Everett1 project protocol

Craniofacial morphology of 14 inbred strains of mice   (2003)

Everett ET
With: Faust D, Hartsfield JK, Ward RE

Mandible measurements are available. Data collection is ongoing; more data are expected.

Sampling procedure

Ten males and ten females of each strain (except SPRET/EiJ where only females were tested) at 48 - 64 days of age (57 ± 4 days) were accessioned and assigned a unique randomized identification number. There was no period of acclimatization. Upon receipt or within 24 h each mouse was euthanized using CO2 gas and weighed (± 0.1 g) and that data recorded along with date of birth and age (days) at the time of euthanasia. Measurements were performed on samples in ascending numeric order and each measurement was taken at least twice and a third time if the differences between measurements were >10%.

Skull preparation

Following euthanasia, the heads were removed, cleaned of skin, fur, loose musculature, and the tongue was removed. Radiographs were taken prior to bleaching which required soaking the head in 1-2% sodium hypochlorite (5.25% sodium hypochorite diluted in 0.9% NaCl) for 14-16 h at room temperature. The cleaned skulls were thoroughly rinsed in fresh 0.9% NaCl and allowed to air dry (2-3 d) prior to varnishing with clear polyurethane spray. The foramen magnum of each bleached, varnished skull was filled with Protech brand glass ionomer band cement (Ormco Corp., Glendora, CA). A lingual button (G&H Wire Co., Greenwood, IN) was then glued to the most posterior aspect of the occiput and used to mount the skull to a vise. Mounting the skulls to a moveable vise permits optimum and consistent orientation of each skull for measurements. The processing of the skulls results in disarticulation of both hemi-mandibles. The hemi-mandibles were varnished as well with no other modifications.

Mandible Analysis

Fifteen variables for each hemi-mandible were analyzed (see Table 1 ). These variables were chosen a priori from many possibilities in order to represent a reasonable mix of non-redundant measurements from all parts of the hemi-mandible (see Figure 1 ). Most variables are Euclidean distances between recognizable landmarks and others are defined by the area between specified landmarks. Each hemi-mandible was placed buccal side up along with a scale bar (stainless steel rectangular arch wire cut to length and measured to ± 0.01 mm using a micrometer). Hemi-mandibles were imaged using a Leica GZ6 stereomicroscope equipped with a Nikon DX1200 digital camera and Dell computer workstation. The digital images were acquired at 3600 by 2880 pixels and saved as high-resolution jpeg files (72 pixels per inch at 50 x 40 inches image size, or 300 pixels per inch at 12 x 9.5 inches image size). Images generated from hemi-mandible samples were subjected to digitizing using Didge software, (Image Digitizing Software, version 2.20) a digitizing program written and designed by Alistair Cullum (Department of Biology, Creighton University, Omaha, NE) to help quickly mark and record coordinates on a series of images. (This program was developed originally for the study of animal locomotion.) Selected points, as pixels, were identified as landmarks and the (X,Y) coordinates were then transferred to a Microsoft Excel spreadsheet. Landmarks were digitized at least two times. Basic geometry formulae were applied to determine distances and areas. Each suite of variables was determined in duplicate and the mean for each hemi-mandible submitted (data for hemi-mandibles are available in the supplemental data set). Measurement averages for right and left hemi-mandibles were computed for each mouse and are used in the project data set.

Table 1. Mandible Variables
short namedescriptionunits definition using landmarks
(see Figure 1)
ant_lengthmandible, infradentale to mentonmmlength 4 - 6
post_lengthmandible, menton to gonionmmlength 1 - 4
mid_lengthmandible, infradentale to posterior ramusmmlength 6 - 20
mid_heightmandible, posterior molar to mentonmmlength 4 - 11
condylemandible, condylar processmmlength 15 - 18
incisor_condylemandible, infradentale to posterior condylemmlength 6 - 18
incisor_gonionmandible, infradentale to gonionmmlength 1 - 6
molar_condylemandible, posterior molar to posterior condylemmlength 11 - 18
molar_archmandible, anterior molar to posterior molarmmlength 10 - 11
incisor_regionmandible, diastema to pogonionmmlength 5 - 9
ramus_heightmandible, antegonion to sigmoidmmlength 3 - 14
ant_areamandible, anterior areamm2area 3, 4, 5, 6, 7, 8, 9, 10, 11
post_areamandible, posterior areamm2area 1, 2, 3, 11, 14, 15, 16, 17, 18, 19, 20
teeth_spacemandible, concavity of incisor to anterior molarmm2area 8, 9, 10
ramus_spacemandible, concavity of posterior ramusmm2area 1, 19, 20

Figure 1. Comparison of mouse and human mandibles.
Upper panel: buccal view of mouse left hemi-mandible with landmarks 1-20 indicated
Lower panel: human cephalometric tracing with selected corresponding landmarks
mouse mandible

mouse mandible
1 = goniona
2 = inferior gonion
3 = antegonion
4 = mentonb
5 = pogonion
6 = infradentalec
7 = incisor alveolus, dorso-posterior point
8 = incisor process
9 = diastema, posterior point
10 = first molar alveolus, dorso-anterior point
11 = molar, posterior
12 = coronoid process, dorsal point
13 = sigmoid, posterior point
14 = sigmoid, ventral point
15 = condylar process, dorsal point
16 = (landmark not named)
17 = condylion
18 = condylar process, ventral point
19 = (landmark not named)
20 = posterior ramus

a mouse, posterior point of angular
process; human, midpoint of coutour
connecting ramus and body of mandible

b mouse, posterior tuberosity of
insertion site of mandibular transverse

c human, termed 'B point'

Craniometric Analysis (data not yet available)

Thirteen paired variables and one midline variable for each skull were analyzed. These variables have been shown to discriminate face shape in a study involving genetic determinants of oral clefting in mice (Everett et al., unpublished). All variables are Euclidean distances between recognizable anatomic landmarks. All measurements were performed using a Leica GZ6 stereomicroscope equipped with a calibrated reticle in the micrometer scale. The suite of variables were measured in triplicate.

Radiography (data not yet available)

Radiographs (either dorsal to ventral or cephalogram with left side of head facing up) were performed using an in- house designed cephalostat to maintain consistent orientation to the film. Heads prepared for bleaching were placed onto an individually wrapped size 2 Kodak ekta-speed plus periapical dental film. Each head was subjected to a voltage output of 18 kV (endpoint energy) with an exposure time set for 24 s using a Hewlett-Packard Faxitron B Model 43855-A02 (Hewlett-Packard-McMinnville Division, McMinnville, OR) with a film to source distance of 61 cm, X- ray tube current 3 mA, with continuously variable voltage output 10-110 kV (endpoint energy). (We have determined that the image on the periapical dental film corresponds to nearly 1:1 with the actual skull.) Radiopaque size calibrators were included with each X-ray. Measurements of Euclidian distances between landmarks and angles were performed on digitized images. Many of the landmarks are homologous to those used for standard orthodontic cephalometry in humans.