X-Ray Fluorescence Imaging: A New Tool for Studying Manganese Neurotoxicity

<div><p>The neurotoxic effect of manganese (Mn) establishes itself in a condition known as <em>manganism</em> or Mn induced parkinsonism. While this condition was first diagnosed about 170 years ago, the mechanism of the neurotoxic action of Mn remains unknown. Moreover, the possibility that Mn exposure combined with other genetic and environmental factors can contribute to the development of Parkinson's disease has been discussed in the literature and several epidemiological studies have demonstrated a correlation between Mn exposure and an elevated risk of Parkinson's disease. Here, we introduce X-ray fluorescence imaging as a new quantitative tool for analysis of the Mn distribution in the brain with high spatial resolution. The animal model employed mimics deficits observed in affected human subjects. The obtained maps of Mn distribution in the brain demonstrate the highest Mn content in the globus pallidus, the thalamus, and the substantia nigra pars compacta. To test the hypothesis that Mn transport into/distribution within brain cells mimics that of other biologically relevant metal ions, such as iron, copper, or zinc, their distributions were compared. It was demonstrated that the Mn distribution does not follow the distributions of any of these metals in the brain. The majority of Mn in the brain was shown to occur in the mobile state, confirming the relevance of the chelation therapy currently used to treat Mn intoxication. In cells with accumulated Mn, it can cause neurotoxic action by affecting the mitochondrial respiratory chain. This can result in increased susceptibility of the neurons of the globus pallidus, thalamus, and substantia nigra pars compacta to various environmental or genetic insults. The obtained data is the first demonstration of Mn accumulation in the substantia nigra pars compacta, and thus, can represent a link between Mn exposure and its potential effects for development of Parkinson's disease.</p> </div

XRF imaging of the substantia nigra of control and treated samples.

<p>XRF images of Mn (<b>A</b>) and Fe (<b>B</b>) for treated and control samples. Note that the maximum Mn intensity for the control sample is 30% of the treated maximum intensity. Numbers given are in µg/g. (<b>C</b>) Tri-colored image of the SN where red, green, and blue represent Mn, Fe, and Cu respectively (same scale for Mn). Scale bar represents a length of 1 mm. (<b>D</b>) Confocal images of tyrosine hydroxylase stained SN area of adjunct sections recorded in identical experimental conditions.</p

Average concentrations for particular brain areas (mean ± SEM).

*<p>Denotes a significant difference from control (p<0.05).</p><p>AB, axon bundle; CPu, caudate putamen; GP, globus pallidus; SN, substantia nigra; Th, thalamus±.</p

Scatter plots and correlations for control and treated groups.

<p>Data points calculated by taking the average of the brain areas of interest for control and treated samples. We performed cluster analysis (2–5 clusters) on the data obtained from treated samples to objectively identify similar data points in terms of metal concentrations. Clustering was done using Cu/Mn, Fe/Mn, or Zn/Mn at equal weights. We found that there was no clear grouping according to Fe/Mn while according to Cu/Mn or Zn/Mn two clusters were appropriate (Group1 and Group2). For Zn/Mn, cluster analysis resulted in two exclusive groups in terms of brain structures. Color matched Pearson's correlation coefficients and <i>p-values</i> are also given. Linear regression parameters and statistical analysis results are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048899#pone.0048899.s011" target="_blank">Table S4</a>.</p

XRF imaging of Mn distribution in brain sections of control and treated rats.

<p>Mn distribution of coronal sections from untreated (control) rats (<b>A</b>) and Mn treated rats (<b>B</b>); from left to right; Bregma −0.48 mm, −2.64 mm, and −5.20 mm. Diagrams of these Bregma sections can be seen to the left where red dashed boxes roughly indicate the area(s) scanned. (<b>A</b>) and (<b>B</b>) demonstrate the increase of Mn concentration in treated samples. (<b>C</b>) The same image as (<b>A</b>) but with the scale adjusted in order to see contrast in the Mn distribution. All values given are in µg/g. Scale bar indicates a length of 2 mm. (<b>D</b>) Typical average pixel spectra for a control (green) and a treated sample (blue). Respective K_α peaks are labeled. The inset displays the Mn Kα peak. (<b>E</b>) T-test results show a statistically significant increase in Mn for treated samples as compared to control. Bars that cross a T-value of 0 indicate that the difference is not statistically significant. (<b>F</b>) ANOVA results of mean values of Mn concentration in treated samples for all areas studied. Overlapping bars indicate that the values are not significantly different. AB, axonal bundle; CPu, caudate putamen; GP, globus pallidus; HPC, hippocampal formation; IC, internal capsule; lv, lateral ventricle; Th, thalamus; SNc, substantia nigra compacta; SNr, substantia nigra reticular.</p

XRF imaging of the caudate putamen, globus pallidus, thalamus, and internal capsule.

<p>(<b>A–C</b>) Mn, Fe, and Zn XRF images in control and treated rats. Maximum intensity for the control Mn images is 30% of the maximum for the treated images. All numbers are given in µg/g. (<b>D</b>) Image displaying the results of cluster analysis. (<b>E & F</b>) Tri-colored plots displaying Mn, Fe, & Cu/Zn as green, red, and blue respectively. Scale bar represents a length of 2 mm.</p

Mn and Fe distributions in the choroid plexus.

<p>XRF images of the Fe (<b>A</b>) and Mn (<b>B</b>) distributions in the choroid plexus (CP) within the lateral ventricle (lv) as identified by increased Fe signal. Images are Bregma −0.48 mm coronal sections of untreated rats (top) and Mn treated (bottom). Note that the images are not displayed on the same intensity scale, in order to see Mn contrast in the control image. Yellow dashed lines indicate the boundary between the ventricle and the labeled structures (CPu and HPC, hippocampal formation). The Fe signal shows the presence of CP (containing blood) within the ventricle. Mn concentration in the ventricle is lower than in adjacent brain structures of the CPu and HPC indicating clearance of Mn from the CP. All values given are in µg/g. Scale bar represents a length of 2 mm.</p