Donahue16 project protocol

Bone mineral density and body composition in C57BL/6J-Chr#A/J/NaJ mouse chromosome substitution strains   (2012)

Donahue L
With: Morgan J

See also: Donahue16 animal documentation


Donahue16_Protocol

Project protocol — Contents
Workflow and sampling
Equipment and supplies
Procedures
Definitions and calculations
Data
References

Workflow and sampling

Workflow

Step
Accomplished procedure
Equipment
Data recorded
1
Body weight measured
scale
bw
2
Mice anesthetized
-
-
3
Mice scanned for body composition using densitometer dual energy X-ray absorptiometry (DXA)
DXA
whole body BMD, mass, % fat, spine & skull BMD

Equipment and supplies

  • Ohaus Portable Navigator Series Electronic Toploading Balance (Model NV-210)
  • Heavy-duty sharp decapitating scissors
  • DXA scanning by PIXImus: The PIXImus small animal DXA system (PIXImus™, Fitchburg, WI) is used to assess whole body areal (a) BMD and body composition at 16 weeks of age. This methodology has been validated in small animals (see Nagy 2000). 
  • Paper towels

    Mouse densitometer dual energy X-ray absorptiometry (PIXImus small animal) DXA system (GE-Lunar, Madison, WI): 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% coefficient of variation (CV) for whole body, approximately 1.5% CV for specialized regions. 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. Full body scans and X-ray absorptiometry data are processed with manufacturer supplied software (Lunar PIXImus 2,vers. 2.1). For additional information: Lunar PIXImus


    Figure 1 A: Lunar PIXImus2 densitometer with integrated PC computer. Figure1 B: Close-up of the Lunar PIXImus2 densitometer with specimen tray.

Reagents and solutions

cleaning and disinfecting solutions

Procedure

I. Collecting image scans
PIXImus scanning of mice for BMC and % fat is both accurate and precise although body size must be considered when comparing inbred strains. Full body scans are obtained and X-ray absorptiometry data gathered and processed with manufacturer supplied software (version 2.1).
a.
The PIXImus densitometer apparatus is first calibrated with a "phantom mouse" according to manufacturer's protocol.
b. Mice are weighed and anesthetized.
c. Then each mouse is placed on the specimen sticky tray (body must be within blue line on the tray) under the PIXImus beam path (see Fig. 1B above). Mice are positioned dorso-ventral with the tail positioned away or alongside from the body, the front legs extended to the side, and the neck and spine are gently straightened.



d.
Trays are positioned such that the area of the head is always oriented toward the left from the investigator's point of view, and the mice are positioned dorso-ventrally in order to scan the entire body and tail. The X-ray process to obtain a single full scan is approximately 5 min; data can be manipulated subsequently to obtain specific regions of interest (ROI's).
e.
The body is scanned (excluding head); then the head is scanned.
f. Disposable plastic trays, with sticky tape for immobilizing mice, can be saved and re-used after a thorough cleaning and disinfecting.

II. Measurement acquisition and image scan analysis
Based on PIXImus validation studies (see Nagy 2000), 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.
a. 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).


Figure 2.
Screen output of image scan analysis for whole body (head-excluded).

b. By using the screen interactive display, "Manual Analysis" (F3) is first clicked to prompt measurement adjustments, and then clicked again for the second time to adjust region of interest (ROI). An outline of the mouse sample is drawn in red rectangular shape to define specific ROI.
c. Then total area to be analyzed is defined within the outlined red box, and areas to be excluded from the calculations are defined within the circled green area. 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.
d. Once the desired ROI is achieved, the Enter key is clicked and resulting data measurement is displayed (Fig. 2). A hard copy of the image and the scan analysis result is printed by pressing F5. Analysis session is ended (F8 or Esc) to return to the main menu screen and ready for the next mice to be tested.
e. Acquired data is saved on a hard drive and on a zip or CD disk for later archiving.

Safety

For safety, gloves must be worn and radiation safety guidelines are strictly adhered to, such that technicians must be 6 feet away from the PIXImus machine during scanning.

Definitions and calculations

ROI = Region of interest

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.


Figure 3.
Screen output of image scan analysis for right femur (outlined in red).

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):

BMC = Bone mineral content [g]

BMD = Bone mineral density = BMC ÷ Area [g/cm2]

Fat tissue mass = all tissues with low density (x-ray scan) [g]

% Fat = (Fat tissue mass ÷ Total body tissue mass ) x 100

Data collected by investigator

  • whole body bone mineral density (BMD)
  • calculated total tissue mass
  • percent of body mass that is fat
  • spine (L2-L5) bone mineral density
  • skull bone mineral density


References

    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. PubMed 10968731

    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. PubMed 8997211