ToxSci Advance Access originally published online on September 12, 2006
Toxicological Sciences 2006 94(2):351-358; doi:10.1093/toxsci/kfl106
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Evidence for Cortical Dysfunction and Widespread Manganese Accumulation in the Nonhuman Primate Brain following Chronic Manganese Exposure: A 1H-MRS and MRI Study
* Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland 21205
Department of Radiology, Johns Hopkins Hospital, Baltimore, Maryland 21205
Department of Neuroscience, Norwegian University of Science and Technology, Trondheim, Norway
Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
1 To whom correspondence should be addressed at Department of Environmental Health Sciences, The Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Room E6622, Baltimore, MD 21205. Fax: (410) 502-2470. E-mail: tguilart{at}jhsph.edu.
Received July 13, 2006; accepted September 9, 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Exposure to high levels of manganese (Mn) is known to produce a complex neurological syndrome with psychiatric disturbances, cognitive impairment, and parkinsonian features. However, the neurobiological basis of chronic low-level Mn exposure is not well defined. We now provide evidence that exposure to levels of Mn that results in blood Mn concentrations in the upper range of environmental and occupational exposures and in certain medical conditions produces widespread Mn accumulation in the nonhuman primate brain as visualized by T1-weighted magnetic resonance imaging. Analysis of regional brain Mn distribution using a "pallidal index equivalent" indicates that this approach is not sensitive to changing levels of brain Mn measured in postmortem tissue. Evaluation of longitudinal 1H-magnetic resonance spectroscopy data revealed a significant decrease (p = 0.028) in the N-acetylaspartate (NAA)/creatine (Cr) ratio in the parietal cortex and a near significant decrease (p = 0.055) in frontal white matter (WM) at the end of the Mn exposure period relative to baseline. Choline/Cr or myo-Inositol/Cr ratios did not change at any time during Mn exposure. This indicates that the changes in the NAA/Cr ratio in the parietal cortex are not due to changes in Cr but in NAA levels. In summary, these findings suggest that during chronic Mn exposure a significant amount of the metal accumulates not only in the basal ganglia but also in WM and in cortical structures where it is likely to produce toxic effects. This is supported by a significantly decreased, in the parietal cortex, NAA/Cr ratio suggestive of ongoing neuronal degeneration or dysfunction.
Key Words: manganese; MRI; MRS; nonhuman primate; brain; basal ganglia; N-acetylaspartate.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Manganese (Mn) is an essential nutrient that is known to produce neurological dysfunction when humans are exposed to high levels. The neurological consequences of occupational exposures to high levels of manganese were first identified in miners exposed to Mn ore (Barbeau, 1984
; Cotzias, 1958; Couper, 1837). Toxic levels of Mn produce a neurological syndrome characterized by psychiatric disturbances, cognitive impairments, and extrapyramidal symptoms with overlapping features to Parkinson's disease (PD) (Olanow, 2004; Pal et al., 1999). Recently, there has been a renewed interest in understanding the neurological consequences of chronic exposure to low levels of Mn. This is based on the concern by regulatory agencies and the scientific community on the potential for adverse neurological health effects to the general population from chronic exposure to increases in ambient levels of Mn (Davis, 1999; Kaiser, 2003). An anthropogenic source that has the potential to markedly increase Mn exposure to the general population is from the automobile combustion of gasoline containing methylcyclopentadienyl manganese tricarbonyl (Davis, 1999; Kaiser, 2003). Also, there are reports that workers in occupations in which chronic exposure to Mn occurs, may have a higher prevalence of developing parkinsonism at an earlier age (Jankovic, 2005; Racette et al., 2001, 2005; Sadek et al., 2003). Further, individuals with certain medical conditions associated with liver dysfunction and total parenteral nutrition exhibit markedly increased blood Mn levels and neurological deficits (Alves et al., 1997; Burkhard et al., 2003). Therefore, increasing our understanding of the long-term effects of increases in brain Mn levels and associated neurological effects is warranted.
