Introduction

The cynomolgus monkey (macaca fascicularis) is one of the most commonly used experimental animals, including its usage as an animal model for Parkinson’s disease.¹ However, due to its small-sized brain,² observing the detailed structures can be a challenging task. Using a 7T MRI is a good solution to reach high resolution leveraging increased MR signal power, but, arising problems regarding system imperfection (e.g. imperfect B₀ shimming) are more severe than 3T, degrading the image quality. In this study, we proposed a sample preparation method for acquiring sub-millimeter resolution MR images of the ex-vivo cynomolgus monkey brain. With the proposed method, the whole-brain T₂*-weighted image and QSM image were acquired with 150 um isotropic resolution, and the structural details were explored and compared to the histology images from references.³

Methods

[Ex-vivo sample preparation]
An ex-vivo brain of the cynomolgus monkey with a history of Parkinson’s disease was prepared for this study. For the preparation of the brain sample, three steps were performed. They were 1) designing the unique shape of the container for better B0 shimming, 2) reducing bubbles for artifact reduction, and 3) agarose embedding for reducing motions.
Firstly, a container was designed to improve B0 shimming performance. Figure 1 shows the 3D image and demonstration images of the designed container. The container consists of the sample container and the shim module. For experiments, the brain sample is placed in the sample container, and the sample container is placed in the shim module. The container was designed to fit the size of the 16-channel human wrist coil, which is used for the following experiments.
Next, bubbles were removed in the sample container. Because the material around the bubble and the air inside the bubble have different susceptibility values, it may induce susceptibility artifacts. We tried to remove the small bubbles existing in samples using a vacuum chamber.
Thirdly, the brain sample was embedded using an agarose gel. Because vibrations can be generated by the machine during an MRI scan, the brain sample may be prone to unexpected motions. Therefore, to fix the position of the brain sample in the sample container, agarose gel was used for embedding the brain sample.

[Data acquisition & image processing]
With the proposed sample preparation method, the ex-vivo sample was scanned at 7T Philips MRI with the 16-channel human wrist coil. The 3D multi-echo gradient-recalled echo (GRE) scan was performed with the following scan parameters: voxel size = 150 um isotropic, TE₁/ΔTE/TR = 5.5/4.7/100 ms, # of echoes = 4, flip angle = 24°, and total scan time = 4.2 hours. From the complex images of DICOM data, the magnitude image was generated by the square root of the sum of squares over echoes, and the QSM map was generated by a series of post-processing steps including Laplacian phase unwrapping, HARPERELLA background field removal, and iLSQR. The magnitude image and QSM image were compared with histology image to explore that the MR images can provide the fine structure in the brain sample.

Results

Figure 2 shows the QSM, T₂*-weighted, and histology images in the axial view of a representative slice. Comparing the T₂*-weighted image and the histology image, the unique structure of the internal capsule and laminar structure of the cortex was consistently observed in both images. However, they were not observed in QSM.
Figure 3 shows the QSM, T₂*-weighted, and histology images in the coronal view of a representative slice. The unique structure between caudate and putamen was consistently observed in QSM, T₂*-weighted, and histology images. Also, the laminar structure of the cortex was consistently observed both in T₂*-weighted and histology images.

Discussion and Conclusion

In summary, we proposed a sample preparation method for acquiring sub-millimeter resolution images of the ex-vivo cynomolgus monkey brain. By acquiring high resolution images, the detailed structure in the brain can be observed even for the small-sized brain. When comparing MR images with the reference histology images, MR images contain abundant information on histological structures.

Acknowledgements

This work has been supported by Heuron Co. Ltd. and by the Korea government (MSIT) (No.NRF-2021R1A2B5B03002783)