Whole-Body γ Irradiation and Sample Collection

Seven-week-old female C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). After acclimation for 1 week at the animal facility of Pacific Northwest National Laboratory (PNNL), they were randomly assigned to groups of four each. Mice were exposed to whole-body γ radiation using a high-activity source (1250 keV ⁶⁰Co) with an LET in the range of 0.2–2 keV/µm. For the irradiations, the animals were isolated in the corner of their polymer cages, placed a minimum of 100 cm from the collimated 6000 Ci (222 TBq) ⁶⁰Co source, and irradiated. After irradiation the isolation barrier was removed and animals were transferred to PNNL Animal Facility. The γ-radiation field at the position of the mice was measured beforehand using a reference-class ionization chamber that was calibrated at the National Institute of Standards and Technology. The resulting absorbed dose rate at a depth of approximately 600 mg/cm² was 0.83 Gy/min relative to tissue. Groups of mice were exposed to doses of 0 (control), 3.0 and 7.8 Gy. At 4 and 11 days after exposure, mice were killed humanely with 70/30 CO₂/O₂, and the right femur from each mouse was immediately removed, frozen in liquid N₂, and stored at −80°C until NMR analysis. The left femur from each mouse was prepared for histology study (see below). All animal work was approved by the Institutional Animal Care and Use Committee (IACUC) at PNNL.

Histopathology Studies

Immediately after excision, the left femur from each mouse was fixed in 10% neutral buffered formalin for histopathological analysis using well-established methods (25,26). Briefly, formalin-fixed femurs were demineralized in formic acid solution, washed, processed in an automated processor, embedded in paraffin, sectioned at 5 µm, mounted on glass microscope slides, and stained with hematoxylin and eosin. The slides were examined by light microscopy.

High-Resolution MAS ¹H NMR Measurements

To prepare samples for high-resolution MAS ¹H NMR, the left femur was thawed slightly, and outside surrounding tissues were removed under magnification. The whole femur was weighed and inserted into a Teflon tube. The Teflon tube assembly was then loaded between two Teflon plugs in a 7.5-mm-outside-diameter, 6-mm-internal-diameter Chemagnetics pencil rotor as shown in Fig. 1. The two solid Teflon plugs can be inserted into the rotor only by precooling the plugs in liquid nitrogen. In this way, a tight seal is created when the plugs warm to the targeted experimental temperature of 2°C. As a result, the marrow spun at the fast sample spinning rate remains inside the sample chamber with no fluid leakage. No fluid leakage was found in more than 100 experiments on femurs using this setup at a sample spinning rate of 4 kHz. In other experiments, femurs with the surrounding tissue removed were also dissected into two sections. One section was a combination of epiphysis (mainly the two femoral heads) and the metaphysis (a short portion of proximal femur beneath the head) while the other section was diaphysis (most of the shaft of the femur). The sample was loaded using the same procedures as for the whole femur.

Figure 1

Schematic of the special liquid-tight sample cell for ¹H MAS NMR experiments with mouse femur. The femur was inserted into a Teflon sleeve and closed tightly with two Teflon plugs inside the zirconium sample rotor.

All ¹H NMR experiments were performed at 2°C on a Varian-Chemagnetics 300 MHz Infinity spectrometer operating at a proton Larmor frequency of 299.97 MHz. A standard Chemagnetics CP/MAS probe with a 7.5-mm pencil-type spinner system was used in all the measurements. The sample spinning rate of 4.0 kHz was controlled automatically by a Chemagnetics MAS speed controller at an accuracy of 1 Hz. Temperature was controlled by a Chemagnetics temperature controller in combination with an FTS heating unit. A rotor-synchronized Carr-Purcel-Meibom-Gill pulse sequence, [90°-(τ-180°-τ)_n-acquisition], was used for acquiring data with a total length of 12.5 ms for the echo segment. The 90° pulse length was adjusted for each sample individually and varied from 6.8 to 7.6 µs. The water suppression segment used in this study was a DANTE (delays alternating with nutations for tailored excitation) sequence (27) consisting of 6000 small tip angle pulses that were separated by 100 µs. A total of 1920 accumulations were acquired with a total acquisition time of ∼1 h for each experiment. The strong lipid (CH₂)_n signal at 1.28 ppm in each spectrum was used as the internal chemical shift reference.

