Churchill1 project protocol

Bone characteristics and body composition of 63 F1-hybrid mouse strains from an 8-way diallel cross   (2004)

Churchill GA, Baldwin C




Churchill1 Protocol

Project protocol — Contents
 Workflow and sampling 
Equipment
Procedures
Data
 References

Workflow and sampling

Workflow

Step
Accomplished procedure
Data recorded
1
 Creation of an 8-way F1 cross -
2
 Morphometric measurement BW, ear pinna length
3
 Densitometer measurement Bone mineral density, different tissues densities
4
 Morphometric measurement Isolated muscle weight

Equipment

  • Ohaus Portable Navigator Series Electronic Toploading Balance (Model NV-210)
  • The 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 specimen.
  • 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 single whole mice and 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% for whole body, approximately 1.5% 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.
  • Dissecting microscope or illuminated magnifying glass
  • Heavy-duty sharp decapitating scissors, micro-dissecting iris scissors for isolating muscle tissues, and precision scale ruler for morphometric measurement

Procedure

Creation of an 8-way F1 cross

I. Measuring ear pinna length
a. First the mice are weighed using a top loading balance.
b. Then they are euthanized by exanguination.
c. Ear pinna length is obtained by measuring from the tip to the base of the pinna.

II. Collecting body composition 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 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. The X-ray process to obtain a single full scan is approximately 5 minutes; data can be manipulated subsequently to obtain specific regions of interest.
d. After the whole body (excluding the head) is scanned, the area of the head is also scanned.
e. 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.

III. Measuring muscle weight
a. The skin and subcutaneous tissue around the hind limbs is removed in order to isolate the quadriceps muscles.
b. Using a small (iris) dissecting scissors, the muscles are removed from their origin in the pelvic bone and their insertion on the knee joint and femoral bone.
c. Any fat depot adjoining the muscles is removed and excluded in the overall weight.

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.

Data collected by investigator

PIXImus data are from the body and tail; head excluded. BMD, BMC, total tissue mass, and % fat were measured. BMD and BMC adjusted for body mass and weights of lean and fat portions are derived and are available in the data set for analysis.

Notes pertaining to the data set:PIXImus scans are analyzed using the PIXImus2 software (version 1.46.007), excluding the head from the analysis. Values for whole body (without the head), both femurs, and spine were obtained. Left femur data are not available for MPD analysis but are provided in the project data set.



References

    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. PubMed 8739896

    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

    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. PubMed 9612238