Recent advances in instrumentation are allowing for in vivo
analysis of body composition in small rodents. Many of these instruments were originally designed for measuring bone mineral density and content in human appendages. For example peripheral DXA instruments originally designed for determining bone density in the human os calcis and the forearm have been validated and used to determine fat, lean, and bone in small rodents (8
). Similarly, pQCT instruments that were designed for determining trabecular and cortical bone density in the human ulna have been adapted and used to measure bone mineral density in rodents (12
). This transfer of technology to animal researchers will lead to exciting new research. In this paper we describe the use of pQCT for determining percent body and liver fat in small rodents, and the potential benefits to both obesity and hepatic steatosis animal models.
One limitation of the technology transfer is that the hardware is seldom changed to specifically accommodate different size rodents. For instance, the GE-Lunar PIXImus DXA has an image area of only 60 × 80 mm. Although this may be ideal for measuring the human os calcis, it is too small to easily measure large mice (ob/ob
, mice over 50g; (24
)). Even scanning smaller mice (25–35 g) is problematic since the length of the mouse often exceeds 80 mm. Thus, the head of the mouse must be excluded from the analysis using an exclusion region of interest.
A second limitation of DXA instruments is that only areal (two-dimensional) measures are possible. Thus, DXA is not ideal for obtaining information on specific fat pads. One study has used small animal DXA to estimate body fat distribution (7
), but little data were shown on the validity of these measures.
Computed tomography is capable of giving information on both absolute adiposity and on specific depots (11
). pQCT may be especially useful to animal researchers given the relatively low cost and portability. The instrument used in the current study has a gantry opening that allows animals up to 90 mm in diameter to be scanned. Thus the machine may be able to scan a rat from birth through adulthood allowing detailed longitudinal studies to be performed.
In the present study, we focused on the ability of pQCT to measure percent body fat and liver fat in small rodents. The precision of the instrument (CV = 3.9%) for determining percent body fat is similar to that of small animal DXA (8
). Although pQCT overestimated percent fat as determined by carcass analysis, the relationship between the two methods was good (r2
= 0.91), suggesting the pQCT may be a useful method for determining body composition of mice.
In a pioneering study by Ross et al
), it was found that CT accurately measured fat mass when compared to chemical carcass analysis and the two methods correlated extremely well (r = 0.99). In that study 12 transverse slices were taken for each rat as compared to only five transverse scans in the present study. A reduction in slice number might be expected to decrease precision and accuracy. We chose 5 slices for practical reasons, since increasing slice number increases total scan time. In our protocol, the entire scan time (scout scan and 5 transverse slices) was approximately 8 minutes. We felt that this was a reasonable period of time to keep animals anesthetized and to allow a fairly high throughput. In studies where greater precision and accuracy are needed, it may be necessary to increase slice numbers.
It is also possible that our selection of the fat threshold was too high. We based our thresholds on visual analysis of selected scans. The attenuation threshold of 0.2773 (1/cm) appeared to give a clear distinction between fat and lean tissue. Therefore we did not change the threshold values in an attempt to lower the percentage fat measured by the instrument. Instead we chose to use a regression technique to correct the pQCT data based on carcass analysis.
In addition to being able to discriminate different tissues (fat and lean), computed tomography should be able to give information on the relative fat infiltration of an organ or muscle. As the fat content of lean tissue increases, the attenuation value should decrease given that fat attenuates X-rays to a lesser extent than pure lean tissue. We therefore used this phenomenon to first, create a regression equation relating percentage liver fat from chemical analysis to the measured attenuation value of the liver. Our findings revealed that the two parameters were highly related and reproducible. With this data in hand, it was then possible to track changes in percent liver fat within the same animal over time by measuring the attenuation value of the liver and then converting this value into percentage liver fat. Although we chose lemmings as our model system, clearly the technique will be useful for other small animal models of hepatic steatosis.
Our results suggest that pQCT may be a useful method for measuring body composition in small rodents. The instrument is small and relatively inexpensive (compared to large clinical-CT instruments) making it feasible for laboratory animal investigations.