The globus pallidus is a brain region in which Mn preferentially accumulates and is one of the earliest brain region in which a hyperintensive signal is prominently observed using T1-weighted magnetic resonance imaging (MRI) due to the paramagnetic properties of Mn. T1-weighted MRI has been widely used to visualize the distribution and accumulation of Mn in the human brain under a variety of occupational and medically related conditions (Choi et al., 2006; Dietz et al., 2001; Iinuma et al., 2003; Josephs et al., 2005; Kim et al., 1999, 2004, 2005, 2006; Lucchini et al., 2000; Park et al., 2003; Tagaki et al., 2001), in nonhuman primate studies (Dorman et al., 2006; Eriksson et al., 1992; Newland, 1999; Newland et al., 1989; Shinotoh et al., 1995) and in rodents (Chaki et al., 2000; Fitsanakis et al., 2006; Kuo et al., 2005). Further, Mn-enhanced MRI has gained popularity to track axonal connections or map neuronal activity in experimental animals (Pautler, 2004).
To semiquantitatively measure Mn accumulation in the brain and because the globus pallidus is a brain region in which Mn is most easily observed upon visual inspection of a T1-weighted MRI image, a "pallidal index" (PI) has been devised. The PI is defined as the ratio of the globus pallidus to frontal white matter (WM) signal intensity (Krieger et al., 1995). Because T1-weighted MRI has exquisite sensitivity in detecting brain Mn accumulation and because of its potential use as a biomarker of Mn exposure, the PI has been used widely in a variety of medical and occupational settings (Choi et al., 2006; Dietz et al., 2001; Kim et al., 2006; Lucchini et al., 2000; Park et al., 2003). Despite these efforts, there is limited information in the association between Mn exposure, whole blood, and brain Mn concentrations and the PI obtained by T1-weighted MRI. Further, there is no study to date in experimental animals such as nonhuman primates that have used 1H-magnetic resonance spectroscopy (1H-MRS) to assess the potential toxic effects of chronic Mn exposure on levels of brain metabolites that are thought to reflect various aspects of neuronal and glial functioning in the brain (Jenkins et al., 1999).
The present report is part of an ongoing multidisciplinary study to assess the behavioral (cognitive and motor), neuroimaging, and neuropathological consequences of chronic exposure to Mn. We have recently shown in the same animals described in the present report, that chronic Mn exposure produces subtle deficits in fine motor function and general activity (Guilarte et al., 2006) as well as in certain aspects of cognitive function (Schneider et al., 2006). Furthermore, positron emission tomography (PET) performed in the same animals showed a marked decrease of striatal in vivo dopamine release in the absence of a change in markers of dopamine terminal integrity in the striatum (Guilarte et al., 2006). The latter suggests that at the level of Mn exposure defined in our studies, there is evidence for an intact but dysfunctional nigrostriatal dopamine system in nonhuman primates. In the present report, we describe results from longitudinal T1-weighted MRI and 1H-MRS studies before and during Mn exposure.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mn administration and general procedures.
Five normal young adult male Cynomolgus macaques (5–6 years of age at the start of the study and housed at Thomas Jefferson University, Philadelphia, PA) were used. Animals were housed one per cage in standard caging and were provided a high protein monkey diet (LabDiet, Richmond, IN) supplemented with fruits and water. All animal studies were reviewed and approved by the Johns Hopkins and the Thomas Jefferson University Animal Care and Use Committee. Three additional normal young adult animals of similar age that did not receive Mn exposure, behavioral evaluations, or imaging studies served as a comparison group to the Mn-exposed animals for postmortem analyses of the brain.
