Analysis of Craniomaxillofacial Malformations in Mice Using Three-dimensional Microcomputed Tomography

Summary

Here, we describe in depth a method, which is based on microcomputed tomography, to segment and measure 3D models of craniomaxillofacial bones in mice, for better assessment of craniomaxillofacial bone development in mice than what is possible with current methods.

Abstract

To model craniofacial malformations caused by vitamin A deficiency (VAD), we expressed a dominant-negative retinoid receptor mutation in osteoblasts to specifically inhibit RAR transcriptional activity in mice. This approach allowed us to investigate the effects of VAD on cranial hypomineralization, mandibular deformity, and clavicular hypoplasia in clinical cases. In this study, microcomputed tomography (microCT) scanning of the craniomaxillofacial region of mice represented a valuable tool for studying the growth and development of this animal model. The manual estimation of images is both time-consuming and inaccurate. Hence, here, we present a straightforward, efficient, and accurate approach for segmenting and quantifying the microCT images of each craniomaxillofacial bone. MicroCT software was used to slice the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone of mice and measure their corresponding lengths and widths. This segmentation method can be applied to study growth and development in developmental biology, biomedicine, and other related sciences and allows researchers to analyze the effects of genetic mutations on individual craniofacial bones.

Introduction

The intricate development of the human skull and face encompasses a sophisticated 3D morphogenetic process, intricately orchestrated by numerous genes. These genes play a pivotal role in regulating the intricate patterns, proliferation, and differentiation of tissues derived from diverse embryonic sources. This highly coordinated process underscores the complexity of human craniofacial growth and development. Craniofacial malformations (including cleft lip and palate, cranial suture closure, and facial hypoplasia) occurring as a result of developmental abnormalities account for more than one-third of all congenital birth defects. As a commonly used model animal in biomedical research, the mouse has a complex and delicate craniomaxillofacial bone structure that is very similar to the human craniomaxillofacial bone in terms of anatomy and physiology. The study of craniomaxillofacial developmental biology has come a long way in recent years with the advent of new techniques in mouse genetics, especially in malformations¹.

Retinoic acid (RA) is the in vivo metabolite of vitamin A². Vitamin A deficiency (VAD) is associated with a range of serious multisystem disorders, such as poor bone remodeling, fractures, as well as craniofacial malformations and skeletal malformations characterized by dwarfism^3,4 . Retinoid receptors (RARs) are crucial transcription factors in retinoid signaling⁵. A dominant-negative RARα403 mutant (dnRARα) was designed⁶ and a mouse model established in which osteoblasts expressed dnRARα. This resulted in the mice exhibiting dwarfism, craniofacial deformities, incomplete cortical bone formation, and increased but poorly remodeled trabecular bone.

Microcomputed tomography (microCT) has great potential for the study of craniomaxillofacial malformations. It possesses the capability to detect and track the evolution of both innate and acquired skeletal abnormalities in rodent models. MicroCT imaging analysis offers an in-depth exploration of craniofacial growth disturbances in genetically modified mouse models^7,8 .Furthermore, 3D imaging emerges as a vital tool for delineating morphological traits, facilitating tailored analysis and visualization approaches⁹. Micro-CT has been used in several studies to analyze craniofacial phenotypes, including defining anatomical landmarks in humans and mice and volumetric analysis of each craniofacial bone^10,11,12. Here, we describe in detail a method based on microCT technology to separate and measure 3D models of mouse craniomaxillofacial bones to enable better evaluation and analysis of mouse craniomaxillofacial skeletal development than is possible with current methods.

Protocol

We have complied with all relevant ethical regulations for animal testing and research. All experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee of the Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University.

Both the Rosa26-loxp-stop-loxp-dnRARα403 strain (R26^dn/dn) and the Osterix-Cre (Osx^Cre) (No.006361) strain of mice used here were maintained on the C57BL/6 background. R26^dn/dn mice were crossed with Osx^Cre mice to generate OSX^Cre;R26^dn/dn mice. All mice were bred and maintained under specific pathogen-free (SPF) conditions.

1. Breeding of mice

NOTE: F means the number of generations of mice; N means the number of generations of mating between zygotic mice and background mice. Thus, F2+N represents the second generation as well as any subsequent mouse breeding programs. F0 means primary mice.

