A water-cooled magnet (WM2) in Chinese High Magnetic Field Laboratory (CHMFL, Hefei, China) was used to generate 3.7 T–24.5 T SMFs. The cylinder bore of the magnet is 50 mm in diameter and 1300 mm long. To investigate the biological effect of the magnetic field provided by the magnet, we designed and constructed a set of mice exposure system with accurate temperature and gas control. We designed two sets of devices to specifically fit in the WM2 magnet, each of which consists of two coaxial nonmagnetic stainless-steel tubes. The outer diameter of the outer tube (OT) is 49 mm and the inner diameter of the inner tube (IT) is 43 mm. Each mouse was housed in a nonmagnetic stainless-steel tube (38.5 mm diameter, 80 mm long) and eight of them were stacked together into the longer tube (41 mm diameter, 700 mm long) before each experiment. To provide proper air supply, we used a nonmagnetic stainless-steel air tube with 10 mm outer diameter held by a height-adjusting nut, which can be adjusted according to the position of the mice in the magnet. Meanwhile, a pump was used to circulate the air inside the cylinder bore, which blew the air into the cylinder bore at 450 L per minute. Furthermore, the air conduit was placed into ice water to avoid overheating caused by the pump. A platinum resistance temperature sensor (PT100) connected to a temperature display was used to monitor the temperature inside the mice tube, which was controlled at 22–24 °C by thermal conduction from temperature-controlled water that flowed through the space between the IT and OT. By adjusting the temperature of the water, the temperature inside the mice tube can be controlled.

After the GIST-T1 tumor volume reached 100–200 mm³, we placed the tumor-bearing mice into individual mice tubes, stacked them into the long tube before they were inserted into the magnet (as SMF-treated group) or left outside of the magnet (as “Sham control” group). Both groups had eight mice. The tube was precisely measured, marked and adjusted to ensure the fifth mouse is precisely located at the center position of the magnet. The MF exposure time was 3 hours in total (increasing field for 15 min, constant 24.49 T field for 2.5 hr and reducing field for 15 min) for each time, three times in total, on day 1, 4, and 7, respectively. It should be noted that although the maximal MF intensity that WM2 can reach is 25 T at the center of the magnet, we maintained it steadily at ∼24.5 T for better stability. The Sham control group and MF group were handled identically during the whole experiment, except for MF exposure. After the last SMF exposure, the mice were fed normally by sterilized food and autoclaved water for another two weeks.

The food and water consumption, body weight and tumor growth were all measured daily during the whole experiment. Tumor volumes were calculated as tumor volume (mm³) = [(W² × L)/2], where width (W) is defined as the smaller diameter of the tumor and length (L) is the larger diameter. Blood samples were collected by removing eyeball on day 22 (endpoint) of the study. blood samples with 0.08% (M/V) ethylenediamine tetraacetic acid disodium salt (EDTA) were used for blood routine examination by an automatic hematology analyzer (Sysmex, Japan). In addition, the heart, liver, spleen, lungs, and kidneys of all mice were collected, weighed and fixed with 4% formaldehyde (#10010018, Sinopharm Chemical Reagent) after blood sampling.

For blood biochemistry analysis, the blood samples were placed at room temperature for 4 hrs, protected from light and collected the serum by centrifugation. The serum was stored at −80 °C or dry ice until it was analyzed by an automatic analyzer (HITACHI 7020, Japan) in the Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China), including alanine aminotransferase (ALT), aspartic transaminase (AST), alkaline phosphatase (AKP), 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).

