High and chronic exposure to manganese (Mn) may lead to symptoms that resemble Parkinson’s disease (PD)^1,2. Mn accumulation in the brain due to exposure may be visualized noninvasively using R1 relaxation rate mapping in MRI³. In our previous studies, a widespread distribution of Mn revealed by MRI was found in basal ganglia, but also motor cortex, parietal cortex, visual cortex and cerebellum in both humans and non-human primates4. However, not much is known about the regional distribution of Mn uptake and release over time in a chronic exposure scenario. In this study, the longitudinal time course of brain Mn accumulation over long-term (66-81 wks) Mn administration, as well as the wash-out of Mn after cessation of Mn administration were evaluated by using whole-brain T1 mapping in non-human primates.

A total of 4 Mn-exposed and 3 control adult male cynomolgus monkeys underwent three MRI exams (1) before Mn exposure (baseline), (2) after 26-41 wks (TP1), and (3) after 66-81 wks (TP2) of injections of MnSO4 (vehicle for controls) 2x/week, resulting in a dose of 1.66-2.5 mg Mn/kg per injection. Two of the Mn-exposed monkeys received a fourth scan acquired 44 wks after the end of a 66-81 wks exposure. Acquisition of the R1 relaxation maps was performed on a 3 T Philips Achieva MRI scanner. High-resolution 3D T1-weighted images (voxel size: 0.5x0.5x0.75 mm3) were obtained using a fast gradient echo pulse sequence (TR/TE=25/3.85 ms, NEX=2) as anatomical reference. Inversion recovery fast spin echo images were acquired using seven different inversion times for quantifying Mn accumulation in the brain (TR = 4000 ms, TI=100, 300, 500, 700, 1000, 1500, 3000 ms, voxel size: 0.5x0.5x2.2 mm³). The R1 rate for each pixel was then calculated by the least-squares method in Matlab (The Mathworks, Natrick, MA) using the following equation for T1 (=1/R1):

$$S = S_{0}\times[1 - f\times \exp(-TI/T1) + \exp(-TR/T1)]$$

where S=signal intensity from images. S₀=Signal of proton density, TI= inversion time, T1=relaxation time, TR=repetition time. f=inversion factor.

Regions of interest (ROI) of diameter 2.5 mm² were placed in the globus pallidus, caudate, putamen, substantia nigra, parietal lobe, occipital lobe and frontal lobe.

For each monkey, different time points were compared to each other by using paired t-tests. For each time point, the exposed group was compared to the control group using a nonparametric Mann-Whitney U test due to the small sample size.As expected, a significant increase of R1, indicative of an increase in brain Mn, was found in all exposed monkeys at 26-41 wks (TP1) and 66-81 wks (TP2) of Mn exposure (p<0.005). In the globus pallidus (GP), R1 remained stable TP2 of exposure (Fig.1). However, in the substantia nigra two exposed monkeys displayed a significant decrease of R1 at TP2 (p<0.05) (Fig 2). In occipital lobe and parietal lobe, the highest R1 value was also found at TP1, decreasing gradually (but not significantly) at TP2 and returning to baseline values after cessation of exposure. In contrast, the optical nerve and visual cortex displayed a slight increase of R1 at TP2 compared to TP1. After cessation of chronic Mn exposure, the R1 rate of the GP decreased almost back to the baseline range, but not fully. The high-resolution T1-weighted images reveal even 44 wks after cessation of Mn exposure still hyperintense regions located in the GP, pituitary gland and the visual area along the temporal lobe (Fig.3).Our results demonstrate regionally different dynamics of Mn uptake and wash-out in a model of chronic Mn exposure. The continuously increasing R1 values in the visual cortex and globus pallidus during exposure suggest the possibility of continuous accumulation of Mn in these regions. The hyperintensities found in T1-weighted images after the cessation of Mn exposure also demonstrate the slowness of Mn elimination in these regions. Structures like the substantia nigra, one of the primary brain structures affected by pathology in PD, seem to have a different saturation and faster wash-out rate, shown by a maximum in R1 at TP1 of exposure and a subsequent decrease in R1 still during exposure. It also is noteworthy that the hyperintensities found after the cessation of Mn exposure are exactly those brain regions that first show hyperintensities in human studies on occupational Mn exposure, possibly reflecting particularly high Mn accumulation in these areas. The study design of chronic exposure provided a condition similar to human overexposure to Mn, such as in occupational exposure settings, drug abuse or liver disease. Understanding the regional differences in Mn uptake and release may help elucidating the relation of Mn exposure to particular neurological symptoms of Mn toxicity⁵.