Behavioral ratings and motor function were monitored on a regular schedule, and animals were trained to perform a variety of cognitive tasks. The effects of Mn exposure on behavior and cognition have been described separately (Guilarte et al., 2006; Schneider et al., 2006). Animals were transferred to the Johns Hopkins Medical Institutions for baseline imaging studies once cognitive training was completed. Imaging studies comprised of T1-weighted MRI, proton magnetic resonance spectroscopy (1H-MRS), and PET studies. The PET results have also been reported elsewhere (Guilarte et al., 2006). Upon their return and once a stable level of behavioral performance was confirmed, Mn exposure was initiated. For injections of Mn, animals were removed from their cages using a pole and collar system, chaired, and then masked and anesthetized with isofluorane (1–3% isofluorane). Animals received injections of manganese sulfate (10–15 mg MnSO4/kg equivalent to 3.26–4.89 mg Mn/kg) into the saphenous vein under the isofluorane anesthesia approximately once per week as follows. A needle and catheter were inserted into the vein and flushed with sterile saline. A sterile and warm MnSO4 solution (50 mg/ml in isotonic physiological saline—approximately a 16.3 mg Mn/ml solution) was administered at a rate of 0.5 ml/min over a 4- to 6-min period, based on body weight. Vital signs were monitored during Mn administration. At the end of the Mn infusion, 1.0 ml of sterile saline was slowly pushed through the catheter. Animals were returned to their home cage and observed for any adverse events.
At the first imaging time point after initiation of Mn exposure (Mn-1) and the second imaging time point (Mn-2), animals received the same MRI/MRS studies as in baseline. Following the last imaging set, animals were anesthetized by ketamine injection (20–30 mg/kg), euthanized by an overdose of pentobarbital (100 mg/kg) and brains collected for postmortem studies.
Imaging Studies
General brain imaging protocol.
Animals were fasted for 12 h prior to any of the imaging studies. On the day of the study, animals were initially anesthetized by intramuscular injection of Saffan (8–10 mg/kg alfadolone and alfaxolone acetate; Pitman-Moore, Middlesex, UK). Two iv catheters were implanted for infusion of anesthesia and injection of radiotracer, respectively. Anesthesia was maintained throughout the study by a continuous iv infusion drip of 6–9 mg/kg/h Saffan. During the preparation of the animal and MRI/MRS studies, an experienced veterinarian technologist continuously monitored anesthesia, pulse, blood pressure, and oxygen saturation for the duration of the studies and remained with the animal until the animal was awake and moving.
Magnetic resonance imaging and spectroscopy.
All experiments were performed on a 1.5T General Electric "Signa" scanner. Sagittal and axial T1-weighted images were recorded using a linear transmit-receive "extremity" coil, which provides a uniform radio frequency field over the volume of the monkey brain. The sagittal T1 images were used for localization purposes and visualization of anatomical structures, while the axial T1 images were used to estimate regional brain Mn concentrations using the methods (i.e., PI) described by Krieger et al., (1995). T1-MRI sequence parameters were TR 400 ms, TE 9 ms, matrix size 192 x 256, slice thickness/gap 2.5/0.5 mm, field of view was 9 x 12 cm, four signal averages, scan time 3 min 53 s. After recording these images, the extremity coil was removed and replaced with a 3 in. receive-only surface coil for MRS (which has higher sensitivity), without moving the head of the animal, which remained fixed in the animal holder (cradle) at all times.
T1-weighted MRI signal intensity was measured in bilateral regions of interest (ROIs) drawn using a standard anatomical template using the program ImageJ. PI values (expressed relative to centrum semiovale WM, and adjacent pericranial muscle) for parenchyma in key ROIs were calculated at each of the imaging time points. Selected ROIs included pituitary, cerebellar WM, globus pallidus, frontal WM, caudate, putamen, substantia nigra and thalamus.
Short echo time single voxel spectroscopy.
Short echo time single voxel spectra from 1 cm3 (1 x 1 x 1 cm voxel size) voxel elements were recorded in striatum, thalamus, parietal cortex, and frontal WM using the "PRESS" ("PROBE-P", GE Medical Systems, Milwaukee, WI) pulse sequence (the globus pallidus was not measured because it is not readily amenable to MRS due to its high iron content which causes excessive line broadening). Sequence parameters were TR = 3 s, TE = 35 ms, 128 averages, for a scan time of 7 min 12 s. Flip angle was 90°, bandwidth 1 kHz, 1024 data points. Spectra were processed with 3 Hz line broadening prior to Fourier transformation. The chosen TR and TE values mean that the spectra are relatively insensitive to any potential changes in metabolite T1 and T2 values that may occur near regions of Mn deposition. A spectrum without water suppression was acquired with 16 averages and the same timing parameters. The water signal was used to correct eddy currents in the water-suppressed (metabolite) spectra and for spectral quantification. The second half of the spin echo was sampled by 2048 data points with a sampling frequency of 2500 Hz. Analysis and quantification of single voxel data were performed using the LCModel (Version 6.0.S.W.Provencher).