Twenty 4-week-old female specific pathogen-free (SPF) BALB/c (nu/nu) mice were from Nanjing Biomedical Research Institute of Nanjing University (Nanjing, China). Each five mice were housed in one polycarbonate cage and all mice were kept in an animal room under SPF condition. The mice were fed with sterilized food and autoclaved tap water freely. The protocol involving animals was approved by the ethical and humane committee of Hefei Institutes of Physical Science, Chinese Academy of Sciences and carried out strictly in accordance with the related regulations. After one week, five million human gastrointestinal stromal tumor (GIST-T1) cells were suspended in PBS and injected into the subcutaneous space on the right flank of nu/nu mice. For SMF exposure, we chose 16 mice whose GIST-T1 tumors had reached 100–200 mm³ after seven days of inoculation. The mice were randomly divided into two groups, Sham control group and SMF exposure group. After MF exposure, we maintained the mice in another room since they were no longer qualified to return back to the SPF animal room, fed them with sterilized food and autoclaved water for another two weeks to monitor their behaviors.

After being fixed in 4% formaldehyde for 24 hr, the heart, liver, spleen, lung, kidney and tumor tissues were placed in the running water overnight to remove formaldehyde. Then the tissues were dehydrated, cleared and immersed in wax before they were embedded in paraffin. The paraffin-embedded specimens were sectioned at thickness and then immersed in xylene twice, 15 min each time. Next, the sections were hydrated in decreasing concentrations of ethanol (100, 95, 85 and 75%) for 3 minutes followed by a 5-min immersion in running water before they were dipped into the hematoxylin (#ZLI-9610, Beijing Zhongshan) for 5 min. After washing the slides in running water for 5 min, they were stained with 1% (V/V) hydrochloric acid solution in absolute ethyl alcohol for 1 s before they were placed into 1% eosin (#ZLI-9610, Beijing Zhongshan) for 3 s. Subsequently, after washing for 5 min in running water, the sections were dehydrated through 85%, 95% alcohol and two changes of 100% alcohol for 3 min each. The alcohol was extracted with two changes of xylene. Finally, one or two drops of mounting medium were added and covered with a coverslip.

Each mouse was placed in a mice container, which was then inserted individually into a long tube (Figs. 1 and S1). For the experimental group, the mice tube was inserted into the water-cooled magnet #2 (WM2) in the Chinese High Magnetic Field Laboratory, Chinese Academy of Sciences, Hefei, China. The Sham control group was kept in the same room, but not inserted in the WM2 magnet. Since we have eight mice in the Sham control group that had essentially the same condition, we took the mean value as control (CT) for all figures in this paper. In the meantime, we used the data of these mice as a “Sham control range” in the figures as references to help justify the influences of the high MF exposure.

Fig. 1.

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Fig. 1. (color online) The mice exposure system for 3.7 T–24.5 T ultra-high magnetic fields. (a) The BALB/c (nu/nu) mouse bearing GIST-T1 tumor, (b) the mice containers, (c) the inner mice tube, (d) the outer mice tube, (e, f) the design of the mice exposure system. Two identical sets were made, one used in the magnet as the experimental set (e) while the other was placed outside of the magnet to serve as the ‘Sham control’ (f). (g) Magnetic field intensity, field gradient, and the magnetic field intensity × field gradient for the center position of each mice container. (h) Magnetic field intensity distribution within the mice tube inside the magnet (water-cooled magnet #2 of Chinese High Magnetic Field Laboratory). (i) Magnetic field gradient distribution within the mice tube inside the magnet.

The experimental and the Sham control sets have identical gas, humidity, and temperature control systems and they were all operated side-by-side. The water-cooled magnet that provides 24.5 T ultra-high SMF in the center, and 3.7 T–23.4 T off the center (Figs. 1(g)–1(i)). The upper part of the magnet mimics the “hypogravity” condition because the MF gradient provides an upward direction force on the mice. The lower part of the magnet mimics the “hypergravity” condition because the MF gradient provides a downward direction force on the mice. The mouse in the center was exposed to a 24.5 T SMF with negligible gradient and magnetic force. The MF intensity, MF gradient as well as the (MF intensity × MF gradient) information for the center position of each mice container are included (Fig. 1(g)). Mice at 14.4 T had the largest , which means that they experienced the strongest magnetic force. For mice at the positions of both 14.4 T and 23.4 T, the values are larger than 1370 T²/m, which is required to lift water and make water “weightless”.