Quantification and Statistical Analysis

The spectrum was integrated with respect to different metabolite peaks in the range of 0.5–4.5 ppm for quantitative data analysis. For comparison within each sample, the integrated peak intensity for each type of metabolite was compared to the most abundant lipid (CH₂)_n peak located at 1.28 ppm in the same spectrum. For comparison between treatment groups, the integrated peak intensity for each type of metabolite in the irradiated group was compared with the same one in the age-matched control group. The reliability of this intertreatment group comparison was validated by performing 12 independent ¹H MAS experiment on a 65-mg PBS + 3 mM TSP sample during a period of 4 days. For each measurement, the probe was removed from and reinserted into the magnet and the 90° pulse was calibrated. Defining the mean value of the integrated intensity for the TSP peak as 100, the mean ± SD was 100 ± 3. This small standard deviation (3%) indicated that both the spectrometer, including the MAS probe, and the pulse sequence were remarkably stable. Therefore, the integrated metabolite peak intensities from the spectra samples that were acquired under similar experimental conditions could be compared directly.

The statistical significance of differences between the metabolite peak intensities of the exposed group and those of the control group was determined using the Student's t test. We used a sample size of four. Furthermore, the t test works under the assumption of a Gaussian distribution of data but also works well for distributions that are not Gaussian (28).

Histopathology Findings

Typical examples of the cell depletion in bone marrow are shown in Fig. 2. Depletion is defined as a decrease in nucleated cells, which have dark blue nuclei when stained with hematoxylin. The depleted marrow has increased numbers of red blood cells that are not nucleated when mature. Thus the color of the depleted marrow cells appears less basophilic, lighter and more eosinophilic (orange) in Fig. 2c. This indicates that there are fewer basophilic nucleated cells, more white adipose cells, and more eosinophilic non-nucleated erythrocytes.

Figure 2

Histology of mouse femurs, illustrating bone marrow hematopoietic cell depletion at high radiation dose. Panel a: Nonirradiated control mouse; panel b: 4 days after whole-body exposure to 3.0 Gy γ radiation; panel c: 4 days after whole-body exposure to 7.8 Gy.

It is evident from Fig. 2 that there was a mild depletion of hematopoietic cells in the bone marrow of C57BL/6 mice at 4 days after irradiation with 3 Gy (Fig. 2b). By day 11 the bone marrow had returned to a normal histology (not shown). However, the bone marrow was severely depleted at 4 days after exposure to 7.8 Gy (Fig. 2c). The bone marrow cavity contains hematopoietic cells, bone marrow stromal and adipose cells, and bone-forming cells such as osteoblasts and osteoclasts. In the studies shown in Fig. 2, the effects on hematopoietic cells were apparent, while the effects on other cellular components were not.

The Effect of High-Resolution MAS ¹H NMR on Whole Femur

A typical example of the effect of MAS on the ¹H NMR spectral resolution of a whole femur is shown in Fig. 3. Both the static (Fig. 3a) and the MAS spectra (Fig. 3b) were obtained on the same femur sample and were acquired using the same experimental parameters except that a sample spinning rate of 4 kHz was used in spectrum b. The data presented in Fig. 3a were acquired first, followed immediately by acquisition of those presented in Fig. 3b.

Figure 3

¹H NMR spectra (300 MHz) of mouse femur acquired at 2°C. (a), Static spectrum; (b), MAS at 4.0 kHz. The assignments of the peaks are as follows: 1, terminal –CH₃; 2, –(CH₂)_n; 3, –O–CO–CH₂–CH₂–; 4, –CH = CH–CH₂–CH₂–; 5, –O–CO–CH₂–CH₂–; 6, –CH = CH–CH₂–CH = CH-; 7, creatine; 8, total cholines, including choline, phosphocholine and phosphatidylcholine; 9, glycogen/glucose; 10 and 11, C1, C3-glycerol ester; and 12, –CH = CH–. The peak in (b) labeled with an asterisk is the residual water signal. The spectrum above to shows the spectral region between 1.5 and 4.5 ppm that is expanded from the spectrum in (b) by about 2.6× horizontally and 5.5× vertically.

