Project protocol — Contents
Workflow and sampling
Equipment and supplies
Reagents and solutions
Procedure: Mouse densitometry using the DXA system
Definitions & formulas
Step Procedure accomplished Data collected 1 Top loading scale for body weight bw 2 Euthanasia: cervical dislocation or CO2 asphyxiation - 3 Mouse body is propped for scanning - 4 PIXImus is calibrated - 5 Whole mouse body is scanned using PIXImus Mouse Densitometer X-ray image of the mouse in 2-dimension 6 Region of interest (ROI) is outlined in red, yellow or blue; the head is excluded and outlined in green BMC, lean tissue mass, body fat mass, % body fat
- Top loading balance: Ohaus Navigator, Model N12120 with internal calibration (Ohaus Corp., Pine Brook, NJ).
Mouse densitometer dual energy X-ray absorptiometry (PIXImus small animal)
- DXA system (LUNAR, Madison, WI), software version 1.43.036.008): The PIXImus mouse densitometer has been reconfigured with lower x-ray energy than in human
- DXA machines in order to achieve optimal contrast in small specimens.
- The Lunar PIXImus for rodents is a fully integrated densitometer designed for the estimation of bone mineral density (BMD) and body composition.The resolution of the PIXImus is 0.18 x 0.18 mm pixels with a usable scanning area of 80 x 65 mm, allowing for measurement of a single mouse or collections of isolated specimens.
- The PIXImus has been calibrated with a phantom utilizing known values, and a QA is performed daily with this same phantom.
- The precision for BMD is less than 1% coefficients of variations (CV) for whole body, approximately 1.5% CV for specialized regions (Nagy et al, 2000). Correlation with pQCT values for 614 isolated spinal vertebrae is significant (p<0.001; r=.704).
- Assessment of accuracy for the PIXImus is done with a set of hydroxyapatite standards (0-2,000 mg), yielding a correlation of 0.999 between standards and PIXImus measurement of mineral.
- For additional information: PIXImus
- Lily tubs: 64-oz paper bucket (Sweetheart Cup Company, Owings Mills, MD).
- Disposable specimen trays with sticky immobilizing tape: Lunar PIXImus Corporation Headquarters, 726 Heartland Trail, Madison, WI 53717.
Figure A: Lunar PIXImus2 densitometer with integrated PC computer. Figure B: Close-up detail of the Lunar PIXImus2 densitometer with specimen tray.
Detergent and cleaning solutions
Acclimation to test conditions
Mice are obtained at 8 and 16 wks of age (DOB +/- 3 days) from production rooms and are acclimated to test conditions for at least half-hour.
Procedure: Mouse densitometry using the DXA system
Collecting image scans:
a. The PIXImus densitometer apparatus is first calibrated with a "phantom mouse" according to manufacturer's protocol.
b. Then each mouse is placed on the specimen sticky tray (body must be within blue line on the tray) under the PIXImus beam path. The tail is placed alongside the body, the front legs are extended to the side, and the neck and spine are gently straightened.
c. Trays are positioned so that the area of the head is always oriented toward the left from the investigator's point of view, and the mice are position dorso-ventral so that the entire body and tail are measured in the scan.
d. The X-ray process to obtain a single full scan is approximately 5 min; specific regions of interest (ROI) are then subsequently defined.
e. Disposable plastic trays, with sticky tape for immobilizing mice, can be saved and re-used after a thorough cleaning and disinfections.
Measurement acquisition and image scan analysis
a. Based on PIXImus validation studies (Nagy, 2000; Johnston, 2005) DXA-estimated measurements of fat tissue correlate well with measurements obtained from chemical extraction. This is made possible by developing software versions with equations that adequately correct raw DXA measurements.
b. Following the completion of an image scan the DXA system automatically implements specialized software to identify bone tissue from either fat tissue or from lean tissue based on the resulting X-ray densities at two distinct energy levels (Pietrobelli, 1996; Johnston, 2005).
c. Visually, following the completion of a scan, the mouse sample is then outlined with red and green colored circle and square to define specific regions of interest.
d. By using the screen interactive display, F3 is first clicked to prompt measurement adjustments, and then clicked again for the second time to adjust ROI (region of interest).
e. The area to be analyzed is defined (red box), and areas to be excluded from the calculations are defined (green). The arrow keys are used to adjust to the desired size, in addition to holding the control key down to enlarge or elongate the circle or square areas.
f. Once the desired ROI is achieved, the Enter key is clicked and resulting data measurement is displayed. By pressing F5 a hard copy of the image and the scan analysis result is printed.
g. To prompt the computer to finish the session, F8 or Esc key is clicked once, and then clicked again to return to the main menu screen where the next subject to be tested begin.
h. Acquired data is saved on the hard drive and on a zip or CD disk for later archiving.
For safety, gloves must be worn and radiation safety guidelines are strictly adhered to, such that technicians must be behind portable shields or 6 feet away from the PIXImus machine during scanning. A dosimetry badge and ring must also be worn. Since both the PIXImus unit and the computer present icons that indicate the "ON" status of the X-ray tube with open shutters, precautions and any possibility of exposure should be avoided.
Bone area measurement is generated by outlining or specifying the limits or dimensions of the entire skeletal bone regions of the body (limbs, neck, spine, and tail), excluding the head, as regions of interest (ROI) following a full body X-ray scan.
Bone mineral content (BMC) is generated from PIXImus density scans which are assessed for accuracy using a set of 0.0 mg to 2,000 mg of hydroxyapatite standards.
According to the DXA system, bone mineral content (measured as the attenuation of the X-ray by the bones being scanned) is divided by the area (also measured by the machine) of the site being scanned to obtain bone mineral density (BMD):
BMD = BMC ÷ bone area
Lean body (non-fat) tissue weight = (Total body tissue weight – body fat weight)
Body fat tissue weight = (Total body tissue weight - lean body tissue weight)
Total body tissue weight = (BMC + Body fat tissue weight + Lean body tissue weight)
Data collected by investigator
PIXImus data are obtained from the entire body and tail, excluding the head area: reported tissue variables included lean tissue weight, fat tissue weight, total body composition, and percent fat, bone mineral density (BMD), bone mineral content (BMC), and bone area.
Beamer WG, Donahue LR, Rosen CJ, Baylink DJ. Genetic variability in adult bone density among inbred strains of mice. Bone. 1996 May;18(5):397-403.
Johnston SL, Peacock WL, Bell LM, Lonchampt M, Speakman JR. PIXImus DXA with different software needs individual calibration to accurately predict fat mass. Obes Res. 2005 Sep;13(9):1558-65.
Nagy TR, Clair AL. Precision and accuracy of dual-energy X-ray absorptiometry for determining in vivo body composition of mice. Obes Res. 2000 Aug;8(5):392-8.
Pietrobelli A, Formica C, Wang Z, Heymsfield SB. Dual-energy X-ray absorptiometry body composition model: review of physical concepts. Am J Physiol. 1996 Dec;271(6 Pt 1):E941-51.
Pietrobelli A, Wang Z, Formica C, Heymsfield SB. Dual-energy X-ray absorptiometry: fat estimation errors due to variation in soft tissue hydration. Am J Physiol. 1998 May;274(5 Pt 1):E808-16.