The mice were exposed to high SMFs for 3 hours each time, three times in total, 3 days apart. To check for possible long-term effects, the mice were maintained and observed for 22 days since the first SMF exposure before they were sacrificed. We recorded their food and water consumptions each day during the whole experiment period and found that the SMF-treated mice had 6.4%–14.8% decreased water and food intake (Figs. 2(a) and 2(b)), although the water intake decrease was not statistically significant (Fig. 2(b)). In fact, some of the mice appeared to be irritated after the first exposure, especially the mouse in the center of the magnet, who was exposed to the highest field SMF (24.5 T). However, this effect was transient because the mice rapidly recovered to their normal alert state a few hours after exposure. In addition, it was interesting that the mice did not show any discomfort after the second or third exposure, which shows that they were adaptive to these exposure conditions.

Fig. 2.

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Fig. 2. (color online) Food and water consumption, gain of body weight and organ weight changes of GIST-T1 tumor-bearing mice caused by high magnetic fields. (a) and (b) Food and water consumption of each mouse per day. The data were collected for each cage of mice and then divided by the number of mice in each cage. Data represent mean ± SD. p values were calculated using unpaired Student’s t test: **p < 0.01. (c) Gain of body weight was measured each day for each mouse. (d) The relative weight of organ to body weight was calculated. For control (CT), the mean value from eight mice was used. The blue shaded area means the mice were under the circumstances of “hypogravity”. The pink shaded area shows the mice were under the circumstances of “hypergravity”. The green shaded area shows the liver reference range defined by eight mice in the Sham control group (0.054–0.065).

We also recorded their body weight each day and found that the gain of body weight in most mice was not obviously affected, except for the 14.4 T-treated mice in the hypogravity condition (Fig. 2(c)). It should be noted that the 14.4 T position has the largest (MF intensity × MF gradient = 1797.3 T²/m), which indicates that mice at this position were exerted the strongest magnetic force. The relative organ weight was also measured for each mouse at the end of the experiment. For heart, spleen, lung, and kidney, there were no noticeable organ changes for all eight SMF exposure conditions (Fig. 2(d)). In contrast, the liver weight was affected in some SMF exposure conditions, although none of them are substantial (Fig. 2(d)).

To get a better assessment of the effects of these ultra-high SMFs on the tumor-bearing mice, we collected their blood and did blood routine test and blood biochemistry examination. We tested 13 different indicators in the blood routine test, including the concentration of red blood cell (RBC), platelet (PLT) (Fig. 3(a)), white blood cell (WBC) (Fig. 3(b)), the mean corpuscular volume (MCV) and mean platelet volume (MPV) (Fig. 3(c)), hemoglobin concentration (HGB) and mean corpuscular hemoglobin concentration (MCHC) (Fig. 3(d)), hematocrit (HCT) and thrombocytocrit (PCT) (Fig. 3(e)), lymphocyte (LYMPH), and monocyte (MONO) (Fig. 3(f)), neutrophil (NEUT) and eosinophil (EO) (Fig. 3(g)). It is interesting that although there are fluctuations in the SMF-treated mice, most of them are within the control range set by the eight mice in the Sham control group, which means that they belong to normal variations among individual mice. The only exceptions are 7.2 T and 14.4 T in the hypogravity condition, which induced decreased white blood cells.

Fig. 3.

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Fig. 3. (color online) Blood routine test results for 3.7 T–24.5 T high magnetic field-treated GIST-T1 tumor-bearing mice. (a), (b) The concentration of red blood cell (RBC), platelet (PLT), white blood cell (WBC); (c) mean corpuscular volume (MCV) and mean platelet volume (MPV); (d) hemoglobin concentration (HGB) and mean corpuscular hemoglobin concentration (MCHC); (e), (f), (g) percentage of hematocrit (HCT), thrombocytocrit (PCT), lymphocyte (LYMPH), monocyte (MONO), neutrophil (NEUT), and eosinophil (EO) were measured for each mouse. For control (CT), the mean value from eight mice was used. The blue shaded area means the mice were under the circumstances of “hypogravity”. The pink shaded area shows the mice were under the circumstances of “hypergravity”. The green shaded area shows the reference range defined by eight mice in the Sham control group. (h) The relative levels of MCV, MCHC, HCT%, RBC, LYMPH%, NEUT%, and WBC were compared between Sham control, 24.5 T (no gradient), and 7.2 T (in hypogravity and hypergravity conditions).

