Comparison of bioimpedance spectroscopy and X-Ray micro-computed tomography for total fat volume measurement in mice.

Obesity and the metabolic syndrome are two pathologies whose prevalence are in a constant increase. Evaluation of the total fat mass but also of the distribution between visceral and subcutaneous adipose tissue are important factors while assessing the pathophysiology of these two pathologies. Computed tomography (CT) and bioimpedance (BIS) are the translational methods the most frequently used in human beings as well as in rodent models in longitudinal studies on adiposity and obesity. Surprisingly, no direct comparison of micro-CT and BIS was reported yet in mice. Therefore, the present study was carried out to evaluate and compare the accuracy and the uncertainty of measurement of micro-CT and BIS in this species. The proportion of fat mass was measured with BIS, micro-CT and direct post-mortem tissue weight, and correlations between the data were established to evaluate the accuracy of the methods but also the uncertainty of BIS and micro-CT. There were significant correlations between weights of fat tissues on scale and proportion of total fat mass determined by BIS or micro-CT (r = 0.81 and 0.86 respectively) but both methods overestimated the total fat mass, especially in the smallest animals; overestimation of fat mass was amplified with BIS compared to micro-CT. In addition BIS and micro-CT were highly correlated (r = 0.94). Test-test reliability showed a greater variability of the BIS with respect to the micro-CT (coefficient of variation = 17.2 vs 5.6% respectively). Hence, as far as subtle differences between groups or changes within one group are awaited, micro-CT may appear as the most reliable method for determination of fat mass in mice. Micro-CT, unlike BIS, will also allow to qualitatively and quantitatively differentiate between subcutaneous and visceral adipose tissues, which is of major importance in studies on adiposity and its complications

Proportion of total adipose tissue in thoracic region.

<p>Proportion of total adipose tissue in thoracic region.</p

The different steps for quantification of total adipose tissue by micro-computed tomography.

<p>Anesthetized mice are placed in a supine position in an imaging cell; three-dimensional x-ray images are acquired on the CT part of a μSPECT-μCT (eXplore speCZT Vision 120, GE, Waukesha, USA). Volumes are reconstructed with a voxel size equal to 100x100x100 μm³; reconstructed images are filtered with a Gaussian filter to reduce noise. (a) Whole body scan. (b) Selection of whole body volume. (c) Choice of threshold level based on fat pad (FP). (d) Representation of total adipose tissue with subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT). (e) Isosurface representation of whole body in grey and fat in yellow.</p

Placement of bioimpedance spectroscopy electrodes on a mouse.

<p>Four 25G needles are placed subcutaneously along the midline of the back and serve to connect the electrodes to the animal. The B electrode (blue) is placed at the intersection between the median line and the line between the ears. The A electrode (black) is placed 1 cm from B towards the muzzle. The C electrode (yellow) is placed between the median line and that joining the thigh muscles. The D electrode (red) is placed 1 cm backwards at the base of the tail. The length between B and C needle electrodes is measured; this parameter, as well as the age and weight of the animal, is required by the software to calculate the proportion of fat mass.</p

The different steps for lung manual correction on fat volumes on micro-computed tomography scans.

<p>Micro-CT scans were performed in 10 mice, with and without respiratory-gated conditions, on the same field of view (thoracic region). (a) 3D representation of the region of interest around the lungs. (b) One slice of representation of total adipose tissue with lung selection through a region of interest (in orange dotted line). (c) Suppression of lung region on the same slice.</p

Relationships between the different methods of determination of total adipose tissue.

<p>Thirty mice (body weight ranging from 26 to 36g) were used. In each animal, the proportion of adipose tissue was measured with bioimpedance spectroscopy (BIS), micro-computed tomography (μCT) and by direct post-mortem tissue weigh as described in Material and Methods. Theoretical adipose tissue weight was calculated from body weight and BIS or μCT data. (a) Correlation between the body weight of animals and the proportion of total fat mass estimated by BIS and μCT. (b-e) Correlations between the weight of harvested adipose tissue and the proportion of fat mass estimated by BIS (b) or μCT (d) and between the weight of harvested adipose tissue and the theoretical calculated adipose tissue weight obtained by BIS (c) or μCT (e). (f) Correlation between total adipose tissue estimated by BIS and μCT. r is the Pearson correlation coefficient.</p

Effect of a normal or high fat diet on body weight and proportion of total fat mass estimated by micro-computed tomography in mice.

<p>Mice received normal (ND) or high fat (HFD) diet for 15 weeks. Animals were weighed and the proportion of total fat mass (FM, %) was determined by micro-computed tomography (μCT) scans as described above before (T0) and at the end (T15) of the treatment period. Evolution of (a) body weight and (b) FM over time. (c) Mean increases in body weight and total FM after 15 weeks of diet. Data are represented as the mean ± standard deviation of 6 animals in each group. Differences in body weight and FM were analyzed by 2-way analysis of variance (with diet and time as factors) (GraphPad Prism 6.0 software). *: p<0.05 HFD <i>versus</i> ND. $: p<0.05 T15 <i>versus</i> T0 within the same group.</p

Total, fat and lean volumes, and calculated fat/total volume and lean/total volume ratios in mice after 15 weeks of normal (ND) or high fat (HFD) diet.

<p>Total, fat and lean volumes, and calculated fat/total volume and lean/total volume ratios in mice after 15 weeks of normal (ND) or high fat (HFD) diet.</p

Scheme of the use of the density meter.

<p>The density meter comprises a graduated tube connected to a tank, filled with an inert liquid (kerosene) (43,44). (a) A sample of mouse fat is weighed and placed slowly in the tank. (b) The fat sample overflows the kerosene that falls into the graduated tube. (c) The volume of kerosene (mL ± 0.05mL) that has flowed into the graduated tube is measured and is equal to the volume of fat.</p