In the static spectrum shown in Fig. 3a, the water peak is so broad that its tail covers a chemical shift range from about 0 to 10 ppm. This is due to the strong magnetic susceptibility variations between the marrow tissue and the bones. As a result, it is impossible to achieve good water suppression without suppressing the corresponding metabolite signals. This prohibits the identification of biologically meaningful metabolites. However, by spinning the sample at 4 kHz (Fig. 3b), water suppression using the same experimental parameters as those in Fig. 3a becomes efficient, and the intensity of the residual water peak at about 4.8 ppm is less than that of the lipid peak at 1.28 ppm. The MAS also greatly enhances the spectral resolution. As shown in Fig. 3b and its corresponding expanded (both horizontally and vertically) insertion spectrum between 1.5 and 4.5 ppm, a number of distinct metabolite peaks were obtained with a narrow peak width. The chemical identities of these major metabolite peaks were assigned according to the published literature (14, 17, 22–24) and were labeled on the spectrum. The effect of spinning at 4 kHz on the sample was also tested; there was no visible difference in the spectrum when the sample was respun at 4 kHz (data not shown). In addition, there was no observable difference when the measurement was repeated a few hours after the first measurement (data not shown), showing that there is minimal sample degradation at the experimental temperature of 2°C used throughout this work.

Metabolic Difference between Diaphysis and Epiphysis + Metaphysis Sections

The spectrum in Fig. 4a was acquired on the diaphysis of a femur from the control group, and the spectrum in Fig. 4b was acquired on the epiphysis + metaphysis sections of the same femur. Both spectra were acquired using the same experimental parameters except that the 90° pulse width was calibrated individually.

Figure 4

¹H NMR spectra (300 MHz) of C57BL/6 mouse femurs exposed to 3 and 7.8 Gy whole-body γ radiation. The spectra were acquired at 2°C with MAS at 4.0 kHz. (a) and (b), Controls; (c) and (d), 4 days after exposure to 3 Gy radiation; (e) and (f), 4 days after exposure to 7.8 Gy. (a), (c) and (e) were acquired on the diaphysis of femur while (b), (d) and (f) were acquired on the epiphysis + metaphysis. The diaphysis samples and the epiphysis + metaphysis samples were always from the same three femurs. All the spectral traces are plotted with the lipid signal at 1.28 ppm at the same height to facilitate the comparison. The insertions above each spectrum show the spectral range between 2.5 and 4.5 ppm that is expanded vertically four times.

The femurs of C57BL/6 mice exposed to 3.0 and 7.8 Gy of γ radiation were analyzed as outlined above; i.e., the diaphysis and the epiphysis + metaphysis of the femur were measured separately with high-resolution MAS ¹H NMR. Typical spectra for irradiated samples are shown in Fig. 4c–f. Quantitative results obtained from statistical analysis of the high-resolution MAS ¹H NMR spectra are summarized in Tables 1 and 2.

Table 1

Quantification of the Metabolite Peaks from the Diaphysis (the shaft) and the Epiphysis + Metaphysis (the two heads) of Femurs Excised from Female C57BL/6 Mice Exposed to Gamma Radiation

Table 2

Within-Sample Summary of the Metabolic Profiling Results from the Diaphysis and Epiphysis + Metaphysis of Femurs Excised from Female C57BL/6 Mice Exposed to Gamma Radiation

For the control groups, the relative ratio of the NMR peaks for the diaphysis and the epiphysis + metaphysis sections of the same femur was significantly different (Table 1). For example, there was a distinct difference in the weight-averaged total peak area between 0.7–4.4 ppm on both day 4 and day 11. High-resolution MAS ¹H NMR is unique for selectively assessing the mobile molecules in tissues. Large molecules such as proteins and rigid phospholipids that form the lipid bilayers of healthy cell membranes are essentially invisible to high-resolution MAS ¹H NMR if the sample spinning rate used is less than about 6 kHz (29). These results clearly showed that the epiphysis + metaphysis contains more mobile molecules than the diaphysis. A careful examination revealed that the increase in the lipid (CH₂)_n signal at 1.28 ppm is the main cause for the increased weight-averaged peak area of the epiphysis + metaphysis relative to the diaphysis. This was further confirmed by the data in Table 1 showing the peak areas for the lipid (CH₂)_n signal at 1.28 ppm, integrated over a spectral range between 1.1 and 1.4 ppm for the control group, for the diaphysis and the epiphysis + metaphysis at both day 4 and day 11. These results indicate unambiguously that the epiphysis + metaphysis contain more mobile lipids. It can also be seen in the vertically expanded spectral regions between 1.5 and 4.5 ppm in Fig. 4 and the data in Table 2 that for the control mice, higher content of lipid moieties (peaks corresponding to 0.7–1.0, 1.4–1.8 and 1.8–2.15 ppm, mainly the fatty acyl groups from triglycerides) and total cholines (choline, phosphocholine and phosphatidylcholine at 3.1–3.3 ppm) were observed in the diaphysis compared with the epiphysis + metaphysis. Similar results were obtained from the irradiated groups. These results indicate that the composition of the lipids in the diaphysis is different from that in the epiphysis + metaphysis, i.e., with increased –CH₃, O–CO–CH₂–, and –CH = CH–CH₂– functional groups accompanied by an increased amount of total cholines. The relative increases in methyl, carboxyl and unsaturated bonds presumably mean that the lipids in the diaphysis contain shorter saturated (CH₂)_n chains compared to the epiphysis + metaphysis. Selective assessment of mobile lipids is important because it has been suggested that mobilization of fatty acids plays an important role in the development of cancer (30), obesity and type II diabetes (31,32). Note that mobile lipids are observed in high-resolution MAS ¹H NMR at a sample spinning rate while the rigid membranes are not. Therefore, high-resolution MAS ¹H NMR can be considered as a selective observation for mobile lipids. The information obtained by high-resolution MAS ¹H NMR complements the standard lipid analysis of cell extractions (33), in which all of the fatty acids from the cells including cell membranes are analyzed. The lack of similar region-specific histological changes suggests that high-resolution MAS ¹H NMR may represent a novel and sensitive means to assess subtle or early events in bone marrow pathology directly in whole bone specimens.