Metal analysis in brain tissue using inductively coupled plasma mass spectrometry.
Concentrated nitric acid (HNO3) (Suprapur, Merck, Darmstadt, Germany) was added to the lyophilized samples in the following amounts: 0.5 ml HNO3 for samples less than 0.05 g tissue, 1 ml HNO3 for 0.05–0.15 g tissue. Samples were placed at room temperature for 24 h prior to digestion either on a heatblock (QBT4, Grant, Cambridge, UK) for 3 h at 70°C or using a microwave oven (Multiwave 3000, Anton Paar, Ashland, VA) using ramp 200 W for 10 min and then held for 10 min. Samples were then diluted with 0.6M HNO3 with 18.2 M
water. Postmortem brain samples were analyzed for trace element content by inductively coupled plasma mass spectrometry using a Thermo-Finnigan model Element 2 instrument (Bremen, Germany), as previously published (Erikson et al., 2004) except that RF-power was set at 1250 W. Briefly, each sample was introduced using a CETAC ASX 510 autosampler (Omaha, NE) with a peristaltic pump (1 ml/min). The instrument is equipped with a concentric Meinhard nebulizer connected to a Scott spray chamber, and a quartz burner with a guard electrode. The nebulizer argon gas flow rate is adjusted daily to give a stable signal with maximum intensity for the nuclide 115In and 238U. The instrument is calibrated using 0.6M HNO3 solutions of multielement standards at appropriate concentrations. After each sample 0.1M HNO3 was flushed through the sample introduction system to reduce memory effects. To check for possible drift in the instrument, a standard solution with known elemental concentrations was analyzed for every 10 samples. In addition, blank samples (0.6M HNO3) were analyzed for approximately every 10 samples. 55Mn, 57Fe, 63Cu, and 67Zn were measured at medium resolution.
Statistics.
Statistical analysis of MRI/MRS results was performed using linear regression with clustering on animal using individual untransformed data to account for repeated measures on the same animal over time using the statistical program STATA version 7.0 (Stata Corporation, College Station, TX). Two-way ANOVA was used on metal concentrations in the brain. Residual analysis justified the use of regression techniques. Student's t-test was used for comparison between two means. Statistical significance was set at p < 0.05.
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RESULTS |
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Cumulative Mn Dose, Time of Exposure, and General Characteristics of Animals at Termination of Studies
The experimental design for behavioral training, Mn exposure, and brain imaging has been described (Guilarte et al., 2006
). Briefly, the cumulative Mn dose from initiation of Mn administration to the first (Mn-1) and second (Mn-2) post-Mn imaging sets were (mean ± SEM): 84.2 ± 7.8 and 156.7 ± 9.5 mg Mn/kg body weight, respectively. The time from initiation of Mn administration to Mn-1 and from Mn-1 to Mn-2 were: 128 ± 12.4 and 157 ± 9.7 days, respectively. The time from the last imaging set (i.e., Mn-2) to euthanasia was 33 ± 8.2 days. No significant differences in body (in kilogram; control: 5.8 ± 0.3 vs. Mn-exposed: 6.5 ± 0.3; p > 0.05) or brain weight (in gram; control: 69.0 ± 1.5 vs. Mn-exposed: 66.0 ± 3.9; p > 0.05) were observed between control naive and Mn-exposed animals at the end of the study. The values provided are the mean ± SEM of n = 4 Mn-exposed animals and n = 3 controls.
T1-Weighted MRI
We used T1-weighted MRI to visualize the distribution of Mn in the nonhuman primate brain in a longitudinal fashion. Figure 1 shows the T1-weighted MRI images of one animal before and after the first post-Mn imaging time point (Mn-1). The figure shows bilateral hyperintensive signals at different levels of the brain consistent with increased Mn accumulation resulting from the cumulative exposure. Although a strong hyperintensive signal was prominent in the globus pallidus, hyperintensive signals were also observed in many other brain regions and in the pituitary gland (Fig. 1). These findings suggest that Mn accumulation is not limited to the basal ganglia and other cortical and subcortical structures also accumulate Mn.
