A longitudinal examination of bone loss in the femurs and tibiae of adult mice was performed following spinal cord injury using sequential low-dose X-ray scans. Tibia bone loss was detected throughout the study, while bone loss in the femur was not detected until 40 days post injury.
Spinal cord injury (SCI) is often accompanied by osteoporosis in the sublesional regions of the pelvis and lower extremities, leading to a higher frequency of fractures 1. As these fractures often occur in regions that have lost normal sensory function, the patient is at a greater risk of fracture-dependent pathologies, including death. SCI-dependent loss in both bone mineral density (BMD, grams/cm2) and bone mineral content (BMC, grams) has been attributed to mechanical disuse 2, aberrant neuronal signaling 3 and hormonal changes 4. The use of rodent models of SCI-induced osteoporosis can provide invaluable information regarding the mechanisms underlying the development of osteoporosis following SCI as well as a test environment for the generation of new therapies 5-7 (and reviewed in 8). Mouse models of SCI are of great interest as they permit a reductionist approach to mechanism-based assessment through the use of null and transgenic mice. While such models have provided important data, there is still a need for minimally-invasive, reliable, reproducible, and quantifiable methods in determining the extent of bone loss following SCI, particularly over time and within the same cohort of experimental animals, to improve diagnosis, treatment methods, and/or prevention of SCI-induced osteoporosis.
An ideal method for measuring bone density in rodents would allow multiple, sequential (over time) exposures to low-levels of X-ray radiation. This study describes the use of a new whole-animal scanner, the IVIS Lumina XR (Caliper Instruments) that can be used to provide low-energy (1-3 milligray (mGy)) high-resolution, high-magnification X-ray images of mouse hind limb bones over time following SCI. Significant bone density loss was seen in the tibiae of mice by 10 days post-spinal transection when compared to uninjured, age-matched control (naïve) mice (13% decrease, p<0.0005). Loss of bone density in the distal femur was also detectable by day 10 post-SCI, while a loss of density in the proximal femur was not detectable until 40 days post injury (7% decrease, p<0.05). SCI-dependent loss of mouse femur density was confirmed post-mortem through the use of Dual-energy X-ray Absorptiometry (DXA), the current “gold standard” for bone density measurements. We detect a 12% loss of BMC in the femurs of mice at 40 days post-SCI using the IVIS Lumina XR. This compares favorably with a previously reported BMC loss of 13.5% by Picard and colleagues who used DXA analysis on mouse femurs post-mortem 30 days post-SCI 9. Our results suggest that the IVIS Lumina XR provides a novel, high-resolution/high-magnification method for performing long-term, longitudinal measurements of hind limb bone density in the mouse following SCI.
1. Mouse spinal transection model
2. Longitudinal assessment of bone density using the IVIS Lumina XR on the same cohort of spinally-transected mice
3. Image analysis of IVIS X-ray scans
4. Post-mortem analysis of bone density:
5. DXA Analysis using a Hologic QDR 4000 Bone Densitometer
6. Representative Results:
The relative bone density loss of a mouse tibia and femur after spinal cord injury when compared to naïve mice is detectable using the above method. There is a detectable significant decrease in bone density after just 10 days (12%, p<0.0005), with up to 15% bone density loss at 40 days (p<0.0005, Figure 4). Bone density loss in the femur was observed at 40 days post injury (7% decrease, p<0.05, Figure 5). These results provide evidence for the use of non-invasive x-ray imaging for the longitudinal observation of bone density change after spinal cord injury.
In order to compare the efficacy of this method to what is currently available; we analyzed the excised femurs of these mice 40 days after injury using DXA imaging. A representation of the data output can be seen in Figure 6. We found that there was a significant loss of bone mineral content in the SCI mice when compared to naïve (12% decrease, p<0.05, Figure 7). Bone mineral density did not change significantly, but did follow a similar trend (Figure 8). These results are similar to those found in the literature; Picard et al observed a 13.5% decrease (p<0.001) in BMC, but no significant decrease in BMD (Picard 2008).
Figure 1. Experimental Timeline.
Figure 2. Representative orientation of left hind-limb: a) photograph and b) x-ray.
Figure 3. ROI dimensions and orientation within the regions of the proximal femur and tibia.
Figure 4. Bone density loss after SCI in the proximal tibia 10, 20, 30 and 40 days after injury (n=5) compared to age-matched naives (n=5). Error bars represent SEM; **p<0.005, *** p<0.0005.
Figure 5. Bone density loss after SCI in the proximal femur 10, 20, 30 and 40 days after injury (n=5) compared to age-matched naives (n=5). Error bars represent SEM; *p<0.05.
Figure 6. Bone density loss after SCI in the distal femur 10, 20, 30 and 40 days after injury (n=5) compared to age-matched naïve controls (n=5). Error bars represent SEM; *p<0.01-0.05; **p<0.001-0.01; ***p<0.0001-0.001; ****p<0.0001.