1. Pair a sexually mature male mouse with a pair of female mice of a comparable age. Commence daily inspections for newborn pups after an initial period of 18 days. Upon identifying pregnant females, isolate them if necessary to ensure optimal maternal care. In the event that no pregnancies are observed within a month of the initial pairing, consider alternating the male mouse among different breeding setups to promote fertilization.
2. Cross R26^dn/dnmice withOSX^Cre mice (F0). Clip the tails for genotyping and keep the male OSX^Cre;R26^dn/+mice in cages until sexually mature, which is ~6 weeks of age (F1).
3. Cross six-week-old male OSX^Cre;R26^dn/+mice with female R26^dn/dnmice (F2). Clip the tails for genotyping and replace the old breeding mice with younger mice in time (F2+N).

2. Preparation

1. Prepare four pairs of 4-week-old Osx^Cre, OSX^Cre;R26^dn/+, and OSX^Cre;R26^dn/dn mice. Individually euthanize the mice through the administration of carbon dioxide asphyxiation.
   NOTE: To ensure efficient euthanasia, the CO₂ flow rate should be adjusted to displace approximately 30% of the cage's volume every minute. For instance, in a cage measuring 45 cm x 30 cm x 30 cm, a flow rate of 40 L/min is recommended.
2. Cut the neck of the mouse with scissors while holding its body, leaving the skull intact.
   NOTE: To obtain a complete skull, open the mouth of the mouse, cut along the corner of the mouth with ophthalmic scissors, pull both hands to both sides, and rotate to peel the skin of the mouse's head.
3. Immerse the mouse skulls in 4% paraformaldehyde for 48 h, then store them in 70% ethanol.
   CAUTION: Paraformaldehyde is toxic; wear appropriate protection.
4. Scan the collected skulls with a microCT scanner- resolution: 4.5 µm; voltage: 70 kV; current: 114 µA; filter: 0.5 mm Al; rotation step: 0.5°.
5. Store CT scan images. Images in DICOM format are selected for import into the software and analyzed.

3. MicroCT imaging and 3D reconstruction

NOTE: All bones utilized in this study were manually segmented.

1. Click the right mouse button on the MASKs page and select New mask; a new layer named Green appears.
2. Select the threshold range of 671-2,566 in the Thresholding page that pops up. Repeat this process for all related layers. (Figure 1A).
3. Use the Eraser tool in the Edit menu to manually erase the bones attached to the mandible in each layer on the 2D sagittal plane (Figure 1B).
4. Select the region of the mandible on the 2D sagittal plane after clicking on the Region Growing option (Figure 1C).
   CAUTION: Ensure that the mandibular region is selected completely in each layer; otherwise, it will result in an incomplete mandible after reconstruction.
5. Click on the newly created layer Mandible in the MASKS page and select Calculate 3D from the dropdown menu (Figure 1D).
6. In the 3D objects interface, right-click Mandible and click Properties. Click to change Color. Color code all the 3D models reconstructed with different bones.
7. Click on Measure in the upper left corner and select distance from the dropdown menu that appears. Calculate the length by marking points in the 3D image (Figure 1E).
   CAUTION: The reference for measuring bone width is the widest part of the bone.
8. In the 3D-objects interface, right-click Mandible and click Properties. Properties-Info-Volume module to read the reconstructed volume values for each craniomaxillofacial bone block. (Figure 1F).
9. Measure the mandible, frontal bone, parietal bone, nasal bone, premaxilla, maxilla, interparietal bone, and occipital bone in the same way as above and mark them individually with different colors.

4. Statistical analysis

1. Perform statistical analysis using the software of choice. Perform two-tailed Student's t-tests (n = 4/group).
2. For all graphs, use error bars to represent standard deviations. Consider P-values < 0.05 statistically significant.

Results

Extensive research underscores the multifaceted impact of genetic mutations on mouse growth, development, and organ systems. A comprehensive evaluation of craniofacial bones in mutant mice necessitates methods beyond single-tissue or 2D image analysis due to their limitations. Therefore, elucidating craniofacial bone development holds paramount importance for investigating human craniofacial disorders.

This method provides a use...

Discussion

MicroCT is a powerful tool for obtaining realistic and isotropic 3D information from dense and opaque biological samples with micrometer resolution. The data obtained from microCT are calibrated for geometry and intensity, making it especially useful for quantitative studies^{13,14,15,16,17}. It is used to study bone and dental microstructure^18<...

Materials

Name	Company	Catalog Number	Comments
GraphPad Prism 6.01 Software	GraphPad Software Inc., La Jolla, CA, USA	/
Micro-CT	Quantum GX micro CT, PerkinElmer, Waltham, MA, USA	/
Mimics Medical 19.0	Materialise, Leuven, Belgium	/
Osterix-Cre (OsxCre)	/	/	from the Jackson Laboratory
Rosa26-loxp-stop-loxp-dnRARα403 strain	/	/	from the Columbia University, USA