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To semiquantitatively measure the level of Mn accumulation in the brain of Mn-exposed nonhuman primates, we used the equivalent of the PI. The PI has been defined as the ratio of the T1-weighted signal intensity from the globus pallidus to the frontal WM signal (Krieger et al., 1995
). We have used this approach to obtain a "PI equivalent" in which the signal intensity from a particular ROI is divided by the signal intensity from the frontal WM. Figure 2A shows PI equivalent values for the different brain regions at baseline and at the Mn-1 and Mn-2 time points. The results indicate that although small increases in PI equivalent were apparent in several brain regions including the globus pallidus, caudate, putamen, and substantia nigra, only the pituitary gland reached statistically significantly higher ratios during Mn exposure relative to baseline (Fig. 2A). Importantly, with Mn exposure, there was a highly significant decrease in the PI equivalent ratio for pericranial muscle relative to baseline. These findings suggest that the T1-weighted signal in WM increases during Mn administration (Fig. 2A). This observation is consistent with hyperintensive signals present in the WM after Mn administration by comparing the baseline image against the post Mn exposure image (Fig. 1). In fact, the images in Figure 1 clearly show that Mn is present throughout the brain as the resolution of the images, including cortical structures, have significantly sharper definition after Mn exposure than at baseline consistent with the ability of Mn to enhance image resolution (Fig. 1).
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In the present studies, we routinely sampled the signal intensity from pericranial muscle in the same image in which the frontal WM signal was sampled, in order to use it as a reference area outside of the brain. Based on the above findings, we expressed the T1-weighted MRI data using the signal intensity from pericranial muscle as the denominator rather than the frontal WM signal since the signal from the pericranial muscle did not appear to change significantly before and during Mn exposure. Figure 2B shows that using this ROI/pericranial muscle approach revealed highly significant increases in the ROI/pericranial muscle ratios during Mn administration in all brain regions examined at both the Mn-1 and Mn-2 time points relative to baseline. Based on the ROI/pericranial muscle ratios, the rank order of Mn accumulation in the brain are: pituitary > globus pallidus > substantia nigra = putamen = caudate = frontal WM = cerebellar WM > thalamus.
To confirm the in vivo MRI findings, we measured Mn levels in the globus pallidus, caudate, putamen, and frontal WM of control and Mn-exposed animals. Table 1 shows that Mn levels increased by 4.6-fold in the globus pallidus, 3.2-fold in the caudate, 3.1-fold in putamen, and 3.3-fold in frontal WM of Mn-treated animals relative to controls. This observation confirms that the increase in Mn levels in frontal WM is likely responsible for the relatively small changes in PI equivalent after Mn exposure relative to baseline.
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We also determine the degree of correlation between regional brain Mn concentrations measured postmortem with the last T1-weighted MRI expressed as either a PI equivalent or ROI/pericranial muscle ratio. We found that while statistically significant linear correlations were present between brain Mn concentrations and both the PI equivalent (r2 = 0.39; p = 0.01) or the ROI/muscle ratios (r2 = 0.26; p = 0.049) (Fig. 3), the correlations were not very strong, most likely due to the length of time that passed between the last MRI study and when the animals were euthanized.