We noticed that for the same MF intensity, the “hypogravity” and “hypergravity” conditions had differential effects. For example, 7.2 T in the hypogravity condition and hypergravity condition sometimes produced opposite effects (Fig. 3(g)). In addition, the highest MF intensity 24.5 T, which is in the center of the magnet and has no MF gradient, did not produce the strongest effects as we predicted (Fig. 3(h)).

We also did blood biochemical test for liver and kidney functions for 3.7 T–24.5 T high MF-treated GIST-T1 tumor-bearing mice. Alanine transaminase (ALT) activity (Fig. 4(a)), aspartate transaminase (AST) (Fig. 4(b)), alkaline phosphatase (AKP) (Fig. 4(c)), total protein (TP), albumin (ALB) (Fig. 4(d)), total bilirubin (TBIL) (Fig. 4(e)), blood urea nitrogen (BUN) (Fig. 4(f)), serum creatinine (CREA) (Fig. 4(g)) were measured for each mouse. For most indicators of liver and kidney functions, high MFs did not severely affect them. However, we noticed that in some specific MF exposure conditions, the liver and kidney functions were affected (Fig. 4). It is interesting that 24.5 T SMF, which is the highest MF intensity, did not affect these indicators. In contrast, some SMFs with gradient produced noticeable effects on the liver and kidney functions. For example, 23.4 T SMF in the hypogravity condition moderately increased the ALT level (Fig. 4(a)), indicating a liver functional abnormality. Moreover, similar to blood routine test above, we found that for the same MF intensity, the “hypogravity” and “hypergravity” conditions had differential effects on these blood biochemistry results. In addition, the highest MF intensity 24.5 T, which has no MF gradient, did not cause obvious changes (Fig. 4(h)).

Fig. 4.

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Fig. 4. (color online) The biochemical test results of liver and kidney functions for 3.7 T–24.5 T high magnetic field-treated GIST-T1 tumor-bearing mice. (a) Alanine transaminase (ALT) activity, (b) aspartate transaminase (AST), (c) alkline phosphatase (AKP), (d) total protein (TP), albumin (ALB), (e) total bilirubin (TBIL), (f) blood urea nitrogen (BUN), (g) serum creatinine (CREA) were measured for each mouse. For control (CT), the mean value from eight mice was used. The blue shaded area means the mice were under the circumstances of “hypogravity”. The pink shaded area shows the mice were under the circumstances of “hypergravity”. The green shaded area shows the reference range defined by eight mice in the Sham control group. (h) The relative levels of ALT, AST, AKP, TP, ALB, TBIL, BUN, and CREA were compared between Sham control, 24.5 T (no gradient), and 7.2 T (in hypogravity and hypergravity conditions).

We also did the blood biochemical test of metabolism-related indicators for 3.7 T–24.5 T high MF-treated GIST-T1 tumor-bearing mice. Blood glucose (GLU) (Fig. 5(a)), calcium (Ca) (Fig. 5(b)), phosphorus (P) (Fig. 5(c)), iron (Fe) (Fig. 5(d)), cholesterol (CHOL) (Fig. 5(e)), triglyceride (TG) (Fig. 5(f)), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) (Fig. 5(g)) were measured for each mouse. Multiple indicators were affected by some high MF conditions, such as blood glucose (Fig. 5(a)), phosphorus (P) (Fig. 5(c)) and iron (Fe) (Fig. 5(d)). Similar to above, the highest MF intensity 24.5 T, which has no MF gradient, did not cause obvious changes while some gradient MFs had effects on metabolisms (Fig. 5(h)).

