at magnetic fields of 14T to 20T: technical feasibility, safety, and neuroscience horizons. Neuroimage 168, 509–531. https://doi.org/10.1016/j.neuroimage.2017.01.067. Eguchi, Y., Ogiue-Ikeda, M., Ueno, S., 2003. Control of orientation of rat Schwann cells using an 8-T static magnetic field. Neurosci. Lett. 351, 130–132. https://doi.org/ 10.1016/s0304-3940(03)00719-5. Emura, R., Ashida, N., Higashi, T., Takeuchi, T., 2001. Orientation of bull sperms in static magnetic fields. Bioelectromagnetics 22, 60–65. https://doi.org/10.1002/1521186x(200101)22:1<60::aid-bem7>3.0.co;2-a. Emura, R., Takeuchi, T., Nakaoka, Y., Higashi, T., 2003. Analysis of anisotropic diamagnetic susceptibility of a bull sperm. Bioelectromagnetics 24, 347–355. https:// doi.org/10.1002/bem.10109. Guevorkian, K., Valles, J.M., 2006. Swimming paramecium in magnetically simulated enhanced, reduced, and inverted gravity environments. Proc. Natl. Acad. Sci. U. S. A 103, 13051–13056. https://doi.org/10.1073/pnas.0601839103. Hammer, B.E., Kidder, L.S., Williams, P.C., Xu, W.W., 2008. Magnetic levitation of MC3T3 osteoblast cells as a ground-based simulation of microgravity. Microgravity Sci. Technol. 21, 311–318. https://doi.org/10.1007/s12217-008-9092-6. Higashi, T., Yamagishi, A., Takeuchi, T., Kawaguchi, N., Sagawa, S., Onishi, S., Date, M., 1993. Orientation of erythrocytes in a strong static magnetic field. Blood 82, 1328–1334. PubMed:8353291. Houpt, T.A., Pittman, D.W., Barranco, J.M., Brooks, E.H., Smith, J.C., 2003. Behavioural effects of high-strength static magnetic fields on rats. J. Neurosci. 23, 1489–1505. https://doi.org/10.1523/JNEUROSCI.23-04-01498.2003. Houpt, T.A., Carella, L., Gonzalez, D., Janowitz, I., Mueller, A., Mueller, K., Neth, B., Smith, J.C., 2011. Behavioral effects on rats of motion within a high static magnetic
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