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1H-Magnetic Resonance Spectroscopy
1H-MRS was used to measure the concentrations of the brain metabolites creatine (Cr), N-acetylaspartate (NAA), choline (Cho), and myo-inositol (mI) in the parietal cortex, striatum, thalamus, and frontal WM. The globus pallidus, a toxicological relevant brain region, was not assessed because it is not readily amenable to MRS due to its high iron content which causes excessive line broadening. The Cr signal was used to normalize the levels of the other metabolites as a ratio as is commonly done in this type of studies. Figure 4A shows the 1H-MR spectra in the parietal cortex of one of the animals at baseline and at the Mn-2 time point, respectively. It shows a smaller NAA peak in the Mn-2 spectra relative to the baseline spectra of the same animal. Statistical analysis of the longitudinal data indicates that while no significant effect in the NAA/Cr ratio was present in the parietal cortex of Mn-exposed animals at the Mn-1 time point, a significant decrease (p = 0.028) was measured at the Mn-2 time point relative to baseline values (Fig. 4B). No significant effect was measured in any other brain region for any of the metabolites or in the parietal cortex for the Cho/Cr and mI/Cr ratios (data not shown). However, a near significant (p = 0.055) decrease in NAA/Cr was found in frontal WM at the Mn-2 time point (Baseline: 1.10 ± 0.01; Mn-1: 1.16 ± 0.10; and Mn-2: 0.96 ± 0.01).
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Metal Analysis in Whole Blood during Mn Exposure and Postmortem Brain Mn Concentrations
We measured the concentration of Mn in whole blood at baseline, on the Mn-1 and Mn-2 time points and at time of euthanizing the animals (termination of study). Postmortem analysis of tissue Mn was performed in the caudate, putamen, globus pallidus, and frontal WM of Mn-exposed and control animals as previously noted. First, as depicted in Table 1, Mn levels were significantly increased in the caudate, putamen, globus pallidus, and WM relative to controls. Consistent with what is known about brain Mn accumulation, the greatest fold increase in Mn levels occurred in the globus pallidus. Further, frontal WM concentrations of Mn-exposed animals increased significantly and to the same extent as in caudate and putamen (Table 1). Whole blood Mn concentrations were significantly increased during Mn exposure relative to baseline (Table 2). It should be noted that the blood measurement for one of the animals at baseline was considered, based on statistical criteria, to be an outlier possibly due to contamination and was excluded from the final baseline results.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, we have assessed metabolite levels and Mn distribution in the brain of nonhuman primates using 1H-MRS and T1-weighted MRI before and during chronic Mn exposure. The present report is part of a larger study in which these same animals were documented to express subtle deficits in motor function and impairment of in vivo dopamine release in the striatum as measured by PET (Guilarte et al., 2006
) as well as small changes in some measures of cognitive function (Schneider et al., 2006). We now describe that 1H-MRS performed in the same animals reveal a significant decrease in the NAA/Cr ratio in the parietal cortex and a near significant effect (p = 0.055) on the NAA/Cr ratio in the frontal WM with cumulative Mn exposure at the Mn-2 time point relative to baseline. No significant changes in metabolite ratios were measured in the striatum. The latter is consistent with a recent report by Kim et al. (2006) in which they also find no significant changes in the metabolite ratios in the basal ganglia of welders with elevated blood Mn levels. We should note, however, that of the brain regions examined in our study, the parietal cortex is the most favorable region for 1H-MRS because it has the best field homogeneity and it is closest to the surface receiver coil. Thus, it is a brain region in which the method is best able to detect changes in metabolite levels. For this reason, we cannot make firm conclusions about the regional dependence of Mn neurotoxicity on metabolite levels. However, the reductions in NAA/Cr ratio in the parietal cortex indicates that at least in this cortical region Mn produces changes that may reflect neuronal loss and/or dysfunction. There is experimental evidence that in nondemented PD patients there is a decrease in the NAA/Cr ratio in the temporoparietal cortex (Hu et al., 1999), and neuronal connections from the parietal cortex to the caudate and putamen have been documented in nonhuman primates (Yeterian and Pandya, 1993). However, the reason for the Mn-induced decrease in parietal cortex NAA/Cr ratios is not currently known and future studies need to investigate this anatomical relationship and its neurological relevance in the context of Mn neurotoxicity.
