Basal ganglia and thalamus pathways are affected in various neurological disorders. Over recent years, non-invasive MRI techniques such as diffusion tensor imaging (DTI) were used to visualize and characterize the white matter architecture and integrity of the normal and diseased brain. In Parkinson’s disease (PD), progressive loss of dopaminergic neurons of the substantia nigra and degeneration of the nigrostriatal pathway induces motor and non-motor impairments characteristic of the disease. We hypothesized that DTI could help to detect dopaminergic lesion of the nigrostriatal pathway to assess the severity and progression of the disease. However, precise delineation of the origins, trajectories and terminations of this pathway is difficult due to poor resolution and sensitivity of these techniques.

Here we present an imaging protocol designed to visualize the thin fibers of the nigrostriatal pathway in a small non-human primate (squirrel monkey). We also focused on the pallidothalamic pathway because of its curved trajectory that is difficult to reconstruct in 3D.

After animal death, the brain was removed, hemisected, postfixed in 4% PFA and cryopreserved in sucrose during 3 days before freezing. Before MRI procedure, the left hemisphere was unfrozen and soaked in phosphate buffer saline containing sodium azide. A 1:200 gadolinium MR contrast agent (Dotarem®, Guerbet, France) was added to the buffer 48 hours before imaging.

An 11.7T Biospec 117/16 system (Bruker, Germany) equipped with a 72-mm diameter transceiver was used within this study.

T1 and T2 were evaluated in gray and white matter (GM and WM respectively) at 3 time points: before Gd- staining, 48 hours after Gd immersion and at the end of the experiment.

Ex vivo diffusion imaging was performed with a 3D diffusion-weighted spin echo sequence. Parameters were: FOV: 5.76*2.96*2.96 cm3; Mtx: 288*148*148 leading to an isotropic resolution of 200 µm; TR/TE=500/21.7 ms; δ=4 ms and Δ=10 ms with a b-value of 2000 mm/s²; one A₀ image (without diffusion gradient) was taken as a reference and 46 directions were acquired. Total scan time for diffusion imaging was 142 hours.

The diffusion-weighted data were then processed with Mrtrix 3 software which estimates the ODF on every voxel. We then used a determinist tracking based on those ODF with the ‘tckgen’ function. Regions of Interest (ROI) were defined manually in the substantia nigra (SN), thalamus (Tha) and pallidum (GP). We seeded from SN to GP and GP to Tha and included a stop criteria when both ROI where reached.

Evaluation of T1 and T2 in GW and WM are summarized in Table 1. As expected, there is a huge decrease of both WM and GM T1 after Gd-staining. Interestingly, GM T1 became shorter than WM T1 after Gd-staining, while WM T2 remained shorter than GM T2. Figure 1 shows the obtained nigrostriatal tracts after processing of the DTI data set and ROI definition. Figure 2 shows the obtained pallidothalimic tracts within the same conditions.

We showed that it is feasible to follow the nigrostriatal and pallidothalamic tracts in the squirrel monkey. These tracts could serve as biomarkers for PD monkey models intoxicated with MPTP, or to monitor a response toward a therapy as these tracts are affected by PD. Moreover, Gd-staining helps to reduce the GM and WM T1, thus allowing to shorter TR without losing too much signal.

Here we used conventional spin echo technique to perform diffusion imaging as we found that EPI-based diffusion technique was not suitable for our study (data not shown), even if scan was shorter (several hours only to acquire the same data sets). These images were too much distorted and the signal to noise ratio was way too low. Spin echo technique gives the advantage of undistorted images as eddy current are negligible in this kspace aquisition technique, whereas it has to be corrected for EPI.

Meanwhile, the main limit of this approach is the time needed to perform diffusion scan (~140 hours for a small monkey half-brain with 46 directions). In the future, we intend to use diffusion-prepared SSFP or diffusion-weighted SSFP as it has been shown to produce diffusion images whose quality is comparable to those obtained with spin-echo diffusion techniques1 within several hours only. Furthermore, to perform in vivo tractography, we intend to use segmented-EPI in order to reduce TE and distortions related to eddy currents within the gradients.