Figure 7. Representative image of DXA data showing BMC and BMD output.
Figure 8. DXA analysis of bone mineral content (grams) in the femurs of SCI mice 40 days post injury (n=5) vs. age-matched naives (n=5). Error bars represent SEM; *p<0.05.
Figure 9. DXA analysis of bone mineral density (mg/cm2) in the femurs of SCI mice 40 days post injury (n=5) vs. age-matched naives (n=5). Error bars represent SEM; no significant difference.
This study presents a novel, non-invasive method for assessing density changes within individual bones (tibiae and femurs) longitudinally, within a single cohort of mice, using the Caliper IVIS Lumina XR imaging system. SCI produced a decrease in bone density in both femurs and tibiae when studied within a single cohort of spinally-transected mice over a 40 day assay period compared to age-matched, uninjured, control mice. Our results in the femur were confirmed, post-mortem, through the use of DXA, suggesting that the application of the Caliper Instruments IVIS Lumina XR can serve as a sensitive measuring tool to assess the long-term and progressive effects of SCI on bone loss.
One potential benefit to investigators using this system is the ability to longitudinally follow the development of pathological changes in the hind limb bones of animals following SCI. The ability to assess a single cohort over long time periods provides strong advantages to the investigator in terms of: 1) costs associated with animal purchase and long-term care. Instead of a study requiring large numbers of animals that need to be sacrificed at specific time points in order to assess time-dependent changes, these pathological outcomes can be followed within the same cohort over that same time period, 2) variability: in addition to comparison with control cohorts, the outcomes generated within the single cohort of injured subjects can be assessed and compared between time points for the same animals throughout the extent of the study; once again, reducing both intra-animal variability as well as the need for larger cohorts of experimental subjects.
While the Caliper IVIS Lumina XR provided us with important longitudinal information regarding the development of SCI-induced osteoporosis, there are limitations that should be considered: 1) while this platform has allowed us to generate high-resolution X-ray data in the hind limb bones of mice following SCI, its utility may be limited to small rodents such as mice. The maximum subject height allowed within the Lumina XR is 2.8 cm. Anything above that height cannot be successfully imaged using the X-ray component of the Lumina XR. In our hands, this has precluded our ability to study rats in the 200-250 gram weight. While it may be possible to image rats under this weight range or other rodents such as hamsters or gerbils, this will require additional testing by the PI. 2) Joint orientation. It is critical that the Investigator carefully arrange the limbs in an easily reproducible orientation that will allow successful comparison between imaging sessions. Standardization of limb placement will allow the Investigator to generate and apply standardized ROI’s that can be saved and applied across both subjects and time.
Overall, we believe that the IVIS Lumina XR provides an excellent platform with which to model the development of SCI-dependent osteoporosis in mice. The ability to study bone loss longitudinally within the same mouse cohort will allow us to: 1) better understand the temporal nature of bone loss and demineralization after SCI, 2) determine whether these changes stabilize over time, 3) explore, with the availability of both transgenic and null mouse lines, the ability to study the molecular mechanisms underlying these pathological changes in a reductionist manner, and 4) quite possibly the most important advantage, begin to test novel interventions designed to either prevent the development of osteoporosis in the early stages of injury, or, to reverse such changes once osteoporosis has already developed. Finally, the Lumina XR, in addition to providing excellent X-ray imaging potential, can also be used to study both bioluminescent and fluorescent signals in living animals. One could easily imagine incorporating the other modalities offered within this system to look at a multitude of factors that determine bone dynamics mechanistically following SCI or any other disease process that results in osteoporosis.
The authors have nothing to disclose.
We would like to thank both Mission Connect and the Gillson-Longenbaugh Foundation for providing the funds for this project. We would also like to thank Dr. Catherine Ambrose for her critical advice and use of her DXA equipment. Finally, we would like to thank Dr. Kaori Ono, Department of Integrative Biology and Pharmacology, for her suggestions in DXA femur bone analysis.
Name of the reagent | Company | Catalogue number | Comments |
---|---|---|---|
Beuthanasia | Butler Schein Animal Health |
001848 | |
Ketathesia (Ketamine Hydrochloride) |
Butler Schein Animal Health |
023061 | |
Xylazine | Butler Schein Animal Health |
037849 | |
Bupivicaine | Butler Schein Animal Health |
021801 | |
Gelfoam; 7MM | Fisher Scientific | NC0085178 | To promote hemostasis during surgery |
IVIS Lumina XR | Caliper Life Sciences | 135400 | |
ZFOV, 2.5 cm field of view lens |
Caliper Life Sciences | 127285 | Absolutely necessary to generate high magnification X-ray images of bone structure |
QDR Bone Densitometer |
Hologic | Model used no longer in production |