Leveraging High Field MRI to Characterize and Quantify 3D Mouse and Rat Knee Anatomy

Introduction: Across species, the knee joint retains a complex anatomy composed of bones, cartilage, ligaments, and menisci [1]. Both qualitative and quantitative methods of characterizing the knee joint are critical to develop a robust understanding of this anatomy. Previous work has characterized rat and mouse knee anatomy using histology and micro-CT to quantify soft tissue anatomy and bone morphology, respectively [2,3]. While histology has been used to characterize tissue composition, it is a destructive technique that does not allow for further testing to be performed on the same joint. Likewise, while micro-CT allows for non-destructive morphometric analysis of bone structure it remains a poor method for visualizing and quantifying the 3D soft tissue anatomy in the knee.
High field MRI is a valuable tool that can provide non-invasive, non-destructive 3D imaging at appropriate resolutions to quantify the morphometry of rat and mouse knee anatomy across tissue types. Rats and mice are common small animal preclinical models used in orthopaedic research, so it is valuable to understand the 3D morphometry of their knees in a healthy state [4]. In this study, high field MRI scans were used to develop high fidelity 3D models of rat and mouse knee joints to quantify their anatomy. The goal of this work was to expand current knowledge of rat and mouse knee morphometry, with specific focus on ligaments and menisci. This will support future work exploring how morphology changes due to growth, injury, and disease.

Materials and

Methods: Tibiofemoral (knee) joints were dissected post-mortem from Long Evans rats and B12 mice and stored at -20°C. The knees were thawed to room temperature and imaged in full extension in a 9.4T Bruker Biospec 94/20 AV Neo MRI machine with a T1 FLASH 3D scan sequence (isotropic voxels of 0.1mm and 0.05mm size for rats and mice, respectively). Images were imported in commercial software for segmentation and 3D models of the four primary ligaments (ACL, PCL, MCL, LCL) and the medial and lateral meniscus were generated [5]. Reconstructions were smoothed with a Gaussian filter, exported as .stl files, and imported into MATLAB.
Using a custom MATLAB code, each tissue was generated as a point cloud and sliced in 0.1mm and 0.05mm increments for the rats and mice, respectively. For the ligaments, the cross-sectional area (CSA) of each slice was calculated and the middle 50% of slices were averaged to determine the CSA of each tissue [5]. The menisci were sliced coronally and sagittally at the same increments. The maximum height and width were recorded at three anatomical locations along the menisci (anterior and posterior horn and a central location) [6].
Statistical analysis was performed in R Studio with custom code and included one-way ANOVAs to compare average CSAs of the ligaments. Two-way mixed ANOVAs were used to compare the height and width of menisci within anatomical locations and between the medial and lateral meniscus. Post-hoc follow-up mean comparisons were performed with non-parametric t-tests. Significance was set at alpha = 0.05.

Results, Conclusions, and Discussions: In rats and mice there was a significant main effect of tissue type on the ligament CSA, respectively (p < 0.001 for both) (Fig 1). Ligaments were ~7 times smaller in mice than in rats (Table 1). In rats, the PCL was significantly larger than the other ligaments (p < 0.05), with an average CSA of 0.35 ± 0.04 mm2. In mice, the LCL was significantly larger than the MCL (p < 0.001) and ACL (p < 0.001), with an average CSA of 0.054 ± 0.017mm2.
The mouse menisci were ~2 times smaller than the rat menisci in both height and width (Table 1). In both species there were significant differences in width between the medial and lateral menisci (rats: p< 0.001, mice: p = 0.016). Specifically, the mice had greater widths in the medial meniscus across locations and the rats had greater medial meniscus widths in the anterior and posterior horns. The posterior horn of the medial meniscus had the largest width at 1.68 ± 0.22 mm and 0.67 ± 0.09 mm for the rats and mice, respectively. There were significant differences at each location (p < 0.001) (Fig 2,3). The posterior horns widths on each meniscus for both species were larger than central and anterior locations. The heights were similar at all three locations for the rats and mice (Table 1).
In this study we quantified 3D morphometric properties of rat and mouse knees using high field MRI, imaging at sub-micron isotropic voxels to resolve soft tissue features at the appropriate length scale. This work leveraged techniques used in previous large animal studies and applied them to common preclinical small animal models – rats and mice. Limitations of this work include an all-female cohort of animals and experimental constraints that do not allow for in vivo imaging. Future work will include expanding understanding of small animal structure function relationships and will include whole joint mechanical testing. Identification and quantification of healthy knee morphometry in small animals will enable future work to evaluate and quantify how soft tissue properties change due to aging, injury, and disease.

Acknowledgements (Optional): This research was supported by the NIH (P20GM103446, P20GM139760).