Fig. 5.

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Fig. 5. (color online) The blood biochemical test results of metabolism-related indicators for 3.7 T–24.5 T high magnetic field-treated GIST-T1 tumor-bearing mice. (a) Blood glucose (GLU), (b) calcium (Ca), (c) phosphorus (P), (d) iron (Fe), (e) cholesterol (CHOL), (f) triglyceride (TG), (g) high density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured for each mouse. For control (CT), the mean value from eight mice was used. The blue shaded area means the mice were under the circumstances of “hypogravity”. The pink shaded area shows the mice were under the circumstances of “hypergravity”. The green shaded area shows the reference range defined by eight mice in the Sham control group. (h) The relative levels of GLU, Ca, P, Fe, CHOL, TG, HDL-C, and LDL-C were compared between Sham control, 24.5 T (no gradient), and 7.2 T (in hypogravity and hypergravity conditions).

Next, we did Hematoxylin-eosin(HE) staining for the liver, kidney and tumor tissues of 3.7 T–24.5 T high MF-treated GIST-T1 tumor-bearing mice. Representative HE stain images of liver, kidney, and tumor in control and MF-treated mice are shown (Figs. 6, S2, and S3). We observed some liver abnormalities in high MF-treated mice, which is consistent with the above-mentioned blood biochemistry results. The kidney and tumor tissue appear normal.

Fig. 6.

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	Fig. 6. (color online) Hematoxylin-eosin (HE) stains of liver, kidney, and tumor tissue of GIST-T1 tumor-bearing mice in Sham control, 23.4 T and 24.5 T high SMF treatment groups. Representative images are shown. Scale bar: .

Besides safety issues, we also investigated the impacts of high MFs on tumor growth (Fig. 7). It is obvious that different MF conditions have differential effects on GIST-T1 tumor growth in mice (Fig. 7(a)). For example, for the same MF intensity of 23.4 T, the GIST-T1 tumor growth in mice was obvious reduced by 23.4 T in hypergravity condition, but not in hypogravity condition (Fig. 7(b)); for 14.4 T, both hypergravity and hypogravity conditions reduced tumor growth, while the hypogravity affected more obviously (Fig. 7(c)); for 7.2 T, the GIST-T1 tumor growth in mice was obviously reduced by 7.2 T in hypergravity condition, but not in hypogravity condition (Fig. 7(d)), which is similar to 23.4 T. For the 24.5 T high SMF with no gradient, the tumor growth was also inhibited (Fig. 7(e)). We took all the data from eight different SMF exposure conditions together and found that they generally inhibited the tumor growth rate in GIST-T1 tumor-bearing mice (Fig. 7(f)). Although the final tumor weight shows that the tumor inhibition efficacy of different SMF exposure conditions are different, in which the tumor growth inhibition (TGI) could reach 42% in 14.4 T in hypogravity condition and 54% in 7.2 T in hypergravity condition (Fig. 7(g)), the overall TGI for all 8 different SMF exposure conditions are about 17% (Fig. 7(h)).

Fig. 7.

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Fig. 7. (color online) GIST-T1 tumor growth in mice was inhibited by static magnetic field. Black arrows show that mice were exposed to static magnetic field for 3 h each time. (a) Gain of tumor volume was measured each day for each mouse under different magnetic field intensity. (b) Gain of tumor volume in mice under the condition of 23.4 T hypogravity and hypergravity was compared. (c) Gain of tumor volume was measured under the condition of 14.4 T hypogravity and hypergravity for each mouse. (d) Gain of tumor volume was measured under the condition of 7.2 T hypogravity and hypergravity for each mouse. (e) Gain of tumor volume was compared between control group and 24.5 T group. (f) Gain of tumor volume was measured each day for each mouse. (g) Each mouse tumor weight in control and different magnetic field exposed groups. (h) Tumor weight in control and exposed groups. Data represent mean ±SD. p value was calculated using unpaired Student’s t test.