One question raised by the Mn-induced decrease in NAA/Cr ratio in the parietal cortex is whether this change represents neuronal loss or dysfunction. NAA is a brain metabolite that is localized in neurons (Demougeot et al., 2001), and studies have shown that the NAA/Cr ratio is decreased in neurological disease states that involve neuronal loss or dysfunction (Block et al., 2002; Clark, 1998; Demougeot et al., 2001; Jenkins and Kraft, 1999). Thus, it is thought that NAA in the brain reflects levels of neuronal density and/or health (Block et al., 2002; Jenkins and Kraft, 1999). Deficits in mitochondrial function have also been shown to reduce NAA levels in the absence of cell loss (Jenkins and Kraft, 1999). Since Mn is known to inhibit mitochondrial function (Hamai and Bondy, 2004), this may contribute to the decrease in the NAA/Cr ratio measured in the parietal cortex of Mn-exposed animals. Thus, our findings point to the possibility of an ongoing neuronal degeneration or at least to some degree of neuronal dysfunction in the parietal cortex.
The T1-weighted MRI studies have also provided valuable information on the applicability of this in vivo imaging technique in Mn neurotoxicity. First, we found that although T1-weighted MRI is a sensitive method to detect Mn in the brain, its expression as a PI equivalent is insensitive to changing brain Mn concentrations (Fig. 2A). This is based on the fact that Mn also accumulates in frontal WM, the brain region used as the denominator in the PI calculation. This was confirmed by postmortem analysis of Mn concentrations in the brain where we found a 3.4-fold increase in WM Mn concentration in exposed animals relative to controls (Table 1). The fold change in WM Mn concentration is similar to those measured in caudate and putamen and slightly less than globus pallidus. These findings may help explain reports in the human literature in which the PI is minimally affected despite significant increases in blood Mn concentrations (Kim et al., 2005). Additional supporting evidence to our findings comes from a recent report by Dorman et al. (2006) in which they also found that Mn accumulates in the WM and produces an underestimation of the PI with increasing levels of Mn exposure. Further, a manuscript by Choi et al. (2006) indicates that the validity of the PI in reflecting brain Mn levels is only accurate in a limited range. In summary, the emerging evidence clearly suggests that the PI may have limitations in its use as a biomarker of Mn exposure. In this context, we found that using pericranial muscle rather than frontal WM as a reference region on the T1-weighted MRI provided a better estimate of regional brain Mn accumulation (Fig. 2). Although this approach has obvious limitations, it more closely estimated the change in brain Mn accumulation than the PI.
Finally, it is important to put into perspective the level of Mn exposure in our current study with those described in environmental or occupational exposures in humans (Gulson et al., 2006). Table 2 shows that blood Mn levels in our animals at the various time points during Mn administration averaged from 42.6 to 67.1 µg/l and ranged from 14.6 to 106.1 µg/l (Table 1). The mean whole blood Mn level at baseline prior to exposure was 9.2 and ranged from 5.1 to 14.2 µg/l. Gulson et al. (2006) have recently provided whole blood Mn levels from a number of studies worldwide in which environmental exposures to the general population or occupationally related exposures have been documented. In this table, the mean average of environmentally exposed individuals of the different studies (defined as nonexposed in the table) ranged from 5.7 to 33.9 µg/l. In their own study of children (Gulson et al., 2006), mean whole blood Mn was 12.3 and 12.2 µg/l for females and males, respectively, with a range from 1.8 to 45.0 µg/l. Takser et al. (2003) in a study of mothers and children at birth have documented mean blood levels as high as 38.6 µg/l with a range from 6.1 to 151.2 µg/l in mothers and 14.9 to 71.2 µg/l in newborns. In a study of the general population in two communities living within a Mn-mining district in Mexico, blood Mn concentrations ranged from 7.5 to 88.0 µg/l with a median concentration of 15 (Santos-Burgoa et al., 2001). Rollin et al. (2005) also reported blood Mn concentrations in the 1.6–32.8 µg/l range in children from two different cities in South Africa. Therefore, it is clear that individuals in the general population are exposed to Mn in their living environment and exhibit blood Mn levels within the range of those achieved in our animals. This suggests that the behavioral and neuroimaging changes observed in the present report and in our other recent publications (Guilarte et al., 2006; Schneider et al., 2006) are likely to occur in individuals under specific living conditions.
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ACKNOWLEDGMENTS |
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This work was supported by a grant from the National Institute of Environmental Health Sciences ES10975 to T.R.G.
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