Impact of Radiation on the Epiphysis + Metaphysis of the Femur

After exposure to 3.0 Gy, the integrated peak area for the most abundant lipid peak centered at 1.28 ppm (1.1–1.4 ppm) showed a nonsignificant increase at both 4 and 11 days after irradiation. However, the peak area was statistically significantly increased at 4 days after exposure to 7.8 Gy radiation. The increased lipid signal at the high radiation dose indicates that radiation changed the lipid profile in the epiphysis + metaphysis of the femur. This conclusion was further supported by the weight-averaged total peak area, which was significantly increased both at 4 days after exposure to 7.8 Gy radiation and at 11 days after exposure to 3.0 Gy radiation.

At 4 days after exposure, the relative integrated peak area for total choline (3.1–3.3 ppm), a combination of choline, phosphocholine and glycerol phosphocholine in the epiphysis + metaphysis of the femur decreased significantly (see the expanded insertion spectra shown in Fig. 4b–d). At 11 days after exposure to 3.0 Gy radiation, the level of total choline in the epiphysis + metaphysis of the femur remained significantly deceased compared with that of the corresponding controls.

For the epiphysis + metaphysis, the relative peak area of creatine (2.9–3.1 ppm) followed a similar trend as that of total choline (Table 2). At 4 days postirradiation, the level of creatine decreased significantly. At 11 days after exposure to 3 Gy, creatine was decreased compared with the corresponding age-matched controls. Creatine is the end product associated with the cellular energy pathway (17). The decreased level of creatine suggests that the basic energy pathway was affected by whole-body exposure to γ radiation.

For the epiphysis + metaphysis, the relative ratios for all other peaks remained approximately the same at 4 days after exposure to 3 Gy (P > 0.05). However, at 11 days after irradiation, statistically significant decreases were observed for many of the peaks (Table 2), indicating that the effect of radiation increased at longer times.

Effect of Radiation on the Diaphysis of the Femur

As in the epiphysis + metaphysis, 4 days after exposure to 7.8 Gy radiation, there were statistically significant decreases in the relative areas of all the peaks (Table 2) for the diaphysis compared with the corresponding controls. However, in contrast to the results obtained for the epiphysis + metaphysis of the femur, there were no significant differences in total choline and creatine 4 days after exposure to 3 Gy radiation. Furthermore, at 11 days postirradiation, except for the peaks corresponding to the terminal methyl groups (0.7–1.0 ppm) and the –CH = CH–CH₂–CH₂–(1.8–2.15 ppm) groups, there were no significant differences for any of the other peaks. This indicated that the epiphysis + metaphysis sections were more sensitive to radiation than the diaphysis of the same femurs. This conclusion was also supported by the results shown in Table 1 for the diaphysis; although both the weight-averaged total peak area and the peak area of long chain lipids (CH₂)_n (1.1–1.4 ppm) were increased in the irradiated animals compared with their corresponding controls, the increase was not statistically significant. In contrast, a significant increase was observed for the corresponding epiphysis + metaphysis sections of the same femur.